Density-dependent microbial gene switch

Abstract

Genetically engineered bacteria which express RNAs or proteins that produce or represent agriculturally useful compounds including fertilizers and plant nutrients upon increases in cell density when grown in plant growth media such as soil are disclosed. Also disclosed are genetically engineered bacteria where expression of RNAs or proteins that produce or represent agriculturally useful compounds including fertilizers and plant nutrients can be inhibited upon reaching increased cell densities when grown in a fermenter or bioreactor.

Description

DENSITY-DEPENDENT MICROBIAL GENE SWITCH CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This international patent application is a continuation-in-part of international patent application PCT / US25 / 14079, filed January 31, 2025, and claims benefit of U.S. Provisional Patent Application Serial Nos.: 63 / 553,528, filed February 14, 2024, and 63 / 552,631, filed February 12, 2024, which is each incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] Not applicable. INCORPORATION OF SEQUENCE LISTING

[0003] A sequence listing contained in the xml file named “P14610WO00.xml” which is 572,876 bytes in size (measured in MS-Windows), which was created on February 9, 2025, and which comprises 400 sequences, is electronically incorporated herein by reference in its entirety. A sequence listing contained in the xml file named “P14610US01.xml” which is 562,836 bytes in size (measured in MS-Windows), which was created on February 14, 2024, and which comprises 389 sequences, is also incorporated herein by reference in its entirety. BACKGROUND

[0004] The expression of certain proteins or production of certain metabolites can be inhibitory to a host strain and reduce its growth rate. Under lab conditions this issue can be overcome by using inducible expression systems with chemical inducers such as tetracycline. However, for bacteria in their natural environments (e.g., soil-borne bacteria in soil) such systems are not suitable either because these inducers are not compatible with environmental requirements and laws, are very expensive, or can simply not reach the host (e.g., in soil). For example, bacteria that are producing and releasing large quantities of ammonia which can be used by crop plants suffer a marked fitness defect that would render them non-competitive and unable to persist in the environment. Therefore, there is a need to provide alternative solutions which allow expression of proteins or production of certain metabolites in bacteria in their natural environments after a desirable delay and / or under certain conditions.Atty. Dkt. No. P14610WO00 SUMMARY

[0005] Genetically engineered bacterium comprising: (a) one or more heterologous gene expression cassette(s) comprising at least one control element which is operably linked to at least one nucleic acid sequence encoding a first quorum sensing synthase protein (QSSP) able to synthesize a quorum sensing signal molecule Q (QSSM Q) and / or at least one nucleic acid sequence encoding a first quorum sensing regulator protein (QSRP) that can bind said QSSM Q; (b) a heterologous gene expression cassette comprising at least one nucleic acid sequence encoding at least one RNA sequence or protein of interest operably linked to a control element comprising a first quorum sensing (QS) promoter, wherein the first quorum sensing promoter is activated by the first quorum sensing regulator protein (QSRP) and the QSSM Q when the population density of the genetically engineered bacterium exceeds a threshold population density and wherein said at least one RNA sequence or protein of interest is or causes the production of at least one agriculturally relevant compound; and (c) at least one heterologous deactivation gene expression cassette comprising a DNA promoter which is operably linked to one or more deactivator(s) which inhibit(s) expression of the at least one RNA sequence or protein of interest at a population density of the genetically engineered bacterium which exceeds the threshold population density when the genetically engineered bacterium (GEB) is: (i) contacted with a quorum quenching compound QQ; or (ii) exposed to a temperature above a threshold temperature are provided.

[0006] Compositions comprising the genetically engineered bacterium and an agriculturally acceptable carrier are also provided.

[0007] Plant parts or plant propagules which are at least partially coated, imbibed, or mixed with the compositions are also provided. Use of the plant parts or plant propagules which are at least partially coated, imbibed, or mixed with the compositions to grow a crop are also provided.

[0008] Agricultural systems comprising: (i) at least one of the engineered bacteria; (ii) at least one plant growth medium; and (iii) at least one crop plant, crop plant seed, or crop plant vegetative propagule; wherein the plant growth medium, crop plant, crop seed, and / or crop plant propagule comprise, are at least partially coated with, imbibed with, and / or are mixed with the engineered bacterium or a composition comprising the engineered bacterium and an agriculturally acceptable carrier, are provided.

[0009] Treated plant seed or plant propagule systems comprising: (i) at least one crop plant seed or crop plant vegetative propagule; and (ii) at least one of the engineered bacteria, wherein the crop plant seed or crop plant propagule are at least partially coated, imbibed, and / or mixed with the engineered bacterium or a composition comprising the engineered bacterium and an agriculturally acceptable carrier, are provided.

[0010] Methods of producing a bacterial culture comprising: (i) growing the genetically engineered bacterium either: (a) in contact with the quorum quenching compound QQ; or (b) in exposure to a temperature above the threshold temperature; and (ii) harvesting the bacterial culture are provided.Atty. Dkt. No. P14610WO00

[0011] Methods of providing at least one agriculturally relevant compound to a plant comprising placing at least one of the genetically engineered bacteria into a plant growth medium, wherein said at least one RNA sequence or protein of interest is or causes the production of said at least one agriculturally relevant compound when the population density of the genetically engineered bacterium exceeds a threshold population density in the plant growth medium and activates expression of said at least one RNA sequence or protein of interest are provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows a gene expression cassette for measuring expression of a green fluorescent protein reporter operably linked to a control element comprising a quorum sensing (QS) promoter. The cognate QSRP transcription factor is expressed constitutively.

[0013] Figure 2 shows a gene expression cassette for measuring expression of a green fluorescent protein (GFP) reporter operably linked to a control element comprising a quorum sensing (QS) promoter. The GFP reporter is expressed when the population of engineered bacteria expressing the QS promoter’s cognate QSSP and QSRP reach a threshold population density in the bacterial growth medium.

[0014] Figure 3 shows the experimental logic for assessment of population density-dependent induction by QS promoters.

[0015] Figure 4 shows population density-dependent induction by strains comprising two heterologous gene expression cassettes: (i) one comprising genes encoding a quorum sensing synthase protein (QSSP) and a quorum sensing regulator protein (QSRP) and (ii) one encoding a GFP reporter protein of interest operably linked to the cognate QS promoters of the QSSP and QSRP in (i). Rows of the figure each contain strains of a specific bacterial host species. Ab = Azospirillum brasilense, Kr = Kosakonia radicincitans; and Ps = Pseudomonas stutzeri. Columns of the figure each show bacterial strains engineered with specific quorum sensing systems. PahlI is the promoter of the AhlRI quorum sensing system, PcinI is the promoter of the CinRI quorum sensing system, and PlasB and PlasI are promoters of the LasRI quorum sensing system. The black “QS” data series came from strains comprising both heterologous gene expression cassettes (i) and (ii) while the gray “WT” data series came from strains comprising only cassette (ii) and missing the cassette encoding the QSSP and QSRP.

[0016] Figure 5 shows bacterial cell counts for cultures grown in + / - 20 mM lactate (for Ab and Ps) or 20 mM glucose (for Kr), as determined by flow cytometry.

[0017] Figure 6 shows relative expression in culture (defined as inducible GFP fluorescence / constitutive LSSmScarlett fluorescence) for strains carrying heterologous QS systems grown in the presence / absence of carbon, as determined by flow cytometry.

[0018] Figure 7 shows bacterial cell counts for bacteria isolated from bulk soil (BS) or from the corn-root rhizoplane (RP), as determined by flow cytometry. Engineered bacterial strains of Azospirillum brasilense (Ab) and Kosakonia radicincitans (Kr) comprised either the cinRI QS system (CinI QSSP, CinR QSRP, andAtty. Dkt. No. P14610WO00 GFP operably linked to PcinI) or the ahlRI QS system (AhlI QSSP, AhlR QSRP, and GFP operably linked to PahlI).

[0019] Figure 8 shows relative expression (defined as inducible GFP fluorescence / constitutive LSSmScarlett fluorescence) for bacteria isolated from bulk soil (BS) or from the corn-root rhizoplane (RP), as determined by flow cytometry. Engineered bacterial strains of Azospirillum brasilense (Ab) and Kosakonia radicincitans (Kr) comprised either the cinRI QS system (CinI QSSP, CinR QSRP, and GFP operably linked to PcinI) or the ahlRI QS system (AhlI QSSP, AhlR QSRP, and GFP operably linked to PahlI).

[0020] Figure 9 shows a system of gene expression cassettes for a quorum sensing system that can be deactivated during fermentation using quorum quenching.

[0021] Figure 10 shows engineered K. radicincitans soil bacteria that exhibit population density-dependent expression of a GFP protein of interest in a plant growth medium that can be conditionally deactivated by contact with an anhydrotetracycline (aTc) quorum quenching compound in culture. The threshold population density for expression of the protein of interest is about 1 * 108cells / g root.

[0022] Figure 11 shows a schematic of constructs with different ribosome binding sites for tuning the expression of a QSRP for density-dependent expression from a QS promoter in engineered systems. The construct comprises two gene expression cassettes. In the first gene expression cassette, a reporter gene (GFP) is operably linked to a quorum sensing promoter (PQS) under control of the ribosome binding site BCD 2. In the second gene expression cassette, the QSRP-expressing gene (Reg.) is operably linked to an inducible promoter (Ptet) under control of ribosome binding sties of differing strength (here BCD17 and BCD22).

[0023] Figure 12 shows the effect of operably linked ribosome binding site on QSRP-mediated expression of a reporter protein of interest. Results for 0.1 µM AHL are at left, for 1 µM AHL are in middle, and for 10 µM AHL are at right for each tested ribosome binding site.

[0024] Figure 13 shows a schematic of constructs with different ribosome binding sites both for tuning the expression of a QSRP and for tuning the expression of a QSSP to alter the rate of AHL production. Each construct comprises three gene expression cassettes. In the first gene expression cassette, a reporter gene (GFP) is operably linked to a quorum sensing promoter (PQS) under control of the ribosome binding site BCD 2. In the second gene expression cassette, the QSRP-expressing gene (Reg.) is operably linked to a repressible promoter (Ptet) under control of ribosome binding sties of differing strength (here BCD17 and BCD22). In the third gene expression cassette, the QSSP-expressing gene (Synthase) is operably linked to a quorum sensing promoter (PQS) under control of ribosome binding sties of differing strength (here BCD1 and BCD8). If strains comprise a separate DNA construct expressing the conditional transcriptional repressor rTetR, addition of the quorum quenching compound (in this system anhydrotetracycline) suppresses expression of the QSRP.Atty. Dkt. No. P14610WO00

[0025] Figures 14A shows cell density (OD600) and cell density-normalized fluorescence over time in Kosakonia radicincitans grown in liquid culture and engineered with a repressible quorum sensing circuit, wherein the QSSP is operably linked to BCD1 and the QRSP is operably linked to BCD22 (ST2712). QQ+ indicates a condition with a quorum quenching compound to suppress GFP protein of interest expression. QQ- indicates a condition without a quorum quenching compound to suppress GFP protein of interest expression.

[0026] Figure 14B shows cell density (OD600) and cell density-normalized fluorescence over time in Kosakonia radicincitans grown in liquid culture and engineered with a repressible quorum sensing circuit, wherein the QSSP is operably linked to BCD1 and the QRSP is operably linked to BCD17 (ST2713). QQ+ indicates a condition with a quorum quenching compound to suppress GFP protein of interest expression. QQ- indicates a condition without a quorum quenching compound to suppress GFP protein of interest expression.

[0027] Figure 14C shows cell density (OD600) and cell density-normalized fluorescence over time in Kosakonia radicincitans grown in liquid culture and engineered with a repressible quorum sensing circuit, wherein the QSSP is operably linked to BCD8 and the QRSP is operably linked to BCD17 (ST2714). QQ+ indicates a condition with a quorum quenching compound to suppress GFP protein of interest expression. QQ- indicates a condition without a quorum quenching compound to suppress GFP protein of interest expression.

[0028] Figure 14D shows cell density (OD600) and cell density-normalized fluorescence over time in Kosakonia radicincitans grown in liquid culture and engineered with a repressible quorum sensing circuit, wherein the QSSP is operably linked to BCD8 and the QRSP is operably linked to BCD22 (ST2715). QQ+ indicates a condition with a quorum quenching compound to suppress GFP protein of interest expression. QQ- indicates a condition without a quorum quenching compound to suppress GFP protein of interest expression.

[0029] Figure 15 shows bacterial cell counts for bacteria isolated from bulk soil (BS) or from the corn-root rhizoplane (RP), as determined by flow cytometry. Engineered bacterial strains of Kosakonia radicincitans (Kr) comprising an ahlRI QS system (AhlI QSSP, AhlR QSRP, and GFP operably linked to PahlI) that can be deactivated by a quorum quenching compound-induced transcriptional deactivator were tested.

[0030] Figure 16 shows relative expression (defined as inducible GFP fluorescence / constitutive LSSmScarlett fluorescence) for bacteria isolated from bulk soil (BS) or from the corn-root rhizoplane (RP), as determined by flow cytometry. Engineered bacterial strains of Kosakonia radicincitans (Kr) comprising an ahlRI QS system (AhlI QSSP, AhlR QSRP, and GFP operably linked to PahlI) that can be deactivated by a quorum quenching compound-induced transcriptional deactivator were tested.

[0031] Figure 17 shows metabolism and regulation of bacterial nitrogen fixation.

[0032] Figure 18 shows a system of gene expression cassettes for a quorum sensing system that can be deactivated during fermentation using quorum quenching. In this system, a constitutively expressed reverseAtty. Dkt. No. P14610WO00 Tet repressor inhibits expression of the AhlR QSRP in the presence of anhydrotetracycline (aTc). In the absence of aTc, the AhlR QSRP, AhlI QSSP, and protein of interest (e.g., SfGFP, PhiC rec (e.g., PhiC31 integrase), or a uAT are induced. DETAILED DESCRIPTION

[0033] Methods, genetically engineered bacteria (GEB), and related systems which use quorum sensing promoters (QS-P) which are conditionally expressed in plant growth media and / or in association with plants when the GEB exceed threshold population densities to drive expression of proteins or RNAs of interest which are agriculturally relevant or which cause the production of at least one agriculturally relevant compound are disclosed herein. In certain embodiments, GEB provided herein will typically and advantageously be below the threshold population density which results in expression of the proteins or RNAs of interest following their placement in plant growth media including soil, permitting more rapid and / or more extensive (e.g., higher density) growth in the plant growth media and / or colonization of crop plants grown therein. The desired production of the proteins, RNAs, and / or agriculturally relevant compounds by the GEB occurs once the GEB exceed certain threshold population densities (e.g. about 6 x 103CFU / mL to about 5 x 108CFU / mL). As growth of the GEB to the threshold population density takes time, a desired delay in production of proteins, RNAs, and / or agriculturally relevant compounds by the GEB is achieved. In certain embodiments, the genetically engineered bacteria are placed in the plant growth medium (e.g., soil) and then grow to a higher titer until the threshold population density is reached and expression of the protein or RNA of interest which is or causes the production of an agriculturally relevant compound is switched on. In comparison, other bacteria which have the protein or RNA of interest under the control of another promoter which is not quorum sensing (e.g., a constitutive promoter) will express the protein, RNA, and / or compound before and / or shortly after placement in the plant growth media and / or in association with plants and will not grow to the higher titers achieved by the genetically engineered bacteria provided herein. Higher titers of the engineered bacteria provided herein can thus provide for higher titers of the desired proteins, RNAs, and / or agriculturally relevant compounds in the plant growth media and / or in association with plants in comparison to bacteria lacking the quorum sensing promoter-controlled genes. In certain embodiments, QS-P regulated genes encoding the RNAs and proteins of interest are activated on plant roots and in the rhizosphere (millimeters from plant roots) where cell density is high, but not in bulk soil (far from plant roots) where cell density is comparatively low.

[0034] Another aspect of the methods and GEB provided herein are genetic systems which can selectively inhibit expression of the proteins or RNAs of interest when the GEB are grown to densities above the threshold population density in fermenters or bioreactors. Such selective inhibition of expression the RNAs and proteins of interest can permit more rapid and economicalAtty. Dkt. No. P14610WO00 production of high-titer GEB cultures used to prepare compositions for agricultural use. Thus, the density-dependent genetic circuit can provide for desired expression of the RNA or protein of interest when the GEB are in plant growth media and / or associated with a plant and for selective inhibition of undesired expression of the RNA or protein of interest when the GEB are in a fermenter or bioreactor.

[0035] Quorum sensing promoter-controlled gene expression systems provided herein are especially useful for the production of ammonia by bacteria which can act as bio-fertilizers. Constitutively active ammonia release can significantly inhibit bacteria growth as protein synthesis is effectively shut down. To obtain effective biofertilizers, bacteria provided herein are engineered to grow first to a high density in soil before the ammonia release mechanism is activated. The quorum sensing gene expression systems provided herein allow a desirable delay in ammonia production. Definitions

[0036] As used herein, the terms “about” or “approximately” indicate values slightly above or below the cited values, e.g., plus or minus 0.1% to 10% of the cited value.

[0037] As used herein, the phrase “agriculturally relevant compound” refers to a compound that provides for plant growth, altered plant morphology (e.g., increased branching / surface area of root systems), plant nutrition, plant growth regulation, or plant protection from abiotic (e.g., drought, salinity, cold, heat, or excess water stress) or biotic stress (e.g., plant pests including bacterial, fungal, insect, nematode, and viral plant pests).

[0038] As used herein, the phrase “agriculturally useful bacteria” or acronym “AUB” refers to bacteria which can grow in plant growth media (e.g., soil) and / or which can colonize crop plants.

[0039] The term "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0040] As used herein, the phrase “constitutive promoter” refers to a promoter, which is active under most growth and / or stationary phase conditions (e.g., in biofilms) in a given organism. Constitutive promoters include the promoters of SEQ ID NO: 390-400 and variants thereof having at least 90%, 95% or 99% sequence identity thereto.

[0041] As used herein, the phrase “control element” refers to a promoter, a 5’ untranslated region (5’ UTR), a ribosome binding site, an enhancer, an insulator, a silencer, or a terminator. Control elements comprising a promoter, 5’ UTR, an enhancer, an insulator, and / or a silencer can containAtty. Dkt. No. P14610WO00 transcriptional repressor binding sites, transcriptional activator binding sites, ribozymes, protein recognition sites, and / or sites for chemical modification of nucleobases.

[0042] The term “gene,” as used herein, refers to a hereditary unit consisting of a sequence of DNA located on a chromosome, plasmid, or other extra-chromosomal element that contains the genetic instruction for a particular characteristic or trait in an organism. The term “gene” thus includes a nucleic acid (for example, DNA or RNA) sequence that comprises coding and / or non-coding sequences necessary for the production of an RNA, a polypeptide, or a precursor of the RNA or protein. A functional polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, DNA-binding activity, transcriptional activation, transcriptional repression, pesticidal activity, ligand binding, and / or signal transduction) of the polypeptide are retained.

[0043] The term “heterologous” is used herein to refer to any polynucleotide (e.g., DNA molecule) that has been introduced into a microorganism (e.g., a bacterium) where the polynucleotide is not sourced from the microorganism and / or has been inserted into a new location (e.g., in a distinct DNA sequence in the chromosome, plasmid, or other extrachromosomal element) in the microorganism (e.g., the bacterium). Non-limiting examples of heterologous DNA molecules that can be introduced into a microorganism include a non-naturally occurring (e.g., synthetic and / or recombinant) DNA molecule, a DNA molecule found in another microorganism (e.g., an intergeneric transfer of a DNA molecule comprising DNA from an organism of a different taxonomic genus), a DNA molecule found in another species (e.g., an intrageneric transfer of a DNA molecule comprising DNA from an organism of the same taxonomic genus), a DNA molecule found in a different location in the same species, and / or a DNA molecule found in the same strain or isolate of a species, where the DNA molecule has been inserted at a new location.

[0044] As used herein, the terms “include,” “includes,” and “including” are to be construed as at least having the features or items to which they refer while not excluding any additional unspecified features or items.

[0045] Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5’ to 3’ direction. Nucleic acid sequences may be provided as DNA or as RNA, as specified; disclosure of a DNA or an RNA obtained therefrom also defines the exact complement of that DNA or RNA.

[0046] As used herein, the term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is or can be regulated by the other. For instance, a promoter is operably linked to an RNA or protein coding sequence if the promoter provides for transcription of the RNA or an mRNA encoding the protein.

[0047] As used herein, the term “phosphate,” when used in the context of phosphate concentration in a plant growth medium and / or phosphate concentrations which can activate a phosphate-sensitive promoter, refers to inorganic and soluble phosphate which is available to plants and microorganisms.Atty. Dkt. No. P14610WO00

[0048] As used herein, the phrase “phosphate-sensitive promoter” refers to a promoter which is activated (e.g., up-regulated) or repressed (e.g., down-regulated) when phosphate concentration changes. In certain embodiments, a phosphate-sensitive promoter is activated or repressed when phosphate concentration crosses a threshold value. In certain embodiments, a phosphate-sensitive promoter is up-regulated or down-regulated proportionally in response to a change in phosphate concentration. In certain embodiments, a phosphate-sensitive promoter is activated when phosphate concentrations decrease below a threshold value of about 50 μM, 40 μM, 30 μM, 20 μM, or 10 μM to about 1 μM. In certain embodiments, a phosphate-sensitive promoter will be active at a concentration of phosphate from 0 μM to about 1 μM, 2 μM, 5 μM, 7 μM, or 10 μM.

[0049] As used herein, the phrase “quorum sensing promoter” refers to a promoter which is activated (e.g., up-regulated) or repressed (e.g., down-regulated) when cell density changes. In certain embodiments, a quorum sensing promoter is activated or repressed when cell density crosses a threshold value. In certain embodiments, a quorum sensing promoter is activated when cell densities increase above a threshold value of about 6 x 103CFU / mL to about 5 x 108CFU / mL.

[0050] As used herein, the term “refactored” refers to a gene, gene cluster, or operon that has been restructured. In some embodiments, restructuring may include changing a DNA coding sequence to a DNA sequence divergent from the wild-type gene while still encoding the same polypeptide. In some embodiments, restructuring may include computationally scanning genes to identify control elements, removing them, and optionally replacing them with different control elements. In some embodiments, “refactored” refers to a gene, gene cluster, or operon wherein the naturally-occurring promoter has been modified to contain a new promoter which exhibits different regulatory characteristics. Examples of such refactored gene clusters include refactored nif and / or fix gene clusters which allow nitrogenase to be expressed without transcriptional down-regulation by fixed nitrogen. Examples of refactoring methods and refactored nif gene clusters include those disclosed in US Patent No, 11479516, incorporated herein by reference in its entirety.

[0051] As used herein, the phrase “segment of a 5’ UTR” refers to one or more nucleotides of DNA encoding a 5’ UTR (5’ untranslated region) of a transcript and / or RNA comprising one or more nucleotides of a 5’ UTR. In certain embodiments, the segment of a 5’ UTR will comprise at least the first nucleotide of the 5’ UTR but can also comprise at least a ribosome binding site in a 5’ UTR or the entire 5’ UTR.

[0052] As used herein, the phrase “transcriptional activator” refers to proteins or ribonucleoprotein (RNP) complexes capable of activating expression of a particular target gene. Transcriptional activators can thus include: (i) transcription factors comprising a DNA binding domain and a transcriptional activation domain; (ii) sigma factors which bind both a target promoter and RNA polymerase; and (iii) a catalytically inactive RNA-guided DNA binding protein which furtherAtty. Dkt. No. P14610WO00 comprises a transcriptional activator domain and a guide RNA which targets the RNP complex to the promoter.

[0053] As used herein, the phrase “transcription factor” refers to proteins or ribonucleoprotein (RNP) complexes capable of altering the rate of transcription of genetic information from DNA to RNA by binding to one or more specific DNA sequence(s). Transcription factors can thus include: (i) transcriptional repressors that decrease the rate of transcription of one or more particular target gene(s) upon binding to a specific DNA sequence, (ii) said transcriptional activators defined above, (iii) proteins or ribonucleoprotein (RNP) complexes that stabilize the rate of transcription, (iv) proteins or ribonucleoprotein (RNP) complexes that selectively accomplish a first function selected from a group comprising (i), (ii), or (iii) under a first set of physical and / or chemical conditions and a different function selected from (i), (ii), or (iii) under a different set of physical and / or chemical conditions, and (v) proteins or ribonucleoprotein (RNP) complexes that selectively accomplish a first function selected from a group comprising (i), (ii), or (iii) upon interaction with a first protein binding partner or first specific DNA sequence and a different function selected from (i), (ii), or (iii) upon interaction with a different protein binding partner or different specific DNA sequence.

[0054] Sequence identity or percent sequence identity can be measured with the BLASTN program (for nucleotide sequence percent identity determinations) or BLASTP program (for protein sequence percent identity determinations) using the BLAST ( Basic Local Alignment Search Tool) available on the internet at blast.ncbi.nlm.nih.gov / Blast.cgi with default settings.

[0055] To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.

[0056] In certain embodiments, one or more bacterial genes are down-regulated to cause the production of the agriculturally relevant compounds. Examples of bacterial genes which can be downregulated to produce ammonia include amtB, glnA, glnB, glnK, glnZ, nifL, and / or draT genes. Target amtB, glnA, glnB, glnK, glnZ, nifL, and / or draT gene sequences which can be down-regulated include those set forth in Table 5, in the sequence listing, and in US Patent Application No. US20210315212, which is incorporated herein by reference in its entirety. Target amtB, glnA, glnB, glnK, glnZ, nifL, or draT gene sequences which can be down-regulated further include sequences having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity across the entire length of the sequences set forth in Table 1, in Table 5, in the sequence listing, and in US Patent Application No. US20210315212. Target amtB, glnA, glnB, glnK, glnZ, nifL, and / or draT gene sequences which can be down-regulated also include sequences which encode AmtB, GlnA, GlnB, GlnK, GlnZ, NifL, or DraT proteins having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity across the entire length of AmtB, GlnA, GlnB, GlnK, GlnZ, NifL, or DraT proteins encoded by genes set forth inAtty. Dkt. No. P14610WO00 Table 1, in Table 5, in the sequence listing, and in US Patent Application No. US20210315212 or to AmtB, GlnA, GlnB, GlnK, GlnZ, NifL, or DraT proteins in US Patent Application No. US20210315212.

[0057] Table 1. Non-limiting summary of target genes for down-regulation and ammonia production. Target Gene Representative Comments Name GenesAtty. Dkt. No. P14610WO00 Target Gene Representative Comments Name Genesn cer a n em o mens, one or more genes se or n a e can e own-regu a e by one or more of the quorum sensing systems or genes set forth in Table 2, Table 3, and / or Table 4. Examples of such combinations include down-regulation of GlnA (glutamine synthetase) by quorum sensing promoter- controlled expression of a modified glnE gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity. Particular embodiments of the systems for down-regulation of genes set forth in Table 1 are also disclosed within numbered embodiments 1-131.Atty. Dkt. No. P14610WO00

[0059] Table 2. Non-limiting summary of systems for down-regulating target genes at cell densities which exceed a threshold density System Non-limiting materials Non-limiting descriptions of which can be used to materials and methods which canAtty. Dkt. No. P14610WO00 System Non-limiting materials Non-limiting descriptions of which can be used to materials and methods which can i l t t b d td f i thAtty. Dkt. No. P14610WO00 System Non-limiting materials Non-limiting descriptions of which can be used to materials and methods which can i l t t b d td f i thAtty. Dkt. No. P14610WO00 System Non-limiting materials Non-limiting descriptions of which can be used to materials and methods which can i l t t b d td f i thAtty. Dkt. No. P14610WO00 System Non-limiting materials Non-limiting descriptions of which can be used to materials and methods which can i l t t b d td f i thAtty. Dkt. No. P14610WO00 System Non-limiting materials Non-limiting descriptions of which can be used to materials and methods which can i l t t b d td f i thAtty. Dkt. No. P14610WO00 System Non-limiting materials Non-limiting descriptions of which can be used to materials and methods which can i l t t b d t d f i thn cer a n em o mens, one or more ac er a genes are up-regua e o cause e pro uc on o the agriculturally relevant compounds. Examples of bacterial genes which can be up-regulated to produce ammonia include nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity (also referred to herein as GlnE-uAT), one or more nif cluster gene(s), and / or one or more fix cluster gene(s). Target nifA, ntrC, glnR, glutaminase encoding genes, GlnE-uAT genes, nif cluster gene(s), and / or fix cluster gene sequences which can be up-regulated include those set forth in Table 3, in the sequence listing, and in US Patent Application No. US20210315212, which is incorporated herein by reference in its entirety. Target nifA, ntrC, glnR, glutaminase encoding genes, GlnE-uAT genes, nif cluster gene(s), and / or fix cluster genes which can be up-regulated further include sequences having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity across the entire length of the sequences set forth in Table 3, in the sequence listing, and in US Patent Application No. US20210315212. Target nifA, ntrC, glnR, glutaminase encoding genes, GlnE-Atty. Dkt. No. P14610WO00 uAT genes, nif cluster gene(s), and / or fix cluster gene sequences which can be up-regulated also include sequences which encode NifA, NtrC, GlnR, glutaminase, GlnE-uAT, nif cluster, and / or fix cluster proteins having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity across the entire length of NifA, NtrC, GlnR, glutaminase, GlnE-uAT, nif cluster, and / or fix cluster proteins encoded by genes set forth in Table 3, in the sequence listing, and / or in US Patent Application No. US20210315212 or to NifA, NtrC, GlnR, Glutaminase, GlnE-uAT, nif cluster, and / or fix cluster proteins in Table 3, in Table 5, in the sequence listing, and / or in US Patent Application No. US20210315212.

[0061] In certain embodiments, one or more bacterial genes are up-regulated to cause the production of phosphate from insoluble forms of phosphate (e.g., present in plant growth media). Examples of bacterial genes which can be up-regulated to produce phosphate include genes encoding a phytase enzyme (e.g., a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase), genes encoding an acid phosphatase enzyme (e.g., an acpA, aphA, phoC, napA, napD, or napE gene), and / or genes encoding a protein which stimulates organic acid release from the bacterium, (e.g., a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase encoding gene). Target phytase-, acid phosphatase- (e.g., an acpA, aphA, phoC, napA, napD, or napE gene), GAD-, GDH-, or pyrroloquinoline (PQQ) synthase-encoding genes which can be up-regulated include those set forth in Table 3, in the sequence listing, and in US Patent Application No. US20210345618, which is incorporated herein by reference in its entirety. Target phytase-, acid phosphatase- (e.g., an acpA, aphA, phoC, napA, napD, or napE gene), GAD-, GDH-, or pyrroloquinoline (PQQ) synthase-encoding genes which can be up-regulated further include sequences having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity across the entire length of the sequences set forth in Table 3, in the sequence listing, and in US Patent Application No. US20210345618. Target phytase-, acid phosphatase- (e.g., an acpA, aphA, phoC, napA, napD, or napE gene), GAD-, GDH-, or pyrroloquinoline (PQQ) synthase-encoding genes which can be up-regulated also include sequences which encode phytase, acid phosphatase (e.g., an AcpA, AphA, PhoC, NapA, NapD, or NapE protein), GAD, GDH, or pyrroloquinoline (PQQ) synthase proteins having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity across the entire length of phytase, acid phosphatase (e.g., an AcpA, AphA, PhoC, NapA, NapD, or NapE protein), GAD, GDH, or pyrroloquinoline (PQQ) synthase proteins encoded by genes set forth in Table 3, in the sequence listing, and in US Patent Application No. US20210345618 or to phytase, acid phosphatase (e.g., an AcpA, AphA, PhoC, NapA, NapD, or NapE protein), GAD, GDH, or pyrroloquinoline (PQQ) synthase proteins in Table 3, in the sequence listing, and / or in US Patent Application No. US20210345618.

[0062] Table 3. Non-limiting summary of target genes for up-regulation and agriculturally relevant compounds produced by up-regulationAtty. Dkt. No. P14610WO00 Target Gene or Target Agriculturally Comment Gene encoded protein relevantAtty. Dkt. No. P14610WO00 Target Gene or Target Agriculturally Comment Gene encoded protein relevantAtty. Dkt. No. P14610WO00 Target Gene or Target Agriculturally Comment Gene encoded protein relevant on nd te. es byAtty. Dkt. No. P14610WO00 Target Gene or Target Agriculturally Comment Gene encoded protein relevantAtty. Dkt. No. P14610WO00 Target Gene or Target Agriculturally Comment Gene encoded protein relevantAtty. Dkt. No. P14610WO00 Target Gene or Target Agriculturally Comment Gene encoded protein relevantAtty. Dkt. No. P14610WO00 Target Gene or Target Agriculturally Comment Gene encoded protein relevantAtty. Dkt. No. P14610WO00 Target Gene or Target Agriculturally Comment Gene encoded protein relevant

[0065] In certain embodiments, one or more genes set forth in Table 3 can be up-regulated by one or more of the quorum sensing systems or genes set forth in Table 4. Particular embodiments of the systems for up- regulation of genes set forth in Table 3 are also disclosed within the following numbered embodiments 1- 131. In certain embodiments, the agriculturally relevant compound is not cellulose and / or genes that are upregulated in the numbered embodiments do not encode a protein involved in the synthesis and / or secretion of cellulose. Such proteins involved in the synthesis and / or secretion of cellulose include bcs operon gene(s), a cmcAx gene, a ccpAx gene, a bglAx gene, pgm gene, galU gene, cdg operon, and / or dgc gene set forth in US Patent Application Publication No. US20230034438.Atty. Dkt. No. P14610WO00

[0066] Table 4. Non-limiting summary for gene up-regulation mechanisms at cell densities which exceed a threshold density System Non-limiting materials Non-limiting descriptions of which can be used to materials and methods which n: 4, in oi: oi: 12 itsAtty. Dkt. No. P14610WO00 System Non-limiting materials Non-limiting descriptions of which can be used to materials and methods whichAtty. Dkt. No. P14610WO00 System Non-limiting materials Non-limiting descriptions of which can be used to materials and methods which ed 0Atty. Dkt. No. P14610WO00 System Non-limiting materials Non-limiting descriptions of which can be used to materials and methods which ed 0Atty. Dkt. No. P14610WO00

[0067] In certain embodiments, target genes encoding an RNA or protein of interest can be upregulated or down regulated by an SSR- or integrase- controlled promoter switch where a control element comprising a promoter can be inverted. Examples of such promoter switches include those in Tables 1-4 or the numbered embodiments. In certain embodiments, inversion of the promoter by the SSR- or integrase- can result in the down-regulation of the RNA sequence or protein of interest by disrupting operable linkage of the promoter to DNA encoding the RNA or protein. In certain embodiments, inversion of the promoter by the SSR- or integrase- can result in the up-regulation of the RNA sequence or protein of interest by operably linking the promoter to DNA encoding the RNA or protein (e.g., an SSR- or integrase- controlled promoter switch). Control elements used in the promoter switch can comprise a constitutive promoter, an inducible promoter, or quorum sensing promoter and at least a segment of a 5’ UTR. In still other embodiments, an SSR- or integrase- controlled promoter switch can be designed to express a first gene before expression of the SSR or integrase and a second gene after expression of the SSR or integrase by the QS-P. In certain embodiments, the first gene can be selected from genes targeted for down-regulation in Table 1 and the second gene can be selected from a gene targeted for up-regulation in Table 3. In certain embodiments, the first gene can be a wild-type or even improved copy of a gene targeted for down- regulation in Table 1 and the second gene can be a wild-type copy of that same first gene from Table 1 which is expressed at levels lower than the wild-type or first gene, a mutated variant of that same first gene from Table 1 with reduced enzymatic or biological activity, or a mutated variant of that same first gene from Table 1 which is also expressed at levels lower than the wild-type or first gene.

