Irreversible condition sensitive microbial gene switches

Abstract

Genetically engineered bacteria which express RNAs or proteins that produce or represent agriculturally useful compounds including fertilizers and plant nutrients upon changes in soil conditions are disclosed.

Description

IRREVERSIBLE CONDITION SENSITIVE MICROBIAL GENE SWITCHESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This international patent application claims benefit of U.S. Provisional PatentApplication Serial Nos.: 63 / 553,528, filed February 14, 2024; 63 / 552,631, filed February 12, 2024; and 63 / 627,673, filed January 31, 2024, which arc each incorporated herein by reference in its entireties.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] Not applicable.INCORPORATION OF SEQUENCE LISTING

[0003] A sequence listing contained in the xml file named ‘P14613WO02.xml” which is 785,948 bytes in size (measured in MS-Windows), which was created on January 31, 2025, and which comprises 622 sequences, is electronically filed herewith and is incorporated herein by reference in its entirety. Sequence listings of the aforementioned related applications to which benefit is claimed and contained in the xml files named: (i) “P14613US00.xml” which is 658,989 bytes in size (measured in MS-Windows), which was created on January 31, 2024, and which comprises 512 sequences; (ii) “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; and (iii) “P14610US00.xml” which is 562.891 bytes in size (measured in MS-Windows), which was created on February712, 2024, and which comprises 389 sequences; are also each incorporated herein by reference in their entireties.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 arc not compatible with environmental requirements and laws, arc 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.SUMMARY

[0005] Methods of providing at least one agriculturally relevant compound to a plant comprising placing at least one genetically engineered bacterium into a plant growth medium, wherein the genetically engineered bacterium is an agriculturally useful bacterium comprising: (i) a first heterologous gene expression cassette comprising at least one DNA molecule encoding a protein or RNA sequence of interest, wherein the DNA molecule is operably linked to at least one DNA molecule comprising a site-specific recombinase recognition sequence (SSRRS) and wherein the first heterologous gene expression cassette lacks an irreversible insertion, excision, or inversion of a DNA molecule in the first heterologous gene expression cassette; and (ii) a second heterologous gene expression cassette comprising at least one DNA molecule encoding at least one recombinase that can catalyze site-specific recombination at said SSRRS, wherein the DNA molecule encoding said recombinase is operably linked to a promoter activated by a change of at least one condition in the plant growth medium, and wherein the site-specific recombination induces an irreversible insertion, excision, or inversion of: (a) a DNA molecule comprising a control element in, from, at. or near the DNA molecule encoding the protein or RNA sequence of interest in the first heterologous gene expression cassette; or (b) a DNA molecule encoding the protein or RNA sequence of interest in the first heterologous gene expression cassette are provided.

[0006] Methods of providing at least one agriculturally relevant compound to a plant comprising placing at least one genetically engineered bacterium into a plant growth medium, wherein the genetically engineered bacterium is an agriculturally useful bacterium comprising: (i) a first part of a first heterologous gene expression cassette comprising a DNA molecule encoding a control element, protein or RNA sequence of interest, or a part thereof, wherein the first part of the first heterologous gene expression cassette has reduced or no function and wherein the DNA molecule is operably linked to at least one sitespecific recombinase recognition sequence (SSRRS); and a second part of the first heterologous gene expression cassette comprising the control element, the protein or RNA sequence of interest, or part thereof absent from the first part of the first heterologous gene expression cassette; and (ii) a second heterologous gene expression cassette comprising at least one nucleic acid sequence encoding at least one recombinase that can catalyze site-specific recombination at said SSRRS, wherein the DNA molecule encoding said recombinase is operably linked to a promoter activated by a change of at least one condition in the plant growth medium, and wherein the site-specific recombination induces an irreversible insertion, excision, or inversion of DNA which results in the operable linkage of the first and second part of the first heterologous gene expression cassette to provide a functional first heterologous gene expression cassette are provided.

[0007] Genetically engineered bacterium comprising: (i) a first heterologous gene expression cassette comprising at least one DNA molecule encoding a protein or RNA sequence of interest, wherein the DNA molecule is operably linked to at least one DNA molecule comprising a site-specific recombinase recognition sequence (SSRRS) and wherein the first heterologous gene expression cassette lacks an irreversible insertion, excision, or inversion of a DNA molecule in the first heterologous gene expression cassette; and (ii) a second heterologous gene expression cassette comprising at least one DNA moleculeencoding at least one recombinase that can catalyze site-specific recombination at said SSRRS, wherein the DNA molecule encoding said recombinase is operably linked to a promoter activated by a change of at least one condition in the plant growth medium, and wherein the site-specific recombination can induce an irreversible insertion, excision, or inversion of: (a) a DNA molecule comprising a control element in, from, at, or near the DNA molecule encoding the protein or RNA sequence of interest in the first heterologous gene expression cassette; or (b) a DNA molecule encoding the protein or RNA sequence of interest in the first heterologous gene expression cassette; wherein the genetically engineered bacterium is an agriculturally useful bacterium are provided.

[0008] Genetically engineered bacterium comprising: (i) a first part of a first heterologous gene expression cassette comprising a DNA molecule encoding a control element, protein or RNA sequence of interest, or a part thereof, wherein the first part of the heterologous gene expression cassette has reduced or no function and wherein the DNA molecule is operably linked to at least one site-specific recombinase recognition sequence (SSRRS); and a second part of the first heterologous gene expression cassette comprising the control element, the protein or RNA of interest, or part of absent from the first part of the first heterologous gene expression cassette; and (ii) a second heterologous gene expression cassette comprising at least one DNA molecule encoding at least one recombinase that can catalyze site-specific recombination at said SSRRS, wherein the DNA molecule encoding said recombinase is operably linked to a promoter activated by a change of at least one condition in the plant growth medium, and wherein the site-specific recombination induces an irreversible insertion, excision, or inversion of DNA which results in the operable linkage of the first and second part of the first heterologous gene expression cassette to provide a functional first heterologous gene expression cassette, wherein the genetically engineered bacterium is an agriculturally useful bacterium are provided.

[0009] Compositions comprising the genetically engineered bacterium as well as agricultural systems, treated plants, plant parts, and plant propagules obtained with the compositions and genetically engineered bacteria are also provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 shows three modes of recombinase mediated DNA recombination. Recombinases such as PhiC, Bxbl. or CreR catalyze recombination between imperfect repeat attachment (all) sites (site-specific recombinase recognition sites (SSRRS); triangles) and flanking DNA regions (associated lines) that can exist in four different configurations all / ,. attR, attB, and attP. When only the recombinase is expressed, the directionality of recombination strongly favors attB / attP attL / attR. The three modes of recombination depicted are integration, inversion, and excision.

[0011] Figure 2 shows four modes (labeled 1, 2, 3, and 4) of activating or increasing expression of a gene of interest (“geneA”) encoding a protein or RNA of interest with a recombinase and SSRRS. Promoters and the direction of transcription directed by the promoters are shown with arrows. Transcriptional terminators are shown with the “T” shaped icon.

[0012] Figure 3 shows five modes (labeled 5, 6, 7, 8, and 9) of inactivating or decreasing expression of a gene of interest (“geneA”) encoding a protein or RNA of interest by a recombinase and imperfect repeat attachment (att) sites (SSRRS; triangles). Promoters and the direction of transcription directed by the promoters arc show with arrows. Transcriptional terminators arc shown with the “T” shaped icon.

[0013] Figure 4 shows five additional modes (labeled 10, 11, 12, 13, and 14) of inactivating or decreasing expression of a gene of interest (“geneA") encoding a protein or RNA of interest by a recombinase and imperfect repeat attachment (a / Z) sites (SSRRS; triangles). In mode 14, the site-specific recombination induces insertion of the blocking DNA (e g., labeled “junk”) into the DNA molecule to reduce expression and / or activity of the protein or RNA sequence of interest.

[0014] Figure 5 shows a mode for inactivation of a first gene encoding a first protein or RNA (“geneA”) and activation of a second gene (“geneB”) encoding a second protein or RNA with a recombinase and imperfect repeat attachment (att) sites (SSRRS; triangles). Embodiments where the first gene is inactivated and a second gene is activated to provide for ammonia production are listed in the figure.|0015| Figure 6 shows a first promoter and a second promoter directed opposite one another in a control element flanked by two SSRRS in an inverted configuration, where the first promoter of the control element is operably linked to the DNA molecule encoding at least one protein of interest (e.g., GlnA) prior to inversion by the site-specific recombinase. In the lower panel, the transcription promoting-activity of the second promoter is at least 14%, 28%, 42%, or 53% less than the transcription promoting-activity of the first promoter and is operably linked to the DNA molecule encoding the at least one protein of interest upon inversion by the site-specific recombinase. In the upper panel, the transcription promoting-activity of the second promoter is at least 2-, 5-, or 10-fold greater than the transcription promoting-activity of the first promoter and is operably linked to the DNA molecule encoding the at least one protein of interest upon inversion by the site-specific recombinase.

[0016] Figure 7 shows a genetic reporter circuit for measuring expression of a green fluorescent protein reporter under control of the phosphate-sensitive pstS and Pliar53 promoters when there is a decrease in phosphate concentration in a bacterial growth medium).

[0017] Figure 8 shows low-Pi dependent induction of expression (defined as GFP / constitutive LSS mScarlett fluorescence) from the pstS reporters in model bacterial strains incubated for 15 h in minimal media with or without supplementation of 500 pM potassium phosphate. Error bars represent one standard deviation from mean calculated from 3 biological replicates.

[0018] Figure 9 shows low-Pi dependent induction of expression (defined as GFP / constitutive LSS mScarlett fluorescence) from the Pliar53 reporters in model bacterial strains incubated for 15 h in minimal media with or without supplementation of 500 pM potassium phosphate. Error bars represent one standard deviation from mean calculated from 3 biological replicates.

[0019] Figure 10 A. B, C. D show that decreasing phosphate concentration led to expression of a GFP reporter protein of interest operably linked to control elements comprising the phosphate-sensitive Pliar53 promoter and promoters of the indicated pstS gene. Model soil bacterial strains are shown. The solid blackline data series shows phosphate concentration over time while the dotted line data series shows per-cell GFP fluorescence over time. Error bars represent one standard deviation from mean calculated from 3 biological replicates.

[0020] Figure 11 shows phosphate switch activation in the rhizosphere of corn inoculated with Azospirilum brasilense (pstS promoter controlling GFP expression) in a sterile system containing sand (upper panel) and phosphate concentration in sand in the rhizosphere of com plants inoculated with Azospirillum brasilense (strain containing pstS promoter controlling GFP expression) in sterile conditions (lower panel

[0021] Figure 12 shows low P concentration activated GFP expression in Kosakonia sacchari (Ks) and Klebsiella variicola (Kv) soil isolates transformed with a PpstS-GFP construct under field conditions at various locations in North America.

[0022] Figure 13 shows low-Pi inducible expression of GFP by PstS promoters in gamma-proteobacterial soil isolates (Root data). Rahnella aceris, Klebsiella variicola, and Kosakonia sacchari soil isolates were tested with either native PstS or heterologous PstS promoters fused to GFP.

[0023] Figure 14 shows low-Pi inducible expression of GFP by two pstS promoters in Paenibacillus graminis. In the figure for each tested construct (Ppstl GFP, Ppst2.1 GFP, Ppst2.2 GFP) or negative control (WT SM532), GFP expression normalized to the OD600 was determined from left to right as follows: 24 hours at 50 pM Pi, 24 hours at 0 pM Pi, 48 hours at 50 pM Pi, and 48 hours at 0 pM Pi.

[0024] Figure 15 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.

[0025] Fi gurc 16 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 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.

[0026] Figure 17 shows the experimental logic for assessment of population density-dependent induction by QS promoters.

[0027] Figure 18 shows population density-dependent induction by strains comprising tw o 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).

[0028] Figure 19 show s 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.

[0029] Figure 20 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.

[0030] Figure 21 shows bacterial cell counts for bacteria isolated from bulk soil (BS) or from the com- 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 (Ahll QSSP, AhlR QSRP, and GFP operably linked to Pahll).

[0031] Figure 22 shows relative expression (defined as inducible GFP fluorescence I constitutive LSSmScarlett fluorescence) for bacteria isolated from bulk soil (BS) or from the com-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 (Ahll QSSP, AhlR QSRP, and GFP operably linked to Pahll).

[0032] Figure 23 shows a system of gene expression cassettes for a quorum sensing system that can be deactivated during fermentation using quorum quenching. The GFP gene is a reporter used to show quorum sensing induction in the absence of a quorum quenching compound (e.g., aTc) and quorum sensing inhibition in the presence of the quomm quenching compound. In other embodiments, GFP is replaced with a recombinase which is repressed during fermentation with the quomm quenching compound.

[0033] Figure 24 shows engineered K. radicincitans soil bacteria that exhibit population densitydependent 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.

[0034] Figure 25 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 quomm 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 BCD 17 and BCD22).

[0035] Figure 26 shows the effect of operably linked ribosome binding site on QSRP -mediated expression of a reporter protein of interest.

[0036] Figure 27 shows a schematic of constmcts 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 BCD 17 and BCD22). In the third gene expression cassette, the QSSP-expressing gene (Synthase) is operably linked to a quomm sensing promoter (PQS) under control of ribosome binding sties of differing strength (hereBCD1 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.

[0037] Figure 28A, B, C, and D show cell density (ODeoo) and cell density-normalized fluorescence over time in Kosakonia radicincitans grown in liquid culture and engineered with a repressible quorum sensing circuit. In Fig. 14A, 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. Figure 14B shows cell density (ODeoo) 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 BCD 17 (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. Figure 14C shows cell density (ODeoo) 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. Figure 14D shows cell density (ODeoo) 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.

[0038] Figure 29 shows bacterial cell counts for bacteria isolated from bulk soil (BS) or from the com- root rhizoplane (RP), as determined by flow cytometry. Engineered bacterial strains of Kosakonia radicincitans (Kr) comprising an ahlRI QS sy stem (Ahll QSSP, AhlR QSRP, and GFP operably linked to Pahll) that can be deactivated by a quorum quenching compound-induced transcriptional dcactivator were tested.

[0039] Figure 30 shows relative expression (defined as inducible GFP fluorescence / constitutive LSSmScarlett fluorescence) for bacteria isolated from bulk soil (BS) or from the com-root rhizoplane (RP), as determined by flow cytometry . Engineered bacterial strains of Kosakonia radicincitans (Kr) comprising an ahlRI QS system (Ahll QSSP, AhlR QSRP, and GFP operably linked to Pahll) that can be deactivated by a quorum quenching compound-induced transcriptional deactivator were tested.

[0040] Figure 31 shows metabolism and regulation of bacterial nitrogen fixation.

[0041] Figure 32 shows the logic of a reporter gene expression cassette comprising a green fluorescent protein (GFP) protein of interest that can be expressed upon recombinase-mediated inversion of a controlelement comprising the constitutive promoter J23104. When an integrase catalyzes inversion of the control element, the J23104 promoter becomes operably linked to the GFP gene of interest, permitting its expression and the fluorescence of cells that comprise the reporter gene expression cassette. As shown in the bottom schematic, a recombinase can be expressed constitutively to test this reporter construct.

[0042] Figure 33 shows functionality of the reporter construct of Figure 32 across numerous taxa of agriculturally useful bacteria. Only the reporter gene expression cassette was introduced into the bacteria, no fluorescence was observed. When a recombinase was also expressed, fluorescence was observed.

[0043] Figure 34 shows confirmation by polymerase chain reaction that fluroescence of the reporter gene expression cassette of Figure 32 reflects inversion of the DNA sequence of the control element.

[0044] Figure 35 shows the sequences of PCR primers used to conduct the method validation assay of Figure 34. Pl is SEQ ID NO: 620, P2 is SEQ ID NO: 621, and P3 is SEQ ID NO: 622.

[0045] Figure 36 shows a schematic of chromosomal integration of the recombinase reporter construct of Figure 32 into a fitness-neutral genomic locus. Transcriptional terminators (shown with the “T” shaped icon) flank the recombinase reporter construct in order to minimize interaction with the surrounding DNA.

[0046] Figure 37 shows a schematic of a library of chromosomally integrated gene expression cassettes designed to induce recombinase expression upon the decrease of phosphate concentration. Library combinations of ribosome binding site and start codon operably linked to a gene encoding the PhiC31 recombinase are listed below those elements in the schematic. Right angle arrows represent promoters, half circle represent ribosome binding sites, “T” icons represent transcriptional terminators, and large bolded straight arrows represent protein-coding regions.

[0047] Figure 38 shows the effect of ribosome binding site and start codon operably linked to a recombinase on phosphate-inducible inversion of a GFP reporter gene of interest in bacterial culture, measured by flow cytometry.

[0048] Figure 39 shows the effect of ribosome binding site and start codon operably linked to a recombinase on phosphate-inducible inversion of a GFP reporter gene of interest on com roots, measured by flow cytometry.

[0049] Figure 40 shows the effect of ribosome binding site and start codon operably linked to a recombinase on phosphatc-induciblc inversion of a GFP reporter gene of interest on com roots, measured by flow cytometry .

[0050] Figure 41 shows a schematic of a library of chromosomally integrated gene expression cassettes designed to induce recombinase expression upon the decrease of nitrogen concentration. Library' combinations of ribosome binding site and start codon operably linked to a gene encoding the PhiC31 recombinase are listed below those elements in the schematic. Right angle arrows represent promoters, half circle represent ribosome binding sites. “T” icons represent transcriptional terminators, and large bolded straight arrows represent protein-coding regions.

[0051] Figure 42 shows the effect of ribosome binding site and start codon operably linked to a recombinase on nitrogen-inducible nitrogenase enzyme activity in Kosakonia sacchari bacterial culture, measured by gas chromatography.

[0052] Figure 43 shows the effect of ribosome binding site and start codon operably linked to a recombinase on nitrogen-inducible inversion of a GFP reporter gene of interest in bacterial culture, measured by flow cytometry.

[0053] Figure 44 shows a schematic of a library of chromosomally integrated gene expression cassettes designed to induce recombinase expression through quorum sensing upon the increase in bacterial population density. Library combinations of ribosome binding site and start codon operably linked to a gene encoding the PhiC31 recombinase are listed below those elements in the schematic. Right angle arrows represent promoters, half circle represent ribosome binding sites, “T” icons represent transcriptional terminators, and large bolded straight arrows represent protein-coding regions.

[0054] Figure 45 shows flow cytometry results demonstrating aTc quorum quenching compound- mediated deactivation of quorum sensing-mediated inversion of a gene of interest by a recombinase in an engineered strain of the agriculturally useful bacterium Enterobacter chinensis.

[0055] Figure 46 shows flow cytometry results demonstrating aTc quorum quenching compound- mediated deactivation of quorum sensing-mediated inversion of a gene of interest by a recombinase in an engineered strain of the agriculturally useful bacterium Klebsiella variicola.

[0056] Figure 47 shows flow cytometry results demonstrating aTc quorum quenching compound- mediated deactivation of quorum sensing-mediated inversion of a gene of interest by a recombinase in an engineered strain of the agriculturally useful bacterium Kosakonia sacchari.

[0057] Figure 48 A, B shows flow cytometry results demonstrating (A) aTc quorum quenching compound-mediated deactivation of quorum sensing-mediated inversion of a gene of interest by a recombinase in an engineered strain of the agriculturally useful bacterium Rahnella aceris and (B) durable quorum sensing-mediated inversion of a GFP gene of interest by a recombinase in an engineered strain of the agriculturally useful bacterium Kosakonia sacchari on maize roots.

[0058] Figure 49 shows flow cytometry results demonstrating durable quorum sensing-mediated inversion of a GFP gene of interest by a recombinase in an engineered strain of the agriculturally useful bacterium Rahnella aceris on maize roots.

[0059] Figure 50 shows a schematic of a library of chromosomally integrated gene expression cassettes designed to determine the optimal promoter strength for glnA expression that results in ammonia release from agriculturally useful bacteria. Right angle arrows represent promoters, half circle represent ribosome binding sites, “T” icons represent transcriptional terminators, and large bolded straight arrows represent protein-coding regions.

[0060] Figure 51 shows growth curves of engineered Kosakonia sacchari bacteria, each comprising one of the chromosomally integrated gene expression cassettes of the library depicted in Figure 50.

[0061] Figure 52 shows growth cur es of engineered Klebsiella variicola bacteria, each comprising one of the chromosomally integrated gene expression cassettes of the library depicted in Figure 50.

[0062] Figure 53 shows growth curves of engineered Rahnella aceris bacteria, each comprising one of the chromosomally integrated gene expression cassettes of the library' depicted in Figure 50.

[0063] Figure 54 shows ammonia release from engineered Kosakonia sacchari bacteria, each comprising one of the chromosomally integrated gene expression cassettes of the library' depicted in Figure 50.

[0064] Figure 55 shows ammonia release from engineered Klebsiella variicola bacteria, each comprising one of the chromosomally integrated gene expression cassettes of the library depicted in Figure 50.

[0065] Figure 56 shows ammonia release from engineered Rahnella aceris bacteria, each comprising one of the chromosomally integrated gene expression cassettes of the library depicted in Figure 50.

[0066] Figure 57 shows a schematic of a library of chromosomally integrated gene expression cassettes designed to optimize phosphate-inducible ammonia release from agriculturally useful bacteria via inversion of a control element by a recombinase to reduce glnA expression. Right angle arrows represent promoters, half circle represent ribosome binding sites. "T” icons represent transcriptional terminators, and large bolded straight arrows represent protein-coding regions.

[0067] Figure 58 shows ammonia release from engineered Kosakonia sacchari bacteria, each comprising one of the chromosomally integrated gene expression cassettes of the library depicted in Figure 57.

[0068] Figure 59 shows ammonia release from engineered Rahnella aceris bacteria, each comprising one of the chromosomally integrated gene expression cassettes of the library depicted in Figure 57.DETAILED DESCRIPTION

[0069] It has been found that direct activation or inactivation of target gene expression by systems based on plant growth media (e.g., soil)-based conditions including bacterial density (e.g., through quorum sensing systems), soil nitrogen (N) concentrations, or soil phosphate (P) concentrations can result in an unstable, fluctuating expression of the target gene as the condition (e.g., density', N concentration, P concentration) can vary' over time. It is therefore desirable to establish systems which irreversibly activate or inactivate expression of the target gene once the condition reaches a certain threshold. Disclosed herein arc sy stems comprising at least two steps where the plant growth media (e.g., soil)-bascd condition results in expression of a recombinase which irreversibly activates or inactivates expression of a target gene. At least one benefit of tire systems described herein is that they can uncouple a desired effect of target gene activation or inactivation (e.g., production of an agriculturally useful compound including ammonia or phosphorus) from the variability of the plant growth media (e.g., soil)-based condition.

[0070] Methods, genetically engineered bacteria (GEB), and related systems which use conditionsensitive promoters ( CS-P) which are expressed under certain plant growth media (e.g, soil) conditions to drive expression of a recombinase (e.g., a site-specific recombinase (SSR) or integrase (INT)) which induces an irreversible insertion, excision, or inversion of a DNA molecule comprising a control element or DNA encoding a gene of interest to activate or inactivate a target gene of interest are disclosed herein.Conditions which can activate condition-sensitive promoters include : (i) plant nutrient concentrations (e.g. , phosphate, potassium, carbon, nitrate, and ammonia concentrations); (ii) bacterial density (e.g., quorum sensing promoters); (iii) gaseous element concentrations (e.g., oxygen, oxygen / nitrogen); (iv) phytohormonc or plant signaling molecule concentrations (e.g., auxins including IAA and PAA, jasmonatc, salicy lic acid); (v) amino acids (e.g., tryptophan, phenylalanine, proline, lysine, methionine, glutamate);(vi) sugars (e.g., trehalose, maltose, mannitol, sucrose, glucose, fructose, arabinose, xylose, galactose);(vii) trace minerals (e.g., copper, zinc, iron, magnesium); (viii) plant or microbial metabolites (e.g., naringenin, quercetin, luteolin, apigenin, octopine, nopaline, Scyllo-inosamine); and (ix) other conditions including oxygen levels, light, temperature. pH, osmotic stress, and peroxide concentrations. Conditions and associated CS-P contemplated for use in the methods, GEB, and related systems include those set forth in TABLE 7

[0071] In certain, embodiments the CS-P promoters used in the systems disclosed herein are phosphatesensitive promoters (PS-P). In plant growth media including soil, phosphate is consumed over time by plants and microorganisms. Once phosphate concentrations fall below a certain threshold level (e.g. about 50 pM, 40 pM, 30 pM, 20 pM. or 10 pM to about 1 pM), the desired production of proteins, RNAs. and / or agriculturally relevant compounds by the genetically engineered bacteria occurs. As the consumption of phosphate by the plants and microorganisms takes time, a desired delay in production of proteins, RNAs. and / or agriculturally relevant compounds by the genetically engineered bacteria 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 phosphate is consumed 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 condition-sensitive (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 will not grow to the higher titers achieved by tire 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 in comparison to bacteria lacking the condition-sensitive promoter controlled genes.

[0072] Methods, genetically engineered bacteria (GEB), and related sy stems where the condition-sensing promoter (CS-P) is a quorum sensing promoter (QS-P) are also provided. 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 a recombinase (e.g., a site-specific recombinase (SSR) or integrase (INT)) which induces an irreversible insertion, excision, or inversion of a DNA molecule comprising a control element to activate or inactivate a target gene of interest 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 recombinase following their placement in plant growth media including soil, permitting more rapid and / or more extensive (e.g.. higher density) growth inthe plant growth media and / or colonization of crop plants grown therein. The desired production of the 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) which induces expression of the recombinase which irreversibly activates or inactivates the target gene, resulting in production of the agriculturally relevant compound. 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 recombinase which irreversibly activates or inactivates the target gene to cause 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 recombinase. In certain embodiments. QS-P regulated genes encoding the recombinases (which activate or inactivate the target 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.

[0073] Another aspect of the methods and GEB provided herein are QS-P based genetic systems which can selectively inhibit expression of the recombinase which activates or inactivates the target genes when the GEB are grown to densities above the threshold population density in fermenters or bioreactors. Such selective inhibition of expression of the recombinase can permit more rapid and economical 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 recombinase which activates or inactivates the target gene when the GEB are in plant growth media and / or associated with a plant and for selective inhibition of undcsircd expression of the recombinase when the GEB arc in a fermenter or biorcactor.

[0074] Condition-sensitive 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 condition-sensing gene expression systems provided herein allow a desirable delay in ammonia production. In certain embodiments, the condition-sensing promoter controlled gene expression system is a phosphate-sensitive promoter system that provides the desirable delay as phosphate is only slowly consumed by the plant and bacteria. In certain embodiments, the condition-sensingpromoter controlled gene expression system is a Quorum sensing promoter (QS-P)-controlled gene expression that provides the desirable delay as the engineered bacteria grow first to a high density in soil before the ammonia release mechanism is activated.Definitions

[0075] As used herein, tire 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.

[0076] 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).

[0077] 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.

[0078] 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).

[0079] As used herein, the phrase “blocking DNA” refers to DNA that either (a) reduces or prevents transcription or translation of DNA encoding a protein or RNA when inserted into the DNA or (b) encodes an additional amino acid sequence within the protein-coding region of a gene of interest that has the effect of diminishing the biological activity of the protein produced by the gene of interest. Examples of blocking DNA include DNA with translational stop codons in one, two, or three reading frames on one or both DNA strands. Examples of blocking DNA also include DNA with one or more transcription terminators on one or both strands.

[0080] 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.

[0081] As used herein, tire 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 contain transcriptional repressor binding sites, transcriptional activator binding sites, ribozymes, protein recognition sites, and / or sites for chemical modification of nucleobases.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] As used herein, the phrase “irreversible insertion, excision, or inversion” refers to an insertion, excision, or inversion of a DNA molecule catalyzed by a recombinase acting at one or more site-specific recombinase recognition sequences (SSRRS) which cannot be reversed (i.e., by excision, insertion, or inversion, respectively) by the recombinase without introducing additional co-enzymes, co-factors, or chemically modifying the recombinase.

[0086] 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.

[0087] As used herein, tire 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, acted upon, or restored 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. In another instance, a nucleic acid comprising a site-specific recombinase recognition sequence (SSRRS) or sequences is / are operably linked to DNA comprising a control element (e.g., a promoter or tenninator) or a coding region (e.g., DNA encoding an RNA or mRNA) when the SSRRS sequence(s) can provide for an insertion, excision, or inversion of, or at. the DNA comprising thecontrol element or the coding region that is catalyzed by a recombinase which recognizes the SSRRS sequence(s). In another instance, a first non-functional part of a nucleic acid encoding a protein and a second non-functional part of the nucleic acid encoding a protein can be operably linked to one another to restore their respective functions.

[0088] 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.

[0089] 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 pM. 40 pM. 30 pM, 20 pM, or 10 pM to about 1 pM. In certain embodiments, a phosphate-sensitive promoter will be active at a concentration of phosphate from 0 pM to about 1 pM. 2 pM, 5 pM, 7 pM, or 10 pM.

[0090] As used herein, the phrase “quorum sensing promoter” (“QS-P”) 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.

[0091] 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 tire 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 .

[0092] As used herein, the phrase “ribosome binding site” and corresponding acronym “RBS” refers to RNA comprising a Shine-Dalgamo sequence. In certain embodiments, a ribosome binding site can comprise a Shine-Dalgamo sequence and the translational initiation codon.

[0093] 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 morenucleotides 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.

[0094] As used herein, the phrase “transcriptional activator’’ refers to proteins or ribonuclcoprotcin (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 further comprises a transcriptional activator domain and a guide RNA which targets the RNP complex to the promoter.

[0095] 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 Aligmnent Search Tool) available on the internet at blast.ncbi.nlm.nih.gov / Blast.cgi with default settings.

[0096] 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.

[0097] 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 glnA. glnB. glnK, glnZ, nifL. and / or draT genes. Target glnA, glnB, glnK, glnZ. nifL, and / or draT gene sequences which can be down-regulated include those set forth in TABLE 5, TABLE 6, in the sequence listing, and in US Patent Application No. US20210315212, which is incorporated herein by reference in its entirety. Target 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 TABLE 6, in the sequence listing, and in US Patent Application No. US20210315212. Target glnA, glnB, glnK, glnZ, nifL, and / or draT gene sequences which can be down-regulated also include sequences which encode 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 GlnA, GlnB, GlnK, GlnZ, NifL, or DraT proteins encoded by genes set forth in TABLE 1, in TABLE 5, in TABLE 6, in the sequence listing, and in US Patent Application No. US20210315212 or to GlnA, GlnB, GlnK, GlnZ, NifL, or DraT proteins in US Patent Application No. US20210315212.TABLE 1. Non-limiting summary of target genes for down-regulation and ammonia production.

[0098] In certain embodiments, one or more genes set forth in TABLE 1 can be down-regulated by one or more of the condition-sensitive 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 conditionsensitive 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 1-138.TABLE 2. Non-limiting summary of systems for down-regulating and / or up-regulating target genes in response to changes in plant growth media conditions.

[0099] In certain embodiments, one or more bacterial genes are up-regulated to cause the production of 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, TABLE 5 AND 6. in the sequence listing, and in US Patent Application No. US20210315212. Target nifA. ntrC. glnR, glutaminase encoding genes, GlnE-uAT genes, ^ / / '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 AND 6, in the sequence listing, and / or in US Patent Application No. US20210315212.

[0100] In certain embodiments, one or more bacterial genes arc 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 pyrrolo quinoline (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., tm cpA. aphA, phoC, napA. napD. or napEgene), GAD-, GDH-. or pyrrolo quinoline (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.TABLE 3. Non-limiting summary of target genes for up-regulation and agriculturally relevant compounds produced by up-regulation

[0103] In certain embodiments, one or more genes set forth in TABLE 3 can be up-regulated by one or more of the condition-sensitive systems or genes set forth in TABLE 2 and 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 1-138.TABLE 4. Non-limiting summary for gene up-regulation mechanisms triggered by condition changes

[0104] In certain embodiments, target genes encoding an RNA or protein of interest can be uprcgulatcd or down regulated by a recombinase (e.g., 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 condition-sensitive 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 CS-P. In certain embodiments, the first gene can be selected from genes targetedfor down-regulation in TABLE 1 and the second gene can be selected from a gene targeted for upregulation 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-ty pe or first gene, a mutated variant of that same first gene from TABLE 1 with reduced enzy matic 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.

[0105] A variety of recombinases can be used in conjunction with the site-specific recombinase recognition sites (SSRRS) which they recognize to catalyze the irreversible insertion, excision, and inversion of DNA molecules in the methods, genetically engineered bacteria, and systems provided herein. In certain embodiments including those illustrated in Fig. 1, the recombinase is a serine integrase and the SSRRS are attB and attP sites. Where irreversible integration is desired, the DNA molecule which is to be integrated by the recombinase (e.g., DNA comprising a promoter, terminator, or coding region) can be located in a circular DNA that further comprises an attP site and an attB site will be located in the target integration site (e.g., in the bacterial chromosome or in a bacterial plasmid; both as shown in Figure 1. item A). Once integrated, the inserted DNA molecule is flanked by attL and attR sites oriented as direct repeats (e.g., as illustrated in Fig. 1, item A). Where irreversible inversion is desired, the DNA molecule which is to be inverted by the recombinase (e.g., DNA comprising a promoter or coding region) can be flanked by attB and attP sites disposed in the DNA molecule lacking the inversion as inverted repeats (e g., as shown in Figure 1, item B). Once inverted, the inverted DNA molecule is flanked by attL and attR sites oriented as inverted repeats (e g., as illustrated in Fig. 1, item A). Where irreversible excision is desired, the DNA molecule of interest (e.g., DNA comprising a promoter, terminator, gene, or coding region) is flanked by attL and attR sites as directed repeats (e.g., as shown in Fig. 1C). Suitable integrases which can be used in such embodiments include 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 or attL or attR sites recognized respectively by the PhiC31, IntS, IntM, IntG, YdcL, or Sxt / R39 integrase. Other suitable recombinases and SSRRS sites which include: (1) Crc recombinase and their LoxP recognition sites; (2) FLP recombinases and their FRT recognition sites; (3) xerC / xerD recombinases of E. coli and their 28 bp dif site (Leslie and Sherratt (1995) EMBO J. 14: 1561); (4) Dre recombinase and their roxP recognition sites; (5) aforementioned and other recombinase and SSRRS pairs and variants thereof disclosed in US Patent No. 10.614,353 and WO 2023 / 220697, both incorporated herein by reference in their entireties; and (6) systems which employ a catalytically inactive RNA-directed DNA binding protein (e.g., dCas9, dCasl2a, and the like) which bind and block recombinase activity at the SSRRS (e.g., Huang et al., 2024, doi.org / 10.1038 / s41467-024-46755- 1) .

[0106] 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 cataly tic 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 CS-P promoter activation of the SSR or integrase. GEB comprising this promoter switch will express wildtype or catalytically improved GS prior to CS-P promoter-mediated activation of the SSR or integrase, promoting growth of the GEB in plant growth media. After CS-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, 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.

[0107] In one embodiment, the SSR- or integrase- controlled promoter switch is placed between a first gene comprising a glnE gene encoding a wild-ty pe 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 CS- P promoter activation of the SSR or integrase. GEB comprising this promoter switch will express wild-type GlnE protein prior to CS-P promoter-mediated activation of the SSR or integrase, promoting growth of the GEB in plant growth media. After CS-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.

[0108] 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 CS-P promoter activation of the SSR or integrase. GEB comprising this promoter switch will express wild-type NifL protein prior to CS-P promoter-mediated activation of the SSR or integrase, promoting growth of the GEB in plant growth media. After CS-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-rcgulation of nifA, and ammonia production.

[0109] 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 CS-P controlled cascade of repressors / repressor-controlled genes encoding repressors and / or a CS-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 CS- 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., the 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 CS-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.

[0110] 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 tire 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.

[0111] 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, Herbaspir ilium, 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, Kosakonia, Klebsiella, Paenibacillus, Phytobacter, Pseudomonas, Rahnella, 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 incorporated 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. In certain embodiments, the aforementioned GEB are agriculturally useful bacteria (AUB). In certain embodiments the AUB are diazotrophic bacteria (i.e., bacteria having a nitrogenase enzyme capable of reducing inert atmospheric di-nitrogen gas into ammonia under micro-aerobic conditions). In certain embodiments, the AUB are bacteria which can solubilize phosphate (i.e., phosphate-solubilizing bacteria). Phosphate solubilizing bacteria can include Alcaligenes spp., Aerobactor aerogenes, Achromobacter spp., Actinomadura oligospora, Agrobacterium spp.. Azospirillum brasilense, Bacillus spp., Bacillus circulans, Bacillus cereus,Bacillus fusiformis, Bacillus pumils, Bacillus megaterium, Bacillus mycoides, Bacillus polymyxa, Bacillus coagulans, Bacillus chitinolyticus, Bacillus subtilis,Bradyzhizobium spp., Brevibacterium spp., Citrobacter spp., Pseudomonas spp., Pseudomonas putida, Pseudomonas striata, Pseudomonas fluorescens, Pseudomonas calcis, Flavobacterium spp., Nitrosomonas spp., Erwinia spp., Mirococcus spp., Escherichia intermedia, Enterobacter asburiae, Serratia phosphoticum, Nitrobacter spp., Thiobacillus ferroxidans, Thiobacillus thiosidans, Rhizobium meliloti, and Xanthomonas spp.