[0068] In one embodiment, the SSR- or integrase- controlled promoter switch is placed between a first gene encoding a GlnA (glutamine synthetase (GS)) protein with wild-type or improved catalytic activity in comparison to wild-type GS and a second gene encoding: (i) a wild-type GS with reduced levels of expression in comparison to the wild-type glnA gene, (ii) a GS variant with decreased catalytic activity in comparison to wild-type GS, or (iii) a GS variant with decreased catalytic activity in comparison to wild-type GS and reduced levels of expression in comparison to the wild-type glnA gene, where the promoter in the SSR- or integrase- controlled promoter switch is operably linked to the first gene prior to SSR- or integrase-mediated promoter inversion resulting from QS-P promoter activation of the SSR or integrase. GEB comprising this promoter switch will express wild-type or catalytically improved GS prior to QS-P promoter-mediated activation of the SSR or integrase, promoting growth of the GEB in plant growth media. After QS-P promoter-mediated activation of the SSR or integrase, the promoter in the promoter switch is inverted and operably linked to the wild- type GS with reduced expression, variant GS with reduced catalytic activity, or variant GS with reduced catalytic activity and reduced expression, resulting in ammonia production. Reductions in expression of the second gene encoding a wild-type or GS variant can be achieved in a variety of ways including use of weak ribosome binding sites in the 5’ UTR, use of non-preferred codons,Atty. Dkt. No. P14610WO00 substitution of the ATG start codon with a GTG start codon, mutations that result in decrease protein or mRNA and / or protein stability, and combinations thereof. Certain methods of reducing expression of a glnA gene disclosed in US20200331820 can be adapted for use in embodiments disclosed herein.

[0069] In one embodiment, the SSR- or integrase- controlled promoter switch is placed between a first gene comprising a glnE gene encoding a wild-type GS adenylyltransferase protein and a second gene comprising a glnE gene encoding a GS adenylyltransferase protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity (e.g., a GlnE-uAT gene), where the promoter in the SSR- or integrase- controlled promoter switch is operably linked to the first gene prior to SSR- or integrase-mediated promoter inversion resulting from QS-P promoter activation of the SSR or integrase. GEB comprising this promoter switch will express wild-type GlnE protein prior to QS-P promoter-mediated activation of the SSR or integrase, promoting growth of the GEB in plant growth media. After QS-P promoter-mediated activation of the SSR or integrase, the promoter in the promoter switch is inverted and operably linked to the GlnE-uAT gene, resulting in the adenylation and inactivation of GS and ammonia production. Sources of GlnE-uAT genes are set forth in Table 3.

[0070] In one embodiment, the SSR- or integrase- controlled promoter switch is placed between a first gene comprising a nifL gene and a second gene comprising a nifA gene, where the promoter in the SSR- or integrase- controlled promoter switch is operably linked to the first nifL gene prior to SSR- or integrase-mediated promoter inversion resulting from QS-P promoter activation of the SSR or integrase. GEB comprising this promoter switch will express wild-type NifL protein prior to QS- P promoter-mediated activation of the SSR or integrase, promoting growth of the GEB in plant growth media. After QS-P promoter-mediated activation of the SSR or integrase, the promoter in the promoter switch is inverted and operably linked to the nifA gene, resulting in the down-regulation of nifL, the up-regulation of nifA, and ammonia production.

[0071] In certain embodiments, it is desirable to delay expression of genes encoding an RNA or protein of interest (e.g., genes disclosed in Table 1 or 3) by constructing a QS-P controlled cascade of repressors / repressor-controlled genes encoding repressors and / or a QS-P controlled cascade of transcriptional activators / transcriptional activator-controlled genes encoding transcriptional activators in the GEB. In certain embodiments, a gene encoding the RNA or protein of interest or which regulates expression of the gene encoding the RNA or protein of interest is at the end of the cascade. At the head of the repressor and / or transcriptional-activator cascade is a QS-P promoter that drives expression of the first repressor or transcriptional activator, respectively. In certain embodiments, a repressor cascade can further comprise a gene encoding a transcriptional activator which is controlled by a repressor in the cascade (e.g., the terminal repressor in the cascade which regulates ). In certain embodiments, a transcriptional activator cascade can further comprise a gene encoding a repressor which is controlled by a transcriptional activator in the cascade (e.g., theAtty. Dkt. No. P14610WO00 terminal repressor in the cascade). In a repressor cascade, each repressor represses the subsequent repressor in the cascade. In a transcriptional activator cascade, each transcriptional activator activates the subsequent transcriptional activator in the cascade. Descriptions of such regulatory cascades which can be adapted for use in the QS-P controlled regulatory cascades described herein include regulatory cascades disclosed in Tables 2 and 4 as well as in those disclosed in Hooshangi et al., 2004, doi: 10.1073pnas.0408507102 and Pinto et al., doi: 10.1093 / nar / gky614.

[0072] Without being limited by theory, it is believed that the delay in expression of the gene encoding the RNA or protein of interest is a factor of the number of repressor modules and / or transcriptional activator modules are in the cascade, the rate at which each of the repressors and / or transcriptional activator degrade and / or accumulate in the cell. It is further believed that delaying expression of RNAs or proteins of interest is beneficial insofar as expression of those RNAs, proteins, or the agriculturally relevant compounds they produce can in certain embodiments reduce the GEB’s ability to compete against other microorganisms in the plant growth media and / or on the plant (e.g., root system). It is thus believed that such delays in expression can promote growth of the GEB to sufficient cell density in the plant growth media and / or plant before the agriculturally relevant compound is produced.

[0073] Bacteria selected from the groups of bacteria of the taxonomic classes of alphaproteobacteria, betaproteobacteria, and gammaproteobacteria can be modified to obtain the GEB disclosed herein. In certain embodiments, the bacteria selected for modification to obtain the GEB are bacteria in the taxonomic genera of Acetobacter, Acidothermus, Acinetobacter, Agrobacterium, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Burkholderia, Conexibacter, Curtobacterium, Ensifer, Enterobacter, Erwinia, Escherichia, Flavobacterium, Frankia, Gaiella, Gluconacetobacter, Gluconobacter, Herbaspirillum, Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimibacter, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrobacter, Paenibacillus, Panotea, Pediococcus, Peribacillus, Phytobacter, Priestia, Pseudarthrobacter, Pseudomonas, Rahnella, Rhizobium, Rhodococcus, Rhodoplanes, Rhodopseudomonas, Rhodospirillum, Serratia, Solirubrobacter, Sphingobacterium, Sphingomonas, Stenotrophomonas, Streptomyces, Stutzerimonas, Variovorax, Xanthobacter, and Yoonia, optionally wherein the bacteria are selected from at least one of the taxonomic genera selected from the group consisting of Azospirillum, Enterobacter, Herbaspirillum, Klebsiella, Kosakonia, Paenibacillus, Phytobacter, Pseudomonas, Ranhella, Sphingomonas, or Variovorax. In certain embodiments, the GEB and / or bacteria used to obtain the GEB are bacteria which have been modified and / or selected for increased potential to colonize the roots of target crop plants (e.g., maize, rice, wheat, and the like). Genes which have been modified for improved colonization include bcsll, bcslll, yjbE, fhaB, pehA, glgA, otsB, treZ, and cysZ genes (US Patent. Applic. Publ. No. 20210315212 and WO2019032926, both incorporatedAtty. Dkt. No. P14610WO00 herein by reference in their entireties). In certain embodiments, the genetically engineered strains disclosed herein can be obtained by modification of Klebsiella, Kosakonia, or Rahnella strains, and include such strains deposited as NCMA 201701002, a bacterium deposited as NCMA 201708004, a bacterium deposited as NCMA 201708003, a bacterium deposited as NCMA 201708002, a bacterium deposited as NCMA 201712001, or a bacterium deposited as NCMA 201712002. These strains were deposited with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Me.04544, USA under the terms of the Budapest Treaty as described in US Patent. Applic. Publ. No.20210315212. In certain embodiments, the genetically engineered bacteria disclosed herein can be selected for improved plant colonization and / or improved production of nitrogenous compounds (e.g., ammonia) by various methods including those disclosed in US Patent Application No. 20240010576, incorporated herein by reference in its entirety. Examples of other bacteria that can be modified to obtain the GEB disclosed herein also include Rahnella aquatilis and Enterobacter sacchari strains were deposited with the American Type Culture Collection and assigned ATTC Patent Deposit Designation numbers PTA-122293 and PTA- 122294, respectively, as described in US Patent Application No.20240010576.

[0074] A variety of control elements comprising QS-P promoters and optionally 5’ UTR segments can be used in the GEB, methods, and systems provided herein. Desirable characteristics of such control elements include activation of expression of operably linked RNAs or proteins in agriculturally useful bacteria in response to population densities which exceed threshold population densities in the plant growth medium. In certain embodiments, the control elements comprising QS- P promoters will be derived in whole or in part from agriculturally useful bacteria which include: (i) alphaproteobacteria, betaproteobacteria, and gammaproteobacteria; (ii) bacteria in the genus Acetobacter, Acidothermus, Acinetobacter, Agrobacterium, Aliivibrio, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Burkholderia, Chromobacterium, Conexibacter, Curtobacterium, Ensifer, Enterobacter, Erwinia, Escherichia, Flavobacterium, Frankia, Gaiella, Gluconacetobacter, Gluconobacter, Herbaspirillum, Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimibacter, Mesorhizobium, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrobacter, Paenibacillus, Pantoea, Pediococcus, Peribacillus, Priestia, Pseudarthrobacter, Pseudomonas, Rahnella, Rhizobium, Rhodococcus, Rhodoplanes, Rhodopseudomonas, Rhodospirillum, Serratia, Solirubrobacter, Sphingobacterium, Sphingomonas, Stenotrophomonas, Streptomyces, Stutzerimonas, Variovorax, Vibrio, Xanthobacter, and Yoonia; (iii) Aliivibrio, Burkholderia, Chromobacterium, Mesorhizobium, Pantoea, Pseudomonas, and Rhizobium. In certain embodiments, the control elements comprising the QS-P are used in bacteria of the genus or the species from which they were derived in whole or in part.Atty. Dkt. No. P14610WO00

[0075] In certain embodiments, the control elements comprising QS-P promoters and optionally 5’ UTR segments will comprise elements which can be bound by an AhlR, CCiR, CinR, CviR, EsaR D91G, EsaR, LasR, LuxR, or TraR QSRP.

[0076] In certain embodiments, the control elements comprising QS-P promoters and optionally 5’ UTR segments will comprise elements which can be bound by an AhlR, CCiR, CinR, CviR, EsaR D91G, EsaR, LuxR, TraR, or LasR QSRP or fragment thereof (specific regulator-binding DNA sequences that facilitate the activation or inhibition of gene expression in a quorum sensing manner) or the corresponding QS-P promoters are obtained in whole or in part from various agriculturally useful bacteria (AUB). Agriculturally useful bacteria (AUB) can include bacteria which can grow in plant growth media and / or which can colonize crop plants. AUB include bacteria in the taxonomic genera of Acetobacter, Acidothermus, Acinetobacter, Agrobacterium, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Burkholderia, Chromobacterium, Conexibacter, Curtobacterium, Ensifer, Enterobacter, Erwinia, Escherichia, Flavobacterium, Frankia, Gaiella, Gluconacetobacter, Gluconobacter, Herbaspirillum, Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimibacter, Mesorhizobium, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrobacter, Paenibacillus, Pantoea, Pediococcus, Peribacillus, Phytobacter, Priestia, Pseudarthrobacter, Pseudomonas, Rahnella, Rhizobium, Rhodococcus, Rhodoplanes, Rhodopseudomonas, Rhodospirillum, Serratia, Solirubrobacter, Sphingobacterium, Sphingomonas, Stenotrophomonas, Streptomyces, Stutzerimonas, Variovorax, Xanthobacter, and Yoonia. Such AUB QS-P sequences can comprise those set forth in SEQ ID NO: 356-364, 373, and 374 and variants thereof comprising 1, 2, or 3 nucleotide substitutions. Such AUB QS-P promoters can comprise DNA molecules having at least 85%, 90%, 95%, 98%, or 99% sequence identity across the entire length of any one of SEQ ID NO: 356-364, 373, and 374. Such QS-P promoters can also include those which are operably linked to a gene encoding a protein having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a QSSP protein (e.g., an AhlI, CciI, CinI, CviI, EsaI, LuxI, TraI, or LasI protein) set forth in Table 5 and the sequence listing. In certain embodiments, the control elements comprising the AUB sequences and AUB QS-P promoters provided herein can activate expression of operably linked RNAs or gene-encoding proteins of interest in agriculturally useful bacteria including Gluconacetobacter sp., Azorhizobium sp., Azospirillum sp., Herbaspirillum sp., Kosakonia sp., Paenibacillus sp., and Pseudomonas sp. in response to increases in cell density above a threshold population density. In certain embodiments, the control elements comprising the AUB sequences and AUB QS-P promoters provided herein can activate expression of operably linked RNAs or proteins in a Gluconacetobacter diazotrophicus, Azorhizobium caulinodans, Azospirillum brasilense, Herbaspirillum seropedicae, Kosakonia radicincitans, Paenibacillus azotofixans, and Pseudomonas stutzeri isolate, strain, or derivative thereof. In certain embodiments, a control elementAtty. Dkt. No. P14610WO00 comprising an AUB sequence or AUB QS-P promoters derived in whole or in part from Gluconacetobacter sp., Azorhizobium sp., Azospirillum sp., Herbaspirillum sp., Kosakonia sp., Paenibacillus sp., or Pseudomonas sp. (e.g., the corresponding AUB sequences and AUB QS-P promoters set forth in Table 5) are respectively used in a GEB obtained from a Gluconacetobacter sp., Azorhizobium sp., Azospirillum sp., Herbaspirillum sp., Kosakonia sp., Paenibacillus sp., or Pseudomonas sp. In certain embodiments, a control element comprising an AUB sequences or AUB QS-P promoter derived in whole or in part from Gluconacetobacter diazotrophicus, Azorhizobium caulinodans, Azospirillum brasilense, Herbaspirillum seropedicae, Kosakonia radicincitans, Paenibacillus azotofixans, or Pseudomonas stutzeri (e.g., the corresponding AUB sequences and AUB QS-P promoters set forth in Table 5) are respectively used in a GEB obtained from a Gluconacetobacter diazotrophicus, Azorhizobium caulinodans, Azospirillum brasilense, Herbaspirillum seropedicae, Kosakonia radicincitans, Paenibacillus azotofixans, or Pseudomonas stutzeri isolate, strain, or derivative thereof.

[0077] In certain embodiments, the AUB provided herein comprising the control element comprising an AUB sequences or AUB QS-P promoter which is operably linked to a nucleic acid sequence encoding the RNA or protein of interest can further comprise one or more heterologous gene expression cassettes encoding a quorum sensing synthase protein (QSSP) and / or a quorum sensing regulator protein (QSRP), where the QSSP can produce a quorum sensing signal molecule (QSSM, e.g., an AHL) which can activate the QSRP and the QS-P. Combinations of QSSP, QSRP, and QS-P which can be used together in the agriculturally useful bacteria include: i. an AhlI protein, an AhlR protein, and an ahlI QS-P, respectively; ii. a CinI protein, CinR protein, and a cinI QS-P, respectively; iii. a CciI protein, a CciR protein, and a cciI QS-P, respectively; iv. a CviI protein, a CviR protein, and a cviI QS-P, respectively; v. an EsaI protein, an EsaR D91G protein, and an esaR repressable QS-P, respectively; vi. an EsaI protein, an EsaR protein, and an esaI QS-P, respectively; vii. a LuxI protein, a LuxR protein, and a luxI QS-P, respectively; viii. a TraI protein, a TraR protein, and a traI QS-P, respectively; or ix. a LasI protein, a LasR protein, and a lasI or lasB QS-P; In certain embodiments, the combinations can be used either in an AUB of the same genus or species from which they were derived (e.g., in a Pseudomonas sp., a Burkholderia sp., a Rhizobium sp., a or a Pantoea sp.). In certain embodiments, the combinations are used in an AUB of a different genus from which they were derived (e.g., in an Acetobacter, Acidothermus, Acinetobacter, Agrobacterium, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum, Azotobacter, Bacillus,Atty. Dkt. No. P14610WO00 Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimibacter, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrobacter, Paenibacillus, Panotea, Pediococcus, Peribacillus, Priestia, Pseudarthrobacter, Rahnella, Rhodococcus, Rhodoplanes, Rhodopseudomonas, Rhodospirillum, Serratia, Solirubrobacter, Sphingobacterium, Sphingomonas, Stenotrophomonas, Streptomyces, Stutzerimonas, Variovorax, Xanthobacter, and Yoonia sp.).

[0078] In certain embodiments, the heterologous gene expression cassette comprising the control elements comprising the sequences and QS-P promoters (e.g., the corresponding AUB sequences and AUB QS-P promoters set forth in Table 5) is integrated at a location in the chromosome of the genetically engineered bacterium which does not comprise the location of an endogenous quorum sensing promoter in the unmodified agriculturally useful bacterium. In certain embodiments, a control element comprising a QS-P promoter (e.g., QS-P promoter in Table 5) is integrated in the genome of the GEB at a location other than the location of the endogenous (e.g., wild-type) QS-P promoter in the GEB. In certain embodiments, a control element comprising an ahlI QS-P promoter (e.g., an ahlI in Table 5) is integrated in the genome of the GEB at a location other than the location of the endogenous (e.g., wild-type) ahlI QS-P promoter in the GEB. In certain embodiments, a control element comprising a cciI QS-P promoter (e.g., a cciI promoter in Table 5) is integrated in the genome of the GEB at a location other than the location of the endogenous (e.g., wild-type) cciI QS- P promoter in the GEB. In certain embodiments, a control element comprising a cinI_V1 or cinI_V2 QS-P promoter (e.g., a cinI_V1 or cinI_V2 QS-P promoter in Table 5) is integrated in the genome of the GEB at a location other than the location of the endogenous (e.g., wild-type) cinI_V1 or cinI_V2 QS-P promoter in the GEB. Methods for inserting control elements comprising QS-P promoters at sites distinct from the endogenous gene include Tn7 transposon mediated insertion (McKenzie and Craig, N.L., 2006, doi: 10.1186 / 1471-2180-6-39).

[0079] Methods of producing preparations of the genetically engineered bacterium (GEB) for use in treating plant growth media, plants, plant parts, plant propagules, and / or for formulating a composition comprising the GEB are provided herein. In certain embodiments, the methods can comprising growing the GEB in a bacterial growth medium comprising a carbon and nitrogen source and harvesting the GEB from the media. Conditions for growing the GEB include axenic growth in continuous stirred tank reactors, batch fermentation reactors, and the like. In certain embodiments, the GEB are grown either (a) in contact with the quorum quenching compound QQ; or (b) in exposure to a temperature above the threshold temperature to suppress expression of the RNA or protein of interest encoded by the gene which is operably linked to the quorum sensing promoter. Such contact with the quorum quenching compound QQ or in exposure to a temperature above the threshold temperature result in production of one or more deactivators of the quorum sensing system which comprises the quorum sensing synthase protein (QSSP) able to synthesize a quorum sensing signal molecule, the quorum sensing regulator protein (QSRP), and quorum sensing promoter in the GEB.Atty. Dkt. No. P14610WO00 In certain embodiments, the deactivator can comprise an enzyme which catalyzes the degradation of the quorum sensing signal molecule, where the expression of the enzyme is induced by the addition of the quorum quenching compound QQ or the increase in temperature. In certain embodiments, the deactivator is an inducible repressor which: (i) inhibits expression of the QSSP and / or the first QSRP when the GEB is contacted with the quorum quenching compound QQ or the increase in temperature; and / or (ii) inhibits expression of the RNA or protein of interest control element comprising the first quorum sensing promoter. In certain embodiments, the quorum quenching compound QQ is inorganic phosphate or soluble phosphate, wherein the control element operably linked to the gene encoding the first QSRP comprises a phosphate-sensitive promoter. In certain embodiments where a temperature above the threshold temperature is used to suppress expression of the recombinase which is operably linked to the quorum sensing promoter, temperature induced de-repression of the deactivator or temperature induced de-activation of the first QSSP and / or the first QSRP can be used.

[0080] In certain embodiments, QS systems are deactivated by quorum quenching enzymes (QEs) that degrade AHL quorum sensing signal molecules (QSSMs). QEs span several classes of QSSM- degrading enzymes, including AHL acylase enzymes (SEQ ID NO: 320-322, and 323), alpha-beta hydrolase fold lactonase proteins (SEQ ID NO: 324, 325, and 326), metallo-beta-lactamase-like lactonase proteins (SEQ ID NO: 327-330, and 331), phosphotriesterase-like lactonase proteins (SEQ ID NO: 332-335, and 336), and variants of the QEs having at least 70%, 80%, 85%, 90%, 95%, 98%, and 99% sequence identity to SEQ ID NO: 320 to 335, or 336).

[0081] In certain embodiments, the quorum quenching compound (QQ) is a compound that inhibits a repressor protein (e.g., anhydrotetracycline (aTc), an inhibitor of the rTetR repressor or IPTG, an inhibitor of the LacI repressor). Examples of QQ systems based on repressor inhibitors and repressors include those illustrated in Figure 18. In Figure 18, bacteria containing the illustrated genetic circuit are grown to final desired titer in presence of aTC to suppress expression of the RNA or protein of interest (e.g., a GFP reporter, a recombinase, or uAT). The bacteria are harvested and then placed into a plant growth media (e.g., soil) in absence of aTC to allow the QS circuit to activate expression of the RNA or protein of interest when the bacteria reach a threshold population density. The system illustrated in Figure 18 can be adapted for use with LacI by replacing rTetR with LacI repressor and placing ahlR under control of the Plac promoter. In such LacI-based systems, the bacteria are harvested and then placed into a plant growth media (e.g., soil) in absence of IPTG to allow the QS circuit to activate expression of the RNA or protein of interest when the bacteria reach a threshold population density.

[0082] Various deactivators for inhibiting expression of QS-P and quorum-sensing systems that can be adapted for use in the methods, GEB, and systems provided herein are disclosed in US Patent Appln. Pub. Nos. US20110124522, US2015133339, US20220411769, and US Patent No. 7098014; each of which is incorporated herein by reference in their entireties.Atty. Dkt. No. P14610WO00

[0083] Compositions comprising one or more of the genetically engineered bacterium (GEB) are also provided herein. Such compositions can be adapted for storage of the GEB and / or for use of the GEB in the methods and agricultural systems disclosed herein. In certain embodiments, the compositions comprising the GEB can be used to treat a plant part including a leaf, stem, root, and / or seed by least partially coating the plant part with the composition. In certain embodiments, the compositions comprising the GEB can placed in plant growth medium (e.g., soil and / or water) prior to, during, and / or after depositing or establishing a seed, a seedling, plant, or vegetative propagule in the plant growth medium. In certain embodiments, a composition comprising the GEB is in a solid form. Such solid compositions can include those comprising a wettable powder, granules, a gel, pellets, or microencapsulated particles. In certain embodiments, a composition comprising the GEB is in a liquid form. Such liquid compositions can include those comprising an aqueous solution, aqueous suspension, water-in-oil emulsion, an oil, or an alcohol. Descriptions of compositions and components thereof which can be adapted for use with the GEB, related methods, agricultural systems, seedlings, plant parts, and vegetative propagules provided herein include those described below and / or in US Patent Application Publication Nos. US20210315212A1 and 20230148607, which are incorporated herein by reference in their entirety.

[0084] Compositions comprising the GEB provided herein can comprise an agriculturally acceptable carrier. Such carriers include liquid carriers comprising water, aqueous solutions, plant oils, and combinations thereof. Such carriers can in other embodiments comprise one of more solids including diatomaceous earth, loam, silica, clay, bentonite, vermiculite, seed cases, plant products (e.g., ground hulls, husk, stalks, stems, leaves, and the like), animal products, or combinations thereof.

[0085] Compositions comprising the GEB provided herein can comprise an agriculturally acceptable adjuvant.

[0086] In certain embodiments, the agriculturally acceptable adjuvant is an adhesive agent (e.g., an agent which promotes adherence of the composition and / or GEB to a plant part). In certain embodiments, the adhesive agents comprise one or more alginates, gums, starches, lecithins, formononetin, polyvinyl alcohol, alkali formononetinate, hesperetin, polyvinyl acetate, cephalins, Gum Arabic, Xanthan Gum, Mineral Oil, Polyethylene Glycol (PEG), Polyvinyl pyrrolidone (PVP), Arabino-galactan, Methyl Cellulose, PEG 400, Chitosan, Polyacrylamide, Polyacrylate, Polyacrylonitrile, Glycerol, Triethylene glycol, Vinyl Acetate, Gellan Gum, Polystyrene, Polyvinyl, Carboxymethyl cellulose, Gum Ghatti, and / or polyoxyethylene-polyoxybutylene block copolymers. In certain embodiments, the adhesive agents can comprise one or more waxes (e.g., carnauba wax, beeswax, or Chinese wax, shellac wax, spermaceti wax, candelilla wax, castor wax, ouricury wax, or rice bran wax), a polysaccharide (e.g., starch, dextrins, maltodextrins, alginate, and chitosans), a fat, oil, a protein (e.g., gelatin and zeins), gum ambles, and / or a shellac. In certain embodiments,Atty. Dkt. No. P14610WO00 adhesive agents can comprise one or more polymers or copolymers including polyvinyl acetates, polyvinyl acetate copolymers, ethylene vinyl acetate (EVA) copolymers, polyvinyl alcohols, polyvinyl alcohol copolymers, celluloses (e.g., ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses, and carboxymethylcelluloses), polyvinylpyrolidones, vinyl chloride, vinylidene chloride copolymers, calcium lignosulfonates, acrylic copolymers, polyvinylacrylates, polyethylene oxide, acylamide polymers and copolymers, polyhydroxyethyl acrylate, methylacrylamide monomers, and / or polychloroprene.

[0087] In certain embodiments, the agriculturally acceptable adjuvant is a desiccant or mixture thereof. In certain embodiments, the desiccant comprises one or more of trehalose, sucrose, glycerol, and / or methylene glycol. In certain embodiments, the desiccant comprises one or more non-reducing sugars and sugar alcohols (e.g., mannitol or sorbitol). In certain embodiments, desiccants are provided at about 5% to about 50% by weight / volume (w / v), about 10% to about 40% (w / v), about 15% to about 35% (w / v), or about 20% to about 30% (w / v) in the composition.

[0088] In certain embodiments, the agriculturally acceptable adjuvant is a dispersant (e.g., a surfactant). In certain embodiments, the dispersant comprises one or more of a nitrogen-surfactant blend (e.g., Prefer 28 (Cenex), Surf-N(US), Inhance (Brandt), P-28 (Wilfarm) and Patrol (Helena)), an esterified seed oil (e.g., Sun-It II (AmCy), MSO (UAP), Scoil (Agsco), Hasten (Wilfarm) and Mes-100 (Drexel)), and / or an organo-silicone surfactant (e.g., Silwet L77 (UAP), Silikin (Terra), Dyne-Amic (Helena), Kinetic (Helena), Sylgard 309 (Wilbur-Ellis) and Century (Precision)). In one embodiment, the surfactant or mixture of surfactants are at a concentration of about 0.01% volume / volume (v / v) to about 10% v / v in the composition. In another embodiment, the surfactant or mixture of surfactants are at a concentration of 0.1% (v / v) to 1% (v / v) in the composition.

[0089] In certain embodiments, the agriculturally acceptable adjuvant is a fungicide, insecticide, a nematicide, a rodenticide, and / or a bacteriocide. When the adjuvant is a bacteriocide, it is a bacteriocide selected for compatibility with the GEB and / or provided at a concentration or in a form which does not compromise the viability of the GEB. Numbered Embodiments

[0090] Additionally, the following numbered embodiments are included in the disclosure.

[0091] 1. A genetically engineered bacterium comprising: (a) one or more heterologous gene expression cassette(s) comprising at least one control element which is operably linked to at least one nucleic acid sequence encoding a first quorum sensing synthase protein (QSSP) able to synthesize a quorum sensing signal molecule Q (QSSM Q) and / or at least one nucleic acid sequence encoding a first quorum sensing regulator protein (QSRP) that can bind said QSSM Q; (b) a heterologous gene expression cassette comprising at least one nucleic acid sequence encoding at least one RNA sequence or protein of interest operably linked to a control element comprising a first quorum sensing promoter, wherein the first quorum sensing promoter is activated by the first quorumAtty. Dkt. No. P14610WO00 sensing regulator protein (QSRP) and the QSSM Q when the population density of the genetically engineered bacterium exceeds a threshold population density and wherein said at least one RNA sequence or protein of interest is or causes the production of at least one agriculturally relevant compound; and (c) at least one heterologous deactivation gene expression cassette comprising a DNA promoter which is operably linked to one or more deactivator(s) which inhibit(s) expression of the at least one RNA sequence or protein of interest at a population density of the genetically engineered bacterium which exceeds the threshold population density when the genetically engineered bacterium (GEB) is: (i) contacted with a quorum quenching compound QQ; or (ii) exposed to a temperature above a threshold temperature.

[0092] 2. The genetically engineered bacterium of embodiment 1, wherein the deactivator is triggered by: (a) QQ induced de-repression of the deactivator, wherein the GEB comprises a repressor protein which binds the DNA promoter which is operably linked to the deactivator in the absence of QQ, wherein the repressor protein is released from the DNA promoter in the presence of QQ, and wherein the deactivator is expressed in the presence of QQ, optionally wherein the deactivator comprises an enzyme which catalyzes the degradation of the quorum sensing signal molecule Q; (b) QQ induced activation of the deactivator, wherein the GEB comprises an activator protein which fails to bind the DNA promoter which is operably linked to deactivator in the absence of QQ, wherein the activator protein binds the DNA promoter in the presence of QQ, and wherein the deactivator is expressed in the presence of QQ, optionally wherein the deactivator comprises an enzyme which catalyzes the degradation of the quorum sensing signal molecule Q; (c) QQ induced repression of the first QSSP, the first QSRP, and / or the RNA or protein of interest, wherein the deactivator comprises a repressor protein which binds one or more promoter(s) which are operably linked to the gene(s) encoding the first QSSP, the first QSRP, and / or the RNA or protein of interest in the presence of QQ, wherein the repressor protein is released from the promoter(s) in the absence of QQ, and wherein the first QSSP, the first QSRP, and the RNA or protein of interest are expressed in the absence of QQ; (d) QQ induced de-activation of the first QSSP, the first QSRP, and / or the RNA or protein of interest, wherein the deactivator comprises an activator protein which binds one or more promoter(s) which are operably linked to the gene(s) encoding the first QSSP, the first QSRP, and / or the RNA or protein of interest in the absence of QQ, wherein the activator protein is released from the promoter(s) in the presence of QQ, and wherein the gene(s) operably linked to the promoter(s) is / are transcribed in the absence of QQ; (e) temperature induced de-repression of the deactivator, wherein the GEB comprises a repressor protein which binds a DNA promoter which is operably linked to the deactivator below a threshold temperature, wherein the repressor protein is released from the DNA promoter above the threshold temperature, and wherein the deactivator is expressed above the threshold temperature, optionally whereinAtty. Dkt. No. P14610WO00 the deactivator comprises an enzyme which catalyzes the degradation of the quorum sensing signal molecule Q; (f) temperature induced activation of the deactivator, wherein the GEB comprises an activator protein which fails to bind a DNA promoter that is operably linked to a gene encoding the deactivator below a threshold temperature, wherein the activator protein binds the DNA promoter above the threshold temperature, and wherein the deactivator is expressed above the threshold temperature, optionally wherein the deactivator comprises an enzyme which catalyzes the degradation of the quorum sensing signal molecule Q; (g) temperature induced repression of the first QSSP, the first QSRP, and / or the RNA or protein of interest, wherein the deactivator comprises a repressor protein which binds one or more promoter(s) which is / are operably linked to the gene(s) encoding the first QSSP, the first QSRP, and / or the RNA or protein of interest above a threshold temperature, wherein the repressor protein is released from the promoter(s) below the threshold temperature, and wherein the first QSSP, the first QSRP, and the RNA or protein of interest are expressed below the threshold temperature; (h) temperature induced de-activation of the first QSSP, the first QSRP, and / or the RNA or protein of interest, wherein the deactivator comprises an activator protein which binds one or more promoter(s) which is / are operably linked to the gene(s) encoding the first QSSP, the first QSRP, and / or the RNA or protein of interest below a threshold temperature, wherein the activator protein is released from the promoter(s) above the threshold temperature, and wherein the gene(s) operably linked to the promoter(s) is / are transcribed below the threshold temperature.