[0112] A variety of control elements comprising CS-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 (AUB) in response to: (i) decreases in phosphate concentration in the plant growth medium (e.g., through use of phosphate-sensitive promoters (PS-P)); and (ii) population densities which exceed threshold population densities in the plant growth medium (e.g., through use of quorum sensing promoters (QS-P). Such agriculturally useful bacteria include bacteria capable of growth in plant growth media and in particular growth in plant growth media used to grow crop plants and / or growth on and / or in crop plants. In certain embodiments, the control elements comprising Pho boxes and / or PS-P promoters or 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, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Burkholderia, Conexibacter, Curtobacterium, Ensifer, Enterobacter, Erwinia, Escherichia, Flavobacterium, Frankia, Gaiella, Gluconacetobacter, Gluconob act r, Herbaspirillum, Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimibacter, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrob acter, 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; (iii) Azospirillum, Herbaspirillum, Pseudomonas, and Kosakonia. In certain embodiments, the bacteria are selected from at least one of the taxonomic genera selected from the group consisting of Azospirillum, Enterobacter, Herbaspirillum, Kosakonia, Klebsiella, Paenibacillus, Phytobacter, Pseudomonas, Rahnella, Sphingomonas, or Variovorax. In certain embodiments, the control elements comprising the CS-P (e.g., PS-P or QS-P) are used in bacteria of the genus or the species from which they were derived in whole or in part.

[0113] In certain embodiments, the control elements comprising PS-P promoters and optionally 5’ UTR segments will comprise “Pho box” elements or the corresponding PS-P promoters obtained from E. coli. Such E.coli Pho Box sequences can comprise those set forth in SEQ ID NO: 234-345,378-382, 525-529, and variants thereof comprising 1, 2, or 3 nucleotide substitutions. Such E.coli PS-P promoters can comprise DNA molecules having at least 85%, 90%, 95%, 98%, or 99% sequence identity across the entire length of any of SEQ ID NO: 419 and 420. In certain embodiments, the control elements comprising the E. coli Pho Box sequences provided herein can activate expression of operably linked RNAs or proteins in agriculturally useful bacteria including various gram-negative bacteria (e g., Klebsiella, Escherichia, or Serratid) in response to decreases in phosphate concentration.

[0114] In certain embodiments, the control elements comprising PS-P promoters and optionally 5’ UTR segments will comprise “Pho box” elements or the corresponding PS-P promoters obtained from 5. coelicolor. Such S. coelicolor Pho box sequences can comprise those set forth in SEQ ID NO: 346-375. and variants thereof comprising 1, 2, or 3 nucleotide substitutions. In certain embodiments, the control elements comprising the S. coelicolor Pho Box sequences provided herein can activate expression of operably linked RNAs or proteins in agriculturally useful bacteria including various gram-positive bacteria and actinomycetes (e.g., Streptomyces sp.) in response to decreases in phosphate concentration.

[0115] In certain embodiments, the control elements comprising PS-P promoters and optionally 5’ UTR segments will comprise “Pho box” elements (specific regulator-binding DNA sequences that facilitate the activation or inhibition of gene expression in a phosphate-sensitive manner) or the corresponding PS-P promoters are obtained in whole or in part from various agriculturally useful bacteria (AUB). AUB include bacteria in the taxonomic genera of Acetobacter, Aci do thermits, Acinetobacter, Agrob acterium, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Burkholderia, Conexibacter, Curtob acterium, Ensifer, Enterobacter, Erwinia, Escherichia, Flavobacterium, Frankia, Gaiella, Gluconacetobacter, Gluconob act r, Herbaspirillum, Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimibacter, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrob acter, Paenibacillus, Panotea, Pediococcus, Peribacillus, Priestia, P seudarthrobacter, Pseudomonas, Rahnella, Rhizobium, Rhodococcus, Rhodoplanes, Rhodopseudomonas, Rhodospirillum, Serratia, Solirubrobacter, Sphingobacterium, Sphingomonas, Stenotrophomonas, Streptomyces, Stutzerimonas, Variovorax, Xanthobacter, and Yoonia. Such AUB Pho Box sequences can comprise those set forth in SEQ ID NO: 376, 377, 383-397, 525-529, and variants thereof comprising 1, 2, or 3 nucleotide substitutions. Such AUB PS-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: 417-418, 421-428, 515, 517, or 518. Such Pho boxes and PS-P promoters can also include those which are operably linked to a pho A, phoX, phy, or pstS gene encoding a protein having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a PhoA, PhoX. Phy (phytase), or PstS protein set forth in TABLE 5 AND 6 and the sequence listing. In certain embodiments, the control elements comprising the AUB Pho box sequences and AUB PS-Ppromoters provided herein can activate expression of operably linked RNAs or proteins in agriculturally useful bacteria including Gluconacetobacter sp., Azorhizobium sp., Azospirillum sp., Herbaspirillum sp., Kosakonia sp., Paenibacillus sp., Enterobacter sp., Klebsiella sp., Phytobacter sp., Pseudomonas sp., Rahnella sp., Sphingomonas sp., Variovorax sp., and Pseudomonas sp. in response to decreases in phosphate concentration. In certain embodiments, the control elements comprising the AUB Pho box sequences and AUB PS-P promoters provided herein can activate expression of operably linked RNAs or proteins in a Gluconacetobacter diazo trophicus, Azorhizobium caulinodans, Azospirillum brasilense, Herbaspirillum seropedicae, Kosakonia radicincitans, Paenibacillus azotofixans, Kosakonia sp., Paenibacillus sp., Enterobacter sp., Klebsiella sp., Phytobacter sp., Pseudomonas sp., Rahnella sp., Sphingomonas sp., Variovorax sp., and Pseudomonas stutzeri isolate, strain, or derivative thereof. In certain embodiments, a control element comprising an AUB Pho box sequence or AUB PS-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 Pho box sequences and AUB PS-P promoters set forth in TABLE 5 and TABLE 6) 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 Pho box sequences or AUB PS-P promoter derived in whole or in part from Gluconacetobacter diazotrophicus. Azorhizobium caulinodans, Azospirillum brasilense. Herbaspirillum seropedicae, Kosakonia radicincitans, Klebsiella sp., Paenibacillus azotofixans, or Pseudomonas stutzeri (e.g., the corresponding AUB Pho box sequences and AUB PS-P promoters set forth in TABLE 5 and TABLE 6) are respectively used in a GEB obtained from a Gluconacetobacter diazotrophicus, Azorhizobium caulinodans, Azospirillum brasilense. Herbaspirillum seropedicae, Kosakonia radicincitans, Paenibacillus azotofixans, Klebsiella sp., or Pseudomonas stutzeri isolate, strain, or derivative thereof. In certain embodiments, the ARD Pho Box sequence can comprise a DNA molecule set forth in SEQ ID NO: 383-391, 397, and variants thereof comprising 1, 2, or 3 nucleotide substitutions. In certain embodiments, the ARD Pho Box sequence can comprise a DNA molecule having at least 85%, 90%, 95%, 98%, or 99% sequence identity across the entire length of any one of SEQ ID NO: 417-418, and 421-425, or 428.

[0116] In certain embodiments, the heterologous gene expression cassette comprising the control elements comprising the Pho box sequences and PS-P promoters (e.g., the corresponding AUB Pho box sequences and AUB PS-P promoters set forth in TABLE 5 and TABLE 6) is integrated at a location in the chromosome of the genetically engineered bacterium which does not comprise the location of an endogenous phosphate-sensitive promoter in the unmodified agriculturally relevant bacterium. In certain embodiments, a control element comprising a phoA Pho box or phoA PS-P promoter (e.g., a phoA box or phoA PS-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) phoA box or phoA PS-P promoter in the GEB. In certain embodiments, a control element comprising a phoX Pho box or phoX PS-P promoter (e.g., a PhoX box or phoX PS-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-ty pe) phoX box or phoX PS-P promoter in the GEB. In certain embodiments, a control element comprising a phy Pho box or phy PS-P promoter (e.g., a phy Pho box or phy PS-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) phy Pho box or phy PS-P promoter in the GEB. In certain embodiments, a control element comprising a pstS Pho box or pstS PS-P promoter (e.g., a pstS Pho box or pstS PS-P promoter in TABLE 5 and TABLE 6) is integrated in the genome of the GEB at a location other than the location of the endogenous (e.g., wild-type) pstS Pho box or pstS PS-P promoter in the GEB. Methods for inserting control elements comprising Pho boxes or PS-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).|00117] 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.

[0118] 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, 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: 573-581, 590, and 591 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: 573-581, 590, and 591. Such QS-P promoters can also include those which are operably linked to agene encoding a protein having at least 80%, 85%, 90%. 95%, 98%, or 99% sequence identity to a QSSP protein (e.g., an Ahll, Ceil, CinI, Cvil, Esal, LuxI, Tral, or LasI protein) set forth in Table 6 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., Pctenibacillus 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 element 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 6) 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 shitzeri (e.g., the corresponding AUB sequences and AUB QS-P promoters set forth in Table X) 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.

[0119] 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 sy nthase protein (QSSP) and / or a quorrmr 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 Ahll protein, an AlilR protein, and an ahll QS-P, respectively; ii. a CinI protein, CinR protein, and a cinl QS-P, respectively; iii. a Ceil protein, a CciR protein, and a ceil QS-P, respectively; iv. a Cvil protein, a CviR protein, and a evil QS-P, respectively; v. an Esal protein, an EsaR D91G protein, and an esaR repressable QS-P, respectively;vi. an Esal protein, an EsaR protein, and an esal QS-P, respectively; vii. a LuxI protein, a LuxR protein, and a luxl QS-P, respectively; viii. a Tral protein, a TraR protein, and a tral QS-P, respectively; or ix. a Last protein, a LasR protein, and a Iasi 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 P ntoea sp.). In certain embodiments, tire 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, Bifidobacterium, Bradyrhizobium, Conexibacter, Curtobacterium, Ensifer, Enterobacter, Erwinia, Escherichia, Flavobacterium, Frankia, Gaiella, Gluconacetobacter, Gluconob acter, Herbaspirillum, Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimib acter, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrob acter, Paenibacillus, Panotea, Pediococcus, Peribacillus, Priestia, Pseudarthrobacter, Rahnella, Rhodococcus, Rhodoplanes, Rhodopseudomonas, Rhodospirillum, Serratia, Solirubrobacter, Sphingobacterium, Sphingomonas, Stenotrophomonas, Streptomyces, Stutzerimonas, Variovorax, Xanthobacter, and Yoonia sp.).

[0120] 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 X) 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 X) 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 ahll QS-P promoter (e.g., an ahll in Table X) is integrated in the genome of the GEB at a location other than the location of the endogenous (e.g., wild-type) ahll QS-P promoter in the GEB. In certain embodiments, a control element comprising a ceil QS-P promoter (e.g, a ceil promoter in Table X) is integrated in the genome of the GEB at a location other than the location of the endogenous (e.g., wild-type) ceil QS-P promoter in the GEB. In certain embodiments, a control element comprising a cinI Vl or cinI_V2 QS-P promoter (e.g., a cinI Vl or cinI_V2 QS-P promoter in Table X) is integrated in the genome of the GEB at a location other than the location of die endogenous (e.g., wild-type) cinI Vl 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).

[0121] Mediods 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 cancomprise 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, GEB that arc used in the methods provided herein arc placed in the plant growth media prior to having an irreversible insertion, excision, or inversion of a DNA molecule in the first heterologous gene expression cassette that is catalyzed by the recombinase. In such embodiments, the GEB are established (e.g., before and after transformation with the gene expression cassettes encoding the recombinase), maintained, and fermented for bulk placement in plant growth media under conditions where the CS-P which is operably linked to the recombinase is not activated and the recombinase expression is inhibited. In certain embodiments where the CS-P is a phosphate-sensitive promoter (PS-P), recombinase expression is inhibited during establishment, maintenance, and fermentation by using bacterial growth media having phosphate concentrations sufficient to inhibit PS-P expression (e.g., phosphate concentrations of at least about 100 pM, 1 mM, 10 mM, 20 mM, or 50 mM). Other GEB comprising CS-P including oxygen, nitrogen / oxygen, potassium, carbon, nitrate, naringenin, and other CS-P listed in TABLE 7 are similarly established, maintained, and fermented under oxygen, nitrogen / oxygen, potassium, carbon, nitrate, naringenin. and other conditions listed in TABLE 7 which inhibit activation of the CS-P and expression of the operably linked recombinase.

[0122] In certain embodiments where the recombinase is operably linked to a quorum sensing promoter (QS-P) and the GEB further comprise 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 recombinase at a population density of the genetically engineered bacterium which exceeds the threshold population density when the genetically engineered bacterium (GEB), the GEB are grown either (a) in contact with the quorum quenching compound QQ; or (b) in exposure to a temperature above tire threshold temperature to suppress expression of the recombinase 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. In certain embodiments, the deactivator can comprise an enzyme which catalyzes tire degradation of tire quorum sensing signal molecule, where the expression of the enzyme is induced by the addition of tire 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 recombinase. 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- M -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 derepression of the deactivator or temperature induced de-activation of the first QSSP and / or the first QSRP can be used.

[0123] 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: 537-539, and 540), alpha-beta hydrolase fold lactonase proteins (SEQ ID NO: 541, 542, and 543), metallo-beta-lactamase-like lactonase proteins (SEQ ID NO: 544-547, and 548), phosphotriesterase-like lactonase proteins (SEQ ID NO: 549-552, and 553), and variants of the QEs having at least 70%, 80%, 85%, 90%, 95%, 98%, and 99% sequence identity to SEQ ID NO: 537 to 552, or 553).

[0124] 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.

[0125] 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.

[0126] 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, venniculite. seed cases, plant products (e.g, ground hulls, husk, stalks, stems, leaves, and the like), animal products, or combinations thereof.

[0127] Compositions comprising the GEB provided herein can comprise an agriculturally acceptable adjuvant.

[0128] 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, 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, acrylamide polymers and copolymers, polyhydroxyethyl acrylate, methylacrylamide monomers, and / or polychloroprene.

[0129] 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.

[0130] 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.

[0131] In certain embodiments, the agriculturally acceptable adjuvant is a fungicide, insecticide, a nematicide, a rodenticide, and / or a bactcriocidc. When the adjuvant is a bactcriocidc, 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

[0132] Additionally, the following numbered embodiments are included in the disclosure.

[0133] 1. A method of providing at least one agriculturally relevant compound to a plant comprising placing at least one genetically engineered bacterium into a plant growth medium, wherein the genetically engineered bacterium is an agriculturally useful bacterium comprising: (i) a first heterologous gene expression cassette comprising at least one DNA molecule encoding a protein or RNA sequence of interest, wherein the DNA molecule is operably linked to at least one DNA molecule comprising a site-specific recombinase recognition sequence (SSRRS) and wherein the first heterologous gene expression cassette lacks an irreversible insertion, excision, or inversion of a DNA molecule in the first heterologous gene expression cassette; and (ii) a second heterologous gene expression cassette comprising at least one DNA molecule encoding at least one recombinase that can catalyze site-specific recombination at said SSRRS. wherein the DNA molecule encoding said recombinase is operably linked to a promoter activated by a change of at least one condition in the plant growth medium, and wherein the site-specific recombination induces an irreversible insertion, excision, or inversion of: (a) a DNA molecule comprising a control element in, from, at. or near the DNA molecule encoding the protein or RNA sequence of interest in the first heterologous gene expression cassette; or (b) a DNA molecule encoding the protein or RNA sequence of interest in the first heterologous gene expression cassette.

[0134] 2. A method of providing at least one agriculturally relevant compound to a plant comprising placing at least one genetically engineered bacterium into a plant growth medium, wherein the genetically engineered bacterium is an agriculturally useful bacterium comprising: (i) a first part of a first heterologous gene expression cassette comprising a DNA molecule encoding a control element, protein or RNA sequence of interest, or a part thereof, wherein the first part of the first heterologous gene expression cassette has reduced or no function and wherein die DNA molecule is operably linked to at least one site-specific recombinase recognition sequence (SSRRS); and a second part of the first heterologous gene expression cassette comprising the control element, the protein or RNA sequence of interest, or part thereof absent from the first part of the first heterologous gene expression cassette; and (ii) a second heterologous gene expression cassette comprising at least one nucleic acid sequence encoding at least one recombinase that can catalyze site-specific recombination at said SSRRS, wherein the DNA molecule encoding said recombinase is operably linked to a promoter activated by a change of at least one condition in the plant growthmedium, and wherein the site-specific recombination induces an irreversible insertion, excision, or inversion of DNA which results in the operable linkage of the first and second part of the first heterologous gene expression cassette to provide a functional first heterologous gene expression cassette.

[0135] 3. The method of embodiment 2, wherein the first part and the second part of the first heterologous gene expression cassette are located on separate DNA molecules which are not covalently linked.

[0136] 4. The method of embodiment 2, wherein the first part and the second part of the first heterologous gene expression cassette are located on a single DNA molecule.

[0137] 5. The method of embodiment 1, wherein the DNA molecule encoding the recombinase comprises an ATG, ACG, or ATT translation initiation codon which is operably linked to the open reading frame of the DNA molecule encoding the recombinase.

[0138] 6. The method of embodiment 1, wherein the irreversible insertion, excision, or inversion of the DNA molecule comprising the control element or the DNA molecule encoding the protein or RNA sequence of interest reduces expression of the protein or RNA sequence of interest in comparison with the first heterologous gene expression cassette lacking the irreversible insertion, excision, or inversion of the DNA molecule.

[0139] 7. The method of embodiment 6. wherein the irreversible insertion, excision, or inversion of the DNA molecule causes the expression of the protein or RNA sequence of interest to be reduced by at least 10%, 20%, 30%. 40%. 50%, 60%, 70%, 80%. 90%. 95%, 98%, or 99% or by 10% or 15% to 20%, 30%, 40%, 50%. 60%, 70%, 80%, 90%. 95%, 98%, or 99% in comparison to the first heterologous gene expression cassette of (i) lacking the irreversible insertion, excision, or inversion of a DNA molecule.

[0140] 8. The method of embodiment 6. wherein the control element comprises a first promoter and a second promoter directed opposite one another, wherein the control element is flanked by two SSRRS in an inverted configuration, wherein the first promoter of the control element is operably linked to the DNA molecule encoding at least one protein or RNA sequence of interest prior to inversion by the site-specific recombinase, wherein transcription promoting-activity of the second promoter is: (i) at least: 14%, 28%, 42%, or 53% , (ii) 10% or 15% to 20%, 30%, 40%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, or (iii) 2-, 5-, or 10-fold less than the transcription promoting-activity of the first promoter, and wherein the second promoter of the control element is operably linked to the DNA molecule encoding the at least one protein or RNA sequence of interest upon inversion by the site-specific recombinase.

[0141] 9. The method of embodiment 8, wherein: (i) the first promoter comprises a constitutive promoter, a native promoter of the native gene encoding the protein or RNA sequence of interest, or a promoter having activity equivalent to or greater than the native promoter: and / or (ii) theprotein of interest comprises a GlnA, AmtB, GlnB, GlnK, GlnZ, NifL, and / or DraT protein and the agriculturally relevant compound is ammonia.

[0142] 10. The method of embodiment 8, wherein:(i) the first promoter comprises a J23100 constitutive promoter of SEQ ID NO: 609. a variant thereof having at least 95%, 98%, or 99% sequence identity' thereto, and / or a promoter having equivalent activity thereof; (ii) the second promoter comprises a J23106 constitutive promoter of SEQ ID NO: 613, J23102 constitutive promoter of SEQ ID NO: 611, J23111 constitutive promoter of SEQ ID NO: 614, 123104 constitutive promoter of SEQ ID NO: 612, a variant thereof having at least 95%, 98%, or 99% sequence identity thereto, and / or a promoter having equivalent activity thereof: (iii) the protein of interest comprises a GlnA protein and the agriculturally relevant compound is ammonia, optionally wherein the GlnA protein is set forth in Table 5, Table 6. or the sequence listing; and (iv) the agriculturally useful bacterium is a member of the genus Azospirillum, Enterob acter, Klebsiella, Kosakonia, Rahnella, Paenibacillus, Phytobacter, Sphingomonas, or Variovorax. wherein the bacterium is optionally a member of the genus Azospirillum, Klebsiella, Kosakonia, or Rahnella.

[0143] 11. The method of embodiment 6. wherein the nucleic acid molecule encoding the protein or RNA sequence of interest is flanked by two SSRRS in a direct configuration and wherein the site-specific recombination induces excision of said nucleic acid molecule encoding a protein or RNA sequence of interest to reduce expression of the protein or RNA sequence of interest.

[0144] 12. The method of embodiment 6, wherein the first heterologous gene expression cassette comprises a promoter flanked by two SSRRS in a direct configuration, wherein said promoter is operably linked to the DNA molecule encoding a protein or RNA sequence of interest, and wherein the site-specific recombination induces excision of said promoter to reduce expression of the protein or RNA sequence of interest.

[0145] 13. The method of embodiment 11. wherein: (i) the SSRRS comprise attB and attP sites and / or wherein the DNA molecule comprises 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 (ii) the agriculturally relevant compound is ammonia.

[0146] 14. The method of embodiment 8, wherein: (i) the SSRRS comprise attB and attP sites and / or wherein the DNA molecule comprises 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 (ii) the agriculturally relevant compound is ammonia.

[0147] 15. The method of embodiment 6, wherein the first heterologous gene expression cassette comprises a promoter flanked by two SSRRS in an inverted configuration, wherein said promoter is operably linked to the DNA molecule encoding a protein or RNA sequence of interest, and wherein the site-specific recombination induces inversion of said promoter to uncouple the promoter from the DNA molecule and to reduce expression of the protein or RNA sequence of interest.

[0148] 16. The method of embodiment 6. wherein the DNA molecule encoding the protein or RNA sequence of interest is flanked by two SSRRS in an inverted configuration, wherein the DNA molecule is operably linked to a promoter, and wherein the site-specific recombination induces inversion of said DNA molecule encoding the protein or RNA sequence of interest to uncouple the DNA molecule from the promoter, thereby reducing expression of the protein or RNA sequence of interest.

[0149] 17. The method of embodiment 6, wherein the first heterologous gene expression cassette comprises a promoter which is: (i) inoperably linked to a terminator flanked by two SSRRS in an inverted configuration; and (ii) operably linked to a DNA molecule encoding the protein or RNA sequence of interest, and wherein the site-specific recombination induces inversion of said terminator to operably link the promoter to the terminator and to uncouple the DNA molecule from the promoter, thereby reducing expression of the protein or RNA sequence of interest.

[0150] 18. The method of embodiment 6, wherein the DNA molecule encoding the protein or RNA sequence of interest comprises a first SSRRS comprising an in-frame open reading frame (ORF), a part of the DNA molecule encoding a part of the protein or RNA sequence of interest, and a second SSRRS comprising an in-frame ORF, wherein the protein or RNA sequence is functional, and wherein the site-specific recombination induces excision of the part of the DNA molecule and one SSRRS to reduce activity of the protein or RNA sequence of interest.

[0151] 19. The method of embodiment 6, wherein: (i) the DNA molecule encoding the protein or RNA sequence of interest comprises an inserted first SSRRS comprising an in-frame open reading frame (ORF) and wherein the protein or RNA sequence is functional; and (ii) the genetically engineered bacterium further comprises a blocking DNA covalently linked to at least one SSRRS; wherein the site-specific recombination induces insertion of the blocking DNA into the DNA molecule to reduce expression and / or activity of the protein or RNA sequence of interest.

[0152] 20. The method of embodiment 6, wherein the protein of interest comprises a GlnA, AmtB. GlnB, GlnK, GlnZ, NifL, and / or DraT protein and the agriculturally relevant compound is ammonia.

[0153] 21. The method of embodiment 1, wherein the irreversible insertion, excision, or inversion of the DNA molecule comprising the control element or the DNA molecule encoding the protein or RNA sequence of interest increases expression of the protein or RNA sequence of interest in comparison to the first heterologous gene expression cassette lacking the irreversible insertion, excision, or inversion of the DNA molecule.

[0154] 22. The method of embodiment 21, wherein the irreversible insertion, excision, or inversion of the DNA molecule causes the expression of the protein or RNA sequence of interest to be increased: (i) at least 2- fold, 5-fold or at least 10-fold; or (ii) about 2-fold or 5-fold to about 15-fold, both in comparison to the first heterologous gene expression cassette lacking the irreversible insertion, excision, or inversion of a DNA molecule.

[0155] 23. The method of embodiment 21. wherein the control element comprises a promoter, wherein the promoter is flanked by two SSRRS in an inverted configuration, and wherein the promoter is operably linked to the at least one protein or RNA sequence of interest upon inversion by the sitespecific recombinase.

[0156] 24. The method of embodiment 21, wherein the control element comprises a first promoter and a second promoter directed opposite one another, wherein the control element is flanked by two SSRRS in an inverted configuration, wherein the first promoter of the control element is operably linked to the DNA molecule encoding at least one protein or RNA sequence of interest prior to inversion by the site-specific recombinase, wherein transcription promoting-activity of the second promoter is at least 2-, 5-, or 10-fold greater than the transcription promoting-activity of the first promoter, and wherein the second promoter of the control element is operably linked to the at least one protein or RNA sequence of interest upon inversion by the site-specific recombinase.

[0157] 25. The method of embodiment 24. wherein: (i) the first promoter comprises a constitutive promoter, a native promoter of the native gene encoding the protein or RNA sequence of interest, or a promoter having activity equivalent to or lower than the native promoter: and / or (ii) the protein of interest is a NifA protein. NtrC protein. GlnR protein, or modified GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity.

[0158] 26. The method of embodiment 21. wherein the control element comprises a promoter, wherein the DNA molecule encoding the protein or RNA sequence of interest is flanked by two SSRRS in an inverted configuration, and wherein the DNA molecule encoding the protein or RNA sequence of interest is operably linked to the control element upon inversion by the site-specific recombinase.

[0159] 27. The method of embodiment 21, wherein the control element comprises a terminator, wherein the terminator is flanked by two SSRRS in a direct configuration, wherein the terminator and two SSRRS are between the at least one DNA molecule encoding protein or RNA sequence of interest and a promoter in the first heterologous gene expression cassette, and wherein the DNA molecule encoding the protein or RNA sequence of interest is operably linked to the promoter in the first heterologous gene expression cassette upon excision of said terminator.

[0160] 28. The method of embodiment 21, wherein the control element comprises a terminator, wherein the terminator is flanked by two SSRRS in an inverted configuration, wherein the terminator and two SSRRS are betw een the DNA molecule encoding at least one protein or RNA sequence of interest and a promoter in the first heterologous gene expression cassette, and wherein the DNA molecule encoding the protein or RNA sequence of interest is operably linked to the promoter in the first heterologous gene expression cassette upon inversion of said terminator.

[0161] 29. The method of embodiment 2. wherein the first part of first heterologous gene expression cassette comprises the first part of the DNA molecule encoding the first part of the protein or RNA sequence of interest, wherein the SSRRS is located at the beginning of, within, or at the end of thefirst part of the DNA molecule, wherein die genetically engineered bacterium further comprises a third heterologous gene expression cassette comprising a site-specific recombinase recognition site and the second part of the DNA molecule encoding the second part of the protein or RNA of interest absent from the first heterologous gene expression cassette, and wherein the first and second part of the DNA molecule encoding the protein or RNA sequence of interest are operably linked to provide a functional protein or RNA sequence of interest upon insertion of die second part of the DNA molecule into the first heterologous gene expression cassette.

[0162] 30. The method of embodiment 2, wherein the first part of the first heterologous gene expression cassette comprises the DNA molecule encoding the protein or RNA sequence of interest but lacks a control element, wherein the SSRRS is located at the beginning of. within, or at the end of the first part of the DNA molecule, wherein the genetically engineered bacterium further comprises a third heterologous gene expression cassette comprising a site-specific recombinase recognition site and a control element, and wherein the control element and the DNA molecule encoding the protein or RNA sequence of interest are operably linked to provide a functional first heterologous gene expression cassette upon insertion of the control element into the first heterologous gene expression cassette.

[0163] 31. The method of embodiment 21, 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 agriculturally relevant compound 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 agriculturally relevant compound is ammonia; (c) a GlnR protein and the agriculturally relevant compound is ammonia; (d) a glutaminase enzyme and the agriculturally relevant compound is ammonia; (e) a protein product of a refactored nif or fix gene cluster and the agriculturally relevant compound 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 inliibits 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 (cl), 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 agriculturally relevant compound is ammonia; or (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 gcnc(s) is / arc a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the agriculturally relevant compound is ammonia.

[0164] 32. The method of embodiment 1, wherein: (i) the first heterologous gene expression cassette comprises a control element comprising a promoter flanked by two SSRRS oriented as inverted repeats, wherein the promoter is operably linked to a first DNA molecule encoding a first protein or RNA sequence of interest, wherein the promoter flanked by two SSRRS is uncoupled from a second DNA molecule encoding a second protein or RNA sequence of interest; and (ii) the sitespecific recombination induces the inversion of the DNA molecule comprising the promoter to uncouple the promoter from the first DNA molecule, to operably link the promoter to the second DNA molecule, and to reduce expression of the first protein or RNA sequence of interest and increase expression of the second protein or RNA sequence of interest, both in comparison to the first heterologous gene expression cassette of (i) lacking the irreversible inversion of the DNA molecule.

[0165] 33. The method of embodiment 2. wherein:(i) the first heterologous gene expression cassette comprises a control element comprising a promoter which is operably linked to a first DNA molecule encoding a first protein or RNA sequence of interest which is operably linked to an SSRRS, wherein the promoter is uncoupled from a second DNA molecule encoding a second protein or RNA sequence of interest which is operably linked to a terminator element and an SSRRS; and (ii) the site-specific recombination induces the insertion of the second DNA molecule encoding the second protein or RNA sequence of interest to uncouple the promoter from the first DNA molecule, to operably link the promoter to the second DNA molecule, and to reduce expression of the first protein or RNA sequence of interest and increase expression of the second protein or RNA sequence of interest, both in comparison to the first heterologous gene expression cassette of (i) lacking the irreversible inversion of the DNA molecule.

[0166] 34. The method of embodiment 32, whcrcin:(a) the first protein of interest comprises a wild-type glutamine synthetase (GS) or variant thereof with improved catalytic activity in comparison to wild-type GS and the second protein of interest comprises a wild-type GS with reduced levels of expression in comparison to the wild-type GS or a GS variant with decreased catalytic activity in comparison to wild-type GS, and the agriculturally relevant compound is ammonia; (b) the first protein of interest comprises a GS adenylyltransferase protein and the second protein of interest comprises a GS adenylyltransferase protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, and the agriculturally relevant compound is ammonia; or (c) the first target gene is a nifL gene and the second target gene is a nijA' gene, and the agriculturally relevant compound is ammonia.

[0167] 35. The method of any of embodiments 1 to 34 wherein said change of at least one condition in the plant growth medium condition comprises: (a) growth of the genetically engineered bacterium to exceed a threshold population density in the plant growth medium;(b) reduction of at least one fertilizer or plant nutrient in the soil, 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;(c) growth of a plant in the plant growth media;(d) change in soil temperature; (e) change in motility of the bacterium; (1) change in light levels in the soil; (g) change in oxygen concentration in the soil; (h) change in concentration of bioavailable nitrogen in the soil; (i) change in pH in the soil; (j) change in overall solute concentration (osmotic pressure) in the soil; (k) change in phytohormone or plant signaling molecule concentrations; (1) change in an amino acid concentration; (m) change in a sugar concentration; and / or (n) a change in plant or microbial metabolite concentrations, optionally wherein the plant or microbial metabolite is naringenin, quercetin, luteolin, apigenin, octopine, nopaline, or Scyllo-inosamine.

[0168] 36. The method of any one of embodiments 1 to 35, wherein said promoter activated by a change of at least one plant growth medium condition comprises: (i) a phosphate-sensitive promoter; (ii) a nitrogen -sensitive promoter; (iii) a quorum sensing promoter; (iv) a promoter which is induced or repressed by decreased oxygen levels; (v) a promoter which is induced or repressed by increased oxygen levels; (vi) a promoter which is induced by decreased oxygen and decreased nitrogen levels, optionally wherein the promoter is a nifH promoter; (vii) a promoter which is induced or repressed by increased or decreased potassium levels;(viii) a promoter which is induced by decreased carbon levels, optionally wherein the promoter is a Pfic promoter;(ix) a promoter that is induced by increased nitrate levels, optionally wherein the promoter is an oxygen insensitive PnarG promoter; (x) a promoter that is induced by increased nitrate levels, optionally wherein the promoter is a narK or narX promoter; (xi) a promoter which is upregulated by an increase in the concentration of naringenin; and / or (xii) a promoter or system comprising a promoter set forth in Table 5, Table 6, or Table 7.

[0169] 37. The method of embodiment 36, wherein the promoter which is induced by decreased oxygen and decreased nitrogen levels is a NifH promoter.

[0170] 38. The method of embodiment 37, wherein the NifH promoter comprises a DNA molecule having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 595 or 596.

[0171] 39. The method of embodiment 36, wherein tire genetically engineered bacterium comprises the phosphate-sensitive promoter which is operably linked to the recombinase and wherein:(a) the threshold concentration of phosphate in the plant growth medium which activates expression of the recombinase is about 0 pM to about 50 pM;(b) the phosphate-sensitive promoter and / or a segment of the 5’ untranslated region (UTR) which is operably linked to the phosphate-sensitive promoter comprises at least one copy of an operably linked Pho box, wherein the Pho boxcomprises the sequence of SEQ ID NO: 525-529. 234-396, or 397 and sequences with at least 95% identity to any one of SEQ ID NO: 234-397, optionally wherein the Pho box comprises SEQ ID NO: 525-529, 376, 377, 383-396, or 397;(c) the phosphate -sensitive promoter and / or a segment of the 5’ UTR comprises a promoter and / or a segment of a 5‘UTR of a phoA, phoX, phy, pstS gene, a variant thereof, or a combination thereof; and / or(d) the phosphate-sensitive promoter and / or the segment of the 5’ UTR comprises a promoter of a gene encoding:® a PstS protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 523, 399, 401, 403, 405, 407, 409, 415, 416, 513, 523, and 524 or having an identity of at least 76%, 80%, 85%, 90%, 95%, 98%, or 99% with any one of SEQ ID NO: 399, 401, 403. 405, 407, 409, 415, 416, 513, or 524; (ii) a PhoX protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 408. 410, 411, 412, and 413 or having an identity of at least 80% with any one of SEQ ID NO: 408, 410, 411, 412. and 413; (iii) a Phy protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 414, 57-138, and 139 or having an identity of at least 80% with any one of SEQ ID NO: 414, 57-138, or 139; and / or (iv) a PhoA protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 398, 400, 402. 404, and 406 or having an identity of at least 80% with any one of SEQ ID NO: 398, 400, 402, 404. or 406.

[0172] 40. The method of embodiment 39, wherein the phosphate-sensitive promoter and / or the segment of the 5’ UTR comprises: i. a pstS promoter comprising a DNA sequence selected from the group consisting of SEQ ID NO: 515, 417, 418, 420. 421, 422, 423. 424, 425, 428, 517, and 518; ii. a phoX promoter comprising the DNA sequence of SEQ ID NO: 426; iii. a phy promoter comprising the DNA sequence of SEQ ID NO: 427; iv. a phoA promoter comprising the DNA sequence of SEQ ID NO: 419; and / or v. a Pliar53 promoter comprising the DNA sequence of SEQ ID NO: 459;vi. a variant of the promoters under i. to v. having an identity of at least 76%, 80%, 90%, 95%, 98%, or 99% with any of SEQ ID NO: 515, 417, 418-428, 459, 517, or 518, wherein said variant comprises at least one Pho box and / or retains at least one Pho box present in each promoter and wherein said variant promoters are activated by a decrease in phosphate concentration to about 0 pM to about 1 pM, 2 pM, 5 pM, 7 pM, 10 pM, 20 pM, 30 pM, 40 pM, or 50 pM.

[0173] 41. The method of embodiment 39, wherein the bacterial pstS gene encodes a protein having at least 76%, 80%, 85%, 90%, 95%, 98%. or 99% sequence identity to SEQ ID NO: 513 and comprises the polypeptide of SEQ ID NO: 514.