[0093] 3. The genetically engineered bacterium of embodiment 2, wherein the repressor in the QQ induced de-repression of the deactivator comprises: (a) a TetR repressor having at least 95% sequence identity to SEQ ID NO: 27 and wherein the DNA promoter operably linked to the gene encoding the deactivator comprises a Ptet promoter having at least 95% sequence identity to SEQ ID NO: 32, and optionally wherein the QQ compound is doxycycline, tetracycline, and / or anhydrotetracycline; (b) a LacI repressor having at least 95% sequence identity to SEQ ID NO: 29 and wherein the DNA promoter comprises a Plac promoter having at least 95% sequence identity to SEQ ID NO: 33, and optionally wherein the QQ compound is allolactose, isopropyl-β-D-thiogalactopyranoside (IPTG), or thiomethylgalactoside; or (c) a PhlF repressor having at least 95% sequence identity to SEQ ID NO: 30 and wherein the DNA promoter operably linked to the gene encoding the deactivator comprises a PphlA promoter having at least 95% sequence identity to SEQ ID NO: 34, and optionally wherein the QQ compound is 2,4- diacetylphoroglucinol (DAPG).

[0094] 4. The genetically engineered bacterium of embodiment 2, wherein the activator protein used in the QQ induced activation of the deactivator comprises a second quorum sensing regulator protein, theAtty. Dkt. No. P14610WO00 DNA promoter comprises a second quorum sensing promoter, and the QQ compound is an N-acyl homoserine lactone molecule (AHL) that binds the second quorum sensing regulator protein, wherein said AHL does not bind the first quorum sensing regulator protein and said second quorum sensing regulator protein binds the second quorum sensing promoter but not the first quorum sensing promoter.

[0095] 5. The genetically engineered bacterium of embodiment 2, wherein the repressor in the QQ induced repression comprises an rTetR repressor having at least 95% sequence identity to SEQ ID NO: 28, and wherein the DNA promoter(s) which is / are operably linked to the gene(s) encoding the first QSSP and / or the first QSRP comprise(s) a Ptet promoter having at least 95% sequence identity to SEQ ID NO: 32, optionally wherein the quorum sensing promoter which is operably linked to the RNA or protein of interest comprises an operably linked rTetR binding site element of a Ptet promoter having at least 95% sequence identity to SEQ ID NO: 32 and / or optionally wherein the QQ compound is doxycycline, tetracycline, and / or anhydrotetracycline.

[0096] 6. The genetically engineered bacterium of embodiment 2, wherein the deactivator is triggered by: (i) temperature induced de-repression of the deactivator, wherein the repressor comprises a cIts2 protein having at least 95% sequence identity to SEQ ID NO: 337 and wherein the DNA promoter which is operably linked to the deactivator comprises a PLpromoter having at least 95% sequence identity to SEQ ID NO: 31; or (ii) temperature induced de-activation of the first QSSP and / or the first QSRP wherein the deactivator comprises a cIts2 protein having at least 95% sequence identity to SEQ ID NO: 337 and the DNA promoter which is operably linked to the gene encoding the first QSSP and / or the first QSRP comprises a PRMpromoter having at least 95% sequence identity to SEQ ID NO: 338.

[0097] 7. The genetically engineered bacterium of any one of embodiments 1 to 6, wherein the deactivator comprises an enzyme which catalyzes the degradation of the quorum sensing signal molecule Q and wherein the expression of the enzyme is induced by the addition of the quorum quenching compound QQ or the increase in temperature.

[0098] 8. The genetically engineered bacterium of any one of embodiments 1 to 7, wherein the quorum sensing signal molecule Q is an acyl homoserine lactone (AHL) molecule and the enzyme which catalyzes its degradation comprises an AHL acylase enzyme, an AHL lactonase enzyme, or an AHL metallo-beta-lactamase enzyme.

[0099] 9. The genetically engineered bacterium of embodiment 8, wherein: (i) the AHL acylase enzyme comprises a PvdQ, AiiD, AigC, or QuiP protein, optionally wherein the PvdQ, AiiD, AigC, or QuiP protein has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 320, 321, 322, or 323, respectively;Atty. Dkt. No. P14610WO00 (ii) the AHL lactonase enzyme comprises an alpha-beta hydrolase fold lactonase protein, optionally wherein the lactonase comprises an AiiM, QqlM, or AidH protein, and optionally wherein the AiiM, QqlM, or AidH protein has at least 75% sequence identity to SEQ ID NO: 324, 325, or 326, respectively; (iii) the AHL lactonase enzyme comprises a phosphotriesterase-like lactonase protein, optionally wherein the lactonase comprises a Pph, SsoPox, Sislac, Gkl, or QsdA protein and optionally wherein the Pph, SsoPox, Sislac, Gkl, or QsdA protein has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 332, 333, 334, 335, or 336, respectively; or (iv) the AHL lactonase enzyme comprises a metallo-beta-lactamase protein, optionally wherein the metallo-beta-lactamase protein comprises a GcI, AttM, AidC, AiiB, or AiiA protein and optionally wherein the GcI, AttM, AidC, AiiB, or AiiA protein has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 327, 328, 329, 330, or 331, respectively.

[0100] 10. The genetically engineered bacterium of any one of embodiments 1 to 9, wherein the deactivator comprises an inducible repressor or transcription factor which: (i) decreases or inhibits expression of the first QSSP and / or the first QSRP when the GEB is contacted with the quorum quenching compound QQ or the temperature is increased; and / or (ii) decrease or inhibits expression of the RNA or protein of interest control element comprising the first quorum sensing promoter.

[0101] 11. The genetically engineered bacterium of any one of embodiments 1 to 10, wherein: (i) the QSSM Q molecule comprises N-(3-Hydroxytetradecanoyl)-DL-homoserine lactone and the first QSSP comprises a CinI protein; (ii) the QSSM Q molecule comprises N-(3-Hydroxytetradecanoyl)-DL-homoserine lactone and the first QSRP comprises a CinR protein; (iii) the QSSM Q molecule comprises N-(β-Ketocaproyl)-L-homoserine lactone and the first QSSP comprises an AhlI protein; or (iv) the QSSM Q molecule comprises N-(β-Ketocaproyl)-L-homoserine lactone and the first QSRP comprises an AhlR protein.

[0102] 12. The genetically engineered bacterium of any one of embodiments 1-11, wherein the quorum quenching compound QQ is inorganic phosphate or soluble phosphate, wherein the control element operably linked to the gene encoding the first QSRP comprises a phosphate-sensitive promoter.

[0103] 13. The genetically engineered bacterium of any one of embodiments 1 to 12, wherein: i. the first QSSP comprises an AhlI protein, the first QSRP comprises an AhlR protein, and the first quorum sensing promoter comprises an ahlI promoter, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum, Enterobacter, Klebsiella, Kosakonia, Pseudomonas, or Rahnella.;Atty. Dkt. No. P14610WO00 ii. the first QSSP comprises an CinI protein, the first QSRP comprises a CinR protein, and the first quorum sensing promoter comprises a cinI promoter, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum, Pseudomonas, or Kosakonia; iii. the first QSSP comprises an CciI protein, the first QSRP comprises a CciR protein, and the first quorum sensing promoter comprises a cciI promoter, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum or Pseudomonas; iv. the first QSSP comprises an CviI protein, the first QSRP comprises a CviR protein, and the first quorum sensing promoter comprises a cviI promoter, optionally wherein the bacterium is a member of the taxonomic genus Herbaspirillum, Kosakonia, or Pseudomonas; v. the first QSSP comprises an first EsaI protein, the first QSRP comprises a EsaR_D91G protein, and the first quorum sensing promoter comprises an easR_repressable promoter; vi. the first QSSP comprises an EsaI protein, the first QSRP comprises a EsaR protein, and the q first uorum sensing promoter comprises an esaI promoter; vii. the first QSSP comprises a LasI protein, the first QSRP comprises a LasR protein, and the first quorum sensing promoter comprises a lasI or lasB promoter, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum, Kosakonia, or Pseudomonas; viii. the first QSSP comprises an LuxI protein, the first QSRP comprises a LuxR protein, and the first quorum sensing promoter comprises a luxI promoter, optionally wherein the bacterium is a member of the taxonomic genus Pseudomonas; or ix. the first QSSP comprises an TraI protein, the QSRP comprises a TraR protein, and the quorum sensing promoter comprises a traI promoter.

[0104] 14. The genetically engineered bacterium of embodiment 13, wherein: (i) the AhlI protein comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 348; (ii) the CciI protein comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 349; (iii) the CinI protein comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 350; (iv) the CinI protein comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 351; (v) the CviI protein comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 352; (vi) the EsaI protein comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 353; (vii) LasI protein comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 376;Atty. Dkt. No. P14610WO00 (viii) the LuxI protein comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 354; or (ix) the TraI protein comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 355.

[0105] 15. The genetically engineered bacterium of any one of embodiments 1 to 14, wherein the first QSRP activates the first quorum sensing promoter by binding the QSSM and the first quorum sensing promoter, optionally wherein the first QSRP comprises an AhlR, CciR, CinR, CviR, LasR, LuxR, or TraR QSRP.

[0106] 16. The genetically engineered bacterium of any one of embodiments 1-14, wherein the first QSRP is a repressor which is released from the first quorum sensing promoter when the first QSRP binds the QSSM, optionally wherein the first QSRP comprises an EsaR QSRP with or without the amino acid change D91G.

[0107] 17. The genetically engineered bacterium of any one of embodiments 1 to 16, wherein the QSRP comprises an AhlR, CciR, CinR, CviR, EsaR, LasR, LuxR, or TraR QSRP.

[0108] 18. The genetically engineered bacterium of embodiment 17, wherein the AhlR, CciR, CinR, CviR, Esa_D91G, EsaR, LasR, LuxR, or TraR QSRP comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 339, 340 or 342, 343, 344 or 345, 346, or 347, respectively.

[0109] 19. The genetically engineered bacterium of any one of embodiments 1 to 18, wherein the control element(s), the first quorum sensing promoter, and / or the DNA promoter further comprises at least a segment of a 5’ untranslated region (5’ UTR) which is operably linked to the control element(s), quorum sensing promoter, and / or the DNA promoter, optionally wherein the 5’ UTR which is operably linked to the first quorum sensing promoter comprises a 5’ UTR sequence which has at least 85% sequence identity to an endogenous 5’ UTR sequence which is operably linked to an endogenous quorum sensing promoter.

[0110] 20. The genetically engineered bacterium of embodiment 19, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR which is operably linked to the quorum sensing promoter comprises at least one copy of an operably linked DNA binding site which can be bound by an AhlR, CciR, CinR, CviR, EsaR, LasR, LuxR, or TraR QSRP, optionally wherein said QSRP has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 339 to 347, or 375.

[0111] 21. The genetically engineered bacterium of embodiment 19 or 20, wherein the first quorum sensing promoter and / or segment the of the 5’ UTR is a promoter and / or 5’ UTR derived from at least one gram-negative bacterium.

[0112] 22. The genetically engineered bacterium of embodiment 19 or 20, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR is a promoter and / or 5’ UTR derived from at least one bacterium of the taxonomic classes of alphaproteobacteria, betaproteobacteria, and gammaproteobacteria.Atty. Dkt. No. P14610WO00

[0113] 23. The genetically engineered bacterium of embodiment 19 or 20, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR is a promoter and / or segment of a 5’ UTR derived from at least one bacterium of a genus selected from the group consisting of Acetobacter, Acidothermus, Acinetobacter, Agrobacterium, Aliivibrio, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Burkholderia, Chromobacterium, Conexibacter, Curtobacterium, Ensifer, Enterobacter, Erwinia, Escherichia, Flavobacterium, Frankia, Gaiella, Gluconacetobacter, Gluconobacter, Herbaspirillum, Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimibacter, Mesorhizobium, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrobacter, Paenibacillus, Pantoea, Pediococcus, Peribacillus, Priestia, Pseudarthrobacter, Pseudomonas, Rahnella, Rhizobium, Rhodococcus, Rhodoplanes, Rhodopseudomonas, Rhodospirillum, Serratia, Solirubrobacter, Sphingobacterium, Sphingomonas, Stenotrophomonas, Streptomyces, Stutzerimonas, Variovorax, Vibrio, Xanthobacter, and Yoonia, optionally wherein the quorum sensing promoter and / or the segment of the 5’ UTR is derived from at least one bacterium of a genus selected from the group consisting of Aliivibrio, Burkholderia, Chromobacterium, Mesorhizobium, Pantoea, Pseudomonas, and Rhizobium .

[0114] 24. The genetically engineered bacterium of any one of embodiments 19 to 23, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR comprises a promoter and / or a segment of a 5’ UTR of an ahlI, cciI, cinI, cviI, esaI, esaR, lasB, lasI, luxI, or traI gene, a variant thereof, or a combination thereof.

[0115] 25. The genetically engineered bacterium of any one of embodiments 19 to 23, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR comprises at least 50 nucleotides of DNA located upstream of a start codon of a gene encoding an AhlI, CciI, CinI, CviI, EsaI, EsaR, LasB, LasI, LuxI, or TraI protein, optionally wherein the AhlI, CciI, CinI, CviI, EsaI, EsaR, LasB, LasI, LuxI, or TraI protein protein has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NO: 348 to 355 or 376 and / or optionally wherein the promoter region and / or the segment of the 5’ UTR comprises about 50 to about 250 nucleotides of DNA located upstream of said start codon of said gene encoding said AhlI, CciI, CinI, CviI, EsaI, EsaR, LasB, LasI, LuxI, or TraI protein.

[0116] 26. The genetically engineered bacterium of any one of embodiments 19 to 25, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR comprises a promoter and / or a segment of a 5’ UTR of a promoter comprising a DNA sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 356- 364 , 373, or 374.

[0117] 27. The genetically engineered bacterium of any one of embodiments 19 to 26, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR comprises:Atty. Dkt. No. P14610WO00 (i) an ahlI promoter comprising the DNA sequence of SEQ ID NO: 356, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum, Enterobacter, Klebsiella, Kosakonia, Pseudomonas, or Rahnella; (ii) a cciI promoter comprising the DNA sequence of SEQ ID NO: 357, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum or Pseudomonas; (iii) a cinI promoter comprising the DNA sequence of SEQ ID NO: 358 or 359, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum, Pseudomonas, or Kosakonia; (iv) a cviI promoter comprising the DNA sequence of SEQ ID NO: 360, optionally wherein the bacterium is a member of the taxonomic genus Herbaspirillum, Kosakonia, or Pseudomonas; (v) an esaR_repressable promoter comprising the DNA sequence of SEQ ID NO: 361; (vi) an esaI promoter comprising the DNA sequence of SEQ ID NO: 362; (vii) a lasB promoter comprising the DNA sequence of SEQ ID NO: 374; (viii) a lasI promoter comprising the DNA sequence of SEQ ID NO: 373, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum, Kosakonia, or Pseudomonas; (ix) a luxI promoter comprising the DNA sequence of SEQ ID NO: 363, optionally wherein the bacterium is a member of the taxonomic genus Pseudomonas; (x) a traI promoter comprising the DNA sequence of SEQ ID NO: 364; or (xi) a variant of the promoters under i) to (ix) comprising a nucleic acid molecule having a sequence identity of at least 80%, 85%, 90%, 95%, 98%, or 99% with any of SEQ ID NO: 356-364, 373, or 374, optionally wherein said variant promoters are activated by an increase in the population density of the genetically engineered bacterium above the threshold population density at which the promoters under (i) to (x) are activated.

[0118] 28. The genetically engineered bacterium of any one of embodiments 19 to 27, wherein the quorum sensing promoter and / or the segment of the 5’ UTR or its variant is derived from the same genus or species as the genetically engineered bacterium.

[0119] 29. The genetically engineered bacterium of any one of embodiments 1 to 28, wherein the heterologous gene expression cassette is integrated at a location in the chromosome of the genetically engineered bacterium which does not comprise the location of an endogenous quorum sensing promoter.

[0120] 30. The genetically engineered bacterium of any one of embodiments 1 to 29, wherein one or more of the heterologous gene expression cassette(s) further comprises one or more elements comprising: a. a ribosome binding site (RBS), wherein the RBS is operably linked to the nucleic acid sequence coding for the QSSP, QSRP, and / or the protein of interest and optionally wherein the RBS is an RBS having at least 95% sequence identity to SEQ ID NO:287-313, 366-371, or 372; and / or b. a terminator sequence (TS), wherein the TS is operably linked to the nucleic acid sequence coding for the QSSP, QSRP, and / or the RNA sequence or protein of interest and optionally wherein the TS is a TS having at least 95% sequence identity to SEQ ID NO: 314, 315, or 316.Atty. Dkt. No. P14610WO00

[0121] 31. The genetically engineered bacterium of any one of embodiments 1 to 30, wherein the at least one agriculturally relevant compound: (i) is selected from the group consisting of a. at least one fertilizer or plant nutrient, optionally wherein the fertilizer or plant nutrient is selected from the group consisting of ammonia, ammonium, bioavailable carbon, calcium, iron, nitrate, nitrite, nitrogen, potassium, phosphate, sulfur, urea, zinc, a combination thereof, and a mixture thereof; b. at least one pesticide, optionally wherein the pesticide is an RNA- and / or protein-based fungicide, insecticide, nematicide, antibacterial agent, and / or antiviral agent; c. at least one phytohormone or plant growth regulator, optionally wherein the phytohormone or plant growth regulator is an auxin, a cytokinin, a gibberellin, abscisic acid, a brassinosteroid, jasmonic acid, a polyamine, a strigolactone, trehalose, and / or a volatile organic compound, or optionally wherein the phytohormone or plant growth regulator is cytokinin, indole butyric acid, and / or combinations thereof; and d. at least one carbon-containing compound, optionally wherein the carbon-containing compound is bicarbonate, carbonate, CaCO3, MgCO3, CaMg(CO3)2, polyhydroxybutyrate, melanin, chitin, and / or combinations thereof; or (ii) is not cellulose and / or wherein the protein of interest is not a protein involved in the synthesis and / or secretion of cellulose.

[0122] 32. The genetically engineered bacterium of embodiment 31, wherein the RNA sequence or protein of interest encoded by the heterologous gene expression cassette and operably linked to the control element comprising the first quorum sensing promoter comprises: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of- function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild- type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; (f) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more first target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein products of the first target genes and / or wherein the repressor protein optionally comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof and / or optionally wherein theAtty. Dkt. No. P14610WO00 first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (g) a non-coding synthetic small RNA (sRNA); optionally wherein the non-coding synthetic small RNA (sRNA) binds a natural or synthetic DNA and / or RNA motif in the promoter, 5’ UTR, and / or coding region of any one or more first target gene(s) of the genetically engineered bacterium, optionally wherein the non-coding synthetic small RNA (sRNA) comprises a guide RNA that additionally binds an RNA- guided DNA endonuclease, RNA-guided RNA endonuclease, or variant thereof and / or optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (h) a site-specific recombinase (SSR) or integrase protein: wherein any one or more first target gene(s) of the genetically engineered bacterium and / or a promoter operatively linked thereto are flanked by site-specific recombinase recognition sites (SSRRS) in a direct configuration, optionally wherein the SSRRS comprise attL and attR sites and the genetically engineered bacterium comprises a gene encoding a recombinase directionality factor (RDF) and / or optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, segment of glnE encoding an adenylyl-removing domain of a glutamine synthetase adenylyltransferase, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (i) a site-specific recombinase (SSR) or integrase protein: wherein any one or more first target gene(s), the promoter(s) thereof, and / or the 5’ UTR(s) thereof of the genetically engineered bacterium comprise(s) one or more internal synthetic SSRRS and wherein the gene(s), promoter(s), the 5’ UTR(s), and / or a segment(s) thereof is / are excised or inactivated after a recombination event; optionally wherein the genetically engineered bacterium further comprises a plasmid comprising an SSRRS; optionally wherein the genetically engineered bacterium further comprises one or more genes encoding a recombinase directionality factor (RDF); and / or optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnE, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (j) a site-specific recombinase (SSR) or integrase protein, wherein any one or more first target gene(s), the promoter(s) thereof, and / or the 5’ UTR(s) thereof of the genetically engineered bacterium comprise(s) one or more internal integrative SSRRS and retain(s) activity of said first target gene(s), promoter(s), and / or 5’ UTR(s) and wherein the genetically engineered bacterium further comprises an integrative element comprising an SSRRS; optionally wherein the internal integrative SSRRS is / are an attB site(s) and the integrative element comprises an SSRRS comprising an attP site; and / or optionally wherein the target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (k) a site-specific recombinase (SSR) or integrase protein, wherein a control element comprising a promoter and / or at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase recognition sites (SSRRS) in an inverted configuration; and (ii) is operably linked to one or more first target gene(s) of the genetically engineered bacterium; optionally wherein the control element which is operably linked to said first target gene(s) comprises a constitutive promoter, an inducible promoter, or quorum sensing promoter;Atty. Dkt. No. P14610WO00 and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (l) a site-specific recombinase (SSR) or integrase protein, wherein a control element comprising a promoter and at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase recognition sites (SSRRS) in an inverted configuration; and (ii) is operably linked to one or more first target gene(s) of the genetically engineered bacterium upon inversion by the SSR or integrase; optionally wherein the control element which is operably linked to said first target gene(s) upon inversion by the SSR or integrase comprises a constitutive promoter, an inducible promoter, or a quorum sensing promoter; and / or optionally wherein the first target gene(s) is / are: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase, and the agriculturally relevant compound is phosphate; (m) a site-specific DNA endonuclease, wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) one or more specific DNA sequence(s) recognized by the site-specific DNA endonuclease, optionally wherein the site-specific DNA endonuclease comprises an RNA-guided DNA endonuclease, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or a restriction endonuclease, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (n) a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) comprising DNA encoding the PSRS in the protein coding region of the first target gene(s), wherein the target protein product comprising the one or more in-frame insertion(s) has activity, and wherein cleavage of the target protein product(s) by the protease deactivates the target protein product(s), optionally wherein the location of the one or more in-frame insertion(s) is given by Table 6, optionally wherein the first target gene(s) is / are under the control of a constitutive promoter, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (o) a first protein of interest comprising a ClpAP ATP-dependent protease and a second protein of interest comprising a ClpS Leu / N-recognin, wherein any one or more first target gene(s) of the genetically engineered bacterium encodes a protein comprising an N-terminal -Leu, -Phe, -Trp, or -Tyr residue, andAtty. Dkt. No. P14610WO00 optionally wherein the first and second protein of interest are operably linked to distinct control elements, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (p) a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion(s) of DNA encoding the PSRS at the N-terminus of the protein coding region of the gene followed by a -Leu, - Phe, -Trp, or -Tyr residue and wherein cleavage of the PSRS from the N-terminus of the protein(s) encoded by the gene(s) by the protease results in a protein comprising an N-terminal -Leu, -Phe, -Trp, or -Tyr residue which is degraded by native ClpS and ClpAP, optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (q) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and activates expression of the protease, wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) of DNA encoding a PSRS, optionally wherein the transcriptional activator protein comprises a tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, optionally wherein the transcriptional activator domain is VP16 , and / or optionally wherein the first target gene(s) is / are: glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (r) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and inhibits expression of the protease, wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion of DNA encoding an N-terminal amino acid tag designed to render the protein product(s) of the first target gene(s) inactive, followed by a PSRS, optionally wherein the repressor protein comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (s) a nanobody, wherein the nanobody binds a protein product of any one or more first target gene(s) of the genetically engineered bacterium, wherein binding of the nanobody to such protein product inhibits function of the protein product, optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (t) an aptamer, wherein the aptamer binds a protein product of any one or more first target gene(s) of the genetically engineered bacterium and inhibits function of any one or more of the protein product(s)Atty. Dkt. No. P14610WO00 of the first target gene(s), optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; or (u) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of any one or more first target gene(s) and increases expression of any one or more of the first target gene(s), optionally wherein the transcriptional activator protein comprises the tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator- like effector (TALE), or any variant thereof, optionally wherein the transcriptional activator domain is VP16 and / or optionally wherein the first target gene(s) is / are any one or more of: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, and / or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase and the agriculturally relevant compound is phosphate.

[0123] 33. The genetically engineered bacterium of embodiment 32, wherein the integrase is a serine integrase, optionally wherein the serine integrase is a phage PhiC31 serine integrase, IntS, IntM, IntG – ICEMcSym 1271, YdcL - ICEBs, or Int - ICE SXT / R39 integrase, and the SSRRS are attB and attP sites recognized respectively by the PhiC31, IntS, IntM, IntG, YdcL, or Sxt / R39 integrase.

[0124] 34. The genetically engineered bacterium of embodiment 32, wherein: (i) one or more SSR is a yeast flippase (FLP) recombinase and the SSRRS are FRT sites; or (ii) one or more SSR is a Cre- recombinase and the SSRRS are loxP sites.

[0125] 35. The genetically engineered bacterium of embodiment 32, wherein a PSRS is inserted between any one or more of the pairs of amino acid residues corresponding to: (i) amino acid residues 98 and 99, 121 and 122, 279 and 280, and / or 285 and 286 of the glutamine synthetase (GS) polypeptide of SEQ ID NO: 229; (ii) amino acid residues 98 and 99, 119 and 120, 283 and 284, and / or 298 and 299 of the glutamine synthetase (GS) polypeptide of SEQ ID NO: 230; (iii) amino acid residues 448 and 449, 480 and 481, 505 and 506, 528 and 529, and / or 624 and 625 of the GlnE polypeptide of SEQ ID NO: 231;Atty. Dkt. No. P14610WO00 (iv) amino acid residues 527 and 528, 537 and 538, and / or 547 and 548 of the GlnE polypeptide of SEQ ID NO: 232; or (v) amino acid residues 422 and 423 and / or 608 and 609 of the GlnE polypeptide of SEQ ID NO: 233.

[0126] 36. The genetically engineered bacterium of embodiment 32 or 35, wherein the protease or heterologous protease: a. comprises a tobacco etch virus (TEV) protease and the PSRS comprises the peptide EXXYXQ- (S / G) or ENLYFQ-(S / G / A / M / C / H), wherein X is any amino acid and the TEV protease cleaves between the Q and S, G, A, M, C, or H residues; b. comprises a tobacco vein mottling virus (TVMV) protease and the PSRS comprises the peptide ETVRFQ-(G / S), wherein the TVMV protease cleaves between the Q and S or G residues; c. comprises a sunflower mild mosaic virus (SMMV) protease and the PSRS comprises the peptide EEIHLQ-(S / G), wherein the SMMV protease cleaves between the Q and S or G residues; d. comprises a turnip mosaic virus (TrMV) protease and the PSRS comprises the peptide VXHQ or VRHQ-S, wherein X is any amino acid and the TrMV protease cleaves C-terminal to the Q residue; e. comprises a soybean mosaic virus (SMV) protease and the PSRS comprises the peptide XVXXQ-(G / S), wherein X is any amino acid and the SMV protease cleaves between Q and S or G residues; f. comprises a plum pox virus (PPV) protease and the PSRS comprises the peptide NVVVHQ-A, wherein the PPV protease cleaves between the Q and the A residue; g. comprises a hepatitis C virus (HCV) NS3 protease and the PSRS comprises the peptide (D / E)XXXXC(A / S), wherein X is any amino acid and the HCV protease cleaves between the C and the A or S residues; h. comprises an enterokinase and the PSRS comprises the peptide DDDDK, wherein the enterokinase cleaves C-terminal to the K residue; i. comprises a Factor Xa protease and the PSRS comprises the peptide I(D / E)GR, wherein the Factor Xa protease cleaves C-terminal to the R residue; or j. comprises a furin protease and the PSRS comprises the peptide RX(K / R)R, wherein the furin protease cleaves C-terminal to the C-terminal R residue.

[0127] 37. The genetically engineered bacterium of embodiment 31, wherein: (i) the plant nutrient is zinc or potassium and the protein of interest is a gluconate dehydrogenase (GAD) enzyme; (ii) the plant nutrient is iron and the protein of interest is a siderophore biosynthetic and transport proteins optionally selected from a dhbACDEBF gene cluster; a non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and NRPS-independent siderophore synthetase (NIS); and major facilitator superfamily (MFS) transporters (ymfE), TonB, ExbD, and / or ExbB; (iii) the phytohormone is auxin and the protein of interest is an IpdC or IaaM protein;Atty. Dkt. No. P14610WO00 (iv) the plant nutrient is ammonia and α-ketobutyrate and the protein is ACC deaminase; (v) the plant growth regulator is a volatile organic compound and the proteins of interest are Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and 2,3-butanediol Dehydrogenase (BDH); (vi) the agriculturally relevant compound is trehalose and the protein(s) of interest is OtsA, OtsB, and TreS or TreS; or (vii) the carbon containing compound is calcium carbonate and the protein of interest is a beta- carbonic anhydrase or alpha-carbonic anhydrase.

[0128] 38. The genetically engineered bacterium of embodiment 31, wherein the protein of interest comprises a site-specific recombinase (SSR) or integrase protein, wherein a control element comprising a promoter and / or at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase recognition sites (SSRRS) in an inverted configuration; (ii) is operably linked to one or more first target gene(s) of the genetically engineered bacterium; and (iii) is operably linked to one or more second target gene(s) of the genetically engineered bacterium upon inversion by the SSR or integrase; and wherein: (a) the first target gene is a glnA gene encoding a wild-type glutamine synthetase (GS) or variant thereof with improved catalytic activity in comparison to wild-type GS and the second target gene is a glnA gene encoding a wild-type GS with reduced levels of expression in comparison to the wild-type glnA gene or a GS variant with decreased catalytic activity in comparison to wild-type GS, and the fertilizer is ammonia; (b) the first target gene is a glnE gene encoding a wild-type GS adenylyltransferase protein and the second target gene is a glnE gene encoding a GS adenylyltransferase protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, and the fertilizer is ammonia; or (c) the first target gene is a nifL gene and the second target gene is a nifA gene, and the fertilizer is ammonia.

[0129] 39. The genetically engineered bacterium of embodiment 32, wherein the control element which is operably linked to said target genes comprises a constitutive promoter, an inducible promoter, a repressible promoter, and / or a quorum sensing promoter.

[0130] 40. The genetically engineered bacterium of embodiment 32, wherein the integrase is a serine integrase, optionally wherein the serine integrase is a phage PhiC31 serine integrase, IntS, IntM, IntG – ICEMcSym 1271, Yd–L - ICEBs, or I–t - ICE SXT / R39 integrase, and the SSRRS are attB and attP sites recognized respectively by the PhiC31, IntS, IntM, IntG, YdcL, or Sxt / R39 integrase.

[0131] 41. The genetically engineered bacterium of embodiment 32, wherein: (i) one or more SSR is a yeast flippase (FLP) recombinase and the SSRRS are FRT sites; or (ii) one or more SSR is a Cre- recombinase and the SSRRS are loxP sites.

[0132] 42. The genetically engineered bacterium of embodiment 31, wherein the protein of interest is a first repressor protein and wherein the genetically engineered bacterium further comprises:Atty. Dkt. No. P14610WO00 (i) a second control element comprising a promoter which is repressed by the first repressor protein and operably linked to a gene encoding a second repressor protein; (ii) optionally a third control element comprising a promoter which is repressed by the second repressor protein and operably linked to a gene encoding a third repressor protein; (iii) optionally a fourth control element comprising a promoter which is repressed by the third repressor protein and operably linked to a gene encoding a fourth repressor protein; and (iv) a control element comprising a promoter which is repressed by the second, third, or fourth repressor protein and which is operably linked to a first target gene, optionally wherein the first, second, third, and / or fourth repressor protein(s) optionally comprise(s) the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof.

[0133] 43. The genetically engineered bacterium of embodiment 42, wherein the genetically engineered bacterium lacks (ii) and (iii), and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s), and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase, and the agriculturally relevant compound is phosphate.

[0134] 44. The genetically engineered bacterium of embodiment 42, wherein the genetically engineered bacterium further comprises (ii) and (iii), and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase, and the agriculturally relevant compound is phosphate.Atty. Dkt. No. P14610WO00

[0135] 45. The genetically engineered bacterium of embodiment 42, wherein the genetically engineered bacterium further comprises (ii) and lacks (iii), and the first target gene is amtB, draT, glnA, glnB, glnK, glnZ, and / or nifL, and the agriculturally relevant compound is ammonia.

[0136] 46. The genetically engineered bacterium of embodiment 42, wherein first target gene encodes a transcriptional activator protein and wherein the genetically engineered bacterium further comprises a control element comprising a promoter which is activated by the transcriptional activator protein and operably linked to at least one second target gene.

[0137] 47. The genetically engineered bacterium of embodiment 42, wherein the genetically engineered bacterium comprises an even number of repressors and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), a pyrroloquinoline (PQQ) synthase, and the agriculturally relevant compound is phosphate.