[0174] 42. The method of embodiment 39, wherein the phosphate-sensitive promoter comprises a sequence having at least 95% sequence identity to SEQ ID NO: 517.

[0175] 43. The method of embodiment 36, wherein the genetically engineered bacterium comprising the quorum sensing promoter which is operably linked to the recombinase further comprises: (a) one or more heterologous gene expression cassette(s) comprising at least one control element which isoperably linked to at least one DNA molecule encoding a first quorum sensing synthase protein (QSSP) able to synthesize a quorum sensing signal molecule Q (QSSM Q) and / or at least one DNA molecule encoding a first quorum sensing regulator protein (QSRP) that can bind said QSSM Q, wherein the first quorum sensing promoter can be activated by the first quorum sensing regulator protein (QSRP) and the QSSM Q when the population density of tire genetically engineered bacterium exceeds a threshold population density; and (b) 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 recombinase 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.

[0176] 44. The method of embodiment 43, 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.

[0177] 45. The method of embodiment 44, 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.

[0178] 46. The genetically engineered bacterium of embodiment 45. 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: 537, 538. 539, or 540, 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: 541, 542, or 543, 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: 549, 550, 551, 552, or 553, respectively ; or

[0179] (iv) the AHL lactonase enzy me comprises a mctallo-bcta-lactamasc protein, optionally wherein the metallo-beta-lactamase protein is a Gel, AttM, AidC, AiiB, or AiiA protein and optionally wherein the Gel, AttM, AidC, AiiB, or AiiA protein has at least 75% sequence identity to SEQ ID NO: 544, 545, 546, 547, or 548, respectively.

[0180] 47.The method of embodiment 43, wherein the deactivator comprises an inducible transcription factor which: (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.

[0181] 48. The method of embodiment 43, 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-(P-Kctocaproyl)- L-homoserine lactone and the first QSSP comprises an Ahll protein; or (iv) the QSSM Q molecule comprises N-(P-Ketocaproyl)-L-homoserine lactone and the first QSRP comprises an AlilR protein.

[0182] 49. The method of embodiment 43, 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.

[0183] 50. The method of embodiment 43, wherein: i. the first QSSP comprises an Ahll protein, the first QSRP comprises an AhlR protein, and the first quorum sensing promoter comprises an ahll promoter; ii. the first QSSP comprises an CinI protein, the first QSRP comprises a CinR protein, and the first quorum sensing promoter comprises a cinl promoter; iii. the first QSSP comprises an Ceil protein, the first QSRP comprises a CciR protein, and the first quorum sensing promoter comprises a ceil promoter; iv. the first QSSP comprises an Cvil protein, the first QSRP comprises a CviR protein, and the first quorum sensing promoter comprises a evil promoter; v. the first QSSP comprises an Esal 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 Esal protein, the first QSRP comprises a EsaR protein, and the first quorum sensing promoter comprises an esal promoter; vii. the first QSSP comprises a LasI protein, the first QSRP comprises a LasR protein, and the first quorum sensing promoter comprises a Iasi or lasB promoter; viii. the first QSSP comprises an LuxI protein, the first QSRP comprises a LuxR protein, and the first quorum sensing promoter comprises a hixl promoter; or ix. the first QSSP comprises an Tral protein, the first QSRP comprises a TraR protein, and the first quorum sensing promoter comprises a tral promoter.

[0184] 51. The method of embodiment 50, wherein:(i) the Ahll protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 565 ;(ii) the Ceil protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 566; (iii) the CinI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 567;(iv) the CinI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 568;(v) the Cvil protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 569;(vi) the Esal protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 570;(vii) the LasI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 593;(viii) the LuxI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 571; or(ix) the Tral protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 572.

[0185] 52. The method of embodiment 43, 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.

[0186] 53. The method of embodiment 43, 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.

[0187] 54. The method of embodiment 43, wherein the first QSRP comprises an AhlR, CciR, CinR, CviR, EsaR, LasR, LuxR, or TraR QSRP.

[0188] 55. The method of embodiment 54, 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: 556, 557, 558 or 559, 560, 561, or 562, 592, 563. or 564, respectively.

[0189] 56. The method of any one of embodiments 1 to 55, wherein the AUB is a diazotrophic and / or phosphate-solubilizing bacterium.|00190] 57. The method of any one of embodiments 1 to 56. wherein the AUB is a member of the genus Azospirillum, Enterobacter, Klebsiella, Kosakonia, Rahnella, Paenibacillus, Phytobacter, Rahnella, Sphingomonas, or Variovorax. wherein the bacterium is optionally a member of the genus Azospirillum, Klebsiella, Kosakonia, or Rahnella.

[0191] 58. The method of any one of embodiments 1 to 56, wherein the AUB is selected from the taxonomic class of Gammaproteobacteria.

[0192] 59. The method of embodiment 58. wherein the Gammaproteobacteria is a Kosakonia sp.. Enterobacter sp., Klebsiella sp., Rahnella sp., Pseudomonas sp.. or Acinetobacter sp.

[0193] 60. The method of any one embodiments 1 to 59, wherein the recombinase 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: (i) attB and attP sites; or (ii) attL or attR sites; wherein the (i) attB and attP sites; or (ii) attL or attR sites recognized respectively by the PhiC31, IntS. IntM, IntG, YdcL, or Sxt / R39 integrase.

[0194] 61. The method of embodiment 60, wherein the recombinase is a phage PhiC31 serine integrase having at least 90%, 95% , or 99% sequence identity to SEQ ID NO: 41 and the SSRRS comprise an attP of SEQ ID NO: 42 and an attB recombinase recognition site of SEQ ID NO: 43, or functional equivalents thereof recognized by the phage PhiC31 serine integrase.

[0195] 62. The method of any one of embodiments 1 to 59, wherein: (i) the recombinase is a yeast flippase (FLP) recombinase and the SSRRS are FRT sites; or (ii) the recombinase is a Cre-recombinase and the SSRRS are loxP sites.

[0196] 63. The method of any one of embodiments 1 to 62, 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 thegenetically 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.

[0197] 64. The method of any one of embodiments 1 to 63, 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.

[0198] 65. A genetically engineered bacterium comprising: (i) a first heterologous gene expression cassette comprising at least one DNA molecule encoding a protein or RNA sequence of interest, wherein the DNA molecule is operably linked to at least one DNA molecule comprising a sitespecific recombinase recognition sequence (SSRRS) and wherein the first heterologous gene expression cassette lacks an irreversible insertion, excision, or inversion of a DNA molecule in the first heterologous gene expression cassette; and (ii) a second heterologous gene expression cassette comprising at least one DNA molecule encoding at least one recombinase that can catalyze sitespecific recombination at said SSRRS, wherein the DNA molecule encoding said recombinase is operably linked to a promoter activated by a change of at least one condition in the plant growth medium, and wherein the site-specific recombination can induce an irreversible insertion, excision, or inversion of: (a) a DNA molecule comprising a control element in, from, at, or near the DNA molecule encoding the protein or RNA sequence of interest in the first heterologous gene expression cassette; or (b) a DNA molecule encoding the protein or RNA sequence of interest in the first heterologous gene expression cassette; wherein the genetically engineered bacterium is an agriculturally useful bacterium.

[0199] 66. A genetically engineered bacterium comprising: (i) a first part of a first heterologous gene expression cassette comprising a DNA molecule encoding a control element, protein or RNA sequence of interest, or a part thereof, wherein the first part of the heterologous gene expression cassette has reduced or no function and wherein the DNA molecule is operably linked to at least one site-specific recombinase recognition sequence (SSRRS); and a second part of the first heterologous gene expression cassette comprising the control element, die protein or RNA of interest, or part of absent from the first part of the first heterologous gene expression cassette; and (ii) a second heterologous gene expression cassette comprising at least one DNA molecule encoding at least one recombinase that can catalyze site-specific recombination at said SSRRS, wherein the DNA molecule encoding said recombinase is operably linked to a promoter activated by a change of at least one condition in die plant growth medium, and wherein the site-specific recombination induces an irreversible insertion, excision, or inversion of DNA which results in the operable linkage of the first and second part of the first heterologous gene expression cassette toprovide a functional first heterologous gene expression cassette, wherein the genetically engineered bacterium is an agriculturally useful bacterium.

[0200] 67. The genetically engineered bacterium of embodiment 66, wherein the first part and the second part of the first heterologous gene expression cassette arc located on separate DNA molecules which are not covalently linked.

[0201] 68. The genetically engineered bacterium of embodiment 66, wherein the first part and the second part of the first heterologous gene expression cassette are located on a single DNA molecule.

[0202] 67. The genetically engineered bacterium of embodiment 65, wherein the DNA molecule encoding the recombinase comprises an ATG, ACG, or ATT translation initiation codon which is operably linked to the open reading frame of the DNA molecule encoding the recombinase.

[0203] 68. The genetically engineered bacterium of embodiment 65, wherein the irreversible insertion, excision, or inversion of the DNA molecule comprising the control element or the DNA molecule encoding the protein or RNA sequence of interest reduces expression of the protein or RNA sequence of interest in comparison with the first heterologous gene expression cassette lacking the irreversible insertion, excision, or inversion of the DNA molecule.

[0204] 69. The genetically engineered bacterium of embodiment 68. wherein the irreversible insertion, excision, or inversion of the DNA molecule causes the expression of the protein or RNA sequence of interest to be reduced by at least 10%, 20%, 30%, 40%. 50%. 60%, 70%, 80%, 90%. 95%, 98%. or 99% or by 10% or 15% to 20%, 30%, 40%. 50%, 60%, 70%. 80%, 90%, 95%, 98%. or 99% in comparison to the first heterologous gene expression cassette of (i) lacking the irreversible insertion, excision, or inversion of a DNA molecule.

[0205] 70. The genetically engineered bacterium of embodiment 68, wherein the control element comprises a first promoter and a second promoter directed opposite one another, wherein the control element is flanked by two SSRRS in an inverted configuration, wherein the first promoter of the control element is operably linked to the DNA molecule encoding at least one protein or RNA sequence of interest prior to inversion by the site-specific recombinase, wherein transcription promoting-activity of the second promoter is at least: (i) at least: 14%, 28%, 42%, or 53% , (ii) 10% or 15% to 20%, 30%, 40%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, or (iii) 2- , 5-, or 10-fold less than tire transcription promoting-activity of the first promoter, and wherein tire second promoter of the control element is operably linked to the DNA molecule encoding the at least one protein or RNA sequence of interest upon inversion by the site-specific recombinase.

[0206] 71. The genetically engineered bacterium of embodiment 70. wherein:(i) the first promoter comprises a constitutive promoter, a native promoter of the native gene encoding the protein or RNA sequence of interest, or a promoter having activity equivalent to or greater than the native promoter; and / or(ii) the protein of interest comprises a GlnA, AmtB, GlnB, GlnK, GlnZ, NifL, and / or DraT protein and the agriculturally relevant compound is ammonia.

[0207] 72. The genetically engineered bacterium of embodiment 70, wherein:(i) the first promoter comprises a J23100 constitutive promoter of SEQ ID NO: 609, a variant thereof having at least 95%, 98%, or 99% sequence identity thereto, and / or a promoter having equivalent activity thereof; (ii) the second promoter comprises a J23106 constitutive promoter of SEQ ID NO: 613, J23102 constitutive promoter of SEQ ID NO: 611, J23111 constitutive promoter of SEQ ID NO: 614, J23104 constitutive promoter of SEQ ID NO: 612, a variant thereof having at least 95%, 98%, or 99% sequence identity thereto, and / or a promoter having equivalent activity thereof(iii) the protein of interest comprises a GlnA protein, optionally wherein the GlnA protein is set forth in Table 5, Table 6, or the sequence listing, and the agriculturally relevant compound is ammonia; and(iv) the agriculturally useful bacterium is a member of the genus Azospirillum, Enterobacter, Klebsiella, Kosakonia, Rahnella, Paenibacillus, Phytobacter, Rahnella, Sphingomonas, or Variovorax. wherein the bacterium is optionally a member of the genus Azospirillum, Klebsiella, Kosakonia, or Rahnella.

[0208] 73. The genetically engineered bacterium of embodiment 68. wherein the nucleic acid molecule encoding the protein or RNA sequence of interest is flanked by two SSRRS in a direct configuration and wherein the site-specific recombination can induce excision of said nucleic acid molecule encoding a protein or RNA sequence of interest to reduce expression of the protein or RNA sequence of interest.

[0209] 74. The genetically engineered bacterium of embodiment 68. wherein the first heterologous gene expression cassette comprises a promoter flanked by two SSRRS in a direct configuration, wherein said promoter is operably linked to the DNA molecule encoding a protein or RNA sequence of interest, and wherein the site-specific recombination can induce excision of said promoter to reduce expression of the protein or RNA sequence of interest.

[0210] 75. The genetically engineered bacterium of embodiment 73, wherein: (i) the SSRRS comprise attB and attP sites and / or wherein the DNA molecule comprises 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 (ii) the agriculturally relevant compound is ammonia.

[0211] 76. The genetically engineered bacterium of embodiment 73, wherein: (i) the SSRRS comprise attB and attP sites and / or wherein the DNA molecule comprises 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 (ii) the agriculturally relevant compound is ammonia.

[0212] 77. The genetically engineered bacterium of embodiment 68, wherein the first heterologous gene expression cassette comprises a promoter flanked by two SSRRS in an inverted configuration,wherein said promoter is operably linked to the DNA molecule encoding a protein or RNA sequence of interest, and wherein the site-specific recombination can induce inversion of said promoter to uncouple the promoter from the DNA molecule and to reduce expression of the protein or RNA sequence of interest.

[0213] 78. The genetically engineered bacterium of embodiment 68, wherein the DNA molecule encoding the protein or RNA sequence of interest is flanked by tw o SSRRS in an inverted configuration, wherein the DNA molecule is operably linked to a promoter, and wherein the site-specific recombination can induce inversion of said DNA molecule encoding the protein or RNA sequence of interest to uncouple the DNA molecule from the promoter, thereby reducing expression of the protein or RNA sequence of interest.

[0214] 79. The genetically engineered bacterium of embodiment 68, wherein the first heterologous gene expression cassette comprises a promoter which is: (i) inoperably linked to a terminator flanked by two SSRRS in an inverted configuration; and (ii) operably linked to a DNA molecule encoding the protein or RNA sequence of interest, and wherein the site-specific recombination can induce inversion of said terminator to operably link the promoter to the terminator and to uncouple the DNA molecule from the promoter, thereby reducing expression of the protein or RNA sequence of interest.

[0215] 80. The genetically engineered bacterium of embodiment 68, wherein the DNA molecule encoding the protein or RNA sequence of interest comprises a first SSRRS comprising an in-frame open reading frame (ORF), a part of the DNA molecule encoding a part of the protein or RNA sequence of interest, and a second SSRRS comprising an in-frame ORF, wherein the protein or RNA sequence is functional, and wherein the site-specific recombination can induce excision of the part of the DNA molecule and one SSRRS to reduce activity of the protein or RNA sequence of interest.

[0216] 81. The genetically engineered bacterium of embodiment 68. wherein: (i) the DNA molecule encoding the protein or RNA sequence of interest comprises an inserted first SSRRS comprising an in-frame open reading frame (ORF) and wherein the protein or RNA sequence is functional; and (ii) the genetically engineered bacterium further comprises a blocking DNA covalendy linked to at least one SSRRS; wherein the sitc-spccific recombination can induce insertion of the blocking DNA into the DNA molecule to reduce expression and / or activity of the protein or RNA sequence of interest.

[0217] 82. The genetically engineered bacterium of embodiment 68, wherein the protein of interest comprises a GlnA. AmtB, GlnB, GlnK, GlnZ, NifL, and / or DraT protein and the agriculturally relevant compound is ammonia.

[0218] 83. The genetically engineered bacterium of embodiment 65, wherein the irreversible insertion, excision, or inversion of the DNA molecule comprising the control element or the DNA molecule encoding the protein or RNA sequence of interest increases expression of the protein orRNA sequence of interest in comparison to the first heterologous gene expression cassette lacking the irreversible insertion, excision, or inversion of the DNA molecule.

[0219] 84. The genetically engineered bacterium of embodiment 83, wherein the irreversible insertion, excision, or inversion of the DNA molecule causes the expression of the protein or RNA sequence of interest to be increased: (i) at least 2-fold, 5-fold or at least 10-fold; or (ii) about 2-fold or 5-fold to about 15-fold, both in comparison to the first heterologous gene expression cassette lacking the irreversible insertion, excision, or inversion of a DNA molecule.

[0220] 85. The genetically engineered bacterium of embodiment 83, wherein the control element comprises a promoter, wherein the promoter is flanked by two SSRRS in an inverted configuration, and wherein the promoter is operably linked to the at least one protein or RNA sequence of interest upon inversion by the site-specific recombinase.

[0221] 86. The genetically engineered bacterium of embodiment 83, wherein the control element comprises a first promoter and a second promoter directed opposite one another, wherein the control element is flanked by two SSRRS in an inverted configuration, wherein the first promoter of the control element is operably linked to the DNA molecule encoding at least one protein or RNA sequence of interest prior to inversion by the site-specific recombinase, wherein transcription promoting-activity of the second promoter is at least 2-, 5-, or 10-fold greater than the transcription promoting-activity of the first promoter, and wherein the second promoter of the control element is operably linked to the at least one protein or RNA sequence of interest upon inversion by the site-specific recombinase.

[0222] 87. The genetically engineered bacterium of embodiment 86, wherein:(i) the first promoter comprises a constitutive promoter, a native promoter of the native gene encoding the protein or RNA sequence of interest, or a promoter having activity equivalent to or lower than the native promoter; and / or (ii) the protein of interest is a NifA protein, NtrC protein, GlnR protein, or modified GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity.

[0223] 88. The genetically engineered bacterium of embodiment 83, wherein the control element comprises a promoter, wherein the DNA molecule encoding the protein or RNA sequence of interest is flanked by two SSRRS in an inverted configuration, and wherein the DNA molecule encoding the protein or RNA sequence of interest is operably linked to the control element upon inversion by the site-specific recombinase.

[0224] 89. The genetically engineered bacterium of embodiment 83, wherein the control element comprises a terminator, wherein the terminator is flanked by two SSRRS in a direct configuration, wherein the terminator and two SSRRS are between the at least one DNA molecule encoding protein or RNA sequence of interest and a promoter in the first heterologous gene expression cassette, and wherein the DNA molecule encoding the protein or RNA sequence of interest isoperably linked to the promoter in the first heterologous gene expression cassette upon excision of said terminator.

[0225] 90. The genetically engineered bacterium of embodiment 83, wherein the control element comprises a terminator, wherein the terminator is flanked by two SSRRS in an inverted configuration, wherein the terminator and two SSRRS are between the DNA molecule encoding at least one protein or RNA sequence of interest and a promoter in the first heterologous gene expression cassette, and wherein the DNA molecule encoding the protein or RNA sequence of interest is operably linked to the promoter in the first heterologous gene expression cassette upon inversion of said terminator.

[0226] 91. The genetically engineered bacterium of embodiment 66, wherein the first part of first heterologous gene expression cassette comprises the first part of the DNA molecule encoding the first part of the protein or RNA sequence of interest, wherein the SSRRS is located at the beginning of, within, or at the end of the first part of the DNA molecule, wherein the genetically engineered bacterium further comprises a third heterologous gene expression cassette comprising a sitespecific recombinase recognition site and the second part of the DNA molecule encoding the second part of the protein or RNA of interest absent from the first heterologous gene expression cassette, and wherein the first and second part of the DNA molecule encoding the protein or RNA sequence of interest are operably linked to provide a functional protein or RNA sequence of interest upon insertion of the second part of the DNA molecule into the first heterologous gene expression cassette.

[0227] 92. The genetically engineered bacterium of embodiment 66, wherein the first part of the first heterologous gene expression cassette comprises the DNA molecule encoding the protein or RNA sequence of interest but lacks a control element, wherein the SSRRS is located at the beginning of, within, or at the end of the first part of the DNA molecule, wherein the genetically engineered bacterium further comprises a third heterologous gene expression cassette comprising a sitespecific recombinase recognition site and a control element, and wherein the control element and the DNA molecule encoding the protein or RNA sequence of interest are operably linked to provide a functional first heterologous gene expression cassette upon insertion of the control element into the first heterologous gene expression cassette.

[0228] 93. The genetically engineered bacterium of embodiment 83, 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 adenylyltransf erase (uAT) activity and the agriculturally relevant compound 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 agriculturallyrelevant compound is ammonia;(c) a GlnR protein and the agriculturally relevant compound is ammonia; (d) a glutaminase enzy me and the agriculturally relevant compound is ammonia;(e) a protein product of a refactored nif or fix gene cluster and the agriculturally relevant compound 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 (cl), 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, niJL, and / or draT gene and the agriculturally relevant compound is ammonia; or (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 agriculturally relevant compound is ammonia.

[0229] 94. The genetically engineered bacterium of embodiment 65, wherein: (i) the first heterologous gene expression cassette comprises a control element comprising a promoter flanked by two SSRRS oriented as inverted repeats, wherein the promoter is operably linked to a first DNA molecule encoding a first protein or RNA sequence of interest, wherein the promoter flanked by two SSRRS is uncoupled from a second DNA molecule encoding a second protein or RNA sequence of interest; and (ii) the site-specific recombination can induce the inversion of the DNA molecule comprising the promoter to uncouple the promoter from the first DNA molecule, to operably link the promoter to the second DNA molecule, and to reduce expression of the first protein or RNA sequence of interest and increase expression of the second protein or RNA sequence of interest, both in comparison to the first heterologous gene expression cassette of (i) lacking the irreversible inversion of the DNA molecule.

[0230] 95. The genetically engineered bacterium of embodiment 66, wherein: (i) the first heterologous gene expression cassette comprises a control element comprising a promoter which is operably linked to a first DNA molecule encoding a first protein or RNA sequence of interest which is operably linked to an SSRRS, wherein the promoter is uncoupled from a second DNA molecule encoding a second protein or RNA sequence of interest which is operably linked to a terminator element and an SSRRS; and (ii) the site-specific recombination can induce the insertion of the second DNA molecule encoding the second protein or RNA sequence of interest to uncouple the promoter from the first DNA molecule, to operably link the promoter to the second DNA molecule,and to reduce expression of the first protein or RNA sequence of interest and increase expression of the second protein or RNA sequence of interest, both in comparison to the first heterologous gene expression cassette of (i) lacking the irreversible inversion of the DNA molecule.

[0231] 96. The genetically engineered bacterium of embodiment 94, wherein: (a) the first protein of interest comprises a wild-type glutamine synthetase (GS) or variant thereof with improved catalytic activity in comparison to wild-type GS and the second protein of interest comprises a wild-type GS with reduced levels of expression in comparison to the wild-type GS or a GS variant with decreased catalytic activity in comparison to wild-type GS, and the agriculturally relevant compound is ammonia; (b) the first protein of interest comprises a GS adenylyltransferase protein and the second protein of interest comprises a GS adenylyltransferase protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, and the agriculturally relevant compound is ammonia; or (c) the first target gene is a nifL gene and the second target gene is a nifA gene, and the agriculturally relevant compound is ammonia.|00232|97. The genetically engineered bacterium of any of embodiments 65 to 96 wherein said change of at least one condition in the plant growth medium condition comprises: (a) growth of the genetically engineered bacterium to exceed a threshold population density in the plant growth medium; (b) reduction of at least one fertilizer or plant nutrient in the soil, 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;(c) growth of a plant in the plant growth media;(d) change in soil temperature;(e) change in motility of the bacterium;(f) change in light levels in the soil;(g) change in oxygen concentration in the soil;(h) change in concentration of bioavailable nitrogen in the soil; (i) change in pH in the soil;(j) change in overall solute concentration (osmotic pressure) in the soil; (k) change in phytohormone or plant signaling molecule concentrations ;(1) change in an amino acid concentration;(m) change in a sugar concentration; and / or(n) a change in plant or microbial metabolite concentrations, optionally wherein the plant or microbial metabolite is naringenin, quercetin, luteolin, apigenin, octopine, nopaline, or Scyllo-inosamine.

[0233] 98. The genetically engineered bacterium of any one of embodiments 65 to 97, wherein said promoter activated by a change of at least one plant growth medium condition comprises: (i) a phosphate-sensitive promoter; (ii) a nitrogen -sensitive promoter; (iii) a quorum sensing promoter; (iv) a promoter which is induced or repressed by decreased oxygen levels; (v) a promoter which is induced or repressed by increased oxygen levels; (vi) a promoter which is induced by decreased oxygen and decreased nitrogen levels, optionally wherein the promoter is a nifH promoter; (vii) a promoter which is induced or repressed by increased or decreased potassium levels; (viii) a promoter which is induced by decreased carbon levels, optionally wherein the promoter is a Pfic promoter;(ix) a promoter that is induced by increased nitrate levels, optionally wherein the promoter is an oxygen insensitive PnarG promoter; (x) apromoter that is induced by increased nitrate levels, optionally wherein the promoter is a narK or narX promoter; (xi) a promoter which is upregulated by an increase in the concentration of naringenin; and / or (xii) a promoter or system comprising a promoter set forth in Table 5, Table 6, or Table 7.

[0234] 99. The genetically engineered bacterium of embodiment 98, wherein the promoter which is induced by decreased oxy gen and decreased nitrogen levels is a NifH promoter.

[0235] 100. The genetically engineered bacterium of embodiment 99, wherein the NifH promoter comprises a DNA molecule having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 595 or 596.

[0236] 101. The genetically engineered bacterium of embodiment 98, wherein the genetically engineered bacterium comprises the phosphate-sensitive promoter which is operably linked to the recombinase and wherein: (a) the threshold concentration of phosphate in the plant growth medium which activates expression of the recombinase is about 0 pM to about 50 pM; (b) the phosphate-sensitive promoter and / or a segment of the 5’ untranslated region (UTR) which is operably linked to the phosphate-sensitive promoter comprises at least one copy of an operably linked Pho box, wherein the Pho box comprises the sequence of SEQ ID NO: 525-529, 234-396, or 397 and sequences with at least 95% identity to any one of SEQ ID NO: 234-397. optionally wherein the Pho box comprises SEQ ID NO: 525-529, 376. 377, 383-396. or 397; (c) the phosphate-sensitive promoter and / or a segment of the 5’ UTR comprises a promoter and / or a segment of a 5’UTR of a phoA, phoX, phy, pstS gene, a variant thereof, or a combination thereof; and / or (d) the phosphate-sensitive promoter and / or the segment of the 5’ UTR comprises a promoter of a gene encoding: (i) a PstS protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 523, 399, 401. 403, 405, 407, 409. 415, 416, 513, 523, and 524 or having an identity of at least 76%, 80%, 85%. 90%, 95%, 98%. or 99% with any one of SEQ ID NO: 399. 401, 403, 405, 407, 409, 415, 416, 513, or 524; (ii) a PhoX protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 408, 410, 411, 412, and 413 or haying an identity of at least 80% yvith any one of SEQ ID NO: 408, 410, 411, 412, and 413; (iii) a Phy protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 414, 57-138, and 139 or haying an identity of at least 80% yvith any one of SEQ ID NO: 414, 57-138, or 139; and / or (iv) a PhoA protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 398, 400, 402, 404, and 406 or having an identity of at least 80% with any one of SEQ ID NO: 398, 400. 402, 404, or 406.

[0237] 102. The genetically engineered bacterium of embodiment 101, wherein the phosphate-sensitive promoter and / or the segment of the 5‘ UTR comprises: i. a pstS promoter comprising a DNA sequence selected from the group consisting of SEQ ID NO: 515, 417, 418, 420. 421, 422, 423, 424, 425, 428. 517, and 518; ii. a phoX promoter comprising the DNA sequence of SEQ ID NO: 426; iii. a phy promoter comprising the DNA sequence of SEQ ID NO: 427; iv. a phoA promoter comprising the DNA sequence of SEQ ID NO: 419; and / or v. a Pliar53 promotercomprising the DNA sequence of SEQ ID NO: 459; vi. a variant of the promoters under i. to v. having an identity of at least 76%, 80%, 90%, 95%, 98%, or 99% with any of SEQ ID NO: 515, 417, 418-428, 459, 517, or 518, wherein said variant comprises at least one Pho box and / or retains at least one Pho box present in each promoter and wherein said variant promoters arc activated by a decrease in phosphate concentration to about 0 pM to about 1 pM. 2 pM, 5 pM, 7 pM, 10 pM, 20 pM, 30 pM, 40 pM, or 50 pM.

[0238] 103. The genetically engineered bacterium of embodiment 101, wherein the bacterial pstS gene encodes a protein having at least 76%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 513 and comprises the polypeptide of SEQ ID NO: 514.

[0239] 104. The genetically engineered bacterium of embodiment 101, wherein the phosphate-sensitive promoter comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 517.

[0240] 105. The genetically engineered bacterium of embodiment 98, wherein the genetically engineered bacterium comprising the quorum sensing promoter which is operably linked to the recombinase further comprises: (a) one or more heterologous gene expression cassette(s) comprising at least one control element which is operably linked to at least one DNA molecule encoding a first quorum sensing synthase protein (QSSP) able to synthesize a quorum sensing signal molecule Q (QSSM Q) and / or at least one DNA molecule encoding a first quorum sensing regulator protein (QSRP) that can bind said QSSM Q, wherein the first quorum sensing promoter can be 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 (b) 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 recombinase 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.

[0241] 106. The genetically engineered bacterium of embodiment 105, wherein the deactivator comprises an enzyme which catalyzes the degradation of the quorum sensing signal molecule Q and wherein the expression of the enzy me is induced by the addition of the quorum quenching compound QQ or the increase in temperature.

[0242] 107. The genetically engineered bacterium of embodiment 106, 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.

[0243] 108. The genetically engineered bacterium of embodiment 107, 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: 537, 538, 539, or 540,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: 541, 542, or 543, 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: 549, 550, 551. 552, or 553, respectively; or (iv) the AHL lactonase enzyme comprises a metallo-beta-lactamase protein, optionally wherein the metallo-beta-lactamase protein is a Gel, AttM, AidC, AiiB, or AiiA protein and optionally wherein the Gel, AttM. AidC, AiiB. or AiiA protein has at least 75% sequence identity to SEQ ID NO: 544, 545, 546, 547, or 548, respectively.

[0244] 109. The genetically engineered bacterium of embodiment 105, wherein the deactivator comprises an inducible transcription factor which:|00245] (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

[0246] (ii) decreases expression of the RNA or protein of interest control element comprising the first quorum sensing promoter.

[0247] 110. The genetically engineered bacterium of embodiment 105, 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-((3- Ketocaproyl)-L-homoserine lactone and the first QSSP comprises an Ahll protein; or (iv) the QSSM Q molecule comprises N-(P-Ketocaproyl)-L-homoserine lactone and the first QSRP comprises an AhlR protein.

[0248] 111. The genetically engineered bacterium of embodiment 105, wherein the quorum quenching compound QQ is inorganic phosphate or soluble phosphate, wherein the control element operably linked to the gene encoding tire first QSRP comprises a phosphate-sensitive promoter.

[0249] 112. The genetically engineered bactcrirmr of embodiment 105, wherein: i. the first QSSP comprises an Ahll protein, the first QSRP comprises an AhlR protein, and the first quorum sensing promoter comprises an ahll promoter; ii. the first QSSP comprises an CinI protein, tire first QSRP comprises a CinR protein, and the first quorum sensing promoter comprises a cinl promoter; iii. the first QSSP comprises an Ceil protein, the first QSRP comprises a CciR protein, and the first quorum sensing promoter comprises a ceil promoter; iv. the first QSSP comprises an Cvil protein, the first QSRP comprises a CviR protein, and the first quorum sensing promoter comprises a evil promoter; v. the first QSSP comprises an Esal 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 Esal protein, the first QSRPcomprises a EsaR protein, and the first quorum sensing promoter comprises an esal promoter; vii. the first QSSP comprises a Last protein, the first QSRP comprises a LasR protein, and the first quorum sensing promoter comprises a Iasi or lasB promoter; viii. the first QSSP comprises an LuxI protein, the first QSRP comprises a LuxR protein, and the first quorum sensing promoter comprises a luxl promoter; or ix. the first QSSP comprises an Tral protein, the first QSRP comprises a TraR protein, and the first quorum sensing promoter comprises a tral promoter.

[0250] 113. The genetically engineered bacterium of embodiment 112, wherein: (i) the Alill protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 565; (ii) the Ceil protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 566; (iii) the CinI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 567; (iv) the CinI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 568;(v) the Cvil protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 569; (vi) the Esal protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 570; (vii) the LasI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 593; (viii) the Luxl protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 571; or (ix) the Tral protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 572.

[0251] 114. The genetically engineered bacterium of embodiment 105. 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.

[0252] 115. The genetically engineered bacterium of embodiment 105, 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.

[0253] 116. The genetically engineered bacterium of embodiment 105, wherein the first QSRP comprises an AhlR, CciR, CinR, CviR, EsaR, LasR, LuxR, or TraR QSRP.

[0254] 117. The genetically engineered bacterium of embodiment 116, 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: 556, 557, 558 or 559, 560, 561, or 562, 592, 563. or 564, respectively.

[0255] 118. The genetically engineered bacterium of any one of embodiments 65 to 117, wherein the AUB is a diazotrophic and / or phosphate-solubilizing bacterium.

[0256] 119. The genetically engineered bacterium of any one of embodiments 65 to 118, wherein the AUB is a member of the genus Azospirillum, Enterobacter, Klebsiella, Kosakonia, Rahnella, Paenibacillus, Phytobacter, Rahnella, Sphingomonas, or Variovorax, wherein the bacterium is optionally a member of the genus Azospirillum, Klebsiella, Kosakonia, or Rahnella.

[0257] 120. The genetically engineered bacterium of of any one of embodiments 65 to 96, wherein the AUB is selected from the taxonomic class of Gammaproteobacteria.

[0258] 121. The genetically engineered bacterium of embodiment 120, wherein the Gammaproteobacteria is a Kosakonia sp., Entcrobactcr sp., Klebsiella sp., Ralmclla sp., Pseudomonas sp., or Acinetobacter sp..

[0259] 122. The genetically engineered bacterium of any one embodiments 65 to 121, wherein the recombinase 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: (i) attB and attP sites; or (ii) attL or attR sites; wherein the (i) attB and attP sites; or (ii) attL or attR sites recognized respectively by the PhiC31, IntS, IntM, IntG, YdcL, or Sxt / R39 integrase.

[0260] 123. The genetically engineered bacterium of embodiment 122, wherein the recombinase is a phage PhiC31 serine integrase having at least 90%. 95% . or 99%sequence identity to SEQ ID NO: 41 and the SSRRS comprise an attP of SEQ ID NO: 42 and an attB recombinase recognition site of SEQ ID NO: 43. or functional equivalents thereof recognized by the phage PhiC31 serine integrase.

[0261] 124. The genetically engineered bacterium of any one of embodiments 65 to 123, wherein: (i) the recombinase is a yeast flippase (FLP) recombinase and the SSRRS are FRT sites; or (ii) the recombinase is a Cre-recombinase and the SSRRS are loxP sites.

[0262] 125. A method of producing a bacterial culture comprising: (i) growing the genetically engineered bacterium of any one of embodiments 65 to 124 either: (a) in contact with the quorum quenching compound QQ; (b) in exposure to a temperature above the threshold temperature; or (c) or under conditions where expression or activity7of the recombinase is suppressed; and (ii) harvesting the bacterial culture.

[0263] 126. An agricultural system comprising: (i) a plant growth medium; (ii) at least one plant of an agronomically relevant plant species rooted in said plant growth medium; and (iii) the genetically engineered bacterium of any one of embodiments 65 to 124.

[0264] 127. The system of embodiment 126, wherein the plant is an alfalfa, apple, banana, barley, bean, buckwheat, cabbage, cassava, chili, clover, coffee, com, 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, tye, safflower, sorghum, soybean, sugar beet, sugar cane, sunflower, tangerine, tobacco, tomato, triticale, turnip, wheat, or yam plant, seed, or vegetative propagule.

[0265] 128. The system of embodiment 127, wherein the plant growth medium comprises soil and / or water, optionally wherein the soil and / or water is non-axenic.

[0266] 129. The system of embodiment 127, wherein the vegetative propagule comprises a cutting, tuber, or stolon.

[0267] 130. A composition comprising the genetically engineered bacterium of any one of embodiments 65 to 124 and an agriculturally acceptable carrier.

[0268] 131. The composition of embodiment 130, wherein the composition further comprise s:(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.

[0269] 132. The composition of embodiment 130, wherein the composition is in a solid form, optionally wherein the solid form comprises a wettable powder, granules, a gel, pellets, or microencapsulated particles.

[0270] 133. The composition of embodiment 130, 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.