[0138] 48. The genetically engineered bacterium of embodiment 42, wherein the genetically engineered bacterium comprises an even number of repressors and the first target gene encodes an RNA sequence or protein comprising: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of- function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, optionally wherein the genetically engineered bacterium comprises one or more heterologous genes from a wild-type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; (f) a phytase enzyme and the agriculturally relevant compound is phosphate, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase; (g) an acid phosphatase enzyme and the agriculturally relevant compound is phosphate, optionally wherein the acid phosphatase enzyme is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof;Atty. Dkt. No. P14610WO00 (h) a protein which stimulates organic acid release from the bacterium and the agriculturally relevant compound is phosphate, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase; (i) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more second target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein product(s) of the second target gene(s) and / or wherein the repressor protein optionally comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof and / or optionally wherein the second target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (j) a non-coding synthetic small RNA (sRNA); optionally wherein the non-coding synthetic small RNA (sRNA) binds a natural or synthetic DNA and / or RNA motif in the promoter, 5’ UTR, and / or coding region of any one or more second target gene(s) of the genetically engineered bacterium, optionally wherein the non-coding synthetic small RNA (sRNA) comprises a guide RNA that additionally binds an RNA- guided DNA endonuclease, RNA-guided RNA endonuclease, or variant thereof and / or optionally wherein the second target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (k) a site-specific recombinase (SSR) or integrase protein: wherein any one or more second target gene(s) of the genetically engineered bacterium and / or a promoter operatively linked thereto are flanked by site-specific recombinase recognition sites (SSRRS) in a direct configuration, optionally wherein the SSRRS comprise attL and attR sites and the genetically engineered bacterium comprises a gene encoding a recombinase directionality factor (RDF) and / or optionally wherein the second target gene(s) is / are a glnA, amtB, glnB, segment of glnE encoding an adenylyl-removing domain of a glutamine synthetase adenylyltransferase, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (l) a site-specific recombinase (SSR) or integrase protein: wherein any one or more second target gene(s), the promoter(s) thereof, and / or the 5’ UTR(s) thereof of the genetically engineered bacterium comprise(s) one or more internal synthetic SSRRS and wherein the gene(s), promoter(s), the 5’ UTR(s), and / or a segment(s) thereof is / are excised or inactivated after a recombination event; optionally wherein the genetically engineered bacterium further comprises a plasmid comprising an SSRRS; optionally wherein the genetically engineered bacterium further comprises one or more genes encoding a recombinase directionality factor (RDF); and / or optionally wherein the second target gene(s) is / are a glnA, amtB, glnB, glnE, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (m) a site-specific recombinase (SSR) or integrase protein, wherein any one or more second target gene(s), the promoter(s) thereof, and / or the 5’ UTR(s) thereof of the genetically engineered bacteriumAtty. Dkt. No. P14610WO00 comprise(s) one or more internal integrative SSRRS and retain(s) activity of said second target gene(s), promoter(s), and / or 5’ UTR(s) and wherein the genetically engineered bacterium further comprises an integrative element comprising an SSRRS; optionally wherein the internal integrative SSRRS is / are an attB site(s), the integrative element comprises an SSRRS comprising an attP site; and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (n) a site-specific recombinase (SSR) or integrase protein, wherein a control element comprising a promoter and / or at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase recognition sites (SSRRS) in an inverted configuration; and (ii) is operably linked to one or more second target gene(s) of the genetically engineered bacterium; optionally wherein the control element which is operably linked to said second target gene(s) comprises a constitutive promoter, an inducible promoter, or a quorum sensing promoter; and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (o) a site-specific recombinase (SSR) or integrase protein, wherein a control element comprising a promoter and at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase recognition sites (SSRRS) in an inverted configuration; and (ii) is operably linked to one or more second target gene(s) of the genetically engineered bacterium upon inversion by the SSR or integrase; optionally wherein the control element which is operably linked to said second target gene(s) upon inversion by the SSR or integrase comprises a constitutive promoter, an inducible promoter, or a quorum sensing promoter; and / or optionally wherein the second target gene(s) is / are: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase, and the agriculturally relevant compound is phosphate; (p) a site-specific DNA endonuclease, wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) one or more specific DNA sequence(s) recognized by the site-specific DNA endonuclease, optionally wherein the site-specific DNA endonuclease comprises an RNA-guided DNA endonuclease, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or a restriction endonuclease, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia;Atty. Dkt. No. P14610WO00 (q) a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) comprising DNA encoding the PSRS in the protein coding region of the second target gene(s), wherein the target protein product comprising the one or more in-frame insertion(s) has activity, and wherein cleavage of the target protein product(s) by the protease deactivates the target protein product(s), optionally wherein the location of the one or more in-frame insertion is given by Table 6, optionally wherein the second target gene is under the control of a constitutive promoter, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (r) a first protein comprising a ClpAP ATP-dependent protease and a second protein comprising a ClpS Leu / N-recognin, wherein any one or more second target gene(s) of the genetically engineered bacterium encodes a third protein comprising an N-terminal -Leu, -Phe, -Trp, or -Tyr residue, and optionally wherein the first and second protein are operably linked to distinct control elements, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (s) a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion(s) of DNA encoding the PSRS at the N-terminus of the protein coding region of the gene(s) followed by a -Leu, -Phe, -Trp, or -Tyr residue and wherein cleavage of PSRS from the N-terminus of the protein(s) encoded by the gene(s) by the protease results in a protein comprising an N-terminal -Leu, -Phe, -Trp, or -Tyr residue which is degraded by native ClpS and ClpAP, optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (t) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and activates expression of the protease, wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) of DNA encoding a PSRS, optionally wherein the transcriptional activator protein comprises a tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA- binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, optionally wherein the transcriptional activator domain is VP16 , and / or optionally wherein the second target gene(s) is / are: glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (u) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and inhibits expression of the protease, wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion of DNA encoding an N-terminal amino acid tag designed to render the protein product(s) of the second target gene(s) inactive, followed by a PSRS, optionally wherein the repressor protein comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), aAtty. Dkt. No. P14610WO00 catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (v) a nanobody, wherein the nanobody binds a protein product of any one or more second target gene(s) of the genetically engineered bacterium, wherein binding of the nanobody to such protein product inhibits function of the protein product, optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (w) an aptamer, wherein the aptamer binds a protein product of any one or more second target gene(s) of the genetically engineered bacterium and inhibits function of any one or more of the protein product(s) of the second target gene(s), optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; or (x) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of any one or more second target gene(s) and increases expression of any one or more of the second target gene(s), optionally wherein the transcriptional activator protein comprises the tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, wherein the DNA targeting protein is optionally a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, wherein the transcriptional activator domain is optionally VP16 and / or optionally wherein the second target gene(s) is / are any one or more of: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, and / or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase and the agriculturally relevant compound is phosphate.

[0139] 49. The genetically engineered bacterium of embodiment 42, wherein the genetically engineered bacterium comprises an odd number of repressors and the second target gene is amtB, draT, glnA, glnB, glnK, glnZ, and / or nifL, and the agriculturally relevant compound is ammonia.

[0140] 50. The genetically engineered bacterium of embodiment 31, wherein the protein of interest is a first transcriptional activator protein and wherein the genetically engineered bacterium further comprises: (i) a control element comprising a promoter which is activated by the first transcriptional activator protein and operably linked to a gene encoding a second transcriptional activator protein;Atty. Dkt. No. P14610WO00 (ii) optionally a control element comprising a promoter which is activated by the second transcriptional activator protein and operably linked to a gene encoding a third transcriptional activator protein; (iii) optionally a control element comprising a promoter which is activated by the third transcriptional activator protein and operably linked to a gene encoding a fourth transcriptional activator protein; and (iv) a control element comprising a promoter which is activated by the second, third, or fourth transcriptional activator protein and which is operably linked to a first target gene, optionally wherein the first, second, third, and / or fourth transcriptional activator protein comprise(s) the tet responsive element- binding tTA transcription factor or an activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), and / or any variant thereof, optionally wherein the activator domain is VP16.

[0141] 51. The genetically engineered bacterium of embodiment 50, wherein the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase, and the agriculturally relevant compound is phosphate.

[0142] 52. The genetically engineered bacterium of embodiment 31, wherein the protein of interest is a repressor protein and wherein the genetically engineered bacterium further comprises: (i) a control element comprising a promoter which is repressed by the repressor protein and operably linked to a gene encoding a transcriptional activator protein; and (ii) a control element comprising a promoter which is activated by the transcriptional activator protein and operably linked to a target gene.

[0143] 53. The genetically engineered bacterium of embodiment 52, wherein the target gene is a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the agriculturally relevant compound is ammonia.

[0144] 54. The genetically engineered bacterium of embodiment 31, wherein the protein of interest is a transcriptional activator protein and wherein the genetically engineered bacterium further comprises: (i) a second control element comprising a promoter which is activated by the transcriptional activator protein and operably linked to a gene encoding a repressor protein; andAtty. Dkt. No. P14610WO00 (ii) a third control element comprising a promoter which is repressed by the repressor and operably linked to a target gene.

[0145] 55. The genetically engineered bacterium of embodiment 54, wherein the target gene is a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the agriculturally relevant compound is ammonia.

[0146] 56. The genetically engineered bacterium of embodiment 32, wherein the glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene in (n), (o), (p), or (q) is under the control of a heterologous constitutive promoter.

[0147] 57. The method of embodiment 48, wherein the glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene in (q), (r), (s), or (t) is under the control of a heterologous constitutive promoter.

[0148] 58. The genetically engineered bacterium of embodiment 31, wherein the plant nutrient is phosphate and the protein of interest operably linked to the quorum sensing promoter comprises: a. a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase; b. an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof; c. a protein which stimulates organic acid release from the bacterium, optionally wherein the protein comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), a pyrroloquinoline (PQQ) synthase including pqqFABCDEG, or any combination of GAD, GDH, and PQQ; or, d. any combination of proteins of a, b, or c.

[0149] 59. The genetically engineered bacterium of any one of embodiments 1 to 58, wherein: (i) the genetically engineered bacterium comprises the heterologous gene expression cassettes in a bacterium originally isolated from a plant growth medium or a plant; and (ii) wherein the bacterium originally isolated from the plant growth medium or the plant lacks the heterologous gene expression cassettes.

[0150] 60. The genetically engineered bacterium of any one of embodiments 1 to 59, wherein the bacteria are selected from the group of gram-negative bacteria.

[0151] 61. The genetically engineered bacterium of any one of embodiments 1 to 59, wherein the bacteria are selected from the groups of bacteria of the taxonomic classes of alphaproteobacteria, betaproteobacteria, and gammaproteobacteria.

[0152] 62. The genetically engineered bacterium of any one of embodiments 1 to 59, wherein the bacteria are selected from the groups of bacteria in the taxonomic genera of Acetobacter, Acidothermus, Acinetobacter, Agrobacterium, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum,Atty. Dkt. No. P14610WO00 Rhodococcus, Rhodoplanes, Rhodopseudomonas, Rhodospirillum, Serratia, Solirubrobacter, Sphingobacterium, Sphingomonas, Stenotrophomonas, Streptomyces, Stutzerimonas, Variovorax, Xanthobacter, and Yoonia, optionally wherein the bacteria are selected from at least one of the taxonomic genera selected from the group consisting of Herbaspirillum, Azospirillum, Kosakonia, or Pseudomonas.

[0153] 63. A composition comprising the genetically engineered bacterium of any one of any one of embodiments 1 to 62 and an agriculturally acceptable carrier.

[0154] 64. The composition of embodiment 63, wherein the composition further comprises: (i) an agriculturally acceptable adjuvant, optionally wherein the adjuvant comprises an adhesive agent, a desiccant, and / or a dispersant; (ii) a fungicide, an insecticide, a nematicide, a rodenticide, and / or a bacteriocide; and / or (iii) a fertilizer, optionally wherein the fertilizer comprises nitrogen, phosphorous, potassium, calcium, sulfur, magnesium, boron, chloride, manganese, iron, zinc, copper, molybdenum, and / or selenium.

[0155] 65. The composition of embodiment 63 or 64, wherein the composition is in a solid form, optionally wherein the solid form comprises a wettable powder, granules, a gel, pellets, or microencapsulated particles.

[0156] 66. The composition of embodiment 63 or 64, wherein the composition is in a liquid form, optionally wherein the liquid form comprises an aqueous solution, aqueous suspension, water-in-oil emulsion, an oil, or an alcohol.

[0157] 67. A plant part or plant propagule which is at least partially coated, imbibed, or mixed with the composition of any one of embodiments 63 to 66.

[0158] 68. The plant part of embodiment 67, wherein the part is a leaf, stem, root, or seed.

[0159] 69. The plant propagule of embodiment 67, wherein the propagule comprises a cutting, tuber, or stolon.

[0160] 70. Use of the plant part or plant propagule of embodiment 67 to grow a crop.

[0161] 71. The use of embodiment 70, wherein fertilizer input is reduced in comparison to a crop grown from a plant part or plant propagule which has not been treated.

[0162] 72. An agricultural system comprising: (i) at least one engineered bacterium of any one of embodiments 1 to 62; (ii) at least one plant growth medium; and (iii) at least one crop plant, crop plant seed, or crop plant vegetative propagule; wherein the plant growth medium, crop plant, crop seed, and / or crop plant propagule comprise, are at least partially coated, imbibed, and / or are mixed with the engineered bacterium or a composition comprising the engineered bacterium and an agriculturally acceptable carrier.

[0163] 73. The system of embodiment 72, wherein the crop plant, seed, or vegetative propagule is an alfalfa, apple, banana, barley, bean, buckwheat, cabbage, cassava, chili, clover, coffee, corn, cotton, cowpea, cucumber, fonio, garlic, herb, lettuce, maize, melon, millet, nut, oat, oilseed rape, olive, onion, orange,Atty. Dkt. No. P14610WO00 sunflower, pea, Phaseolus bean, plantain, potato, quinoa, rice, rye, safflower, sorghum, soybean, sugar beet, sugar cane, sunflower, tangerine, tobacco, tomato, triticale, turnip, wheat, or yam plant, seed, or vegetative propagule.

[0164] 74. The system of embodiment 72 or 73, wherein the plant growth medium comprises soil and / or water, optionally wherein the soil and / or water is non-axenic.

[0165] 75. The system of embodiment 72, 73, or 74, wherein the vegetative propagule comprises a cutting, tuber, or stolon.

[0166] 76. A treated plant seed or plant propagule system comprising: (i) at least one crop plant seed or crop plant vegetative propagule; and (ii) at least one engineered bacterium of any one of embodiments 1 to 62, wherein the crop plant seed or crop plant propagule are at least partially coated, imbibed, and / or mixed with the engineered bacterium or a composition comprising the engineered bacterium and an agriculturally acceptable carrier.

[0167] 77. The system of embodiment 76, wherein the crop plant, seed, or vegetative propagule is an alfalfa, apple, banana, barley, bean, buckwheat, cabbage, cassava, chili, clover, coffee, corn, cotton, cowpea, cucumber, fonio, garlic, herb, lettuce, maize, melon, millet, nut, oat, oilseed rape, olive, onion, orange, sunflower, pea, Phaseolus bean, plantain, potato, quinoa, rice, rye, safflower, sorghum, soybean, sugar beet, sugar cane, sunflower, tangerine, tobacco, tomato, triticale, turnip, wheat, or yam plant, seed, or vegetative propagule.

[0168] 78. The system of embodiment 76 or 77, wherein the vegetative propagule comprises a cutting, tuber, or stolon.

[0169] 79. A method of producing a bacterial culture comprising: (i) growing the genetically engineered bacterium of any one of embodiments 1 to 62 either: (a) in contact with the quorum quenching compound QQ; or (b) in exposure to a temperature above the threshold temperature; and (ii) harvesting the bacterial culture.

[0170] 80. The method of embodiment 79, wherein growing the genetically engineered bacterium in the presence of QQ and / or at a temperature above the threshold temperature enables said genetically engineered bacteria to grow to a greater population density and / or at a faster rate to a maximal population density than in the absence of QQ and / or at a temperature below the threshold temperature, respectively.

[0171] 81. The method of embodiment 79 or 80, wherein the genetically engineered bacterium is grown to a cell density in excess of the cell density where the RNA or protein is expressed in the absence of the compound QQ and / or temperatures in excess of the threshold temperature.

[0172] 82. The method of embodiment 79, 80, or 81, wherein the genetically engineered bacterium is grown to a cell density in excess of about 1 x 107, 1 x 108, or 1 x 109colony forming units per milliliter (CFU / mL) of the culture prior to harvesting.Atty. Dkt. No. P14610WO00

[0173] 83. The method of any one of embodiments 79 to 82, wherein the bacterial culture is grown in the presence of: (i) excess nitrogen; and / or (ii) excess phosphorus, optionally wherein the quorum quenching compound QQ is inorganic phosphate or soluble phosphate, optionally wherein the control element operably linked to the gene encoding the first QSRP comprises a phosphate-sensitive promoter.

[0174] 84. The method of any one of embodiments 79 to 83, wherein the repressor in the QQ induced de-repression of the deactivator comprises: (a) a TetR repressor having at least 95% sequence identity to SEQ ID NO: 27 and wherein the DNA promoter operably linked to the gene encoding the deactivator comprises a Ptet promoter having at least 95% sequence identity to SEQ ID NO: 32, and optionally wherein the QQ compound is doxycycline, tetracycline, and / or anhydrotetracycline; (b) a LacI repressor having at least 95% sequence identity to SEQ ID NO: 29 and wherein the DNA promoter operably linked to the gene encoding the deactivator comprises a Plac promoter having at least 95% sequence identity to SEQ ID NO: 33, and optionally wherein the QQ compound is allolactose, isopropyl-β-D-thiogalactopyranoside (IPTG), or thiomethylgalactoside; or (c) a PhlF repressor having at least 95% sequence identity to SEQ ID NO: 30) and wherein the DNA promoter operably linked to the gene encoding the deactivator comprises a PphlA promoter having at least 95% sequence identity to SEQ ID NO: 34, and optionally wherein the QQ compound is 2,4- diacetylphoroglucinol (DAPG).

[0175] 85. The method of any one of embodiments 79 to 84, wherein: (i) the RNA or protein of interest is not expressed in the bacterial culture when harvested in step (ii); and / or (ii) the agricultural compound of interest is not produced by the bacterial culture when harvested in step (ii).

[0176] 86. The method of any one of embodiments 79 to 85, wherein harvesting comprises separating the bacteria from the culture media to obtain a solid bacterial culture, optionally wherein the separating comprises filtering the liquid bacterial culture from the culture media and / or centrifuging the bacterial culture and decanting the liquid bacterial culture.

[0177] 87. The method of any one of embodiments 79 to 86, further comprising combining the harvested bacterial culture with an agriculturally acceptable carrier and optionally (i) an agriculturally acceptable adjuvant, optionally wherein the adjuvant comprises an adhesive agent, a desiccant, and / or a dispersant; (ii) a fungicide, an insecticide, a nematicide, a rodenticide, and / or a bacteriocide; and / or (iii) a fertilizer, optionally wherein the fertilizer comprises nitrogen, phosphorous, potassium, calcium, sulfur, magnesium, boron, chloride, manganese, iron, zinc, copper, molybdenum, and / or selenium, thereby forming a composition.Atty. Dkt. No. P14610WO00

[0178] 88. The method of embodiment 87, wherein the composition is made in a solid form, optionally wherein the solid form is made by formulating the composition into a wettable powder, granules, a gel, pellets, or microencapsulated particles.

[0179] 89. The method of embodiment 87 or 88, further comprising drying and / or lyophilizing the composition.

[0180] 90. The method of embodiment 87, wherein the composition is made in a liquid form, optionally wherein the liquid form is made by formulating the composition as an aqueous solution, aqueous suspension, water-in-oil emulsion, an oil, or an alcohol.

[0181] 91. A method of providing at least one agriculturally relevant compound to a plant comprising placing at least one genetically engineered bacterium of any one of embodiments 1 to 62 into a plant growth medium, wherein said at least one RNA sequence or protein of interest is or causes the production of said at least one agriculturally relevant compound when the population density of the genetically engineered bacterium exceeds a threshold population density in the plant growth medium and activates expression of said at least one RNA sequence or protein of interest.

[0182] 92. The method of embodiment 91, wherein the plant growth medium comprises soil and / or water, optionally wherein the soil and / or water is non-axenic.

[0183] 93. The method of embodiment 91, wherein the placing is prior to, during, and / or after depositing a seed in the plant growth medium.

[0184] 94. The method of embodiment 91, wherein the placing is prior to, during, and / or after depositing a vegetative propagule in the plant growth medium.

[0185] 95. The method of embodiment 91, wherein the placing comprises depositing a seed which is at least partially coated with the genetically engineered bacterium in the plant growth medium or depositing both the seed and a composition comprising the genetically engineered bacterium in the plant growth medium.

[0186] 96. The method of embodiment 91, wherein the placing comprises depositing the seed in furrow and contacting the seed in the furrow with a composition comprising the genetically engineered bacterium.

[0187] 97. The method of embodiment 91, wherein the placing of the genetically engineered bacterium in the plant growth medium is prior to, during, and / or after establishment of a plant in the plant growth medium.

[0188] 98. The method of embodiment 91, wherein the genetically engineered bacterium is placed into the plant growth medium and / or in contact with the plant: (i) by foliar application to the plant; (ii) by an in furrow application, fumigation, and / or soil drench; (iii) with a seed in form of a seed treatment wherein the seed is at least partially coated with a composition comprising the genetically engineered bacterium;Atty. Dkt. No. P14610WO00 (iv) with a seed in the form of bio-priming where the seed is imbibed with an aqueous composition comprising the genetically engineered bacterium before planting; and / or (v) with a root dip transplant whereby a seedling root system is dipped in an aqueous composition comprising the genetically engineered bacterium.

[0189] 99. The method of any one of embodiments 91 to 98, wherein the at least one agriculturally relevant compound is selected from the group consisting of: (a) at least one fertilizer or plant nutrient, optionally wherein the fertilizer or plant nutrient is selected from the group consisting of ammonia, ammonium, bioavailable carbon, calcium, iron, nitrate, nitrite, nitrogen, potassium, phosphate, sulfur, urea, zinc, a combination thereof, and a mixture thereof; (b) at least one pesticide, optionally wherein the pesticide is an RNA- and / or protein-based fungicide, insecticide, nematicide, antibacterial agent, and / or antiviral agent; (c) at least one phytohormone or plant growth regulator, optionally wherein the phytohormone or plant growth regulator is an auxin, a cytokinin, a gibberellin, abscisic acid, a brassinosteroid, jasmonic acid, a polyamine, a strigolactone, trehalose, and / or a volatile organic compound, or optionally wherein the phytohormone or plant growth regulator is cytokinin, indole butyric acid, and / or combinations thereof; and (d) at least one carbon-containing compound, optionally wherein the carbon-containing compound is bicarbonate, carbonate, CaCO3, MgCO3, CaMg(CO3)2, polyhydroxybutyrate, melanin, chitin, and / or combinations thereof.

[0190] 100. The method of embodiment 99, wherein the RNA sequence or protein of interest encoded by the heterologous gene expression cassette and operably linked to the control element comprises: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of- function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild- type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; (f) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more first target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein products of the first target genes and / or wherein the repressor protein optionally comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof and / or optionally wherein theAtty. Dkt. No. P14610WO00 first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (g) a non-coding synthetic small RNA (sRNA); optionally wherein the non-coding synthetic small RNA (sRNA) binds a natural or synthetic DNA and / or RNA motif in the promoter, 5’ UTR, and / or coding region of any one or more first target gene(s) of the genetically engineered bacterium, optionally wherein the non-coding synthetic small RNA (sRNA) comprises a guide RNA that additionally binds an RNA- guided DNA or RNA endonuclease or variant thereof and / or optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (h) a site-specific recombinase (SSR) or integrase protein: wherein any one or more first target gene(s) of the genetically engineered bacterium and / or a promoter operatively linked thereto are flanked by site-specific recombinase recognition sites (SSRRS) in a direct configuration, optionally wherein the SSRRS comprise attL and attR sites and the genetically engineered bacterium comprises a gene encoding a recombinase directionality factor (RDF) and / or optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, segment of glnE encoding an adenylyl-removing domain of a glutamine synthetase adenylyltransferase, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (i) a site-specific recombinase (SSR) or integrase protein: wherein any one or more first target gene(s), the promoter(s) thereof, and / or the 5’ UTR(s) thereof of the genetically engineered bacterium comprise(s) one or more internal synthetic SSRRS and wherein the gene(s), promoter(s), the 5’ UTR(s), and / or a segment(s) thereof is / are excised or inactivated after a recombination event; optionally wherein the genetically engineered bacterium further comprises a plasmid comprising an SSRRS; optionally wherein the genetically engineered bacterium further comprises one or more genes encoding a recombinase directionality factor (RDF); and / or optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnE, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (j) a site-specific recombinase (SSR) or integrase protein, wherein any one or more first target gene(s), the promoter(s) thereof, and / or the 5’ UTR(s) thereof of the genetically engineered bacterium comprise(s) one or more internal integrative SSRRS and retain(s) activity of said first target gene(s), promoter(s), and / or 5’ UTR(s) and wherein the genetically engineered bacterium further comprises an integrative element comprising an SSRRS; optionally wherein the internal integrative SSRRS is / are an attB site(s) and the integrative element comprises an SSRRS comprising an attP site; and / or optionally wherein the target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (k) a site-specific recombinase (SSR) or integrase protein, wherein a control element comprising a promoter and / or at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase recognition sites (SSRRS) in an inverted configuration; and (ii) is operably linked to one or more first target gene(s) of the genetically engineered bacterium; optionally wherein the control element which is operably linked to said first target gene(s) comprises a constitutive promoter, an inducible promoter, quorum sensing, or phosphateAtty. Dkt. No. P14610WO00 sensitive promoter; and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (l) a site-specific recombinase (SSR) or integrase protein, wherein a control element comprising a promoter and at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase recognition sites (SSRRS) in an inverted configuration; and (ii) is operably linked to one or more first target gene(s) of the genetically engineered bacterium upon inversion by the SSR or integrase; optionally wherein the control element which is operably linked to said first target gene(s) upon inversion by the SSR or integrase comprises a constitutive promoter, an inducible promoter, or quorum sensing promoter; and / or optionally wherein the first target gene(s) is / are: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase, and the agriculturally relevant compound is phosphate; (m) a site-specific DNA endonuclease, wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) one or more specific DNA sequence(s) recognized by the site-specific DNA endonuclease, optionally wherein the site-specific DNA endonuclease comprises an RNA-guided DNA endonuclease, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or a restriction endonuclease, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (n) a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) comprising DNA encoding the PSRS in the protein coding region of the first target gene(s), wherein the target protein product comprising the one or more in-frame insertion(s) has activity, and wherein cleavage of the target protein product(s) by the protease deactivates the target protein product(s), optionally wherein the location of the one or more in-frame insertion(s) is given by Table 6, optionally wherein the first target gene(s) is / are under the control of a constitutive promoter, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (o) a first protein of interest comprising a ClpAP ATP-dependent protease and a second protein of interest comprising a ClpS Leu / N-recognin, wherein any one or more first target gene(s) of the genetically engineered bacterium encodes a protein comprising an N-terminal -Leu, -Phe, -Trp, or -Tyr residue, andAtty. Dkt. No. P14610WO00 optionally wherein the first and second protein of interest are operably linked to distinct control elements, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (p) a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion(s) of DNA encoding the PSRS at the N-terminus of the protein coding region of the gene followed by a -Leu, - Phe, -Trp, or -Tyr residue and wherein cleavage of PSRS from the N-terminus of the protein(s) encoded by the gene(s) by the protease results in a protein comprising an N-terminal -Leu, -Phe, -Trp, or -Tyr residue which is degraded by native ClpS and ClpAP, optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (q) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and activates expression of the protease, wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) of DNA encoding a PSRS, optionally wherein the transcriptional activator protein comprises a tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, optionally wherein the transcriptional activator domain is VP16 , and / or optionally wherein the first target gene(s) is / are: glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (r) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and inhibits expression of the protease, wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion of DNA encoding an N-terminal amino acid tag designed to render the protein product(s) of the first target gene(s) inactive, followed by a PSRS, optionally wherein the repressor protein comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (s) a nanobody, wherein the nanobody binds a protein product of any one or more first target gene(s) of the genetically engineered bacterium, wherein binding of the nanobody to such protein product inhibits function of the protein product, optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (t) an aptamer, wherein the aptamer binds a protein product of any one or more first target gene(s) of the genetically engineered bacterium and inhibits function of any one or more of the protein product(s)Atty. Dkt. No. P14610WO00 of the first target gene(s), optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; or (u) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of any one or more first target gene(s) and increases expression of any one or more of the first target gene(s), optionally wherein the transcriptional activator protein comprises the tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, wherein the DNA targeting protein is optionally a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator- like effector (TALE), or any variant thereof, wherein the transcriptional activator domain is optionally VP16 and / or optionally wherein the first target gene(s) is / are any one or more of: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, and / or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase and the agriculturally relevant compound is phosphate.

[0191] 101. The method of embodiment 100, wherein the integrase is a serine integrase, optionally wherein the serine integrase is a phage PhiC31 serine integrase, IntS, IntM, IntG – ICEMcSym 1271, YdcL - ICEBs, or Int - ICE SXT / R39 integrase, and the SSRRS are attB and attP sites recognized respectively by the PhiC31, IntS, IntM, IntG, YdcL, or Sxt / R39 integrase.

[0192] 102. The method of embodiment 100, wherein: (i) one or more SSR is a yeast flippase (FLP) recombinase and the SSRRS are FRT sites; or (ii) one or more SSR is a Cre-recombinase and the SSRRS are loxP sites.

[0193] 103. The method of embodiment 100, wherein the PSRS is inserted in a glutamine synthetase (GS) polypeptide between any one or more of the pairs of amino acid residues corresponding to: (i) amino acid residues 98 and 99, 121 and 122, 279 and 280, and / or 285 and 286 of the glutamine synthetase (GS) polypeptide of SEQ ID NO: 229; (ii) amino acid residues 98 and 99, 119 and 120, 283 and 284, and / or 298 and 299 of the glutamine synthetase (GS) polypeptide of SEQ ID NO: 230; (iii) amino acid residues 448 and 449, 480 and 481, 505 and 506, 528 and 529, and / or 624 and 625 of the GlnE polypeptide of SEQ ID NO: 231;Atty. Dkt. No. P14610WO00 (iv) amino acid residues 527 and 528, 537 and 538, and / or 547 and 548 of the GlnE polypeptide of SEQ ID NO: 232; or (v) amino acid residues 422 and 423 and / or 608 and 609 of the GlnE polypeptide of SEQ ID NO: 233.

[0194] 104. The method of embodiment 100, wherein the protease or heterologous protease: a. comprises a tobacco etch virus (TEV) protease and the PSRS comprises the peptide EXXYXQ- (S / G) or ENLYFQ-(S / G / A / M / C / H), wherein X is any amino acid and the TEV protease cleaves between the Q and the S, G, A, M, C, or H residues; b. comprises a tobacco vein mottling virus (TVMV) protease and the PSRS comprises the peptide ETVRFQ-(G / S), wherein the TVMV protease cleaves between the Q and S or G residues; c. comprises a sunflower mild mosaic virus (SMMV) protease and the PSRS comprises the peptide EEIHLQ-(S / G), wherein the SMMV protease cleaves between the Q and S or G residues; d. comprises a turnip mosaic virus (TrMV) protease and the PSRS comprises the peptide VXHQ or VRHQ-S, wherein X is any amino acid and the TrMV protease cleaves C-terminal to the Q residue; e. comprises a soybean mosaic virus (SMV) protease and the PSRS comprises the peptide XVXXQ-(G / S), wherein X is any amino acid and the SMV protease cleaves between Q and S or G residues; f. comprises a plum pox virus (PPV) protease and the PSRS comprises the peptide NVVVHQ-A, wherein the PPV protease cleaves between the Q and the A residue;

[0195] g. comprises a hepatitis C virus (HCV) NS3 protease and the PSRS comprises the peptide (D / E)XXXXC(A / S), wherein X is any amino acid and the HCV protease cleaves between the C and the A or S residues; h. comprises an enterokinase and the PSRS comprises the peptide DDDDK, wherein the enterokinase cleaves C-terminal to the K residue; i. comprises a Factor Xa protease and the PSRS comprises the peptide I(D / E)GR, wherein the Factor Xa protease cleaves C-terminal to the R residue; or j. comprises a furin protease and the PSRS comprises the peptide RX(K / R)R, wherein the furin protease cleaves C-terminal to the C-terminal R residue.

[0196] 105. The method of embodiment 99, wherein: (i) the plant nutrient is zinc or potassium and the protein of interest is a gluconate dehydrogenase (GAD) enzyme; (ii) the plant nutrient is iron and the protein of interest is a siderophore biosynthetic and transport protein optionally selected from a dhbACDEBF gene cluster; a non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and NRPS-independent siderophore synthetase (NIS); and major facilitator superfamily (MFS) transporters (ymfE), TonB, ExbD, and / or ExbB; (iii) the phytohormone is auxin and the protein is an IpdC or IaaM protein; (iv) the plant nutrient is ammonia and α-ketobutyrate and the protein is ACC deaminase;Atty. Dkt. No. P14610WO00 (v) the plant growth regulator is a volatile organic compound and the proteins are Glyceraldehyde- 3-Phosphate Dehydrogenase (GAPDH) and 2,3-butanediol Dehydrogenase (BDH); (vi) the agriculturally relevant compound is trehalose and the protein(s) of interest is OtsA, OtsB, and TreS or TreS; or (vii) the carbon containing compound is calcium carbonate and the protein of interest is a beta- carbonic anhydrase or alpha-carbonic anhydrase.

[0197] 106. The method of embodiment 99, wherein the protein of interest comprises a site-specific recombinase (SSR) or integrase protein, wherein a control element comprising a promoter and / or at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase recognition sites (SSRRS) in an inverted configuration; (ii) is operably linked to one or more first target gene(s) of the genetically engineered bacterium; and (iii) is operably linked to one or more second target gene(s) of the genetically engineered bacterium upon inversion by the SSR or integrase; and wherein: (a) the first target gene is a glnA gene encoding a wild-type glutamine synthetase (GS) or variant thereof with improved catalytic activity in comparison to wild-type GS and the second target gene is a glnA gene encoding a wild-type GS with reduced levels of expression in comparison to the wild-type glnA gene or a GS variant with decreased catalytic activity in comparison to wild-type GS, and the fertilizer is ammonia; (b) the first target gene is a glnE gene encoding a wild-type GS adenylyltransferase protein and the second target gene is a glnE gene encoding a GS adenylyltransferase protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, and the fertilizer is ammonia; or (c) the first target gene is a nifL gene and the second target gene is a nifA gene, and the fertilizer is ammonia.

[0198] 107. The method of embodiment 106, wherein the control element which is operably linked to said target genes comprises a constitutive promoter, an inducible promoter, or quorum sensing promoter.

[0199] 108. The method of embodiment 106 or 107, wherein the integrase is a serine integrase, optionally wherein the serine integrase is a phage PhiC31 serine integrase, IntS, IntM, IntG – ICEMcSym 1271, YdcL - ICEBs, or Int - ICE SXT / R39 integrase, and the SSRRS are attB and attP sites recognized respectively by the PhiC31, IntS, IntM, IntG, YdcL, or Sxt / R39 integrase.

[0200] 109. The method of embodiment 106 or 107, wherein: (i) one or more SSR is a yeast flippase (FLP) recombinase and the SSRRS are FRT sites; or (ii) one or more SSR is a Cre-recombinase and the SSRRS are loxP sites.

[0201] 110. The method of embodiment 99, wherein the protein of interest is a first repressor protein and wherein the genetically engineered bacterium further comprises: (i) a second control element comprising a promoter which is repressed by the first repressor protein and operably linked to a gene encoding a second repressor protein;Atty. Dkt. No. P14610WO00 (ii) optionally a third control element comprising a promoter which is repressed by the second repressor protein and operably linked to a gene encoding a third repressor protein; (iii) optionally a fourth control element comprising a promoter which is repressed by the third repressor protein and operably linked to a gene encoding a fourth repressor protein; and (iv) a control element comprising a promoter which is repressed by the second, third, or fourth repressor protein and which is operably linked to a first target gene,optionally wherein the first, second, third, and / or fourth repressor protein(s) comprise(s) the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof.

[0202] 111. The method of embodiment 110, wherein the genetically engineered bacterium lacks (ii) and (iii), and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s), and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase, and the agriculturally relevant compound is phosphate.