[0271] 134. A plant part or plant propagule which is at least partially coated, imbibed, or mixed with the composition of embodiment 130.

[0272] 135. The plant part of embodiment 134, wherein the part is a leaf, stem, root, or seed.

[0273] 136. The plant propagule of embodiment 134. wherein the propagule comprises a cutting, tuber, or stolon.

[0274] 137. Use of the plant part or plant propagule of embodiment 134 to grow a crop.

[0275] 138. The use of embodiment 137, wherein fertilizer input is reduced in comparison to a crop grown from a plant part or plant propagule which has not been treated.

[0276] In the description, tables, and numbered embodiments 1-138 set forth herein, genes and the proteins they encode which are referred to solely by name include genes and proteins identified in and X and tire sequence listing, include genes having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity’ to the genes identified in TABLE 5, 6, and the sequence listing, and include proteins having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to the proteins identified in TABLE 5, 6, and the sequence listing. GlnE proteins lacking an adcnylyl 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%, 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%. 80%, 85%, 90%, 95%, 98%. or 99% identity to SEQ ID NO: 229. 230, or 460-512.Wild-ty pe 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.TABLE 5. Summary' of Biological Sequences in Sequence ListingTABLE 6. Summary of additional biological sequencesTABLE 7. Condition-sensitive promoters for control of recombinases.EXAMPLESExample 1. Identification and selection of phosphate-sensitive promoters and Pho boxes

[0277] The phosphate (Pho) regulon is a bacterial regulatory mechanism that senses and responds to changing availability and concentration of inorganic phosphate (Pi). The Pho regulon activates the expression of extracellular enzymes, phosphate-specific carriers, and enzymes involved in phosphate storage and preservation. The most conserved member of the Pho regulon in bacteria is the high affinity Pi transporter (Pst), and the most common enzymes induced in response to inorganic phosphate starvation in bacteria are alkaline phosphatases (PhoA, PhoX), phospholipases (PhoD), glycerophosphodiester phosphodiesterases, phytases (Phy). 5 ’-nucleotidase, and the Pst itself.

[0278] The Pho regulon is controlled by a two-component regulatory system that includes an inner membrane histidine kinase sensor protein and a regulator of the cytoplasmic transcriptional response. These proteins are named differently in some bacteria, for example, PhoR-PhoB in Escherichia coli. PhoR-PhoP in Bacillus subtilis, PnpR-PnpS in Streptococcus pneumoniae . In all cases of inorganic phosphate deficiency, the response regulator is phosphorylated at an aspartic acid residue by the sensor kinase. The phosphorylated response regulator binds to specific DNA sequences and activates or inhibits gene transcription.

[0279] These specific regulator-binding DNA sequences that facilitate the activation or inhibition of gene expression in a phosphate-sensitive maimer are termed Pho boxes. Pho boxes have been identified using ChlP-Seq, sequence similarity, and RNA-Seq in Escherichia coli and Streptomyces coelicolor (Allenby et al. (doi: 10.1093 / nar / gks766) and Fitzgerald et al. (doi: 10.1128 / mbio.02535-22), respectively; SEQ ID NO: 234-375).

[0280] Beyond the two-component PhoR-PhoB system, phosphate-sensitive bacterial responses involve additional proteins. By way of example, the E. coli inorganic phosphate uptake pathway requires five more proteins, four of them being the components of the Pst and one a component of the PhoU metal-binding protein.

[0281] Depleting inorganic phosphate in a growth medium activates the Pho regulon in bacteria in the medium. When the concentration of inorganic phosphate decreases, PhoB is activated by PhoR acting as a kinase, but under conditions of excess free phosphate, PhoB activation is interrupted by PhoR acting as a phosphatase. PhoU is required for PhoB dephosphorylation under phosphate -rich conditions. Although PhoU is found in many bacterial genomes, this gene is absent in B. subtilis. The inorganic phosphate-signaling network in this bacterium includes a positive feedback loop between the PhoP-PhoR and ResD-ResE two-component systems. ResD does not bind to the phoPR operon and appears to transfer its control through the expression of terminal oxidases.

[0282] To express an RNA sequence or protein of interest in a phosphate-sensitive manner in agriculturally relevant bacteria, promoters were constructed that are activated by decreasing phosphate concentration. Initially, sequence alignments were calculated for published Pho boxes to identify a consensus sequence so that additional phosphate-sensitive promoters could be identified simply by searching for sequences similar to the consensus sequence in agriculturally relevant bacteria. However, poor homology among published Pho boxes in bacteria of the same species made this approach unsuccessful. As a result, two distinct approaches were pursued to determine lists of Pho boxes that could be searched to find phosphate-sensitive promoters in agriculturally relevant bacteria.

[0283] In the first approach adapted from Torres-Bacete et al. (doi: 10.1111 / 1751-7915.13808), a DNA motif WebLogo and position-by -position DNA base probability were calculated from known Pho boxes. Based on these statistics, degenerate primers were synthesized to create a library of alternative Pho boxes via polymerase chain reaction (PCR) amplification of a DNA template comprising the sequences based on the aforementioned WebLogo. To construct a library of phosphate-sensitive reporter circuits, the DNA library’ of alternative Pho boxes above was integrated into the -35 region of a constitutive promoter (BG42), which was operably linked to a strong ribosome binding site (BCD2) and a green fluorescent protein (GFP) gene. The library' of phosphate-sensitive GFP reporter circuits was then screened in Pseudomonas and Escherichia strains at varying phosphate concentrations to determine the Pho box and phosphate-sensitive promoter with the greatest response to changes in extracellular phosphate concentration. The Pho box from the maximally responding promoter was subsequently searched using the US National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) to identify naturally occurring Pho boxes with high sequence similarity to the Pho box of the maximally responding phosphate-sensitive promoter identified above. Those promoters were screened for ability to induce phosphate-sensitive changes in gene expression as in EXAMPLE 2.

[0284] In the second approach, phosphate-sensitive promoter sequences were bioinformatically extracted from agriculturally relevant bacteria. Bacterial species across a range of agriculturally relevant and model taxa were selected, similar to those described by Santos-Beneit (doi: 10.3389 / fmicb.2015.00402), Grillo-Pucrtas et al. (doi: 10.3389 / fmicb.2021.666277), and Torrcs- Bacete et al. (doi: 10.1111 / 1751-7915.13808). Taxa searched for phosphate-sensitive promoters included both gram-positive bacteria such as Bacillus subtilis, Paenibacillus azotofixans, and Paenibacillus durus as well as gram-negative bacteria. Among gram-negative bacteria, several classes of soil bacteria were searched for phosphate-sensitive promoters, including: alphaproteobacteria like Azospirillum brasilense, Azorhizobium caulinodans, Gluconacetobacter diazotrophicus, Maritimibacter alkaliphilus, and Yoonia vestfoldensis', betaproteobacteria like Aromatoleum aromaticum, Azoarcus olearius, and Herbaspirillum seropedicae'. and gammaproteobacteria like Escherichia coli, Kosakonia radicincitans, Pseudomonas aeruginosa, and Pseudomonas stutzeri. Genes of each bacterial Pho regulon were identified because these genes are known to have phosphate-sensitive expression. The presence of Pho boxes in each of the identified Pho regulon genes was confirmed by sequence similarity to Pho boxes described in Allenby et al. (doi: 10.1093 / nar / gks766). Pho boxes present in agriculturally relevant bacteria are included as SEQ ID NO: 376-397. CLUSTAL alignment performed using Mview with default parameters showed that no consensus sequence could be easily constructed for these Pho boxes.

[0285] Since no consensus sequence emerged using the second approach, phosphate-sensitive promoters were identified by determining the DNA regions immediately upstream of the proteincoding regions of Pho regulon genes in the agriculturally relevant bacterial taxa. The proteincoding sequences of certain Pho regulon genes (given by SEQ ID NO: 398-416, TABLE 7) were determined by query ing the US National Center for Biotechnology Information (NCBI) Protein database. The sequence similarity among homologous Pho regulon genes was determined by CLUSTAL alignment using the M-Coffee multiple sequence aligner with default parameters, and multiple sequence alignment statistics were calculated using Mview (SEQ ID NO: 398-416, TABLE 8). Subsequently, promoter DNA sequences were extracted by locating regions immediately upstream of the start codon of the phoA, phoX, phy, and pstS genes associated with SEQ ID NO: 398-416, TABLE 8. DNA regions selected as promoters can comprise about 100 bp upstream of a relevant start codon but at least 50 bp upstream of the start codon and frequently about 50 to about 250 bp upstream of the relevant start codon (SEQ ID NO: 417-428, TABLE 9).TABLE 8. Protein-coding sequences of Pho regulon genes in agriculturally relevant and model bacteria. Rows are organized by different bacterial species or strains. Columns are organized by different Pho regulon proteins. Cells contain the SEQ ID NO of each protein’s amino acid sequence. “Absent” indicates that the bacterial species or strain docs not have a gene encoding a specific Pho regulon protein. “Not tested” indicates that a specific species was not searched for a gene encoding a specific Pho regulon protein.TABLE 9. DNA regions upstream of Pho regulon coding sequences identified as phosphatesensitive promoters. Rows are organized by different bacterial species or strains. Columns are organized by Pho regulon genes. Cells contain promoter SEQ ID NO and any relevant Pho box SEQ ID NO. “Not tested” indicates that a specific species was not searched for a specific Pho regulon gene promoter.Example 2. Engineering Bacteria to Express a Reporter Gene in Response to DecreasingPhosphate Concentration

[0286] To generate strains that express a GFP reporter gene in response to phosphate depletion, the pstS promoter regions of Azospirillum brasilense Sp245 (SEQ ID NO: 418), Herbaspirillumseropedicae Z67 (SEQ ID NO: 422), Pseudomonas stutzeri DSM 4166 (SEQ ID NO: 428), and Kosakonia radicincitans DSM16656 (SEQ ID NO: 423-424) were PCR amplified from genomic DNA, and the synthetic promoter Pliar53 (SEQ ID NO: 459) was synthesized, each with HiFi adapters for cloning into an entry plasmid digested with Noth The cloning site was designed such that the Pi-dependent promoters were positioned upstream of GFP. Plasmids were confirmed by next generation sequencing and conjugated with aid of the helper strain pRK2013 from the E. coli DH5a donor strains into the strains listed above. Plasmid recipients were confirmed by antibiotic selection and by presence of constitutive LSSmScarlett fluorescence which was additionally conveyed by the cloning plasmid.

[0287] To confirm that pstS and Pliar53 promoters carried by Azospirillum brasilense Sp245, Herbaspirillum seropedicae Z67, Pseudomonas stutzeri DSM 4166, and Kosakonia radicincitans DSM 16656 were specifically activated under Pi depleted conditions, expression from these promoters was measured in cultures (n=3) incubated for 15 hours in nitrogen-free basal high phosphate (NFbHP) media with or without addition of 500 uM potassium phosphate. In these experiments, each pstS and Pliar53 promoter was fused to the reporter gene GFP on a plasmid also carrying 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 1). In all strains tested, expression was activated from the yzs / S and Pliar53 promoters greater than 10-fold in the absence of Pi compared to where Pi was added into the media (FIGURES 2-3)

[0288] To test whether depletion of Pi in the growth media over time would result in activation of pstS and Pliar53 promoters in Azospirillum brasilense Sp245, Herbaspirillum seropedicae Z67, Pseudomonas stutzeri DSM 4166, and Kosakonia radicincitans DSM16656, cultures (M=3) were thrice washed and inoculated into flat bottomed 96-well plates containing 100 pL of NFbHP supplemented with 100 uM starter potassium phosphate. Cultures were incubated with shaking and GFP / LSSmScarlett fluorescence was read at defined time intervals. In these experiments, each pstS and Pliar53 promoter was fused to the reporter gene GFP on a plasmid also carrying 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 1). At each interval, cultures were pelleted by centrifugation after and Pi concentration was measured in the cleared supernatant using a Sigma Aldrich Phosphate Assay Kit (Cat MAK308), as per the manufacturer recommendations. In all strains tested. Pi was completely depleted in the media between 0-24 h, whereas expression was induced in all cases only once the Pi concentration dropped below 50 uM (FIGURE 4A, B, C, and D). Greatest phosphate-sensitive activation of the GFP reporter gene occurred when native pstS promoters derived from each tested strain were usedas the control element in the heterologous gene expression cassette, and the heterologous gene expression cassette w as integrated at a chromosomal locus distinct from the locus of the endogenous phosphate-sensitive promoter.Example 3. Inducing Reporter Gene Expression in Model Soil Bacteria in Response to Decreasing Phosphate Concentration in a Plant Growth Medium

[0289] Promoters induced by low Pi concentration activated GFP expression on plant roots following Pi depletion in a sterile sand plant growth medium. Surface sterilized com seeds were sowed into sterilized 250 mL Schott bottles filled with 200 g of washed sand and 30 mL of Pi-free Hoagland solution. Seeds were germinated in the Schott bottles for 3 days in a growth room set with the following parameters: light intensity 250 pmol / nr / s at 7 inches from the floor; 16:8 light cycles with 30 minute sunrise / sunset dimming; 50-55% humidity. Plants were then inoculated with 1 x 105cells of A. brasilense Sp245 carrying a PstS::GFP reporter plasmid (EXAMPLE 2, FIGURE 1) with constitutive LSSmScarlett reporter, as confirmed by viable CFU counts. The inoculant cells were prepared by sub-culturing single colonies into NFbHP media with 50 mM potassium phosphate to repress PstS::GFP, 20 mM ammonium chloride, and relevant antibiotics, then incubating overnight at 30°C with shaking. The following day. cells were harvested by centrifugation and washed twice in NFbHP and resuspended in NFbHP containing 500 uM potassium phosphate to repress the PstS: :GFP reporter and provide starter Pi for the plant experiment. After inoculation, plants were incubated in the growth room and sampled at 1, 3, 6. 20. and 40 days post inoculation (dpi). For each sampling point, three plant replicates were destructively uprooted and loose sand was discarded. The roots were then excised from the shoot at the cotyledon and vortexed in water to create a bacterial cell suspension. This fraction was defined in these experiments as the rhizosphere. To measure Pi concentration in the samples, aliquots of the cell suspensions were centrifuged at high speed to remove all solids, and the cleared supernatant was assayed using a Sigma Aldrich Phosphate Assay Kit (Cat MAK308). To isolate bacterial cells for analysis of the PstS::GFP reporter, plant and sand debris remaining in the rhizosphere suspension were selectively cleared by centrifugation at low speed, and one hundred microliter aliquots of the supernatant containing the bacterial cells were analyzed by flow' cytometry .

[0290] Because the PstS: :GFP reporter plasmid carried a constitutively expressed LSSmScarlett reporter protein, flow' cytometry events corresponding to bacterial cells could be gated based on LSSmScarlett fluorescence above the background (here defined as LSSmScarlett+), as validated by Haskett et al. (doi: 10.3389 / fmicb.2021.690439). The percentage of the LSSmScarlett+ population activated for PstS::GFP expression at each time point was quantified by establishing PstS::GFP and “+” threshold fluorescence values for the bacteria. This was done by supplementing one experimental treatment with 50 mM potassium phosphate at the time of inoculation to repress expression from PstS::GFP reporter, then generating the GFP- / + threshold value as the upper 99th percentile of GFP fluorescence in this repressed state, as validated by Haskett et al. (doi: 10.1073 / pnas.2117465119).

[0291] As shown in FIGURE 5 and FIGURE 6. after 1-dpi, the phosphate concentration in the rhizosphere was determined to be approximately 100 uM, however, the bacterial cell density at this time point was below the detection range for analysis by flow cytometry’. After 3-dpi, the phosphate concentration in the rhizosphere fell to 40 uM, triggering a fraction of about 35% of the bacterial population to activate PstS::GFP and enter the GFP+ state. The concentration of Pi continued to decline to about 10 uM by 20-dpi, at which point approximately 70% of the bacterial population were expressing the PstS: :GFP reporter fusion, entering the GFP+ state. By 40-dpi, the Pi concentration remained at about 10 uM, and LSSmScarlett+ events were no longer detected in tire sample.Example 4. Inducing Reporter Gene Expression in Soil Bacteria in Response to Decreasing Phosphate Concentration in the Field

[0292] Promoters induced by low Pi concentration activated GFP expression in transformed Kosakonia sacchari (Ks) and Klebsiella variicola (Kv) soil isolates following Pi depletion in various North American field locations. The Kosakonia sacchari (Ks) and Klebsiella variicola (Kv) soil isolates carried a PstS: :GFP reporter plasmid (EXAMPLE 2, FIGURE 1) with constitutive LSSmScarlctt reporter essentially as described in Example 3. Pi-induction of GFP was repressed by addition of Pi for the transformed Kosakonia sacchari (Ks). Percentage of transformed Ks and Kv cells with low Pi concentration activated GFP expression is shown in Figure 12.Example 5. PpstS promoters activate GFP in response to low-Pi in sterile cultures of alpha-, beta-, and gamma-pro teobacteria and gram-positive Paenibacillus

[0293] Activation of GFP reporter expression driven by various PpstS promoters in response to low Pi in various bacteria was tested in sterile culture essentially as described in Example 2. The promoters that were tested are disclosed in TABLE 5. Promoters comprising ~300-bp upstream of the endogenous pstS gene in an Azospirillum brasilense, Kosakonia radicincitans. Herbasprillum seropedicae, Pseudomonas stutzeri, Kosakonia sacchari soil isolate, a Klebsiella varicolla soil isolate, and a Rahnella aquatilis soil isolate activated GFP expression in response to low Pi in response to Pi depletion as shown in Figure 13. Phosphate sensitive promoters were also tested in Paenibacillus graminis. Va PstSI and PstsII promoters for a Paenibacillus graminis soil isolate activate GFP expression in liquid media in response to Pi depletion (Ppst 1, Ppst2.1, and Ppst 2.2 in Figure 14).Example 6. Identifying Quorum Sensing Systems for Protein Expression in Soil Bacteria

[0294] 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.

[0295] 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 of 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.

[0296] 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.

[0297] 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 leguminosarunr, betaproteobacteria like Burkholderia cenocepacia and Chromobacterium violaceum and gammaproteobacteria like Aliivibrio flscheri. 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: 565-572, and 593. QSRP protein-coding sequences present in agriculturally relevant bacteria are included as SEQ ID NO: 556-564, and 592. Quorum sensing promoter DNA sequences presentin agriculturally relevant bacteria are included as SEQ ID NO: 573-581, 590, and 591. 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.