[0203] 112. The method of embodiment 110, wherein the genetically engineered bacterium further comprises (ii) and (iii), and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase, and the agriculturally relevant compound is phosphate.

[0204] 113. The method of embodiment 110, wherein the genetically engineered bacterium further comprises (ii) and lacks (iii), and the first target gene is amtB, draT, glnA, glnB, glnK, glnZ, and / or nifL, and the agriculturally relevant compound is ammonia.Atty. Dkt. No. P14610WO00

[0205] 114. The method of embodiment 110, wherein first target gene encodes a transcriptional activator protein and wherein the genetically engineered bacterium further comprises a control element comprising a promoter which is activated by the transcriptional activator protein and operably linked to at least one second target gene.

[0206] 115. The method of embodiment 110, wherein the genetically engineered bacterium comprises an even number of repressors and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), a pyrroloquinoline (PQQ) synthase, and the agriculturally relevant compound is phosphate.

[0207] 116. The method of embodiment 110, wherein the genetically engineered bacterium comprises an even number of repressors and the first target gene encodes an RNA sequence or protein comprising: a. a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; b. a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of- function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild-type or refactored nif or fix gene cluster and the fertilizer is ammonia; c. a GlnR protein and the fertilizer is ammonia; d. a glutaminase enzyme and the fertilizer is ammonia; e. a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; f. a phytase enzyme and the agriculturally relevant compound is phosphate, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase; g. an acid phosphatase enzyme and the agriculturally relevant compound is phosphate, optionally wherein the acid phosphatase enzyme is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof; h. a protein which stimulates organic acid release from the bacterium and the agriculturally relevant compound is phosphate, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase;Atty. Dkt. No. P14610WO00 i. a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more second target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein product(s) of the second target gene(s) and / or wherein the repressor protein optionally comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof and / or optionally wherein the second target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; j. a non-coding synthetic small RNA (sRNA); optionally wherein the non-coding synthetic small RNA (sRNA) binds a natural or synthetic DNA and / or RNA motif in the promoter, 5’ UTR, and / or coding region of any one or more second target gene(s) of the genetically engineered bacterium, optionally wherein the non-coding synthetic small RNA (sRNA) comprises a guide RNA that additionally binds an RNA- guided DNA endonuclease, an RNA-guided RNA endonuclease, or variant thereof and / or optionally wherein the second target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; k. a site-specific recombinase (SSR) or integrase protein: wherein any one or more second target gene(s) of the genetically engineered bacterium and / or a promoter operatively linked thereto are flanked by site-specific recombinase recognition sites (SSRRS) in a direct configuration, optionally wherein the SSRRS comprise attL and attR sites and the genetically engineered bacterium comprises a gene encoding a recombinase directionality factor (RDF) and / or optionally wherein the second target gene(s) is / are a glnA, amtB, glnB, segment of glnE encoding an adenylyl-removing domain of a glutamine synthetase adenylyltransferase, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; l. a site-specific recombinase (SSR) or integrase protein: wherein any one or more second target gene(s), the promoter(s) thereof, and / or the 5’ UTR(s) thereof of the genetically engineered bacterium comprise(s) one or more internal synthetic SSRRS and wherein the gene(s), promoter(s), the 5’ UTR(s), and / or a segment(s) thereof is / are excised or inactivated after a recombination event; optionally wherein the genetically engineered bacterium further comprises a plasmid comprising an SSRRS; optionally wherein the genetically engineered bacterium further comprises one or more genes encoding a recombinase directionality factor (RDF); and / or optionally wherein the second target gene(s) is / are a glnA, amtB, glnB, glnE, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; m. a site-specific recombinase (SSR) or integrase protein, wherein any one or more second target gene(s), the promoter(s) thereof, and / or the 5’ UTR(s) thereof of the genetically engineered bacterium comprise(s) one or more internal integrative SSRRS and retain(s) activity of said second target gene(s), promoter(s), and / or 5’ UTR(s) and wherein the genetically engineered bacterium further comprises an integrative element comprising an SSRRS; optionally wherein the internal integrative SSRRS is / are an attBAtty. Dkt. No. P14610WO00 site(s), the integrative element comprises an SSRRS comprising an attP site; and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; n. a site-specific recombinase (SSR) or integrase protein, wherein a control element comprising a promoter and / or at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase recognition sites (SSRRS) in an inverted configuration; and (ii) is operably linked to one or more second target gene(s) of the genetically engineered bacterium; optionally wherein the control element which is operably linked to said second target gene(s) comprises a constitutive promoter, an inducible promoter, or quorum sensing promoter; and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; o. a site-specific recombinase (SSR) or integrase protein, wherein a control element comprising a promoter and at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase recognition sites (SSRRS) in an inverted configuration; and (ii) is operably linked to one or more second target gene(s) of the genetically engineered bacterium upon inversion by the SSR or integrase; optionally wherein the control element which is operably linked to said second target gene(s) upon inversion by the SSR or integrase comprises a constitutive promoter, an inducible promoter, or quorum sensing promoter; and / or optionally wherein the second target gene(s) is / are: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase, and the agriculturally relevant compound is phosphate; p. a site-specific DNA endonuclease, wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) one or more specific DNA sequence(s) recognized by the site-specific DNA endonuclease, optionally wherein the site-specific DNA endonuclease comprises an RNA-guided DNA endonuclease, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or a restriction endonuclease, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; q. a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) comprising DNA encoding the PSRS in the protein coding region of the second target gene(s), wherein the target protein product comprising the one or more in-frame insertion(s) has activity, andAtty. Dkt. No. P14610WO00 wherein cleavage of the target protein product(s) by the protease deactivates the target protein product(s), optionally wherein the location of the one or more in-frame insertion is given by Table 6, optionally wherein the second target gene(s) is / are under the control of a constitutive promoter, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; r. a first protein comprising a ClpAP ATP-dependent protease and a second protein comprising a ClpS Leu / N-recognin, wherein any one or more second target gene(s) of the genetically engineered bacterium encodes a third protein comprising an N-terminal -Leu, -Phe, -Trp, or -Tyr residue, and optionally wherein the first and second protein are operably linked to distinct control elements, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; s. a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion(s) of DNA encoding the PSRS at the N-terminus of the protein coding region of the gene(s) followed by a -Leu, -Phe, -Trp, or -Tyr residue and wherein cleavage of PSRS from the N-terminus of the protein(s) encoded by the gene(s) by the protease results in a protein comprising an N-terminal -Leu, -Phe, -Trp, or -Tyr residue which is degraded by native ClpS and ClpAP, optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; t. a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and activates expression of the protease, wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) of DNA encoding a PSRS, optionally wherein the transcriptional activator protein comprises a tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA- binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, optionally wherein the transcriptional activator domain is VP16 , and / or optionally wherein the second target gene(s) is / are: glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; u. a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and inhibits expression of the protease, wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion of DNA encoding an N-terminal amino acid tag designed to render the protein product(s) of the second target gene(s) inactive, followed by a PSRS, optionally wherein the repressor protein comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia;Atty. Dkt. No. P14610WO00 v. a nanobody, wherein the nanobody binds a protein product of any one or more second target gene(s) of the genetically engineered bacterium, wherein binding of the nanobody to such protein product inhibits function of the protein product, optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; w. an aptamer, wherein the aptamer binds a protein product of any one or more second target gene(s) of the genetically engineered bacterium and inhibits function of any one or more of the protein product(s) of the second target gene(s), optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; or x. a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of any one or more second target gene(s) and increases expression of any one or more of the second target gene(s), optionally wherein the transcriptional activator protein comprises the tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, wherein the DNA targeting protein is optionally a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, wherein the transcriptional activator domain is optionally VP16 and / or optionally wherein the second target gene(s) is / are any one or more of: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, and / or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase and the agriculturally relevant compound is phosphate.

[0208] 117. The method of embodiment 110, wherein the genetically engineered bacterium comprises an odd number of repressors and the second target gene is amtB, draT, glnA, glnB, glnK, glnZ, and / or nifL, and the agriculturally relevant compound is ammonia.

[0209] 118. The method of embodiment 99, wherein the protein of interest is a first transcriptional activator protein and wherein the genetically engineered bacterium further comprises: (i) a second control element comprising a promoter which is activated by the first transcriptional activator protein and operably linked to a gene encoding a second transcriptional activator protein; (ii) optionally a third control element comprising a promoter which is activated by the second transcriptional activator protein and operably linked to a gene encoding a third transcriptional activator protein;Atty. Dkt. No. P14610WO00 (iii) optionally a fourth control element comprising a promoter which is activated by the third transcriptional activator protein and operably linked to a gene encoding a fourth transcriptional activator protein; and (iv) a control element comprising a promoter which is activated by the second, third, or fourth transcriptional activator protein and which is operably linked to a first target gene, optionally wherein the first, second, third, and / or fourth transcriptional activator protein comprise(s) the tet responsive element- binding tTA transcription factor or an activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), and / or any variant thereof, optionally wherein the activator domain is VP16.

[0210] 119. The method of embodiment 118, wherein the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase, and the agriculturally relevant compound is phosphate.

[0211] 120. The method of embodiment 99, wherein the protein of interest is a repressor protein and wherein the genetically engineered bacterium further comprises: (i) a second control element comprising a promoter which is repressed by the repressor protein and operably linked to a gene encoding a transcriptional activator protein; and (ii) a third control element element comprising a promoter which is activated by the transcriptional activator protein and operably linked to a target gene.

[0212] 121. The method of embodiment 120, wherein the target gene is a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the agriculturally relevant compound is ammonia.

[0213] 122. The method of embodiment 99, wherein the protein of interest is a transcriptional activator protein and wherein the genetically engineered bacterium further comprises: (i) a control element comprising a promoter which is activated by the transcriptional activator protein and operably linked to a gene encoding a repressor protein; and (ii) a control element comprising a promoter which is repressed by the repressor and operably linked to a target gene.Atty. Dkt. No. P14610WO00

[0214] 123. The method of embodiment 122, wherein the target gene is a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the agriculturally relevant compound is ammonia.

[0215] 124. The method of embodiment 100, wherein the glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene in (n), (o), (p), or (q) is under the control of a heterologous constitutive promoter.

[0216] 125. The method of embodiment 116, wherein the glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene in (q), (r), (s), or (t) is under the control of a heterologous constitutive promoter.

[0217] 126. The method of embodiment 99, wherein the plant nutrient is phosphate and the protein of interest comprises: (a) a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase; (b) an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof; (c) a protein which stimulates organic acid release from the bacterium, optionally wherein the protein comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), a pyrroloquinoline (PQQ) synthase including pqqFABCDEG, or any combination of GAD, GDH, and PQQ; or, (d) any combination of proteins of a, b, or c.

[0218] 127. The method of any one of embodiments 91 to 126, wherein the threshold population density of the genetically engineered bacterium (GEB) which activates expression of the RNA or protein of interest is: (i) as set forth in Table 9 or Table 10 for the genetically engineered bacteria listed therein; (ii) about 6 x 103to 2 x 104CFU / mL, wherein the GEB is grown in liquid media, is a gram negative bacterium, an alphaproteobacterium, an Azospirillum sp., or related bacterium, the QSSP comprises an AhlI protein, the QSRP comprises an AhlR protein, and the quorum sensing promoter comprises an ahlI promoter; (iii) about 7 x 104to 3 x 107CFU / mL, wherein the GEB is grown in liquid media, is a gram negative bacterium, a gammaproteobacterium, is a Pseudomonas sp., or is a related bacterium, the QSSP comprises an AhlI protein, the QSRP comprises an AhlR protein, and the quorum sensing promoter comprises an ahlI promoter, or optionally wherein the bacterium is a member of the taxonomic genus Azospirillum, Enterobacter, Klebsiella, Kosakonia, Pseudomonas, or Rahnella; (iv) about 4 x 105to 3 x 107CFU / mL, wherein the GEB is grown in liquid media, is a gram negative bacterium, a gammaproteobacterium, is a Kosakonia sp., or is a related bacterium, the QSSP comprises an CinI protein, the QSRP comprises an CinR protein, and the quorum sensing promoter comprises an cinI promoter; (v) about 1 x 105to 3 x 107CFU / mL, wherein the GEB is grown in liquid media, is a gram negative bacterium, a gammaproteobacterium, is a Pseudomonas sp., or is a related bacterium, the QSSP comprisesAtty. Dkt. No. P14610WO00 a CinI protein, the QSRP comprises a CinR protein, and the quorum sensing promoter comprises a cinI promoter; (vi) about 5 x 103to 7 x 106CFU / gram soil, wherein the GEB is grown in plant growth media, is a gram negative bacterium, an alphaproteobacterium, an Azospirillum sp., or related bacterium, the QSSP comprises an AhlI protein, the QSRP comprises an AhlR protein, and the quorum sensing promoter comprises an ahlI promoter; or (vii) about 5 x 105to 5 x 108CFU / gram soil, wherein the GEB is grown in plant growth media, is a gram negative bacterium, a gammaproteobacterium, is a Kosakonia sp., or is a related bacterium, the QSSP comprises a CinI protein, the QSRP comprises a CinR protein, and the quorum sensing promoter comprises a cinI promoter.

[0219] 128. The method of any one of embodiments 91 to 127, further comprising; a. determining leaf nitrogen and / or chlorophyll concentrations in a plant grown in the plant growth medium; and b. placing or re-applying the genetically engineered bacterium into the plant growth medium and / or in contact with the plant when the leaf nitrogen and / or chlorophyll concentrations in the plant are sub- optimal for yield.

[0220] 129. The genetically engineered bacterium of any one of embodiments 1 to 62 or the method of any one of embodiments 91 to 128, wherein the first QSSP comprises an AhlI protein, the first QSRP comprises an AhlR protein, and the first quorum sensing promoter comprises an ahlI promoter and wherein the bacterium is a member of the taxonomic genus Azospirillum, Enterobacter, Klebsiella, Kosakonia, Pseudomonas, or Rahnella; and wherein the protein of interest is: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of- function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild- type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; (f) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more first target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein products of the first target genes, wherein the repressor protein optionally comprises the tet repressor (TetR) or a lac repressor (LacI), and optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; or (g) a protein that is or causes the production of at least one agriculturally relevant compound.

[0221] 130. The genetically engineered bacterium of any one of embodiments 1 to 62 or the method of any one of embodiments 91 to 128, wherein the first QSSP comprises a CinI protein, the first QSRP comprisesAtty. Dkt. No. P14610WO00 a CinR protein, and the first quorum sensing promoter comprises a cinI promoter, the bacterium is a member of the taxonomic genus Azospirillum, Pseudomonas, or Kosakonia, and wherein the protein of interest is: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of-function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild-type or refactored nif or fix gene cluster and the fertilizer is ammonia;(c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; (f) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more first target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein products of the first target genes, wherein the repressor protein optionally comprises the tet repressor (TetR) or a lac repressor (LacI), and optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; or (g) a protein that is or causes the production of at least one agriculturally relevant compound.

[0222] 131. The genetically engineered bacterium of any one of embodiments 1 to 62 or the method of any one of embodiments 91 to 128, wherein the first QSRP comprises a CciR protein, and the first quorum sensing promoter comprises a cciI promoter, the bacterium is a member of the taxonomic genus Azospirillum or Pseudomonas, and wherein the protein of interest is: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia;(b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of- function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild- type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; (f) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more first target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein products of the first target genes, wherein the repressor protein optionally comprises the tet repressor (TetR) or a lac repressor (LacI), and optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; or (g) a protein that is or causes the production of at least one agriculturally relevant compound.

[0223] In the description, tables, and numbered embodiments 1-131 set forth herein, genes and the proteins they encode which are referred to solely by name include genes and proteins identified in Table 5 and the sequence listing, include genes having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to the genes identified in Table 5 and the sequence listing, and include proteins having at least 70%, 75%,Atty. Dkt. No. P14610WO00 80%, 85%, 90%, 95%, 98%, or 99% identity to the proteins identified in Table 5 and the sequence listing. GlnE proteins lacking an adenylyl removing domain which exhibit unidirectional adenylyltransferase (uAT) activity referred to solely by name in the preceding description, tables, and numbered embodiments include proteins comprising a protein sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 16-24, or 25. Genes encoding GlnE proteins lacking an adenylyl removing domain which exhibit unidirectional adenylyltransferase (uAT) activity referred to solely by name in the preceding description, tables, and numbered embodiments include genes comprising a DNA sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 1-14, or 15. Wild-type GlnA proteins (glutamine synthetase or GS proteins) referred to solely by name in the preceding description, tables, and numbered embodiments include proteins having a protein sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 229-285, or 286. Wild-type glnA genes encoding wild-type GlnA proteins referred to solely by name in the preceding description, tables, and numbered embodiments include glnA genes encoding proteins having a protein sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 229, 230, or 460-512. Phytases referred to solely by name in the preceding description, tables, and numbered embodiments include phytases comprising a protein sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 58-138, or 139. Phytase or phy genes referred to solely by name in the preceding description, tables, and numbered embodiments include phytase genes encoding phytase proteins comprising a protein sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 58-138, or 139.

[0224] The breadth and scope of the present disclosure should not be limited by any of the above-described embodiments.

[0225] Table 5. Summary of Biological Sequences in Sequence Listing SEQ Sequence Type Description ID Organism nameAtty. Dkt. No. P14610WO00 SEQ Sequence Type Description ID Organism name NO DNA / PRT / RNAAtty. Dkt. No. P14610WO00 SEQ Sequence Type Description ID Organism name NO DNA / PRT / RNAAtty. Dkt. No. P14610WO00 SEQ Sequence Type Description ID Organism name NO DNA / PRT / RNA ns sAtty. Dkt. No. P14610WO00 SEQ Sequence Type Description ID Organism name NO DNA / PRT / RNA e ns ns mAtty. Dkt. No. P14610WO00 SEQ Sequence Type Description ID Organism name NO DNA / PRT / RNA nsAtty. Dkt. No. P14610WO00 SEQ Sequence Type Description ID Organism name NO DNA / PRT / RNA s e ns nsAtty. Dkt. No. P14610WO00 SEQ Sequence Type Description ID Organism name NO DNA / PRT / RNA m s sAtty. Dkt. No. P14610WO00 SEQ Sequence Type Description ID Organism name NO DNA / PRT / RNA isAtty. Dkt. No. P14610WO00 SEQ Sequence Type Description ID Organism name NO DNA / PRT / RNA m sAtty. Dkt. No. P14610WO00 SEQ Sequence Type Description ID Organism name NO DNA / PRT / RNAAtty. Dkt. No. P14610WO00 SEQ Sequence Type Description ID Organism name NO DNA / PRT / RNA m mAtty. Dkt. No. P14610WO00 SEQ Sequence Type Description ID Organism name NO DNA / PRT / RNA m mAtty. Dkt. No. P14610WO00 SEQ Sequence Type Description ID Organism name NO DNA / PRT / RNA

[0226] Table 6. Non-limiting examples of protease-specific recognition sequence (PSRS) insertion sites in glnA and glnE target genes for ammonia release. Corresponding insertion sites in other glnA and glnE genes including those set forth in Table 3, Table 5, and the sequence listing are also provided. Name of Cut Target SEQ Site for C- #Atty. Dkt. No. P14610WO00 Name of Cut Target SEQ Site for C- #EXAMPLES Example 1. Identifying Quorum Sensing Systems for Protein Expression in Soil Bacteria

[0227] Quorum sensing (QS) systems allow activation of gene expression in response to cell density in a bacterial population (i.e., population density). For soil bacteria, QS-regulated genes are activated on plant roots and in the rhizosphere (millimeters from plant roots) where cell density is high, but not in bulk soil (far from plant roots) where cell density is comparatively low. This activation is facilitated by a regulatory feedback mechanism dependent on the synthesis, accumulation, and detection of quorum sensing signal molecules (QSSM) by quorum sensing regulator proteins (QSRP). One class of QSSMs are the small molecule acyl-homoserine lactones (AHL), which allosterically bind transcription factor QSRPs to cause population density-dependent changes in bacterial gene expression.

[0228] As such, agriculturally relevant bacteria can be engineered using QS systems to produce agriculturally relevant compounds conditionally: (i) once the bacteria have colonized plant roots and (ii) once the bacteria have grown to exceed a threshold population density. The production ofAtty. Dkt. No. P14610WO00 such agriculturally relevant compounds consumes energy normally required by bacteria for growth. Since the bacteria engineered with QS systems delay production of agriculturally relevant compounds until they have reached a threshold population density, said bacteria grow to higher titers on plant roots compared to wild-type or engineered bacteria without QS systems that produce the same agriculturally relevant compounds. Higher titers of the bacteria engineered with QS systems thus provide for higher titers of the desired agriculturally relevant compounds in plant growth media in comparison to bacteria lacking the QS-regulated genes.

[0229] QS systems are not present in all bacteria, but they can function in heterologous strains provided the QS system comprises: (i) a quorum sensing synthase protein (QSSP) that synthesizes an AHL QSSM, (ii) a QSRP transcription factor that binds the AHL QSSM, and (iii) a cognate quorum sensing promoter from which transcription of an operably linked gene of interest can be initiated upon formation of the QSSM-QSRP complex.

[0230] To determine which QS systems facilitate population density-dependent expression of an RNA sequence or protein of interest in agriculturally relevant bacteria, corresponding QSSP, QSRP, and quorum sensing promoter sequences were bioinformatically extracted from a range of agriculturally relevant and model bacterial taxa. Taxa searched for QS systems included diverse gram-negative bacteria. Among gram-negative bacteria, several classes of soil bacteria were searched for QS systems, including: alphaproteobacteria like Mesorhizobium ciceri and Rhizobium leguminosarum; betaproteobacteria like Burkholderia cenocepacia and Chromobacterium violaceum; and gammaproteobacteria like Aliivibrio fischeri, Panotea stewartii, Pseudomonas aeruginosa, and Pseudomonas syringae. Genes of each bacterial QS system were identified because these genes are known to be related to quorum sensing. QSSP protein-coding sequences present in agriculturally relevant bacteria are included as SEQ ID NO: 348-355, and 376. QSRP protein- coding sequences present in agriculturally relevant bacteria are included as SEQ ID NO: 339-347, and 375. Quorum sensing promoter DNA sequences present in agriculturally relevant bacteria are included as SEQ ID NO: 356-364, 373, and 374. CLUSTAL alignment performed using Mview with default parameters showed that no consensus sequence could be easily constructed for either the set of QSSP protein-coding sequences or the set or QSRP protein-coding sequences.

[0231] Next, species compatibility of heterologous QSRPs was screened across agriculturally relevant bacteria. Gene fragments encoding QSRP transcription factors (SEQ ID NO: 339, 340, 342, 343, 345, 346, 347) and their cognate QS promoters (SEQ ID NO: 356, 357, 359, 360, 362, 363, 364) were synthesized. The gene fragments were assembled into gene expression cassettes, wherein the sequence encoding a QSRP was operably linked to a constitutive promoter, and in the reverse direction, a gene encoding a green fluorescent protein (GFP) reporter was operably linked to a strong ribosome binding site (RBS) (SEQ ID NO: 290, TABLE 7) and the cognate QS promoter of the QSRP (FIGURE 1). Each gene expression cassette was cloned into a broad hostAtty. Dkt. No. P14610WO00 range plasmid and transformed into Azospirillum brasilense Sp245, Herbaspirillum seropedicae Z67, Pseudomonas stutzeri DSM 4166, and Kosakonia radicincitans DSM16656 to assess functionality.

[0232] Table 7. Genetic circuit elements used to construct heterologous gene expression cassettes. “RBS” denotes ribosome binding sites. “TERM” denotes terminators. Name Function SEQ ID NOAtty. Dkt. No. P14610WO00 Name Function SEQ ID NO BBa B0029 RBS 311strains was then screened for QSRP activity in response to a saturating concentration (10 µM) of either N-(β-Ketocaproyl)-L-homoserine lactone (C6-AHL) or N-(3-hydroxytetradecanoyl)-DL-homoserine lactone (C14-AHL). LB Broth (Miller) with or without each AHL inducer was added to a 96-well plate, which was inoculated with the strain library, and cultures were grown overnight at 30 degrees centigrade with orbital shaking for aeration. Following incubation, GFP expression was quantified via fluorimetry (TABLE 8). The AhlR (SEQ ID NO: 339), CinR (SEQ ID NO: 342), and CviR (SEQ ID NO: 343) QSRP transcription factors had activity across multiple agriculturally relevant bacterial strains.

[0234] Table 8. Functionality of QS promoters and QSRP transcription factors in different agriculturally relevant bacteria. Rows are organized by different QSRP transcription factors and cognate QS promoters. Columns are organized by different agriculturally relevant bacterial strains. Cells say “Responsive” if a fluorescent reporter (GFP) protein of interest was induced ≥3-fold upon addition of an AHL QSSM inducer in a certain engineered strain. Cells say “Intermediate” if the fluorescent reporter (GFP) protein of interest was induced 2 to 3-fold upon addition of an AHL QSSM inducer in a certain engineered strain. Cells say “On” if the fluorescent reporter (GFP) protein of interest was expressed constantly in the presence and absence of an AHL QSSM inducer in a certain engineered strain. Cells say “Non-functional” if the fluorescent reporter (GFP)Atty. Dkt. No. P14610WO00 protein of interest was not expressed in the presence or absence of an AHL QSSM inducer in a certain engineered strain. “NT” indicates not tested. QSRP Transcription Factor / QS A. brasilense H. K. P. stutzeri Promoter seropedicae radicincitans, species of agriculturally relevant bacteria. Heterologous gene expression cassettes comprising: (i) genes encoding AhlR (SEQ ID NO: 339) and AhlI (SEQ ID NO: 348) derived from Pseudomonas syringae B7281a, (ii) genes encoding CinR (SEQ ID NO: 342) and CinI (SEQ ID NO: 351) derived from Rhizobium leguminosarum bv viceae 3841, or (iii) genes encoding LasR (SEQ ID NO: 375) and LasI (SEQ ID NO: 376) derived from P. aeruginosa PA01, were synthesized and cloned into the mini-Tn7 delivery plasmid pUC18R6K-mini-Tn7T-Gm digested at the SacI restriction site. These were stably integrated into the chromosomes of Azospirillum brasilense Sp245 (Ab), Kosakonia radicincitans DSM16656 (Kr), and Pseudomonas stutzeri DSM4166 (Ps) at the glmS gene using the helper plasmid pTNS3 (doi: 10.1038 / nprot.2006.24). In a second heterologous gene expression cassette, the quorum sensing promoter PahlI (SEQ ID NO: 356), PcinI (SEQ ID NO: 359), PlasI (SEQ ID NO: 373), or PlasB (SEQ ID NO: 374) was operably linked to a gene encoding a Superfolder GFP (sfGFP) protein of interest on a broad host range reporter plasmid. Said plasmids also carried a constitutively expressed LSSmScarlett reporter gene such that LSSmScarlett fluorescence could be used to track cell density and internally standardize GFP fluorescence as relative expression units (REU), calculated as GFP fluorescence / LSSmScarlett fluorescence (FIGURE 2). These plasmids were mobilized into wild-type strains and those carrying mini-Tn7 integrated QS systems.Atty. Dkt. No. P14610WO00

[0236] Population density-dependent induction of the GFP protein of interest was assessed by growing cultures in liquid LB media in 96-well plates and monitoring GFP / LSSmScarlett fluorescence as the culture density increased over time. The expected experimental results are shown in FIGURE 3. When activation of a QS promoter operably linked to GFP was null or constitutive, the ratio of GFP / LSSmScarlett did not change as the bacterial population density increased. When the QS promoter operably linked to GFP was subject to population density- dependent QS induction, GFP / LSSmScarlett fluorescence increased exponentially as the bacterial population density increased, until reaching saturation. None of the QS reporter plasmids exhibited population density-dependent QS induction when mobilized into wild-type strains lacking a heterologous gene expression cassette encoding a QSSP and a QSRP. In contrast, transfer of (i) a heterologous gene expression cassette comprising genes encoding CinR (SEQ ID NO: 342) and CinI (SEQ ID NO: 351) and (ii) the cognate PcinI::GFP reporter plasmid into three agriculturally relevant bacterial strains permitted population density-dependent QS induction. This was also the case for (i) the gene expression cassette comprising AhlR (SEQ ID NO: 339) and AhlI (SEQ ID NO: 348) and (ii) the cognate PahlI::GFP reporter plasmid transferred into Ab and Ps, but not Kr. The gene expression cassette comprising LasR (SEQ ID NO: 375) and LasI (SEQ ID NO: 376) with cognate reporter plasmids also exhibited population density-dependent induction in the three strains. However, the PlasI and PlasB promoters failed to function in Ps and Kr respectively (FIGURE 4).

[0237] Strains carrying heterologous QS systems were next assessed for population density- dependent expression by inoculating 104cells of each strain into liquid minimal media with or without added carbon to restrict bacterial growth. After overnight incubation, cultures with added carbon had reached stationary phase, whereas little growth was observed for cultures where carbon was not added. Bacterial cells were identified by flow cytometry via gating events that met a threshold for LSSmScarlett fluorescence (here termed S+) above background level. The cell density (S+ events / mL) in the absence of carbon was markedly lower compared to the cell density in samples where carbon was added to each strain (FIGURE 5). GFP / LSSmScarlett fluorescence was also assessed in the S+ population for each carbon treatment (FIGURE 6). The threshold population density at which each QS system caused each bacterial strain to produce the reporter protein of interest is given in TABLE 9.

[0238] Table 9. In-culture threshold population density at which various agriculturally relevant bacteria comprising various quorum sensing systems begin expressing a protein of interest. Rows are organized by different QS systems (QSSP, QSRP, QS promoter triads). Columns are organized by different agriculturally relevant bacterial strains. Cells express a bacterial population density range in cells / mL that represents the threshold population density for each QS system-strain combination. Cells say “On” if the fluorescent reporter (GFP) protein of interest was expressedAtty. Dkt. No. P14610WO00 independently of bacterial population density. Cells say “Non-functional” if the fluorescent reporter (GFP) protein of interest was not significantly expressed at a tested bacterial population density. QSSP + QSRP + QS Promoter A. brasilense K. radicincitans P. stutzeri *3*4*4*7Soil Bacteria in a Plant Growth Medium

[0239] The genetically engineered Ab and Kr strains of EXAMPLE 1 comprising a QSSP, QSRP, and gene encoding a GFP reporter protein of interest operably linked to a control element comprising a QS promoter, were assessed for population density-dependent expression of GFP on corn roots. Inoculant cells were prepared by culturing the Ab and Kr strains on LB agar under antibiotic selection, then incubating overnight at 30°C. The following day, cells were harvested from each plate and washed twice in 1% KCl prior to inoculation into plant growth media. Surface sterilized corn seeds were sowed into sterilized 250 mL Schott bottles filled with plant growth media consisting of 200 g of washed sand and 30 mL of Hoagland solution. Bottles with or without a corn seed were then inoculated with approximately 1 x 104bacterial cells of said Ab and Kr strains. After inoculation, sample bottles were incubated in a growth tent at 30°C for 7 days, until the corn plants grew to the V1 vegetative growth stage.

[0240] After incubation, 5 replicate plants for each experimental condition with planted seeds were destructively uprooted, and loose sand was discarded. The plant roots were excised from the shoot at the cotyledon and vortexed in 1% KCl solution to create a bacterial cell suspension, denoted the rhizosphere and rhizoplane (RP) fraction. Root fresh weight was recorded for normalization of bacterial population density. Separately, 5 experimental replicates for each experimental condition without planted seeds were sampled by flushing sample bottles with 20 mL of 1% KCl and vortexing, denoted the “bulk soil” (BS) fraction. RP and BS fractions were cleared of residual sand and other particles by centrifugation for 30 sec at 1000 x g, and 100 µL aliquots of the resulting supernatants were analyzed by flow cytometry.

[0241] Because the tested Ab and Kr strains carried a constitutively expressed LSSmScarlett reporter protein, flow cytometry events corresponding to bacterial cells were gated based on LSSmScarlett fluorescence above the background (here defined as S+ events), as validated by Haskett et al. (doi: 10.3389 / fmicb.2021.690439). Bacterial cells (S+ events) were counted, and GFP / LSSmScarlett fluorescence was assessed in the S+ population for each fraction. For both strains, the bacterial population density in sand where corn seeds were not planted (measured as cells g-1sand) was about 1000-fold lower than the bacterial population density observed on corn roots (measured as cells g-1root) (FIGURE 7). Ab and Kr demonstrated population density-dependent induction of the GFP protein of interest on plant rootsAtty. Dkt. No. P14610WO00 with the AhlRI and CinRI QS systems respectively (FIGURE 8). The threshold population density at which each QS system caused each bacterial strain to produce the reporter protein of interest is given in TABLE 10.

[0242] Table 10. Threshold population density at which various agriculturally relevant bacteria comprising various quorum sensing systems begin expressing a protein of interest in a plant growth medium. Rows are organized by different QS systems (QSSP, QSRP, QS promoter triads). Columns are organized by different agriculturally relevant bacterial strains. Cells express a bacterial population density range in cells / g root or cells / g soil that represents the threshold population density for each QS system-strain combination. Cells say “On” if the fluorescent reporter (GFP) protein of interest was expressed independently of bacterial population density. “NT” indicates not tested. QSSP + QSRP + QS Promoter A. brasilense K. radicincitans. the Quorum Sensing Signal Molecule

[0243] Bacteria engineered to express an RNA sequence or protein of interest under control of a QS system first colonize and grow on crop roots before beginning energy-intensive production of agriculturally relevant compounds such as fixed nitrogen. Using bacterial population density as a trigger for production of agriculturally relevant compounds facilitates sufficient bacterial growth because it requires a threshold population density of microbes to be reached before any product formation. However, such microbes are manufactured in a fermentor at a higher population density (e.g. greater than about 1 * 1010cells / mL) than the threshold population density at which energy-intensive product formation is initiated by the QS system. In the fermentor setting, product formation above the threshold population density can be abrogated by deactivating the QS system, permitting continued bacterial growth. Such conditional deactivation of the QS system selectively activates population density-dependent product formation in the field and not in the fermentor.

[0244] QS systems are deactivated by quorum quenching enzymes (QEs) that degrade AHL quorum sensing signal molecules (QSSMs). Expression of said QEs reduces the intracellular concentration of QSSMs, the concentration of QSSM-bound QSRPs, and the abundance of activated QS promoters. QEs span several classes of QSSM-degrading enzymes, including AHL acylase enzymes (SEQ ID NO: 320- 322, and 323), alpha-beta hydrolase fold lactonase proteins (SEQ ID NO: 324, 325, and 326), metallo- beta-lactamase-like lactonase proteins (SEQ ID NO: 327-330, and 331), and phosphotriesterase-like lactonase proteins (SEQ ID NO: 332-335, and 336).Atty. Dkt. No. P14610WO00

[0245] To permit population density-triggered expression of a protein of interest in the field and selectively deactivate bacterial QS systems during fermentation, a system of gene expression cassettes was designed (FIGURE 9). The first “QS circuit” gene expression cassette comprised control elements operably linked to a gene encoding the CinI QSSP (SEQ ID NO: 351) and a gene encoding the CinR QSRP (SEQ ID NO: 342) as well as the PcinI quorum sensing promoter (SEQ ID NO: 359) operably linked to a sequence encoding a GFP reporter protein of interest. The second “QQ circuit” gene expression cassette comprised a gene encoding the QqlM QE (SEQ ID NO: 325) operably linked to the tetracycline-inducible Ptet promoter (SEQ ID NO: 32) as well as a constitutively expressed gene encoding TetR (SEQ ID NO: 27). In a plant growth medium, only the “QS circuit” is operational. The CinI QSSP synthesizes an AHL QSSM, which binds the CinR QSRP, activating the PcinI QS promoter from which the GFP protein of interest is expressed in a population density-dependent manner. In a fermentation medium comprising a quorum quenching compound (anhydrotetracycline, aTc, here), the “QQ circuit” is activated. aTc binds TetR, inducing de- repression of the QE deactivator, degradation of the AHL QSSM, and abrogation of population density- dependent expression of the protein of interest.

[0246] A system of gene expression cassettes analogous to that of FIGURE 9 was built in Kosakonia radicincitans. Variant “QQ circuit” gene expression cassettes comprising a weak BCD22 (SEQ ID NO: 307), medium BCD13 (SEQ ID NO: 298), or strong BCD2 (SEQ ID NO: 288) ribosome binding site operably linked to the gene encoding the QqlM QE were cloned into the reporter plasmid of EXAMPLE 1 and FIGURE 2 comprising PcinI. The resulting plasmids were mobilized into a K. radicincitans strain comprising a chromosomally integrated cinRI “QS circuit” (SEQ ID NO: 318) encoding the CinR QSRP and the CinI QSSP.

[0247] Subsequently, the quorum quenching selective QS deactivation system was assessed in bacterial culture conditions and in a plant growth medium (FIGURE 10). On solid bacterial growth media without the aTc quorum quenching compound, K. radicincitans comprising strong, medium, or weak QQ circuits all demonstrated GFP expression. Upon addition of aTc in the solid bacterial growth media and expression of the QqlM QE deactivator, K. radicincitans comprising strong, medium, or weak QQ circuits all did not demonstrate GFP expression. Following the method of EXAMPLE 2, the K. radicincitans comprising QS and QQ circuits were inoculated into plant growth media, bacterial population density was quantified in bulk soil (BS) and on the rhizoplane (RP) of corn roots, and population density-dependent expression of a GFP reporter protein of interest was assessed by flow cytometry (FIGURE 10). The QQ circuit was not active in the absence of the quorum quenching compound aTc, and the three engineered K. radicincitans strains demonstrated population density-dependent expression of the GFP reporter protein of interest with a threshold population density of about 1 * 108cells per gram root on the rhizoplane.Atty. Dkt. No. P14610WO00 Example 4. Deactivation of Quorum Sensing in Engineered Soil Bacteria via Transcriptional Repression of the Quorum Sensing Regulator Protein

[0248] As described in EXAMPLE 3, when bacteria conditionally overexpress a protein of interest above a threshold population density, biomanufacturing benefits from deactivation of product protein expression. When product protein expression is controlled by quorum sensing, density-dependent overexpression can be selectively deactivated in the fermenter by reducing expression of the quorum sensing regulator protein (QSRP). One method to lower QSRP expression is to reduce the transcription rate of the QSRP-encoding gene.

[0249] To selectively reduce transcription of the QSRP-encoding gene in the fermenter, the control element operably linked to the QSRP-encoding gene was replaced with an inducible control element comprising a promoter that is inactive in common fermentation conditions but active in common field conditions. One such type of promoter is an inducible promoter that can be de-repressed or activated through addition of a chemical to fermentation media that is not present in agricultural fields. Examples of this type of promoter include the tetracycline-inducible Ptet promoter (SEQ ID NO: 32), the allolactose-inducible lac promoter (SEQ ID NO: 33), and the diacetylphoroglucinol-inducible PphlA promoter (SEQ ID NO: 34).

[0250] Another type of promoter that is selectively active in a fermenter but not in an agricultural field is a promoter with variable activity based on temperature. Bacterial fermentation is carried out at higher temperatures than the soil temperature in agricultural fields. As such, promoters that bind temperature- sensitive transcription factors permit selective expression of the QSRP at low field temperatures but not at higher fermenter temperatures. On example of such a system is the temperature-sensitive transcription factor cIts2 (SEQ ID NO: 337), which is active below 25 degrees centigrade but inactive above 25 degrees centigrade. Additionally, cIts2 acts as a transcriptional repressor for the promoter PL(SEQ ID NO: 31) but a transcriptional activator for the promoter PRM(SEQ ID NO: 338). In combination with PL, cIts2 achieves de-repression of a target gene at higher fermentation temperatures. In combination with PRM, cIts2 achieves de-activation of a target gene at higher temperatures. Operably linking PRMto the QSRP-encoding gene of a quorum sensing system while constitutively expressing cIts2 causes activation of the QSRP-encoding gene (and the QS system) at lower field temperatures and de-activation of the QSRP-encoding gene (and the QS system) at higher fermentation temperatures.

[0251] A third type of promoter that can be selectively active in a fermenter but not in an agricultural field is a phosphate-sensitive promoter (SEQ ID NO: 378-389). Bacteria are fermented with high-phosphate media to expedite growth. In such growth media, the phosphate-sensitive promoter is inactive. In an agricultural field, phosphate is initially at high concentration due to the application of chemical fertilizer but decreases as crop plants utilize that fertilizer to grow. After fertilizer concentration decreases, the phosphate-sensitive promoter is activated, expressing an operably linked target gene. In the case where a phosphate-sensitive promoter is operably linked to the QSRP-encoding gene of a QS system, the QS systemAtty. Dkt. No. P14610WO00 is inactive in the fermenter (where there is a high concentration of phosphate) but active in the field once there is a low concentration of phosphate.

[0252] When a QSRP is expressed in excess, expression from the cognate QS promoter loses density dependence. In order to maintain density-dependent expression from the QS promoter, the expression level of the QSRP is tuned so that expression from the QS promoter is density-dependent upon QS-promoter induction. To tune expression of the AhlR QSRP (SEQ ID NO: 339), a reporter construct was generated wherein (i) the chemically inducible Ptet promoter (SEQ ID NO: 32) was operably linked to the gene encoding AhlR in one gene expression cassette and (ii) the PahlI promoter (SEQ ID NO: 356) was operably linked to both the strong ribosome binding site BCD2 (SEQ ID NO: 288) and a green fluorescent protein (GFP) reporter gene in a separate gene expression cassette of the same construct. Different variations of said construct were generated where the AhlR-encoding gene was also operably linked to ribosome binding sites of differing strength, wherein a first construct comprised the medium-strength ribosome binding site BCD17 (SEQ ID NO: 302) and a second construct comprised the weak ribosome binding site BCD22 (SEQ ID NO: 308). A schematic of the two separate constructs for tuning density-dependent AhlR expression are shown as FIGURE 11.

[0253] The constructs of FIGURE 11 were synthesized and cloned into the mini-Tn7 delivery plasmid pUC18R6K-mini-Tn7T-Gm digested at the SacI restriction site. These were stably integrated into the chromosome of Kosakonia radicincitans DSM16656 (Kr) to generate two strains to test the level of expression of AhlR best suited to density-dependent expression of the GFP reporter gene from the PahlI QS promoter. Expression of the GFP reporter gene was quantified at different concentrations of exogenously added AHL quorum sensing signal molecule N-(β-Ketocaproyl)-L-homoserine lactone ranging from 0 μM to 10 μM using fluorescence spectroscopy (FIGURE 12). The construct of FIGURE 11 containing BCD22 operably linked to the AhlR-encoding gene demonstrated a greater fold-change of reporter gene expression across a range of AHL concentrations than the construct with BCD17. A greater fold-change of reporter expression across a range of AHL concentrations predicted a greater density-dependent response in a full QS system including a quorum sensing synthase protein (QSSP).

[0254] Subsequently, sets of DNA constructs analogous to those of FIGURE 11 were designed additionally comprising a third gene expression cassette, wherein the QS promoter PahlI (SEQ ID NO: 356) was operably linked to both a gene encoding the QSSP AhlI (SEQ ID NO: 348) and a ribosome binding site of differing strength in each set of constructs. The first set of constructs comprised the strong ribosome binding site BCD1 (SEQ ID NO: 287). The second set of constructs comprised the weak ribosome binding site BCD8 (SEQ ID NO: 293). The resulting constructs (depicted in FIGURE 13) included four permutations: (1) AhlI-encoding gene operably linked to BCD1 with AhlR-encoding gene operably linked to BCD 22; (2) AhlI-encoding gene operably linked to BCD1 with AhlR-encoding gene operably linked to BCD 17; (3) AhlI-encoding gene operably linked to BCD8 with AhlR-encoding gene operably linked toAtty. Dkt. No. P14610WO00 BCD 22; and (4) AhlI-encoding gene operably linked to BCD8 with AhlR-encoding gene operably linked to BCD 17.

[0255] The constructs of FIGURE 13 were synthesized and cloned into the mini-Tn7 delivery plasmid pUC18R6K-mini-Tn7T-Gm digested at the SacI restriction site. These were stably integrated into the chromosome of Kosakonia radicincitans DSM16656 (Kr) to generate four strains to test density-dependent expression of a GFP protein of interest with and without repression of the QSRP. The four strains of Kr were then transformed with a plasmid expressing the anhydrotetracycline (aTc) responsive transcription factor rTetR (SED ID NO: 28) under control of the Pomega2 promoter (SEQ ID NO: 377). The transformed strains were grown in liquid culture under QSRP-repressing conditions where an aTc quorum quenching compound was present (QQ+) and quorum sensing conditions where aTc was absent (QQ-). GFP fluorescence and OD600 were measured using spectroscopy to assess density-dependent induction.

[0256] The four Kr strains comprising the DNA constructs of FIGURE 13 and the rTetR-expressing plasmid demonstrated density-dependent expression of the GFP protein of interest in the absence of the aTc quorum quenching compound with silencing of GFP expression in the presence of aTc at all tested cell densities (FIGURES 14A, 14B, 14C, 14D). Increasing the strength of the ribosome binding sites operably linked to the QSRP-expressing gene and the QSSP-expressing gene increased the rate of per-cell protein of interest accumulation.

[0257] The engineered Kr strains with transcription-mediated quorum sensing deactivation tested in liquid culture were subsequently tested for suppression of density-dependent expression of a GFP protein of interest during fermentation followed by activation of density-dependent expression of GFP by the same bacteria after addition to a plant growth medium containing a plant. The four Kr strains were cultured in the presence of the quorum quenching aTc compound, then inoculated onto non-germinated corn seeds, and isolated for quantification of the GFP protein of interest using flow cytometry according to the method of EXAMPLE 2. The bacterial population density in sand where corn seeds were not planted (measured as cells g-1sand) was about 1000-fold lower than the bacterial population density observed on corn roots (measured as cells g-1root) (FIGURE 15). Kr demonstrated population density-dependent induction of the GFP protein of interest on plant roots with the AhlRI QS system (FIGURE 16). The threshold population density at which each QS system caused each bacterial strain to produce the reporter protein of interest is about 1 * 105cells per gram root / soil to about 5 * 108cells per gram root / soil. Example 5. Producing Ammonia from Engineered Bacteria by Overexpressing a GlnE Protein Lacking an Adenylyl Removing Domain

[0258] Intracellular glutamine concentration is the dominant indicator of nitrogen status in many nitrogen- fixing bacteria. Decreasing intracellular glutamine concentration prevents cells from sensing high ammonia levels in the environment. Intracellular glutamine concentration can be decreased by increasing expression or activity of glutaminase, an enzyme that converts glutamine into glutamate. Separately, intracellularAtty. Dkt. No. P14610WO00 glutamine concentration can be lowered by decreasing expression or activity of glutamine synthase (GS), an enzyme encoded by the gene glnA that converts ammonia into glutamine. In nitrogen-fixing bacteria, fixed ammonia is quickly assimilated into glutamine and glutamate shunted into cellular metabolism. Blocking ammonia assimilation causes fixed nitrogen to be released into the environment.

[0259] Most ammonia is assimilated into glutamine by glutamine synthetase (GS), and subsequently into glutamate by glutamine oxoglutarate aminotransferase (GOGAT). GS is regulated post-translationally by GS adenylyltransferase (GlnE), a bidirectional enzyme encoded by the glnE gene that catalyzes both the adenylylation and de-adenylylation of GS through activity of its adenylyltransferase (AT) and adenylyl- removing (AR) domains, respectively. When nitrogen is abundant, GS is not expressed, and the GlnE AT domain adenylates and deactivates GS. When nitrogen is scarce, GS is expressed, and the GlnE AR domain de-adenylylates and activates GS.

[0260] GS adenylyltransferase can be engineered such that it only adenylates and deactivates GS, lacking the ability to reactivate the enzyme. This change in function is achieved by expressing a truncated variant of the GlnE enzyme that lacks the AR domain and regulatory region of the enzyme. Such a truncated GlnE is termed a unidirectional adenylyltransferase (uAT). When a uAT is overexpressed in nitrogen-fixing bacteria, uAT activity deactivates GS independently of the concentration of ammonia, glutamine, and other regulators of nitrogen assimilation. Reduced GS activity in the cells causes a decrease in intracellular glutamine concentration and increase in intracellular ammonia concentration. Cells interpret decreased intracellular glutamine concentration as a need for additional nitrogen fixation, further increasing the intracellular concentration of ammonia through increased fixation of N2to NH3by nitrogenase. The excess intracellular ammonia is then secreted into the surrounding environment for uptake and assimilation by nearby plants and microbes.

[0261] Unidirectional ATases (uATs) are engineered as follows. Where present, the glnE gene encoding glutamine synthetase adenylyltransferase (ATase) is identified in soil bacteria such as those of the genera Acetobacter, Acidothermus, Acinetobacter, Agrobacterium, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Burkholderia, Conexibacter, Curtobacterium, Ensifer, Enterobacter, Erwinia, Escherichia, Flavobacterium, Frankia, Gaiella, Gluconacetobacter, Gluconobacter, Herbaspirillum, Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimibacter, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrobacter, Paenibacillus, Panotea, Pediococcus, Peribacillus, Priestia, Pseudarthrobacter, Pseudomonas, Rahnella, Rhizobium, Rhodococcus, Rhodoplanes, Rhodopseudomonas, Rhodospirillum, Serratia, Solirubrobacter, Sphingobacterium, Sphingomonas, Stenotrophomonas, Streptomyces, Stutzerimonas, Variovorax, Xanthobacter, and Yoonia. Next, the locations of the adenylyl-transferring (AT) and adenylyl-removing (AR) domains within each ATase are predicted. By one method, since the adenylyl- transferring (AT) and adenylyl-removing (AR) domains within ATase are homologous to each other, their locations are predicted through alignment of the N- and C- terminal halves of ATase to each other beforeAtty. Dkt. No. P14610WO00 designing uAT variants based on this intraprotein homology. By way of a second distinct method, the ATase amino acid sequence in one organism is aligned to that of another to identify the AT and AR domains. After AT and AR domains are identified, a series of uAT candidates is created based on each ATase where each uAT candidate in the series is obtained by selecting a different truncation site in the region between the AR and AT domain (SEQ ID NO: 1-25). In certain embodiments, the native glnE gene in each organism is then scarlessly deleted by double homologous recombination or other methods, and candidate uATs are expressed. In certain embodiments, the uAT gene is operably linked to a QS promoter in bacteria comprising a QSSP and a QSRP as described herein. Ammonia release is measured by growing strains in nitrogen-free liquid minimal media with an atmosphere of less than 3% oxygen, or by growing them in nitrogen-free semi-solid agar where bacteria form a pellicle at an oxygen concentration that permits nitrogenase activity. After incubation under these conditions, ammonia is then quantified in samples of cleared supernatant using an ammonium probe or a colorimetric assay such as the indophenol assay described by Schnabel and Sattely (doi: 10.1128 / AEM.00582-21). Example 6. Producing Ammonia from Engineered Bacteria by Overexpressing Proteins from Nitrogen Fixation and Assimilation Pathways

[0262] Since nitrogen fixation consumes at least 16 mol of adenosine triphosphate (ATP) per mol N2fixed, diazotrophic bacteria have evolved complex regulatory networks to control this energy- intensive metabolic process. To conserve energy, the N2-fixing catalyst nitrogenase is expressed only under conditions of nitrogen starvation, and the same conditions stimulate upregulation of high-affinity ammonia assimilation by the enzyme glutamine synthetase (GlnA, GS), preventing release of excess ammonia for plants (FIGURE 17). Diazotrophs can be engineered to produce and release ammonia by decoupling their ability to repress nitrogenase under nitrogen replete conditions. De-repression of nitrogenase can be achieved by a number of genetic strategies, and in many bacteria, results in more ammonia produced than can be assimilated, causing diffusion of ammonia from the cell.

[0263] A distinct method to stimulate ammonia production and release is to prevent bacteria from assimilating fixed nitrogen derived from N2 into glutamine, the intracellular signal for nitrogen status. This can be accomplished by chemically or genetically inactivating glutamine synthetase by a number of strategies. In addition to preventing assimilation of ammonia derived from nitrogen fixation, low glutamine levels force the cell to engage a nitrogen starvation response, driving de- repression of nitrogenase. High levels of ammonia production paired with inability to assimilate it causes diffusion of ammonia from the cell in high concentrations.

[0264] Whether a) nitrogenase feedback repression is alleviated, b) ammonia assimilation and glutamine production is inhibited or c) both processes are engineered, bacteria that produce and release large quantities of ammonia suffer a marked fitness defect that renders them non-competitive and unable to persist in the environment. Thus, inducible ammonia production releases a greater amount of ammonia from an initialAtty. Dkt. No. P14610WO00 bacterial inoculum than constitutive ammonia production because bacterial growth before ammonia production increases bacterial biomass and overall ammonia generated.

[0265] The NifA protein, encoded by the nifA gene, is the master transcriptional regulator of genes involved in nitrogenase assembly, function and maintenance, which are encoded by nif cluster and fix cluster genes. Specifically, NifA acts in association with the sigma factor σ54 to drive expression of nif cluster and fix cluster genes. The nifA gene in bacteria can be regulated at the transcriptional and posttranslational level by nitrogen, oxygen, and carbon. In Azotobacter vinelandii, overexpression of nifA from a heterologous promoter drives constitutive nitrogenase activity and stimulates ammonia release.

[0266] In nature, intracellular levels of active NifA protein are controlled by two key factors: transcription of the nifLA operon and inhibition of NifA protein activity by protein-protein interaction with the NifL protein. Increasing the transcription level of the nifLA operon leads to a higher intracellular concentration of NifA proteins, which increases expression of nitrogenase, the rate of ammonia production, and resulting ammonia release.

[0267] The nifL gene is common amongst gamma-proteobacteria and acts as an anti-activator of the nitrogenase master regulator NifA. In conditions of low oxygen and low glutamine, NifL represses activity of NifA, preventing nitrogenase expression and nitrogen fixation. However, deletions in nifL stop NifA inhibition and stop nitrogenase activity from repression by glutamine. Removal of nitrogenase feedback inhibition leads to ammonia release. A nifL gene deletion therefore will stop production of NifL protein, free NifA from inhibition by protein-protein interaction, and permit ammonia release.

[0268] glnR acts as a regulator of nitrogen metabolism genes in gram-positive bacteria. In P. polymyxa WLY78, overexpression of glnR drives nitrogenase activity even in the presence of added nitrogen. As such, overexpression of glnR can drive ammonia release in this strain. Example 7. Producing Ammonia from Engineered Bacteria by Reducing Expression of Proteins from Nitrogen Fixation and Assimilation Pathways

[0269] Ammonia uptake from the environment can be reduced by decreasing the expression level of AmtB ammonia transporter protein (FIGURE 17). In Pseudomonas stutzeri A1501, Azotobacter vinelandii DJ, and Gluconobacter diazotrophicus, deletion of the ammonium uptake system encoded by one or more copies of the amtB gene prevents uptake of ammonium from the extracellular environment, and also results in excretion of ammonia from the cells under diazotrophic growth conditions.

[0270] Separately, the PII proteins are global nitrogen response regulators, acting on a suite of nitrogen metabolism proteins. In Rhodobacter capsulatus and Azorhizobium caulinodans ORS 571, deletion of both PII proteins forces the adenylyl transferase (AT) to adenylate the GS protein, leading to de-repressed nitrogenase activity in the presence of ammonia. When both PII genes are deleted, ORS 571 releases ammonia into the growth media when grown under nitrogen fixing conditions.Atty. Dkt. No. P14610WO00

[0271] Separately, nitrogenase is regulated by feedback repression at the transcriptional and post translational level by ADP-ribosylation via the DraT-DraG system. DraT catalyzes the ADP-ribosylation of the nitrogenase Fe protein and shuts off of nitrogenase under nitrogen excessive conditions, whereas DraG catalyzes the removal of ADP ribose and reactivation of nitrogenase under nitrogen starvation. Deletion of DraT in a cell where nitrogenase is not regulated by feedback repression at the transcriptional level results in nitrogenase activity that escapes feedback repression. Example 8. Producing Ammonia from Engineered Bacteria by Inducibly Repressing Glutamine Synthetase Expression

[0272] As described in EXAMPLE 5, down-regulating glutamine synthetase (GS) activity blocks bacteria from assimilating fixed nitrogen and results in ammonia release into the environment. Transcriptionally repressing glnA in response to increases in bacterial population densities above threshold densities can down-regulate GS and lead to conditional ammonia release at those bacterial population densities.

[0273] To transcriptionally repress the native glutamine synthetase gene, the native glnA in soil bacteria such as those of the genera Acetobacter, Acidothermus, Acinetobacter, Agrobacterium, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Burkholderia, Conexibacter, Curtobacterium, Ensifer, Enterobacter, Erwinia, Escherichia, Flavobacterium, Frankia, Gaiella, Gluconacetobacter, Gluconobacter, Herbaspirillum, Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimibacter, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrobacter, Paenibacillus, Panotea, Pediococcus, Peribacillus, Priestia, Pseudarthrobacter, Pseudomonas, Rahnella, Rhizobium, Rhodococcus, Rhodoplanes, Rhodopseudomonas, Rhodospirillum, Serratia, Solirubrobacter, Sphingobacterium, Sphingomonas, Stenotrophomonas, Streptomyces, Stutzerimonas, Variovorax, Xanthobacter, and / or Yoonia (SEQ ID NO: 229-286) is operably linked to a repressible promoter, such as the Ptet promoter (SEQ ID NO: 32), the Plac promoter (SEQ ID NO: 33), or the PphlA promoter (SEQ ID NO: 34). A protein-coding sequence encoding a repressor protein paired with the repressible promoter, TetR (SEQ ID NO: 27), LacI (SEQ ID NO: 29), or PhlF (SEQ ID NO: 30) respectively, is operably linked to a quorum sensing promoter (SEQ ID NO: e.g., SEQ ID NO: 356-364, 373, and 374) in a heterologous gene expression cassette, which is introduced into a soil bacterium along with compatible QSSP and QSRP (e.g., an AhlI protein, a TetR promoter operably linked to an AhlR protein, with an ahlI promoter operably linked to a PhlF encoding gene) to create a genetically engineered bacterium. The genetically engineered bacterium (GEB) is fermented in bacterial growth media such that the bacteria grow to high cell density (e.g., in the presence of doxycycline, tetracycline, and / or anhydrotetracycline to prevent PhlF mediated repression of glnA at high cell densities). The GEB are then introduced into a plant growth medium in an agricultural context. Once the GEB population densities exceed a threshold density in the plant growth medium (e.g., see representative threshold density ranges in Tables 9 and 10 for certain quorum sensing systems in representative bacterialAtty. Dkt. No. P14610WO00 hosts), the repressor protein is expressed from the quorum sensing promoter, binds to the repressible promoter, and down-regulates expression of glnA, leading to ammonia release. Example 9. Producing Ammonia from Engineered Bacteria by Inducibly Cleaving an Engineered Glutamine Synthetase to Abrogate Enzymatic Activity

[0274] Glutamine synthetase (GS) is catalytically inactivated by disrupting the structure of its active site through cleavage of the GS peptide backbone. Hydrolysis of the GS peptide backbone can be catalyzed by proteases, a class of enzymes that recognize specific short amino acid sequences and cleave a protein’s peptide backbone a consistent distance from said amino acid recognition sequences, herein referred to as protease-specific recognition sequences (PSRS). A GS protein variant that is controllably cleaved by a protease is engineered by encoding one or more PSRS within the coding sequence of the glnA gene. By engineering a nitrogen-fixing bacterial strain to contain (i) a GS protein comprising one or more PSRS and (ii) the cognate protease under inducible control, GS is catalytically inactivated in response to environmental changes. In this system, introduction of an environmental stimulus induces expression of a protease. The protease then binds the one or more PSRS in the engineered GS and cleaves the GS backbone, causing a structural change in GS that inactivates it catalytically and causes ammonia accumulation due to GS down- regulation.

[0275] To engineer an organism with a GS protein that is inactivated by an inducible protease, the GS protein is engineered to comprise one or more PSRS. Proteases capable of cleaving the engineered GS protein peptide backbone include the tobacco etch virus (TEV) protease (SEQ ID NO: 45), tobacco vein mottling virus (TVMV) protease (SEQ ID NO: 46), sunflower mild mosaic virus (SMMV) protease (SEQ ID NO: 47), turnip mosaic virus (TuMV) protease (SEQ ID NO: 48), soybean mosaic virus (SMV) protease (SEQ ID NO: 49), plum pox potyvirus (PPV) protease (SEQ ID NO: 50), Hepatitis C virus (HCV) NS3 protease (SEQ ID NO: 51), Coagulation factor Xa (SEQ ID NO: 52), and Furin (SEQ ID NO: 53). The proteases above recognize the following PSRS sequences, respectively: EXXYXQ-(S / G) or ENLYFQ- (S / G / A / M / C / H), wherein X is any amino acid and the TEV protease cleaves between the Q and the S, G, A, M, C, or H residues; ETVRFQ-(G / S), wherein the TVMV protease cleaves between the Q and S or G residues; EEIHLQ-(S / G), wherein the SMMV protease cleaves between the Q and S or G residues; VXHQ or VRHQ-S, wherein X is any amino acid and the TuMV protease cleaves C- terminal to the Q residue; XVXXQ-(G / S), wherein X is any amino acid and the SMV protease cleaves between Q and S or G residues; NVVVHQ-A, wherein the PPV protease cleaves between the Q and the A residue; (D / E)XXXXC(A / S), wherein X is any amino acid and the HCV NS3 protease cleaves between the C and the A or S residues; I(D / E)GR, wherein the Factor Xa protease cleaves C-terminal to the R residue; or RX(K / R)R, wherein the furin protease cleaves C-terminal to the C-terminal R residue.

[0276] The one or more PSRS are incorporated into the GS coding sequence in a number of locations. To ensure that the one or more PSRS will be efficiently bound by a protease, the PSRS is / are located at theAtty. Dkt. No. P14610WO00 solvent-facing surface of the GS protein to increase the probability that the protease molecule will collide with the PSRS in the GS protein variant and bind to it / them. Multiple methods are used to identify the solvent-facing surface of the GS protein, including visualization of external amino acid residues using crystal structure data, alignment of the amino acid sequence of GS variants without a crystal structure to those with one in order to identify homologous regions of the protein, in silico structure prediction for GS variants without a crystal structure and identification of solvent-facing regions of the protein, and mass- spectrometry based protein labeling methods that identify solvent-facing regions of a protein. Additionally, catalytic deactivation of GS is more complete if protease cleavage compromises the structure of the GS enzyme active site. Compromising the structure of the active site is achieved by rationally positioning the PSRS such that cleavage will create multiple protein fragments that each contain a portion of the incomplete active site, rationally positioning the one or more PSRS near or within the active site so that the break in the peptide backbone alters the sterics of the active site, or screening the enzymatic activity of a library of GS variants with the PSRS positioned at random locations.

[0277] In the Azospirillum brasilense Sp245 GS polypeptide (SEQ ID NO: 229), locations to incorporate one or more PSRS that meet the above criteria include between amino acid residues 98 and 99, 121 and 122, 279 and 280, and / or 285 and 286. In the Pseudomonas stutzeri GS polypeptide (SEQ ID NO: 230), locations to incorporate one or more PSRS that meet the above criteria include between amino acid residues 98 and 99, 119 and 120, 283 and 284, and / or 298 and 299. These insertion sites are depicted in Table 6.

[0278] To achieve inducible deactivation of GS, GS must function normally until an environmental stimulus triggers protease expression, at which point enzymatic activity is minimized. To achieve normal function of GS until the protease is expressed, the native glnA gene in a bacterial strain is replaced with an engineered copy of glnA encoding the one or more PSRS so that the engineered GS is expressed by way of the native glnA genetic context of the host strain. To minimize GS activity after protease cleavage, the engineered copy of glnA encoding the one or more PSRS is the only glnA allele in the host strain. The gene encoding GS, glnA, is an essential gene for many bacterial strains. However, replacing a single native allele with a single engineered allele is accomplished by inserting certain site-specific recombinase recognition sites (SSRRS) flanking a bacterium’s native glnA allele in the bacterium’s chromosome (including FRT sites and / or loxP sites), encoding the corresponding site-specific recombinase (a yeast flippase (FLP) recombinase if FRT SSRRS or a Cre-recombinase if loxP SSRRS) under inducible control on a plasmid with the glnA allele encoding a GS variant comprising the one or more PSRS, and inducing recombination to swap the engineered glnA allele into the chromosome while excising the native allele. Such a process achieves exchange of the wild-type glnA allele for the engineered glnA allele encoding the GS that comprises the one or more PSRS without ever requiring deletion of the essential glnA gene.

[0279] Once a bacterial strain is obtained where the native gene encoding GS has been replaced by a GS gene encoding a GS variant comprising the one or more PSRS, a heterologous gene expression cassette comprising a protease-encoding gene (e.g. SEQ ID NO: 54, 55, or 56) operably linked to a quorum sensingAtty. Dkt. No. P14610WO00 promoter (QS-P; e.g., SEQ ID NO: 356-364, 373, and 374) is introduced. Since the protease gene is expressed by a QS-P (e.g., SEQ ID NO: 356-364, 373, and 374), expression of the protease is prevented before the QS-P is activated when the bacterial strain grows to a density which exceeds the threshold population density in plant growth media. When the bacteria are grown in bacterial cultures (e.g., axenically) in contact with the quorum quenching compound QQ and / or in exposure to a temperature above the threshold temperature, expression of the protease is inhibited at densities which exceed the threshold population density to facilitate growth in the culture.

[0280] A genetically engineered bacterium (GEB) comprising, (i) a gene encoding a GS variant comprising one or more PSRS and (ii) a heterologous gene expression cassette (s) comprising a protease gene of interest operably linked to a QS-P, a QSSP encoding gene, and a QSRP gene are obtained. Enzymatic activity of a GS variant comprising one or more PSRS within a GEB is compared to activity of wildtype GS in a strain- matched bacterium containing a wild-type GS variant to confirm enzymatic function of the PSRS- containing GS variant, using a biochemical assay. Subsequently the GEB comprising the PSRS-containing GS variant is evaluated for ammonia release via an indophenol assay.

[0281] In Azospirillum brasilense Sp245, introducing about two to three PSRS into the natively controlled glnA gene followed by inducing TEV protease expression under control of a tetracycline-responsive promoter via anhydrotetracycline yields a significant decrease in GS activity compared to the wild-type. Placing the engineered glnA alleles under control of constitutive promoters at the native locus (instead of the natively regulated promoter) additionally abolishes up-regulation of glnA expression when GS is cleaved by the proteases.

[0282] In its agricultural application, the genetically engineered bacterium (GEB) is fermented in in contact with the quorum quenching compound QQ and / or in exposure to a temperature above the threshold temperature such that the bacteria grow to high cell density. The GEB are then introduced into a plant growth medium in an agricultural context. Once the GEB population densities exceed a threshold density in the plant growth medium (e.g., see representative threshold density ranges in Tables 9 and 10 for certain quorum sensing systems in representative bacterial hosts), the protease is expressed from the QS-P, cleaves the GS variant expressed by the GEB, and down-regulates expression of glnA, leading to ammonia release.

Claims

Atty. Dkt. No. P14610WO00 WHAT IS CLAIMED IS:

1. A genetically engineered bacterium comprising: (a) one or more heterologous gene expression cassette(s) comprising at least one control element which is operably linked to at least one nucleic acid sequence encoding a first quorum sensing synthase protein (QSSP) able to synthesize a quorum sensing signal molecule Q (QSSM Q) and / or at least one nucleic acid sequence encoding a first quorum sensing regulator protein (QSRP) that can bind said QSSM Q; (b) a heterologous gene expression cassette comprising at least one nucleic acid sequence encoding at least one protein or RNA sequence of interest operably linked to a control element comprising a first quorum sensing promoter, wherein the first quorum sensing promoter is activated by the first quorum sensing regulator protein (QSRP) and the QSSM Q when the population density of the genetically engineered bacterium exceeds a threshold population density and wherein said at least one protein or RNA sequence of interest causes the production of ammonia, ammonium, or phosphate; and (c) at least one heterologous deactivation gene expression cassette comprising a DNA promoter which is operably linked to one or more deactivator(s) which inhibit(s) expression of the at least one RNA sequence or protein of interest at a population density of the genetically engineered bacterium which exceeds the threshold population density when the genetically engineered bacterium (GEB) is: (i) contacted with a quorum quenching compound QQ; or (ii) exposed to a temperature above a threshold temperature.

2. The genetically engineered bacterium of claim 1, wherein the deactivator is triggered by: (a) QQ induced repression of the first QSSP, the first QSRP, and / or the protein or RNA sequence of interest, wherein the deactivator comprises a repressor protein which binds one or more promoter(s) which are operably linked to the gene(s) encoding the first QSSP, the first QSRP, and / or the protein or RNA sequence of interest in the presence of QQ, wherein the repressor protein is released from the promoter(s) in the absence of QQ, and wherein the first QSSP, the first QSRP, and the protein or RNA sequence of interest are expressed in the absence of QQ; (b) QQ induced de-repression of the deactivator, wherein the GEB comprises a repressor protein which binds the DNA promoter which is operably linked to the deactivator in the absence of QQ, wherein the repressor protein is released from the DNA promoter in the presence of QQ, and wherein the deactivator is expressed in the presence of QQ, optionally wherein the deactivator comprises an enzyme which catalyzes the degradation of the quorum sensing signal molecule Q; (c) QQ induced activation of the deactivator, wherein the GEB comprises an activator protein which fails to bind the DNA promoter which is operably linked to deactivator in the absence of QQ, wherein theAtty. Dkt. No. P14610WO00 activator protein binds the DNA promoter in the presence of QQ, and wherein the deactivator is expressed in the presence of QQ, optionally wherein the deactivator comprises an enzyme which catalyzes the degradation of the quorum sensing signal molecule Q; (d) QQ induced de-activation of the first QSSP, the first QSRP, and / or the RNA or protein of interest, wherein the deactivator comprises an activator protein which binds one or more promoter(s) which are operably linked to the gene(s) encoding the first QSSP, the first QSRP, and / or the RNA or protein of interest in the absence of QQ, wherein the activator protein is released from the promoter(s) in the presence of QQ, and wherein the gene(s) operably linked to the promoter(s) is / are transcribed in the absence of QQ; (e) temperature induced de-repression of the deactivator, wherein the GEB comprises a repressor protein which binds a DNA promoter which is operably linked to the deactivator below a threshold temperature, wherein the repressor protein is released from the DNA promoter above the threshold temperature, and wherein the deactivator is expressed above the threshold temperature, optionally wherein the deactivator comprises an enzyme which catalyzes the degradation of the quorum sensing signal molecule Q; (f) temperature induced activation of the deactivator, wherein the GEB comprises an activator protein which fails to bind a DNA promoter that is operably linked to a gene encoding the deactivator below a threshold temperature, wherein the activator protein binds the DNA promoter above the threshold temperature, and wherein the deactivator is expressed above the threshold temperature, optionally wherein the deactivator comprises an enzyme which catalyzes the degradation of the quorum sensing signal molecule Q; (g) temperature induced repression of the first QSSP, the first QSRP, and / or the RNA or protein of interest, wherein the deactivator comprises a repressor protein which binds one or more promoter(s) which is / are operably linked to the gene(s) encoding the first QSSP, the first QSRP, and / or the RNA or protein of interest above a threshold temperature, wherein the repressor protein is released from the promoter(s) below the threshold temperature, and wherein the first QSSP, the first QSRP, and the RNA or protein of interest are expressed below the threshold temperature; (h) temperature induced de-activation of the first QSSP, the first QSRP, and / or the RNA or protein of interest, wherein the deactivator comprises an activator protein which binds one or more promoter(s) which is / are operably linked to the gene(s) encoding the first QSSP, the first QSRP, and / or the RNA or protein of interest below a threshold temperature, wherein the activator protein is released from the promoter(s) above the threshold temperature, and wherein the gene(s) operably linked to the promoter(s) is / are transcribed below the threshold temperature.Atty. Dkt. No. P14610WO00 3. The genetically engineered bacterium of claim 2, wherein the repressor in the QQ induced repression comprises an rTetR repressor having at least 95% sequence identity to SEQ ID NO: 28 and wherein the DNA promoter(s) which is / are operably linked to the gene(s) encoding the first QSSP and / or the first QSRP comprise(s) a Ptet promoter having at least 95% sequence identity to SEQ ID NO:

32.

4. The genetically engineered bacterium of claim 2, wherein the repressor in the QQ induced de- repression of the deactivator comprises: (a) a TetR repressor having at least 95% sequence identity to SEQ ID NO: 27 and wherein the DNA promoter operably linked to the gene encoding the deactivator comprises a Ptet promoter having at least 95% sequence identity to SEQ ID NO: 32, and optionally wherein the QQ compound is doxycycline, tetracycline, and / or anhydrotetracycline; (b) a LacI repressor having at least 95% sequence identity to SEQ ID NO: 29 and wherein the DNA promoter operably linked to the gene encoding the deactivator comprises a Plac promoter having at least 95% sequence identity to SEQ ID NO: 33, and optionally wherein the QQ compound is allolactose, isopropyl-β-D-thiogalactopyranoside (IPTG), or thiomethylgalactoside; or (c) a PhlF repressor having at least 95% sequence identity to SEQ ID NO: 30 and wherein the DNA promoter operably linked to the gene encoding the deactivator comprises a PphlA promoter having at least 95% sequence identity to SEQ ID NO: 34, and optionally wherein the QQ compound is 2,4- diacetylphoroglucinol (DAPG).

5. The genetically engineered bacterium of claim 2, wherein the activator protein used in the QQ induced activation of the deactivator comprises a second quorum sensing regulator protein, the DNA promoter comprises a second quorum sensing promoter, and the QQ compound is an N-acyl homoserine lactone molecule (AHL) that binds the second quorum sensing regulator protein, wherein said AHL does not bind the first quorum sensing regulator protein and said second quorum sensing regulator protein binds the second quorum sensing promoter but not the first quorum sensing promoter.

6. The genetically engineered bacterium of claim 2, wherein the deactivator is triggered by: (i) temperature induced de-repression of the deactivator, wherein the repressor comprises a cIts2 protein having at least 95% sequence identity to SEQ ID NO: 337 and wherein the DNA promoter which is operably linked to the deactivator comprises a PLpromoter having at least 95% sequence identity to SEQ ID NO: 31; orAtty. Dkt. No. P14610WO00 (ii) temperature induced de-activation of the first QSSP and / or the first QSRP wherein the deactivator comprises a cIts2 protein having at least 95% sequence identity to SEQ ID NO: 337 and the DNA promoter which is operably linked to the gene encoding the first QSSP and / or the first QSRP comprises a PRMpromoter having at least 95% sequence identity to SEQ ID NO:

338.

7. The genetically engineered bacterium of claim 1, wherein the deactivator comprises an enzyme which catalyzes the degradation of the quorum sensing signal molecule Q and wherein the expression of the enzyme is induced by the addition of the quorum quenching compound QQ or the increase in temperature.

8. The genetically engineered bacterium of claim 7, wherein the quorum sensing signal molecule Q is an acyl homoserine lactone (AHL) molecule and the enzyme which catalyzes its degradation comprises an AHL acylase enzyme, an AHL lactonase enzyme, or an AHL metallo-beta-lactamase enzyme.

9. The genetically engineered bacterium of claim 8, wherein: (i) the AHL acylase enzyme comprises a PvdQ, AiiD, AigC, or QuiP protein, optionally wherein the PvdQ, AiiD, AigC, or QuiP protein has at least 75% sequence identity to SEQ ID NO: 320, 321, 322, or 323, respectively; (ii) the AHL lactonase enzyme comprises an alpha-beta hydrolase fold lactonase protein, optionally wherein the lactonase comprises an AiiM, QqlM, or AidH protein, and optionally wherein the AiiM, QqlM, or AidH protein has at least 75% sequence identity to SEQ ID NO: 324, 325, or 326, respectively; (iii) the AHL lactonase enzyme comprises a phosphotriesterase-like lactonase protein, optionally wherein the lactonase comprises a Pph, SsoPox, Sislac, Gkl, or QsdA protein and optionally wherein the Pph, SsoPox, Sislac, Gkl, or QsdA protein has at least 75% sequence identity to SEQ ID NO: 332, 333, 334, 335, or 336, respectively; or (iv) the AHL lactonase enzyme comprises a metallo-beta-lactamase protein, optionally wherein the metallo-beta-lactamase protein is a GcI, AttM, AidC, AiiB, or AiiA protein and optionally wherein the GcI, AttM, AidC, AiiB, or AiiA protein has at least 75% sequence identity to SEQ ID NO: 327, 328, 329, 330, or 331, respectively.

10. The genetically engineered bacterium of claim 1, wherein the deactivator comprises an inducible transcription factor which:Atty. Dkt. No. P14610WO00 (i) decreases expression of the first QSSP and / or the first QSRP when the GEB is contacted with the quorum quenching compound QQ or the temperature is increased; and / or (ii) decreases expression of the RNA or protein of interest control element comprising the first quorum sensing promoter.

11. The genetically engineered bacterium of claim 1, wherein: (i) the QSSM Q molecule comprises N-(3-Hydroxytetradecanoyl)-DL-homoserine lactone and the first QSSP comprises a CinI protein; (ii) the QSSM Q molecule comprises N-(3-Hydroxytetradecanoyl)-DL-homoserine lactone and the first QSRP comprises a CinR protein; (iii) the QSSM Q molecule comprises N-(β-Ketocaproyl)-L-homoserine lactone and the first QSSP comprises an AhlI protein; or (iv) the QSSM Q molecule comprises N-(β-Ketocaproyl)-L-homoserine lactone and the first QSRP comprises an AhlR protein.

12. The genetically engineered bacterium of claim 1, wherein the quorum quenching compound QQ is inorganic phosphate or soluble phosphate, wherein the control element operably linked to the gene encoding the first QSRP comprises a phosphate-sensitive promoter.

13. The genetically engineered bacterium of claim 1, wherein: i. the first QSSP comprises an AhlI protein, the first QSRP comprises an AhlR protein, and the first quorum sensing promoter comprises an ahlI promoter, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum, Enterobacter, Klebsiella, Kosakonia, Pseudomonas, or Rahnella; ii. the first QSSP comprises an CinI protein, the first QSRP comprises a CinR protein, and the first quorum sensing promoter comprises a cinI promoter, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum, Pseudomonas, or Kosakonia; iii. the first QSSP comprises an CciI protein, the first QSRP comprises a CciR protein, and the first quorum sensing promoter comprises a cciI promoter, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum or Pseudomonas;Atty. Dkt. No. P14610WO00 iv. the first QSSP comprises an CviI protein, the first QSRP comprises a CviR protein, and the first quorum sensing promoter comprises a cviI promoter, optionally wherein the bacterium is a member of the taxonomic genus Herbaspirillum, Kosakonia, or Pseudomonas; v. the first QSSP comprises an EsaI protein, the first QSRP comprises a mutant EsaR protein with the amino acid change D91G, and the first quorum sensing promoter comprises an esaR repressable promoter; vi. the first QSSP comprises an EsaI protein, the first QSRP comprises a EsaR protein, and the first quorum sensing promoter comprises an esaI promoter; vii. the first QSSP comprises a LasI protein, the first QSRP comprises a LasR protein, and the first quorum sensing promoter comprises a lasI or lasB promoter, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum, Kosakonia, or Pseudomonas; viii. the first QSSP comprises an LuxI protein, the first QSRP comprises a LuxR protein, and the first quorum sensing promoter comprises a luxI promoter, optionally wherein the bacterium is a member of the taxonomic genus Pseudomonas; or ix. the first QSSP comprises an TraI protein, the first QSRP comprises a TraR protein, and the first quorum sensing promoter comprises a traI promoter.

14. The genetically engineered bacterium of claim 13, wherein: (i) the AhlI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 348; (ii) the CciI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 349; (iii) the CinI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 350; (iv) the CinI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 351; (v) the CviI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 352; (vi) the EsaI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 353; (vii) the LasI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 376; (viii) the LuxI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 354; or (ix) the TraI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 355.Atty. Dkt. No. P14610WO00 15. The genetically engineered bacterium of claim 1, wherein the first QSRP activates the first quorum sensing promoter by binding the QSSM and the first quorum sensing promoter, optionally wherein the first QSRP comprises an AhlR, CciR, CinR, CviR, LasR, LuxR, or TraR QSRP.

16. The genetically engineered bacterium of claim 1, wherein the first QSRP is a repressor which is released from the first quorum sensing promoter when the first QSRP binds the QSSM, optionally wherein the first QSRP comprises an EsaR QSRP with or without the amino acid change D91G.

17. The genetically engineered bacterium of claim 1, wherein the first QSRP comprises an AhlR, CciR, CinR, CviR, EsaR, LasR, LuxR, or TraR QSRP.

18. The genetically engineered bacterium of claim 17, wherein the AhlR, CciR, CinR, CviR, EsaR, LasR, LuxR, or TraR QSRP comprises a protein having at least 75% sequence identity to SEQ ID NO: 339, 340, 341 or 342, 343, 344 or 345, 375, 346, or 347, respectively.

19. The genetically engineered bacterium of claim 1, wherein the control element(s), the first quorum sensing promoter, and / or the DNA promoter further comprises at least a segment of a 5’ untranslated region (5’ UTR) which is operably linked to the control element(s), first quorum sensing promoter, and / or the DNA promoter, optionally wherein the 5’ UTR which is operably linked to the first quorum sensing promoter comprises a 5’ UTR sequence which has at least 85% sequence identity to an endogenous 5’ UTR sequence which is operably linked to an endogenous quorum sensing promoter.

20. The genetically engineered bacterium of claim 19, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR which is operably linked to the first quorum sensing promoter comprises at least one copy of an operably linked DNA binding site which can be bound by an AhlR, CciR, CinR, CviR, EsaR, LasR, LuxR, or TraR QSRP, optionally wherein said QSRP has at least 95% identity to SEQ ID NO: 339 to 347, or 375.

21. The genetically engineered bacterium of claim 19, wherein the first quorum sensing promoter and / or segment the of the 5’ UTR is a promoter and / or 5’ UTR derived from at least one gram-negative bacterium.Atty. Dkt. No. P14610WO00 22. The genetically engineered bacterium of claim 19, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR is a promoter and / or 5’ UTR derived from at least one bacterium of the taxonomic classes of alphaproteobacteria, betaproteobacteria, and gammaproteobacteria.

23. The genetically engineered bacterium of claim 19, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR is a promoter and / or segment of a 5’ UTR derived from at least one bacterium of a genus selected from the group consisting of Acetobacter, Acidothermus, Acinetobacter, Agrobacterium, Aliivibrio, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Burkholderia, Chromobacterium, Conexibacter, Curtobacterium, Ensifer, Enterobacter, Erwinia, Escherichia, Flavobacterium, Frankia, Gaiella, Gluconacetobacter, Gluconobacter, Herbaspirillum, Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimibacter, Mesorhizobium, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrobacter, Paenibacillus, Pantoea, Pediococcus, Peribacillus, Priestia, Pseudarthrobacter, Pseudomonas, Rahnella, Rhizobium, Rhodococcus, Rhodoplanes, Rhodopseudomonas, Rhodospirillum, Serratia, Solirubrobacter, Sphingobacterium, Sphingomonas, Stenotrophomonas, Streptomyces, Stutzerimonas, Variovorax, Vibrio, Xanthobacter, and Yoonia, optionally wherein the quorum sensing promoter and / or the segment of the 5’ UTR is derived from at least one bacterium of a genus selected from the group consisting of Aliivibrio, Burkholderia, Chromobacterium, Mesorhizobium, Pantoea, Pseudomonas, and Rhizobium.

24. The genetically engineered bacterium of claim 19, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR comprises a promoter and / or a segment of a 5’ UTR of an ahlI, cciI, cinI, cviI, esaI, esaR, lasB, lasI, luxI, or traI gene, a variant thereof, or a combination thereof.

25. The genetically engineered bacterium of claim 19, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR comprises at least 50 nucleotides of DNA located upstream of a start codon of a gene encoding an AhlI, CciI, CinI, CviI, EsaI, EsaR, LasB, LasI, LuxI, or TraI protein, optionally wherein the promoter region and / or the segment of the 5’ UTR comprises about 50 to about 250 nucleotides of DNA located upstream of said start codon of said gene encoding said AhlI, CciI, CinI, CviI, EsaI, EsaR, LasB, LasI, LuxI, or TraI protein.Atty. Dkt. No. P14610WO00 26. The genetically engineered bacterium of claim 19, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR comprises a promoter and / or a segment of a 5’ UTR of a promoter comprising a DNA sequence having at least 75% sequence identity to SEQ ID NO: 356-364, 373, or 374.

27. The genetically engineered bacterium of claim 19, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR comprises: (i) an ahlI promoter comprising the DNA sequence of SEQ ID NO: 356, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum, Enterobacter, Klebsiella, Kosakonia, Pseudomonas, or Rahnella; (ii) a cciI promoter comprising the DNA sequence of SEQ ID NO: 357, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum or Pseudomonas; (iii) a cinI promoter comprising the DNA sequence of SEQ ID NO: 358 or 359, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum, Pseudomonas, or Kosakonia; (iv) a cviI promoter comprising the DNA sequence of SEQ ID NO: 360, optionally wherein the bacterium is a member of the taxonomic genus Herbaspirillum, Kosakonia, or Pseudomonas; (v) an esaR repressable promoter comprising the DNA sequence of SEQ ID NO: 361; (vi) an esaI promoter comprising the DNA sequence of SEQ ID NO: 362; (vii) a lasB promoter comprising the DNA sequence of SEQ ID NO: 374, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum or Pseudomonas; (viii) a lasI promoter comprising the DNA sequence of SEQ ID NO: 373, optionally wherein the bacterium is a member of the taxonomic genus Azospirillum or Kosakonia; (ix) a luxI promoter comprising the DNA sequence of SEQ ID NO: 363, optionally wherein the bacterium is a member of the taxonomic genus Pseudomonas; (x) a traI promoter comprising the DNA sequence of SEQ ID NO: 364; or (xi) a variant of the promoters under (i) to (x) comprising a nucleic acid molecule having a sequence identity of at least 80% with any of SEQ ID NO: 356-364, 373, or 374, optionally wherein said variant promoters are activated by an increase in the population density of the genetically engineered bacterium above the threshold population density at which the promoters under (i) to (x) are activated.Atty. Dkt. No. P14610WO00 28. The genetically engineered bacterium of claim 19, wherein the first quorum sensing promoter and / or the segment of the 5’ UTR or its variant is derived from the same genus or species as the genetically engineered bacterium.

29. The genetically engineered bacterium of claim 1, wherein any one of the heterologous gene expression cassettes is integrated at a location in the chromosome of the genetically engineered bacterium which does not comprise the location of an endogenous quorum sensing promoter.

30. The genetically engineered bacterium of claim 1, wherein one or more of the heterologous gene expression cassette(s) further comprises one or more elements comprising: a. a ribosome binding site (RBS), wherein the RBS is operably linked to the nucleic acid sequence coding for the QSSP, QSRP, and / or the protein of interest and optionally wherein the RBS is an RBS having at least 95% sequence identity to SEQ ID NO: 287-313, 366- 371, or 372; and / or b. a terminator sequence (TS), wherein the TS is operably linked to the nucleic acid sequence coding for the QSSP, QSRP, and / or the RNA sequence or protein of interest and optionally wherein the TS is a TS having at least 95% sequence identity to SEQ ID NO: 314, 315, or 316.

31. The genetically engineered bacterium of any one of claims 1 to 30, wherein the protein or RNA sequence of interest causes the production of at least one fertilizer or plant nutrient is ammonia or phosphate.

32. The genetically engineered bacterium of claim 31, wherein the RNA sequence or protein of interest encoded by the heterologous gene expression cassette and operably linked to the control element comprising the first quorum sensing promoter comprises: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of-function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises oneAtty. Dkt. No. P14610WO00 or more heterologous genes from a wild-type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; (f) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more first target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein products of the first target genes and / or wherein the repressor protein optionally comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof and / or optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (g) a non-coding synthetic small RNA (sRNA); optionally wherein the non-coding synthetic small RNA (sRNA) binds a natural or synthetic DNA and / or RNA motif in the promoter, 5’ UTR, and / or coding region of any one or more first target gene(s) of the genetically engineered bacterium, optionally wherein the non-coding synthetic small RNA (sRNA) comprises a guide RNA that additionally binds an RNA-guided DNA endonuclease, RNA-guided RNA endonuclease, or variant thereof and / or optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (h) a site-specific DNA endonuclease, wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) one or more specific DNA sequence(s) recognized by the site-specific DNA endonuclease, optionally wherein the site-specific DNA endonuclease comprises an RNA-guided DNA endonuclease, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or a restriction endonuclease, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (i) a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) comprising DNA encoding the PSRS in the protein codingAtty. Dkt. No. P14610WO00 region of the first target gene(s), wherein the target protein product comprising the one or more in-frame insertion(s) has activity, and wherein cleavage of the target protein product(s) by the protease deactivates the target protein product(s), optionally wherein the location of the one or more in-frame insertion(s) is given by Table 6, optionally wherein the first target gene(s) is / are under the control of a constitutive promoter, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (j) a first protein of interest comprising a ClpAP ATP-dependent protease and a second protein of interest comprising a ClpS Leu / N-recognin, wherein any one or more first target gene(s) of the genetically engineered bacterium encodes a protein comprising an N- terminal -Leu, -Phe, -Trp, or -Tyr residue, and optionally wherein the first and second protein of interest are operably linked to distinct control elements, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (k) a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) an in- frame insertion(s) of DNA encoding the PSRS at the N-terminus of the protein coding region of the gene followed by a -Leu, -Phe, -Trp, or -Tyr residue and wherein cleavage of the PSRS from the N-terminus of the protein(s) encoded by the gene(s) by the protease results in a protein comprising an N-terminal -Leu, -Phe, -Trp, or -Tyr residue which is degraded by native ClpS and ClpAP, optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (l) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and activates expression of the protease, wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) of DNA encoding a PSRS, optionally wherein the transcriptional activator protein comprises a tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, optionally wherein the transcriptional activator domain is VP16 , and / or optionally wherein the first target gene(s) is / are: glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia;Atty. Dkt. No. P14610WO00 (m) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and inhibits expression of the protease, wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion of DNA encoding an N-terminal amino acid tag designed to render the protein product(s) of the first target gene(s) inactive, followed by a PSRS, optionally wherein the repressor protein comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (n) a nanobody, wherein the nanobody binds a protein product of any one or more first target gene(s) of the genetically engineered bacterium, wherein binding of the nanobody to such protein product inhibits function of the protein product, optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (o) an aptamer, wherein the aptamer binds a protein product of any one or more first target gene(s) of the genetically engineered bacterium and inhibits function of any one or more of the protein product(s) of the first target gene(s), optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; or (p) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of any one or more first target gene(s) and increases expression of any one or more of the first target gene(s), optionally wherein the transcriptional activator protein comprises the tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, optionally wherein the transcriptional activator domain is VP16 and / or optionally wherein the first target gene(s) is / are any one or more of: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the fertilizer is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphataseAtty. Dkt. No. P14610WO00 enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, and / or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase and the fertilizer or plant nutrient is phosphate; or (q) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, and / or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase and the fertilizer or plant nutrient is phosphate.

33. The genetically engineered bacterium of claim 3, wherein the first QSSP comprises an AhlI protein, the first QSRP comprises an AhlR protein, and the first quorum sensing promoter comprises an ahlI promoter and wherein the bacterium is a member of the taxonomic genus Azospirillum, Enterobacter, Klebsiella, Kosakonia, Pseudomonas, or Rahnella.

34. The genetically engineered bacterium of claim 3, wherein the first QSSP comprises an CinI protein, the first QSRP comprises a CinR protein, and the first quorum sensing promoter comprises a cinI promoter and wherein the bacterium is a member of the taxonomic genus Azospirillum, Pseudomonas, or Kosakonia.

35. The genetically engineered bacterium of claim 32, wherein a PSRS is inserted between any one or more of the pairs of amino acid residues corresponding to: (i) amino acid residues 98 and 99, 121 and 122, 279 and 280, and / or 285 and 286 of the glutamine synthetase (GS) polypeptide of SEQ ID NO: 229; (ii) amino acid residues 98 and 99, 119 and 120, 283 and 284, and / or 298 and 299 of the glutamine synthetase (GS) polypeptide of SEQ ID NO: 230; (iii) amino acid residues 448 and 449, 480 and 481, 505 and 506, 528 and 529, and / or 624 and 625 of the GlnE polypeptide of SEQ ID NO: 231;Atty. Dkt. No. P14610WO00 (iv) amino acid residues 527 and 528, 537 and 538, and / or 547 and 548 of the GlnE polypeptide of SEQ ID NO: 232; or (v) amino acid residues 422 and 423 and / or 608 and 609 of the GlnE polypeptide of SEQ ID NO:

233.

36. The genetically engineered bacterium of claim 32, wherein the protease or heterologous protease: a. comprises a tobacco etch virus (TEV) protease and the PSRS comprises the peptide EXXYXQ- (S / G) or ENLYFQ-(S / G / A / M / C / H), wherein X is any amino acid and the TEV protease cleaves between the Q and S, G, A, M, C, or H residues; b. comprises a tobacco vein mottling virus (TVMV) protease and the PSRS comprises the peptide ETVRFQ-(G / S), wherein the TVMV protease cleaves between the Q and S or G residues; c. comprises a sunflower mild mosaic virus (SMMV) protease and the PSRS comprises the peptide EEIHLQ-(S / G), wherein the SMMV protease cleaves between the Q and S or G residues; d. comprises a turnip mosaic virus (TrMV) protease and the PSRS comprises the peptide VXHQ or VRHQ-S, wherein X is any amino acid and the TrMV protease cleaves C-terminal to the Q residue; e. comprises a soybean mosaic virus (SMV) protease and the PSRS comprises the peptide XVXXQ-(G / S), wherein X is any amino acid and the SMV protease cleaves between Q and S or G residues; f. comprises a plum pox virus (PPV) protease and the PSRS comprises the peptide NVVVHQ-A, wherein the PPV protease cleaves between the Q and the A residue; g. comprises a hepatitis C virus (HCV) NS3 protease and the PSRS comprises the peptide (D / E)XXXXC(A / S), wherein X is any amino acid and the HCV protease cleaves between the C and the A or S residues; h. comprises an enterokinase and the PSRS comprises the peptide DDDDK, wherein the enterokinase cleaves C-terminal to the K residue; i. comprises a Factor Xa protease and the PSRS comprises the peptide I(D / E)GR, wherein the Factor Xa protease cleaves C-terminal to the R residue; or j. comprises a furin protease and the PSRS comprises the peptide RX(K / R)R, wherein the furin protease cleaves C-terminal to the C-terminal R residue.

37. The genetically engineered bacterium of claim 31, wherein the plant nutrient is ammonia and α- ketobutyrate and the protein is ACC deaminase.Atty. Dkt. No. P14610WO00 38. The genetically engineered bacterium of claim 31, wherein the first QSSP comprises an AhlI protein, the first QSRP comprises an AhlR protein, and the first quorum sensing promoter comprises an ahlI promoter and wherein the bacterium is a member of the taxonomic genus Azospirillum, Enterobacter, Klebsiella, Kosakonia, Pseudomonas, or Rahnella; and wherein the protein of interest is: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of-function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild-type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; or (f) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more first target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein products of the first target genes, wherein the repressor protein optionally comprises the tet repressor (TetR) or a lac repressor (LacI), and optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia.

39. The genetically engineered bacterium of claim 32, wherein the control element which is operably linked to said target genes comprises a constitutive promoter, an inducible promoter, a repressible promoter, and / or a quorum sensing promoter.

40. The genetically engineered bacterium of claim 31, wherein the first QSSP comprises a CinI protein, the first QSRP comprises a CinR protein, and the first quorum sensing promoter comprises a cinI promoter, the bacterium is a member of the taxonomic genus Azospirillum, Pseudomonas, or Kosakonia, and wherein the protein of interest is: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia;Atty. Dkt. No. P14610WO00 (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of- function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild- type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; or (f) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more first target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein products of the first target genes, wherein the repressor protein optionally comprises the tet repressor (TetR) or a lac repressor (LacI), and optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia.

41. The genetically engineered bacterium of claim 31, wherein the first QSRP comprises a CciR protein, and the first quorum sensing promoter comprises a cciI promoter, the bacterium is a member of the taxonomic genus Azospirillum or Pseudomonas, and wherein the protein of interest is: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of- function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild- type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; or (f) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more first target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein products of the first target genes, wherein the repressor protein optionally comprises the tet repressor (TetR) or a lac repressor (LacI), and optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia.Atty. Dkt. No. P14610WO00 42. The genetically engineered bacterium of claim 31, wherein the protein of interest is a first repressor protein and wherein the genetically engineered bacterium further comprises: (i) a second control element comprising a promoter which is repressed by the first repressor protein and operably linked to a gene encoding a second repressor protein; (ii) optionally a third control element comprising a promoter which is repressed by the second repressor protein and operably linked to a gene encoding a third repressor protein; (iii) optionally a fourth control element comprising a promoter which is repressed by the third repressor protein and operably linked to a gene encoding a fourth repressor protein; and (iv) a control element comprising a promoter which is repressed by the second, third, or fourth repressor protein and which is operably linked to a first target gene, optionally wherein the first, second, third, and / or fourth repressor protein(s) optionally comprise(s) the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA- guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof.

43. The genetically engineered bacterium of claim 42, wherein the genetically engineered bacterium lacks (ii) and (iii), and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s), and the fertilizer is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof.

44. The genetically engineered bacterium of claim 42, wherein the genetically engineered bacterium further comprises (ii) and (iii), and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the fertilizer is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acidAtty. Dkt. No. P14610WO00 release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase, and the fertilizer or plant nutrient is phosphate.

45. The genetically engineered bacterium of claim 42, wherein the genetically engineered bacterium further comprises (ii) and lacks (iii), and the first target gene is amtB, draT, glnA, glnB, glnK, glnZ, and / or nifL, and the fertilizer is ammonia.

46. The genetically engineered bacterium of claim 42, wherein first target gene encodes a transcriptional activator protein and wherein the genetically engineered bacterium further comprises a control element comprising a promoter which is activated by the transcriptional activator protein and operably linked to at least one second target gene.

47. The genetically engineered bacterium of claim 42, wherein the genetically engineered bacterium comprises an even number of repressors and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the fertilizer is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), a pyrroloquinoline (PQQ) synthase, and the fertilizer or plant nutrient is phosphate.

48. The genetically engineered bacterium of claim 42, wherein the genetically engineered bacterium comprises an even number of repressors and the first target gene encodes an RNA sequence or protein comprising: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of-function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, optionally wherein the genetically engineered bacterium comprises one or moreAtty. Dkt. No. P14610WO00 heterologous genes from a wild-type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; (f) a phytase enzyme and the fertilizer or plant nutrient is phosphate, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase; (g) an acid phosphatase enzyme and the fertilizer or plant nutrient is phosphate, optionally wherein the acid phosphatase enzyme is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof; (h) a protein which stimulates organic acid release from the bacterium and the fertilizer or plant nutrient is phosphate, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase; (i) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more second target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein product(s) of the second target gene(s) and / or wherein the repressor protein optionally comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof and / or optionally wherein the second target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (j) a non-coding synthetic small RNA (sRNA); optionally wherein the non-coding synthetic small RNA (sRNA) binds a natural or synthetic DNA and / or RNA motif in the promoter, 5’ UTR, and / or coding region of any one or more second target gene(s) of the genetically engineered bacterium, optionally wherein the non-coding synthetic small RNA (sRNA) comprises a guide RNA that additionally binds an RNA-guided DNA endonuclease, RNA-guided RNA endonuclease, or variant thereof and / or optionally wherein the second target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (k) a site-specific DNA endonuclease, wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) one or more specific DNA sequence(s) recognized by the site-specific DNA endonuclease, optionally wherein the site-specific DNA endonuclease comprises an RNA-guided DNA endonuclease, a protein comprising a DNA-bindingAtty. Dkt. No. P14610WO00 zinc finger domain, a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or a restriction endonuclease, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (l) a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) comprising DNA encoding the PSRS in the protein coding region of the second target gene(s), wherein the target protein product comprising the one or more in-frame insertion(s) has activity, and wherein cleavage of the target protein product(s) by the protease deactivates the target protein product(s), optionally wherein the location of the one or more in-frame insertion is given by Table 6, optionally wherein the second target gene is under the control of a constitutive promoter, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (m) a first protein comprising a ClpAP ATP-dependent protease and a second protein comprising a ClpS Leu / N-recognin, wherein any one or more second target gene(s) of the genetically engineered bacterium encodes a third protein comprising an N-terminal -Leu, -Phe, - Trp, or -Tyr residue, and optionally wherein the first and second protein are operably linked to distinct control elements, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (n) a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion(s) of DNA encoding the PSRS at the N-terminus of the protein coding region of the gene(s) followed by a -Leu, -Phe, -Trp, or -Tyr residue and wherein cleavage of PSRS from the N- terminus of the protein(s) encoded by the gene(s) by the protease results in a protein comprising an N-terminal -Leu, -Phe, -Trp, or -Tyr residue which is degraded by native ClpS and ClpAP, optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (o) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and activates expression of the protease, wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) of DNA encoding a PSRS, optionally wherein the transcriptional activator protein comprises a tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, aAtty. Dkt. No. P14610WO00 transcription activator-like effector (TALE), or any variant thereof, optionally wherein the transcriptional activator domain is VP16 , and / or optionally wherein the second target gene(s) is / are: glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (p) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and inhibits expression of the protease, wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion of DNA encoding an N-terminal amino acid tag designed to render the protein product(s) of the second target gene(s) inactive, followed by a PSRS, optionally wherein the repressor protein comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (q) a nanobody, wherein the nanobody binds a protein product of any one or more second target gene(s) of the genetically engineered bacterium, wherein binding of the nanobody to such protein product inhibits function of the protein product, optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (r) an aptamer, wherein the aptamer binds a protein product of any one or more second target gene(s) of the genetically engineered bacterium and inhibits function of any one or more of the protein product(s) of the second target gene(s), optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; or (s) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of any one or more second target gene(s) and increases expression of any one or more of the second target gene(s), optionally wherein the transcriptional activator protein comprises the tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, wherein the DNA targeting protein is optionally a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, wherein the transcriptional activator domain is optionally VP16 and / or optionally wherein the second target gene(s) is / are any one or more of: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the fertilizer is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidineAtty. Dkt. No. P14610WO00 acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, and / or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase and the fertilizer or plant nutrient is phosphate.

49. The genetically engineered bacterium of claim 42, wherein the genetically engineered bacterium comprises an odd number of repressors and the second target gene is amtB, draT, glnA, glnB, glnK, glnZ, and / or nifL, and the fertilizer is ammonia.

50. The genetically engineered bacterium of claim 31, wherein the protein of interest is a first transcriptional activator protein and wherein the genetically engineered bacterium further comprises: (i) a control element comprising a promoter which is activated by the first transcriptional activator protein and operably linked to a gene encoding a second transcriptional activator protein; (ii) optionally a control element comprising a promoter which is activated by the second transcriptional activator protein and operably linked to a gene encoding a third transcriptional activator protein; (iii) optionally a control element comprising a promoter which is activated by the third transcriptional activator protein and operably linked to a gene encoding a fourth transcriptional activator protein; and (iv) a control element comprising a promoter which is activated by the second, third, or fourth transcriptional activator protein and which is operably linked to a first target gene, optionally wherein the first, second, third, and / or fourth transcriptional activator protein comprise(s) the tet responsive element-binding tTA transcription factor or an activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator- like effector (TALE), and / or any variant thereof, optionally wherein the activator domain is VP16.

51. The genetically engineered bacterium of claim 50, wherein the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif clusterAtty. Dkt. No. P14610WO00 gene(s), and / or one or more fix cluster gene(s) and the fertilizer is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase, and the fertilizer or plant nutrient is phosphate.

52. The genetically engineered bacterium of claim 31, wherein the protein of interest is a repressor protein and wherein the genetically engineered bacterium further comprises: (i) a control element comprising a promoter which is repressed by the repressor protein and operably linked to a gene encoding a transcriptional activator protein; and (ii) a control element comprising a promoter which is activated by the transcriptional activator protein and operably linked to a target gene.

53. The genetically engineered bacterium of claim 52, wherein the target gene is a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia.

54. The genetically engineered bacterium of claim 31, wherein the protein of interest is a transcriptional activator protein and wherein the genetically engineered bacterium further comprises: (i) a second control element comprising a promoter which is activated by the transcriptional activator protein and operably linked to a gene encoding a repressor protein; and (ii) a third control element comprising a promoter which is repressed by the repressor and operably linked to a target gene.

55. The genetically engineered bacterium of claim 54, wherein the target gene is a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia.

56. The genetically engineered bacterium of claim 32, wherein the glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene in (i), (j), (k), or (l) is under the control of a heterologous constitutive promoter.Atty. Dkt. No. P14610WO00 57. The method of claim 48, wherein the glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene in (l), (m), (n), or (o) is under the control of a heterologous constitutive promoter.

58. The genetically engineered bacterium of claim 31, wherein the plant nutrient is phosphate and the protein of interest operably linked to the first quorum sensing promoter comprises: a. a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase; b. an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof; c. a protein which stimulates organic acid release from the bacterium, optionally wherein the protein comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), a pyrroloquinoline (PQQ) synthase including pqqFABCDEG, or any combination of GAD, GDH, and PQQ; or, d. any combination of proteins of a, b, or c.

59. The genetically engineered bacterium of any one of claims 1 to 30, wherein: (i) the genetically engineered bacterium comprises the heterologous gene expression cassettes in a bacterium originally isolated from a plant growth medium or a plant; and (ii) wherein the bacterium originally isolated from the plant growth medium or the plant lacks the heterologous gene expression cassettes.

60. The genetically engineered bacterium of any one of claims 1 to 30, wherein the bacteria are selected from the group of gram-negative bacteria.

61. The genetically engineered bacterium of any one of claims 1 to 30, wherein the bacteria are selected from the groups of bacteria of the taxonomic classes of alphaproteobacteria, betaproteobacteria, and gammaproteobacteria.

62. The genetically engineered bacterium of any one of claims 1 to 30, wherein the bacteria are selected from the groups of bacteria in the taxonomic genera of Acetobacter, Acidothermus, Acinetobacter, Agrobacterium, Aliivibrio, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Burkholderia, Chromobacterium, Conexibacter, Curtobacterium, Ensifer, Enterobacter, Erwinia, Escherichia, Flavobacterium, Frankia, Gaiella,Atty. Dkt. No. P14610WO00 Gluconacetobacter, Gluconobacter, Herbaspirillum, Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimibacter, Mesorhizobium, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrobacter, Paenibacillus, Pantoea, Pediococcus, Peribacillus, Phytobacter, Priestia, Pseudarthrobacter, Pseudomonas, Rahnella, Rhizobium, Rhodococcus, Rhodoplanes, Rhodopseudomonas, Rhodospirillum, Serratia, Solirubrobacter, Sphingobacterium, Sphingomonas, Stenotrophomonas, Streptomyces, Stutzerimonas, Variovorax, Vibrio, Xanthobacter, and Yoonia, optionally wherein the bacteria are selected from at least one of the taxonomic genera selected from the group consisting of Aliivibrio, Burkholderia, Chromobacterium, Mesorhizobium, Pantoea, Pseudomonas, and Rhizobium.

63. A composition comprising the genetically engineered bacterium of any one of any one of claims 1 to 30 and an agriculturally acceptable carrier.

64. The composition of claim 63, wherein the composition further comprises: (i) an agriculturally acceptable adjuvant, optionally wherein the adjuvant comprises an adhesive agent, a desiccant, and / or a dispersant; (ii) a fungicide, an insecticide, a nematicide, a rodenticide, and / or a bacteriocide; and / or (iii) a fertilizer, optionally wherein the fertilizer comprises nitrogen, phosphorous, potassium, calcium, sulfur, magnesium, boron, chloride, manganese, iron, zinc, copper, molybdenum, and / or selenium.

65. The composition of claim 63, wherein the composition is in a solid form, optionally wherein the solid form comprises a wettable powder, granules, a gel, pellets, or microencapsulated particles.

66. The composition of claim 63, wherein the composition is in a liquid form, optionally wherein the liquid form comprises an aqueous solution, aqueous suspension, water-in-oil emulsion, an oil, or an alcohol.

67. A plant part or plant propagule which is at least partially coated, imbibed, or mixed with the composition of claim 63.

68. The plant part of claim 67, wherein the part is a leaf, stem, root, or seed.Atty. Dkt. No. P14610WO00 69. The plant propagule of claim 67, wherein the propagule comprises a cutting, tuber, or stolon.

70. Use of the plant part or plant propagule of claim 67 to grow a crop.

71. The use of claim 70, wherein fertilizer input is reduced in comparison to a crop grown from a plant part or plant propagule which has not been treated.

72. An agricultural system comprising: (i) at least one engineered bacterium of any one of claims 1 to 30; (ii) at least one plant growth medium; and (iii) at least one crop plant, crop plant seed, or crop plant vegetative propagule; wherein the plant growth medium, crop plant, crop seed, and / or crop plant propagule comprise, are at least partially coated, imbibed, and / or are mixed with the engineered bacterium or a composition comprising the engineered bacterium and an agriculturally acceptable carrier.

73. The system of claim 72, wherein the crop plant, seed, or vegetative propagule is an alfalfa, apple, banana, barley, bean, buckwheat, cabbage, cassava, chili, clover, coffee, corn, cotton, cowpea, cucumber, fonio, garlic, herb, lettuce, maize, melon, millet, nut, oat, oilseed rape, olive, onion, orange, sunflower, pea, Phaseolus bean, plantain, potato, quinoa, rice, rye, safflower, sorghum, soybean, sugar beet, sugar cane, sunflower, tangerine, tobacco, tomato, triticale, turnip, wheat, or yam plant, seed, or vegetative propagule.

74. The system of claim 72, wherein the plant growth medium comprises soil and / or water, optionally wherein the soil and / or water is non-axenic.

75. The system of claim 72, wherein the vegetative propagule comprises a cutting, tuber, or stolon.

76. A treated plant seed or plant propagule system comprising: (i) at least one crop plant seed or crop plant vegetative propagule; and (ii) at least one engineered bacterium of any one of claims 1 to 30,Atty. Dkt. No. P14610WO00 wherein the crop plant seed or crop plant propagule are at least partially coated, imbibed, and / or mixed with the engineered bacterium or a composition comprising the engineered bacterium and an agriculturally acceptable carrier.

77. The system of claim 76, wherein the crop plant, seed, or vegetative propagule is an alfalfa, apple, banana, barley, bean, buckwheat, cabbage, cassava, chili, clover, coffee, corn, cotton, cowpea, cucumber, fonio, garlic, herb, lettuce, maize, melon, millet, nut, oat, oilseed rape, olive, onion, orange, sunflower, pea, Phaseolus bean, plantain, potato, quinoa, rice, rye, safflower, sorghum, soybean, sugar beet, sugar cane, sunflower, tangerine, tobacco, tomato, triticale, turnip, wheat, or yam plant, seed, or vegetative propagule.

78. The system of claim 76, wherein the vegetative propagule comprises a cutting, tuber, or stolon.

79. A method of producing a bacterial culture comprising: (i) growing the genetically engineered bacterium of any one of claims 1 to 30 either: (a) in contact with the quorum quenching compound QQ; or (b) in exposure to a temperature above the threshold temperature; and (ii) harvesting the bacterial culture.

80. The method of claim 79, wherein growing the genetically engineered bacterium in the presence of QQ and / or at a temperature above the threshold temperature enables said genetically engineered bacteria to grow to a greater population density and / or at a faster rate to a maximal population density than in the absence of QQ and / or at a temperature below the threshold temperature, respectively.

81. The method of claim 79, wherein the genetically engineered bacterium is grown to a cell density in excess of the cell density where the RNA or protein is expressed in the absence of the compound QQ and / or temperatures in excess of the threshold temperature.

82. The method of claim 79, wherein the genetically engineered bacterium is grown to a cell density in excess of about 1 x 107, 1 x 108, or 1 x 109colony forming units per milliliter (CFU / mL) of the culture prior to harvesting.Atty. Dkt. No. P14610WO00 83. The method of claim 79, wherein the bacterial culture is grown in the presence of: (i) excess nitrogen; and / or (ii) excess phosphorus, optionally wherein the quorum quenching compound QQ is inorganic phosphate or soluble phosphate, optionally wherein the control element operably linked to the gene encoding the first QSRP comprises a phosphate-sensitive promoter.

84. The method of claim 79, wherein the repressor in the QQ induced de-repression of the deactivator comprises: (a) a TetR repressor having at least 95% sequence identity to SEQ ID NO: 27 and wherein the DNA promoter operably linked to the gene encoding the deactivator comprises a Ptet promoter having at least 95% sequence identity to SEQ ID NO: 32, and optionally wherein the QQ compound is doxycycline, tetracycline, and / or anhydrotetracycline; (b) a LacI repressor having at least 95% sequence identity to SEQ ID NO: 29 and wherein the DNA promoter operably linked to the gene encoding the deactivator comprises a Plac promoter having at least 95% sequence identity to SEQ ID NO: 33, and optionally wherein the QQ compound is allolactose, isopropyl-β-D-thiogalactopyranoside (IPTG), or thiomethylgalactoside; or (c) a PhlF repressor having at least 95% sequence identity to SEQ ID NO: 30 and wherein the DNA promoter operably linked to the gene encoding the deactivator comprises a PphlA promoter having at least 95% sequence identity to SEQ ID NO: 34, and optionally wherein the QQ compound is 2,4- diacetylphoroglucinol (DAPG).

85. The method of claim 79, wherein: (i) the RNA or protein of interest is not expressed in the bacterial culture when harvested in step (ii); and / or (ii) the agricultural compound of interest is not produced by the bacterial culture when harvested in step (ii).

86. The method of claim 79, wherein harvesting comprises separating the bacteria from the culture media to obtain a solid bacterial culture, optionally wherein the separating comprises filtering the liquid bacterial culture from the culture media and / or centrifuging the bacterial culture and decanting the liquid bacterial culture.Atty. Dkt. No. P14610WO00 87. The method of claim 86, further comprising combining the harvested bacterial culture with an agriculturally acceptable carrier and optionally (i) an agriculturally acceptable adjuvant, optionally wherein the adjuvant comprises an adhesive agent, a desiccant, and / or a dispersant; (ii) a fungicide, an insecticide, a nematicide, a rodenticide, and / or a bacteriocide; and / or (iii) a fertilizer, optionally wherein the fertilizer comprises nitrogen, phosphorous, potassium, calcium, sulfur, magnesium, boron, chloride, manganese, iron, zinc, copper, molybdenum, and / or selenium, thereby forming a composition.

88. The method of claim 87, wherein the composition is made in a solid form, optionally wherein the solid form is made by formulating the composition into a wettable powder, granules, a gel, pellets, or microencapsulated particles.

89. The method of claim 87, further comprising drying and / or lyophilizing the composition.

90. The method of claim 87, wherein the composition is made in a liquid form, optionally wherein the liquid form is made by formulating the composition as an aqueous solution, aqueous suspension, water-in- oil emulsion, an oil, or an alcohol.

91. A method of providing at least one fertilizer selected from ammonia, ammonium, or phosphate to a plant comprising placing at least one genetically engineered bacterium of any one of claims 1 to 30 into a plant growth medium, wherein said at least one RNA sequence or protein of interest is or causes the production of ammonia, ammonium, or phosphate when the population density of the genetically engineered bacterium exceeds a threshold population density in the plant growth medium and activates expression of said at least one RNA sequence or protein of interest.

92. The method of claim 91, wherein the plant growth medium comprises soil and / or water, optionally wherein the soil and / or water is non-axenic.Atty. Dkt. No. P14610WO00 93. The method of claim 91, wherein the placing is prior to, during, and / or after depositing a seed in the plant growth medium.

94. The method of claim 91, wherein the placing is prior to, during, and / or after depositing a vegetative propagule in the plant growth medium.

95. The method of claim 91, wherein the placing comprises depositing a seed which is at least partially coated with the genetically engineered bacterium in the plant growth medium or depositing both the seed and a composition comprising the genetically engineered bacterium in the plant growth medium.

96. The method of claim 91, wherein the placing comprises depositing the seed in furrow and contacting the seed in the furrow with a composition comprising the genetically engineered bacterium.

97. The method of claim 91, wherein the placing of the genetically engineered bacterium in the plant growth medium is prior to, during, and / or after establishment of a plant in the plant growth medium.

98. The method of claim 91, wherein the genetically engineered bacterium is placed into the plant growth medium and / or in contact with the plant: (i) by foliar application to the plant; (ii) by an in furrow application, fumigation, and / or soil drench; (iii) with a seed in form of a seed treatment wherein the seed is at least partially coated with a composition comprising the genetically engineered bacterium; (iv) with a seed in the form of bio-priming where the seed is imbibed with an aqueous composition comprising the genetically engineered bacterium before planting; and / or (v) with a root dip transplant whereby a seedling root system is dipped in an aqueous composition comprising the genetically engineered bacterium.

99. The method of claim 91, wherein the at least one fertilizer is ammonia, ammonium, or phosphate.Atty. Dkt. No. P14610WO00 100. The method of claim 99, wherein the RNA sequence or protein of interest encoded by the heterologous gene expression cassette and operably linked to the control element comprising a first quorum sensing promoter comprises: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of-function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild-type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; (f) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more first target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein products of the first target genes and / or wherein the repressor protein optionally comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof and / or optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (g) a non-coding synthetic small RNA (sRNA); optionally wherein the non-coding synthetic small RNA (sRNA) binds a natural or synthetic DNA and / or RNA motif in the promoter, 5’ UTR, and / or coding region of any one or more first target gene(s) of the genetically engineered bacterium, optionally wherein the non-coding synthetic small RNA (sRNA) comprises a guide RNA that additionally binds an RNA-guided DNA or RNA endonuclease or variant thereof and / or optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (h) a site-specific DNA endonuclease, wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) one or more specific DNA sequence(s) recognized by the site-specific DNA endonuclease, optionally wherein the site-specificAtty. Dkt. No. P14610WO00 DNA endonuclease comprises an RNA-guided DNA endonuclease, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or a restriction endonuclease, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (i) a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) comprising DNA encoding the PSRS in the protein coding region of the first target gene(s), wherein the target protein product comprising the one or more in-frame insertion(s) has activity, and wherein cleavage of the target protein product(s) by the protease deactivates the target protein product(s), optionally wherein the location of the one or more in-frame insertion(s) is given by Table 6, optionally wherein the first target gene(s) is / are under the control of a constitutive promoter, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (j) a first protein of interest comprising a ClpAP ATP-dependent protease and a second protein of interest comprising a ClpS Leu / N-recognin, wherein any one or more first target gene(s) of the genetically engineered bacterium encodes a protein comprising an N- terminal -Leu, -Phe, -Trp, or -Tyr residue, and optionally wherein the first and second protein of interest are operably linked to distinct control elements, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (k) a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) an in- frame insertion(s) of DNA encoding the PSRS at the N-terminus of the protein coding region of the gene followed by a -Leu, -Phe, -Trp, or -Tyr residue and wherein cleavage of PSRS from the N-terminus of the protein(s) encoded by the gene(s) by the protease results in a protein comprising an N-terminal -Leu, -Phe, -Trp, or -Tyr residue which is degraded by native ClpS and ClpAP, optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (l) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and activates expression of the protease, wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) one or moreAtty. Dkt. No. P14610WO00 in-frame insertion(s) of DNA encoding a PSRS, optionally wherein the transcriptional activator protein comprises a tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, optionally wherein the transcriptional activator domain is VP16 , and / or optionally wherein the first target gene(s) is / are: glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (m) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and inhibits expression of the protease, wherein any one or more first target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion of DNA encoding an N-terminal amino acid tag designed to render the protein product(s) of the first target gene(s) inactive, followed by a PSRS, optionally wherein the repressor protein comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, and / or optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (n) a nanobody, wherein the nanobody binds a protein product of any one or more first target gene(s) of the genetically engineered bacterium, wherein binding of the nanobody to such protein product inhibits function of the protein product, optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (o) an aptamer, wherein the aptamer binds a protein product of any one or more first target gene(s) of the genetically engineered bacterium and inhibits function of any one or more of the protein product(s) of the first target gene(s), optionally wherein the first target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (p) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of any one or more first target gene(s) and increases expression of any one or more of the first target gene(s), optionally wherein the transcriptional activator protein comprises the tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, wherein the DNA targeting protein is optionally a catalytically inactive RNA-guided DNAAtty. Dkt. No. P14610WO00 binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, wherein the transcriptional activator domain is optionally VP16 and / or optionally wherein the first target gene(s) is / are any one or more of: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the fertilizer is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, and / or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase and the fertilizer or plant nutrient is phosphate; or (q) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, and / or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase and the fertilizer or plant nutrient is phosphate is phosphate.

101. The method of claim 99, wherein the first QSSP comprises an AhlI protein, the first QSRP comprises an AhlR protein, and the first quorum sensing promoter comprises an ahlI promoter and wherein the bacterium is a member of the taxonomic genus Azospirillum, Enterobacter, Klebsiella, Kosakonia, Pseudomonas, or Rahnella; and wherein the protein of interest is: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of-function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild-type or refactored nif or fix gene cluster and the fertilizer is ammonia;Atty. Dkt. No. P14610WO00 (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; or (f) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more first target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein products of the first target genes, wherein the repressor protein optionally comprises the tet repressor (TetR) or a lac repressor (LacI), and optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia.

102. The method of claim 99, wherein the first QSSP comprises a CinI protein, the first QSRP comprises a CinR protein, and the first quorum sensing promoter comprises a cinI promoter, the bacterium is a member of the taxonomic genus Azospirillum, Pseudomonas, or Kosakonia, and wherein the protein of interest is: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of-function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild-type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; or (f) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more first target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein products of the first target genes, wherein the repressor protein optionally comprises the tet repressor (TetR) or a lac repressor (LacI), and optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia.

103. The method of claim 100, wherein the PSRS is inserted in a glutamine synthetase (GS) polypeptide between any one or more of the pairs of amino acid residues corresponding to: (i) amino acid residues 98 and 99, 121 and 122, 279 and 280, and / or 285 and 286 of the glutamine synthetase (GS) polypeptide of SEQ ID NO: 229;Atty. Dkt. No. P14610WO00 (ii) amino acid residues 98 and 99, 119 and 120, 283 and 284, and / or 298 and 299 of the glutamine synthetase (GS) polypeptide of SEQ ID NO: 230; (iii) amino acid residues 448 and 449, 480 and 481, 505 and 506, 528 and 529, and / or 624 and 625 of the GlnE polypeptide of SEQ ID NO: 231; (iv) amino acid residues 527 and 528, 537 and 538, and / or 547 and 548 of the GlnE polypeptide of SEQ ID NO: 232; or (v) amino acid residues 422 and 423 and / or 608 and 609 of the GlnE polypeptide of SEQ ID NO:

233.

104. The method of claim 100, wherein the protease or heterologous protease: a. comprises a tobacco etch virus (TEV) protease and the PSRS comprises the peptide EXXYXQ- (S / G) or ENLYFQ-(S / G / A / M / C / H), wherein X is any amino acid and the TEV protease cleaves between the Q and the S, G, A, M, C, or H residues; b. comprises a tobacco vein mottling virus (TVMV) protease and the PSRS comprises the peptide ETVRFQ-(G / S), wherein the TVMV protease cleaves between the Q and S or G residues; c. comprises a sunflower mild mosaic virus (SMMV) protease and the PSRS comprises the peptide EEIHLQ-(S / G), wherein the SMMV protease cleaves between the Q and S or G residues; d. comprises a turnip mosaic virus (TrMV) protease and the PSRS comprises the peptide VXHQ or VRHQ-S, wherein X is any amino acid and the TrMV protease cleaves C-terminal to the Q residue; e. comprises a soybean mosaic virus (SMV) protease and the PSRS comprises the peptide XVXXQ-(G / S), wherein X is any amino acid and the SMV protease cleaves between Q and S or G residues; f. comprises a plum pox virus (PPV) protease and the PSRS comprises the peptide NVVVHQ-A, wherein the PPV protease cleaves between the Q and the A residue; g. comprises a hepatitis C virus (HCV) NS3 protease and the PSRS comprises the peptide (D / E)XXXXC(A / S), wherein X is any amino acid and the HCV protease cleaves between the C and the A or S residues; h. comprises an enterokinase and the PSRS comprises the peptide DDDDK, wherein the enterokinase cleaves C-terminal to the K residue; i. comprises a Factor Xa protease and the PSRS comprises the peptide I(D / E)GR, wherein the Factor Xa protease cleaves C-terminal to the R residue; orAtty. Dkt. No. P14610WO00 j. comprises a furin protease and the PSRS comprises the peptide RX(K / R)R, wherein the furin protease cleaves C-terminal to the C-terminal R residue.

105. The method of claim 99, wherein the plant nutrient is ammonia and α-ketobutyrate and the protein is ACC deaminase.

106. The method of claim 99, wherein the first QSRP comprises a CciR protein, and the first quorum sensing promoter comprises a cciI promoter, the bacterium is a member of the taxonomic genus Azospirillum or Pseudomonas, and wherein the protein of interest is: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of-function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild-type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; or (f) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more first target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein products of the first target genes, wherein the repressor protein optionally comprises the tet repressor (TetR) or a lac repressor (LacI), and optionally wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia.

107. The method of claim 99, wherein the protein of interest is a first repressor protein and wherein the genetically engineered bacterium further comprises: (i) a second control element comprising a promoter which is repressed by the first repressor protein and operably linked to a gene encoding a second repressor protein; (ii) optionally a third control element comprising a promoter which is repressed by the second repressor protein and operably linked to a gene encoding a third repressor protein;Atty. Dkt. No. P14610WO00 (iii) optionally a fourth control element comprising a promoter which is repressed by the third repressor protein and operably linked to a gene encoding a fourth repressor protein; and (iv) a control element comprising a promoter which is repressed by the second, third, or fourth repressor protein and which is operably linked to a first target gene, optionally wherein the first, second, third, and / or fourth repressor protein(s) comprise(s) the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof.

108. The method of claim 107, wherein the genetically engineered bacterium lacks (ii) and (iii), and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s), and the fertilizer is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase, and the fertilizer or plant nutrient is phosphate.

109. The method of claim 107, wherein the genetically engineered bacterium further comprises (ii) and (iii), and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the fertilizer is ammonia.

110. The method of claim 107, wherein the genetically engineered bacterium further comprises (ii) and lacks (iii), and the first target gene is amtB, draT, glnA, glnB, glnK, glnZ, and / or nifL.

111. The method of claim 107, wherein first target gene encodes a transcriptional activator protein and wherein the genetically engineered bacterium further comprises a control element comprising a promoterAtty. Dkt. No. P14610WO00 which is activated by the transcriptional activator protein and operably linked to at least one second target gene.

112. The method of claim 107, wherein the genetically engineered bacterium comprises an even number of repressors and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the fertilizer is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), a pyrroloquinoline (PQQ) synthase, and the fertilizer or plant nutrientis phosphate.

113. The method of claim 107, wherein the genetically engineered bacterium comprises an even number of repressors and the first target gene encodes an RNA sequence or protein comprising: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of-function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild-type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase enzyme and the fertilizer is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; (f) a phytase enzyme and the fertilizer or plant nutrient is phosphate, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase; (g) an acid phosphatase enzyme and the fertilizer or plant nutrient is phosphate, optionally wherein the acid phosphatase enzyme is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof;Atty. Dkt. No. P14610WO00 (h) a protein which stimulates organic acid release from the bacterium and the fertilizer or plant nutrient is phosphate, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase; (i) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more second target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein product(s) of the second target gene(s) and / or wherein the repressor protein optionally comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof and / or optionally wherein the second target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (j) a non-coding synthetic small RNA (sRNA); optionally wherein the non-coding synthetic small RNA (sRNA) binds a natural or synthetic DNA and / or RNA motif in the promoter, 5’ UTR, and / or coding region of any one or more second target gene(s) of the genetically engineered bacterium, optionally wherein the non-coding synthetic small RNA (sRNA) comprises a guide RNA that additionally binds an RNA-guided DNA endonuclease, an RNA-guided RNA endonuclease, or variant thereof and / or optionally wherein the second target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia; (k) p. a site-specific DNA endonuclease, wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) one or more specific DNA sequence(s) recognized by the site-specific DNA endonuclease, optionally wherein the site-specific DNA endonuclease comprises an RNA-guided DNA endonuclease, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or a restriction endonuclease, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (l) a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) comprising DNA encoding the PSRS in the protein coding region of the second target gene(s), wherein the target protein product comprising the one or more in-frame insertion(s) has activity, and wherein cleavage of the target protein product(s) by the protease deactivates the target protein product(s), optionally wherein the location of the one or more in-frame insertion is given by Table 6, optionally wherein the second target gene(s) is / are under the controlAtty. Dkt. No. P14610WO00 of a constitutive promoter, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (m) a first protein comprising a ClpAP ATP-dependent protease and a second protein comprising a ClpS Leu / N-recognin, wherein any one or more second target gene(s) of the genetically engineered bacterium encodes a third protein comprising an N-terminal -Leu, -Phe, - Trp, or -Tyr residue, and optionally wherein the first and second protein are operably linked to distinct control elements, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (n) a protease which cleaves a protease specific recognition sequence (PSRS), wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion(s) of DNA encoding the PSRS at the N-terminus of the protein coding region of the gene(s) followed by a -Leu, -Phe, -Trp, or -Tyr residue and wherein cleavage of PSRS from the N- terminus of the protein(s) encoded by the gene(s) by the protease results in a protein comprising an N-terminal -Leu, -Phe, -Trp, or -Tyr residue which is degraded by native ClpS and ClpAP, optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (o) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and activates expression of the protease, wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) of DNA encoding a PSRS, optionally wherein the transcriptional activator protein comprises a tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, optionally wherein the transcriptional activator domain is VP16 , and / or optionally wherein the second target gene(s) is / are: glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (p) a repressor protein, wherein the repressor protein binds a natural or synthetic DNA motif in a promoter of a heterologous protease gene of the genetically engineered bacterium and inhibits expression of the protease, wherein any one or more second target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion of DNA encoding an N-terminal amino acid tag designed to render the protein product(s) of the second target gene(s) inactive, followed by a PSRS, optionally wherein the repressor protein comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA bindingAtty. Dkt. No. P14610WO00 protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, and / or optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (q) a nanobody, wherein the nanobody binds a protein product of any one or more second target gene(s) of the genetically engineered bacterium, wherein binding of the nanobody to such protein product inhibits function of the protein product, optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; (r) an aptamer, wherein the aptamer binds a protein product of any one or more second target gene(s) of the genetically engineered bacterium and inhibits function of any one or more of the protein product(s) of the second target gene(s), optionally wherein the second target gene(s) is / are glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT and the fertilizer is ammonia; or (s) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of any one or more second target gene(s) and increases expression of any one or more of the second target gene(s), optionally wherein the transcriptional activator protein comprises the tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, wherein the DNA targeting protein is optionally a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof, wherein the transcriptional activator domain is optionally VP16 and / or optionally wherein the second target gene(s) is / are any one or more of: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the fertilizer is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, and / or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase and the fertilizer or plant nutrient is phosphate.

114. The method of claim 107, wherein the genetically engineered bacterium comprises an odd number of repressors and the second target gene is amtB, draT, glnA, glnB, glnK, glnZ, and / or nifL, and the fertilizer is ammonia.Atty. Dkt. No. P14610WO00 115. The method of claim 99, wherein the protein of interest is a first transcriptional activator protein and wherein the genetically engineered bacterium further comprises: (i) a second control element comprising a promoter which is activated by the first transcriptional activator protein and operably linked to a gene encoding a second transcriptional activator protein; (ii) optionally a third control element comprising a promoter which is activated by the second transcriptional activator protein and operably linked to a gene encoding a third transcriptional activator protein; (iii) optionally a fourth control element comprising a promoter which is activated by the third transcriptional activator protein and operably linked to a gene encoding a fourth transcriptional activator protein; and (iv) a control element comprising a promoter which is activated by the second, third, or fourth transcriptional activator protein and which is operably linked to a first target gene, optionally wherein the first, second, third, and / or fourth transcriptional activator protein comprise(s) the tet responsive element-binding tTA transcription factor or an activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator- like effector (TALE), and / or any variant thereof, optionally wherein the activator domain is VP16.

116. The method of claim 115, wherein the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and / or one or more fix cluster gene(s) and the fertilizer is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof, or a gene encoding a protein which stimulates organic acid release from the bacterium, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase, and the fertilizer or plant nutrient is phosphate.

117. The method of claim 99, wherein the protein of interest is a repressor protein and wherein the genetically engineered bacterium further comprises:Atty. Dkt. No. P14610WO00 (i) a second control element comprising a promoter which is repressed by the repressor protein and operably linked to a gene encoding a transcriptional activator protein; and (ii) a third control element comprising a promoter which is activated by the transcriptional activator protein and operably linked to a target gene.

118. The method of claim 117, wherein the target gene is a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene.

119. The method of claim 99, wherein the protein of interest is a transcriptional activator protein and wherein the genetically engineered bacterium further comprises: (i) a control element comprising a promoter which is activated by the transcriptional activator protein and operably linked to a gene encoding a repressor protein; and (ii) a control element comprising a promoter which is repressed by the repressor and operably linked to a target gene.

120. The method of claim 119, wherein the target gene is a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the fertilizer is ammonia.

121. The method of claim 100, wherein the glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene in (i), (j), (k), or (l) is under the control of a heterologous constitutive promoter.

122. The method of claim 113, wherein the glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene in (l), (m), (n), or (o) is under the control of a heterologous constitutive promoter.

123. The method of claim 99, wherein the plant nutrient is phosphate and the protein of interest comprises: (a) a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase; (b) an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof;Atty. Dkt. No. P14610WO00 (c) a protein which stimulates organic acid release from the bacterium, optionally wherein the protein comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), a pyrroloquinoline (PQQ) synthase including pqqFABCDEG, or any combination of GAD, GDH, and PQQ; or, (d) any combination of proteins of a, b, or c.

124. The method of claim 91, wherein the threshold population density of the genetically engineered bacterium which activates expression of the RNA or protein of interest is: (i) as set forth in Table 9 or Table 10 for the genetically engineered bacteria listed therein; (ii) about 6 x 103to 2 x 104CFU / mL, wherein the GEB is grown in liquid media, is a gram negative bacterium, an alphaproteobacterium, an Azospirillum sp., or related bacterium, the QSSP comprises an AhlI protein, the QSRP comprises an AhlR protein, and the quorum sensing promoter comprises an ahlI promoter; (iii) about 7 x 104to 3 x 107CFU / mL, wherein the GEB is grown in liquid media, is a gram negative bacterium, a gammaproteobacterium, is a Pseudomonas sp., or is a related bacterium, the QSSP comprises an AhlI protein, the QSRP comprises an AhlR protein, and the quorum sensing promoter comprises an ahlI promoter; (iv) about 4 x 105to 3 x 107CFU / mL, wherein the GEB is grown in liquid media, is a gram negative bacterium, a gammaproteobacterium, is a Kosakonia sp., is a Pseudomonas sp., or is a related bacterium, the QSSP comprises an CinI protein, the QSRP comprises an CinR protein, and the quorum sensing promoter comprises an cinI promoter; (v) about 1 x 105to 3 x 107CFU / mL, wherein the GEB is grown in liquid media, is a gram negative bacterium, a gammaproteobacterium, is a Pseudomonas sp., or is a related bacterium, the QSSP comprises a CinI protein, the QSRP comprises a CinR protein, and the quorum sensing promoter comprises a cinI promoter; (vi) about 5 x 103to 7 x 106CFU / gram soil, wherein the GEB is grown in plant growth media, is a gram negative bacterium, an alphaproteobacterium, an Azospirillum sp., or related bacterium, the QSSP comprises an AhlI protein, the QSRP comprises an AhlR protein, and the quorum sensing promoter comprises an ahlI promoter; or (vii) about 5 x 105to 5 x 108CFU / gram soil, wherein the GEB is grown in plant growth media, is a gram negative bacterium, a gammaproteobacterium, is a Kosakonia sp., or is a related bacterium, the QSSP comprises a CinI protein, the QSRP comprises a CinR protein, and the quorum sensing promoter comprises a cinI promoter.Atty. Dkt. No. P14610WO00 125. The method of claim 91, further comprising; a. determining leaf nitrogen and / or chlorophyll concentrations in a plant grown in the plant growth medium; and b. placing or re-applying the genetically engineered bacterium into the plant growth medium and / or in contact with the plant when the leaf nitrogen and / or chlorophyll concentrations in the plant are sub-optimal for yield.

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