[0298] Next, species compatibility of heterologous QSRPs was screened across agriculturally relevant bacteria. Gene fragments encoding QSRP transcription factors (SEQ ID NO: 556, 557, 559, 560, 562, 563, 564) and their cognate QS promoters (SEQ ID NO: 573, 574, 576, 577, 579, 580, 581) 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: 432, TABLE 10) and the cognate QS promoter of the QSRP (FIGURE 15). Each gene expression cassette was cloned into a broad host range plasmid and transformed into Azospirillum brasilense Sp245, Herbaspirillum seropedicae Z67, Pseudomonas stutzeri DSM 4166, and Kosakonia radicincitans DSM16656 to assess functionality.TABLE 10. Genetic circuit elements used to construct heterologous gene expression cassettes.“RBS” denotes ribosome binding sites. ‘‘TERM’’ denotes terminators.

[0299] This library of AHL-responsive strains was then screened for QSRP activity in response to a saturating concentration (10 pM) of either N-(0-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 library7, and cultures were grown overnight at 30 degrees centigrade w ith orbital shaking for aeration. Following incubation, GFP expression was quantified viafluorimetry (TABLE 11). The AhlR (SEQ ID NO: 556), CinR (SEQ ID NO: 559), and CviR (SEQ ID NO: 560) QSRP transcription factors had activity across multiple agriculturally relevant bacterial strains.TABLE 11. 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) 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.

[0300] Subsequently, full QS systems including the QSSP and QSRP were built and tested across species of agriculturally relevant bacteria. Heterologous gene expression cassettes comprising: (i) genes encoding AhlR (SEQ ID NO: 556) and Ahll (SEQ ID NO: 565) derived from Pseudomonas syringae B7281a, (ii) genes encoding CinR (SEQ ID NO: 559) and CinI (SEQ ID NO: 568) derived from Rhizobium leguminosarum bv viceae 3841, or (iii) genes encoding LasR (SEQ ID NO: 592) and LasI (SEQ ID NO: 593) 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 (Ah). 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 Pahll (SEQ ID NO: 573), PcinI (SEQ ID NO: 576), PlasI (SEQ ID NO: 590), or PlasB (SEQ ID NO: 591) 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 16). These plasmids were mobilized into wild-type strains and those carrying mini-Tn7 integrated QS systems.

[0301] 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 17. 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 densitydependent 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: 559) and Cinl (SEQ ID NO: 568) 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: 556) and Ahll (SEQ ID NO: 565) and (ii) the cognate PahlI::GFP reporter plasmid transferred into ,4 A and Ps. but not Kr. The gene expression cassette comprising LasR (SEQ ID NO: 592) and LasI (SEQ ID NO: 593) 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 18).

[0302] Strains carrying heterologous QS systems were next assessed for population densitydependent 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 19). GFP / LSSmScarlett fluorescence was also assessed in the S+ population for each carbon treatment (FIGURE 20). The threshold population density at which each QS system caused each bacterial strain to produce the reporter protein of interest is given in TABLE 12.- I l l -TABLE 12. 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 expressed 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.Example 7. Population Density-Dependent Expression of a Reporter Gene by Engineered Soil Bacteria in a Plant Growth Medium

[0303] The genetically engineered Ab and Kr strains of EXAMPLE 6 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 com 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 eachplatcand washed twice in 1% KC1 prior to inoculation into plant growth media. Surface sterilized com 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 com 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 com plants grew to the VI vegetative growth stage.

[0304] 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% KC1 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 20mL of 1% KC1 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 pL aliquots of the resulting supernatants were analyzed by flow cytometry.

[0305] 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 com seeds were not planted (measured as cells g'1sand) was about 1000-fold lower than the bacterial population density observed on com roots (measured as cells g'1root) (FIGURE 21). Ab and Kr demonstrated population densitydependent induction of the GFP protein of interest on plant roots with the AhlRI and CinRl QS systems respectively (FIGURE 22). The threshold population density at which each QS system caused each bacterial strain to produce the reporter protein of interest is given in TABLE 13.TABLE 13. 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.TABLE 13.Example 8. Deactivation of Quorum Sensing in Engineered Soil Bacteria via Degradation of the Quorum Sensing Signal Molecule

[0306] 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 density7asa trigger for production of agriculturally relevant compounds facilitates sufficient bacterial grow th 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 * I O10cells / 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.

[0307] 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: 537-539, and 540), alpha-beta hydrolase fold lactonase proteins (SEQ ID NO: 541, 542, and 543), metallo-beta-lactamase-like lactonase proteins (SEQ ID NO: 544-547, and 548), and phosphotriesterase-like lactonase proteins (SEQ ID NO: 549-552, and 553).

[0308] 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 23). The first “QS circuit” gene expression cassette comprised control elements operably linked to a gene encoding the CinI QSSP (SEQ ID NO: 568) and a gene encoding the CinR QSRP (SEQ ID NO: 559) as well as the Pcinl quorum sensing promoter (SEQ ID NO: 576) 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: 542) 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 Pcinl 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.

[0309] A system of gene expression cassettes analogous to that of FIGURE 23 was built in Kosakonia radicincitans. Variant “QQ circuit” gene expression cassettes comprising a weak BCD22 (SEQ ID NO: 449). medium BCD13 (SEQ ID NO: 440), or strong BCD2 (SEQ ID NO:430) ribosome binding site operably linked to the gene encoding the QqlM QE were cloned into the reporter plasmid of EXAMPLE 1 and FIGURE 16 comprising Pcinl. The resulting plasmids were mobilized into a K. radicincitans strain comprising a chromosomally integrated cinRI “QS circuit” (SEQ ID NO: 535) encoding the CinR QSRP and the Cinl QSSP.

[0310] Subsequently, the quorum quenching selective QS deactivation system was assessed in bacterial culture conditions and in a plant growth medium (FIGURE 24). 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 com roots, and population density-dependent expression of a GFP reporter protein of interest was assessed by flow cytometry (FIGURE 24). 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.Example 9. Deactivation of Quorum Sensing in Engineered Soil Bacteria via Transcriptional Repression of the Quorum Sensing Regulator Protein

[0311] As described in EXAMPLE 8, 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.

[0312] 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).

[0313] 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 clts2 (SEQ ID NO: 554), which is active below 25 degrees centigrade but inactive above 25 degrees centigrade. Additionally, clts2 acts as a transcriptional repressor for the promoter PL (SEQ ID NO: 31) but a transcriptional activator for the promoter PRM (SEQ ID NO: 555). In combination with PL, clts2 achieves de-repression of a target gene at higher fermentation temperatures. In combination with PRM, clts2 achieves deactivation of a target gene at higher temperatures. Operably linking PR to the QSRP-encoding gene of a quorum sensing system while constitutively expressing clts2 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.

[0314] A third ty pe 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, 515, 517, 518). 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 system 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.

[0315] 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 densitydependent upon QS-promoter induction. To tune expression of the AhlR QSRP (SEQ ID NO: 556), 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 Pahll promoter (SEQ ID NO: 573) was operably linked to both the strong ribosome binding site BCD2 (SEQ ID NO: 430) 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 w as also operably linked to ribosome binding sites ofdiffering strength, wherein a first construct comprised the medium-strength ribosome binding site BCD17 (SEQ ID NO: 444) and a second construct comprised the weak ribosome binding site BCD22 (SEQ ID NO: 450). A schematic of the two separate constructs for tuning densitydependent AhlR expression are shown as FIGURE 25.

[0316] The constructs of FIGURE 25 were synthesized and cloned into the mini-Tn7 delivery plasmid pUC18R6K-mini-Tn7T-Gm digested at the Sad 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 Pahll QS promoter. Expression of the GFP reporter gene was quantified at different concentrations of exogenously added AHL quorum sensing signal molecule N-((3-Ketocaproyl)-L -homoserine lactone ranging from 0 pM to 10 pM using fluorescence spectroscopy (FIGURE 26). The construct of FIGURE 25 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).

[0317] Subsequently, sets of DNA constructs analogous to those of FIGURE 11 were designed additionally comprising a third gene expression cassette, wherein the QS promoter Pahll (SEQ ID NO: 573) was operably linked to both a gene encoding the QSSP Ahll (SEQ ID NO: 565) 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: 429). The second set of constructs comprised the weak ribosome binding site BCD8 (SEQ ID NO: 435). The resulting constructs (depicted in FIGURE 27) included four permutations: (1) Ahll-encoding gene operably linked to BCD1 with AhlR-encoding gene operably linked to BCD 22; (2) Ahll-encoding gene operably linked to BCD1 with AhlR-encoding gene operably linked to BCD 17; (3) Ahll- encoding gene operably linked to BCD8 with AhlR-encoding gene operably linked to BCD 22; and (4) Ahll-encoding gene operably linked to BCD8 with AhlR-encoding gene operably linked to BCD 17.

[0318] The constructs of FIGURE 27 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) undercontrol of the Pomega2 promoter (SEQ ID NO: 598). 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 ODeoo were measured using spectroscopy to assess density -dependent induction.

[0319] The four Kr strains comprising the DNA constructs of FIGURE 27 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 28A, 28B, 28C, 28D). 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.

[0320] 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 Ar strains were cultured in the presence of the quorum quenching aTc compound, then inoculated onto non-germinated com 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 com seeds were not planted (measured as cells g'1sand) was about 1000-fold lower than the bacterial population density observed on com roots (measured as cells g1root) (FIGURE 29). Kr demonstrated population density -dependent induction of the GFP protein of interest on plant roots with the AhlRI QS system (FIGURE 30). 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 10. Expression of a Reporter Gene from Diverse Agriculturally Useful Bacteria Upon Site-Specific DNA Recombination

[0321] To generate strains that express a fluorescent reporter gene of interest in response to sitespecific DNA recombination, a first DNA gene fragment was synthesized comprising: the PhiC31 integrase attP recombinase recognition site (SEQ ID NO: 42), the constitutive promoter J23104 (SEQ ID NO: 44), and the PhiC31 integrase attB recombinase recognition site (SEQ ID NO: 43). A second DNA gene fragment was synthesized comprising a BCD 5 ribosome binding site (SEQ ID NO: 432) operably linked to a super-folder green fluorescent protein (sfGFP) coding sequence. Both gene fragments were PCR amplified with HiFi adapters and cloned into an entry plasmid digested with Bsal. The resulting plasmid comprised the first gene expression cassette ofFIGURE 32 (upper left panel). A plasmid with the second gene expression cassette of FIGURE 32 (bottom left panel), comprising a constitutive promoter operably linked to a BCD 2 vL ribosome binding site (SEQ ID NO: 430) operably linked to a Kosakonia radicincitans codon- optimized DNA sequence encoding the PhiC31 integrase protein (SEQ ID NO: 35), was constructed essentially as described above. Plasmids were confirmed by next generation sequencing.

[0322] To assess recombinase-mediated expression of the sfGFP gene of interest, plasmids were conjugated from E. coli ST 18 donor strains into a strain of each of the agriculturally useful bacteria Azospirillum brasilense, Enterobcicter sp., Klebsiella variicola, Kosakonia radicincitans, Kosakonia sacchari, Rahnella aceris, and Rahnella sp., and plated on LB agar with antibiotic selection. After 48 hours, plates were imaged in an ultraviolet light transilluminator to detect fluorescence of the sfGFP reporter. The first heterologous gene expression cassette comprising the reporter was conjugated into all cells. One experimental group also received the second heterologous gene expression cassette encoding the PhiC31 integrase, while a second experimental group did not. All strains receiving both heterologous gene expression cassettes displayed fluorescence, while strains receiving only the sfGFP reporter cassette without the recombinase cassette lacked fluorescence (FIGURE 33). Fluorescence only occurred when the J23104 promoter was operably linked to the sequence encoding the sfGFP gene of interest upon inversion by the site-specific recombinase (PhiC31 integrase), as shown in FIGURE 32 (upper right panel).

[0323] To confirm that sfGFP expression in this system resulted from promoter inversion by the PhiC31 integrase, (i) the first heterologous gene expression cassette depicted in the upper left panel of FIGURE 32 and (ii) a second heterologous gene expression cassette comprising a DNA molecule encoding PhiC31 integrase (SEQ ID NO: 35) were conjugated into the agriculturally useful bacterium Azospirillum brasilense, plated on solid growth media, and incubated for 48 hours essentially as described above. Single colonies were inoculated into 1 mL of lysogeny broth with antibiotic selection, incubated shaking at 30°C for 24 hours, and analyzed for promoter inversion by polymerase chain reaction (OneTaq DNA polymerase, 1 uL of liquid culture for template, 36 second extension time, 54°C annealing temperature). All samples contained plasmid copies with the first heterologous gene expression cassette (FIGURE 34, left schematic and gel electrophoresis result) in its original configuration. Only samples receiving the second heterologous gene expression cassette encoding the PhiC31 integrase contained plasmid copies where the J23104 promoter of the first heterologous gene expression cassette had been inverted and operably linked to the DNA encoding the sfGFP reporter protein of interest (FIGURE 34,right schematic and gel electrophoresis result). Primers for the polymerase chain reaction assay above are given in FIGURE 35.Example 11. Engineering Agriculturally Useful Bacteria to Irreversibly Express a Reporter Gene via Recombinase in Response to Decreasing Phosphate Concentration.

[0324] Agriculturally useful bacterial (AUB) strains of Enterobacter chinensis, Klebsiella variicola. Kosakonia sacchari, and Rahnella aceris were isolated from maize roots harvested from commercial agricultural fields in the midwestem United States. Additionally, a known soil isolate of Azospirillum brasilense was used. A first heterologous gene expression cassette (FIGURE 36) was designed comprising: the PhiC31 integrase attB recombinase recognition site (SEQ ID NO: 43), the J23119 constitutive promoter (SEQ ID NO: 594), the PhiC31 integrase attP recombinase recognition site (SEQ ID NO: 42), and a BCD 5 ribosome binding site (SEQ ID NO: 432) operably linked to a super-folder green fluorescent protein (sfGFP) coding sequence. This first heterologous gene expression cassette was chromosomally integrated into each of the agriculturally useful bacterial strains above via double homologous recombination at location in the chromosome with a neutral effect on bacterial fitness.

[0325] A library of twelve second heterologous gene expression cassettes was designed (FIGURE 37), comprising (i) a phosphate-sensitive promoter (SEQ ID NO: 418 or 423) and (ii) a ribosome binding site (SEQ ID NO: 430, 439, 447, or 449), both operably linked to DNA encoding the PhiC31 integrase protein (SEQ ID NO: 35), wherein the start codon of the PhiC31 integrase protein coding region was any of ATG, ATT, or ACG, as described in Sacerdot C. et al. Molecular Microbiology (1996) 21(2), 331-346. In each of an Azospirillum brasilense and Klebsiella variicola host strain comprising the first heterologous gene expression cassette above, each second heterologous gene expression cassette was integrated into the chromosome in single copy downstream of each host strain’s glmS gene via site-specific Tn7 transposon-mediated recombination. Strains comprising successfully integrated gene expression cassettes were selected via gentamicin resistance, and chromosomal cassette integration was validated by PCR using primers that anneal to junction of the chromosomal DNA and the inserted heterologous gene expression cassettes.

[0326] To test recombinase-mediated irreversible expression of the sfGFP reporter gene of interest induced by low-phosphate, the Klebsiella and Azospirillum strains constructed above were streaked onto selective solid media and grown for 48 hours at 30°C. 10 single colonies were each inoculated into 1 mL of lysogeny broth with antibiotics and grown shaking, overnight at 30°C. Cultures were then centrifuged for 10 minutes and washed with 1% potassium chloride to removeexcess phosphate and resuspended in 1 mL of 1% potassium chloride. These washed cells were then seeded 1: 10 into 1 mL of NFb + N + P (50 mM, high phosphate) or NFb + N + P (100 uM, low phosphate) and incubated for 48 hours at 30°C. Subsequently, 20 uL of each sample was analyzed for GFP and LSSmScarlett fluorescence by flow cytometry (FIGURE 38).

[0327] Under low-phosphate conditions (100 uM), most cells comprising BCD 22 vL operably linked to the PhiC31 integrase demonstrated inversion of the J23119 constitutive promoter control element and increased expression of the sfGFP reporter gene of interest. Under high-phosphate conditions (50 mM), few cells comprising BCD 22 vL operably linked to the PhiC31 integrase demonstrated inversion of the J23119 constitutive promoter control element and increased expression of the sfGFP reporter gene of interest.Example 12. Irreversibly Inducing Reporter Gene Expression in Agriculturally Useful Bacteria in Response to Decreasing Phosphate Concentration in a Plant Growth Medium.

[0328] Selected Azospirillum and Klebsiella strains of EXAMPLE 11 comprising both (i) sfGFP gene of interest and (ii) PhiC31 integrase-encoding heterologous gene expression cassettes were assessed for phosphate-inducible recombinase-mediated expression of the sfGFP reporter gene of interest on maize roots in a plant growth medium, essentially as described in EXAMPLE 3. Experimental groups included inocula w ith differing levels of starting phosphate concentration: 20 uM, 100 uM, and 50 mM. Plants were harvested at 7 days post inoculation.

[0329] Phosphate-dependent expression of the sfGFP gene of interest was maximized in the Azospirillum brasilense strain comprising the A. brasilense phosphate-dependent PstS promoter (SEQ ID NO: 418) and BCD 22 vL (SEQ ID NO: 449), ), both operably linked to DNA encoding the PhiC31 integrase protein (SEQ ID NO: 35), wherein the start codon of the PhiC31 integrase protein coding region was any ATT (FIGURES 39 and 40).Example 13. Engineering Bacteria Isolated from Agricultural Fields to Irreversibly Express a Reporter Gene via Recombinase in Response to Decreasing Nitrogen Concentration.

[0330] Agriculturally useful bacterial (AUB) strains of Enterobacter chinensis, Klebsiella variicola. Kosakonia sacchari. anA Rahnella aceris were isolated from maize roots harvested from commercial agricultural fields in the midwestern United States. Additionally, a known soil isolate of Azospirillum brasilense was used. A first heterologous gene expression cassette (FIGURE 36) was designed comprising: the PhiC31 integrase attB recombinase recognition site (SEQ ID NO: 43), the J23119 constitutive promoter (SEQ ID NO: 594), the PhiC31 integrase attP recombinase recognition site (SEQ ID NO: 42), and a BCD 5 ribosome binding site (SEQ ID NO: 432)operably linked to a super-folder green fluorescent protein (sfGFP) coding sequence. This first heterologous gene expression cassette was chromosomally integrated into each of the agriculturally useful bacterial strains above via double homologous recombination at location in the chromosome with a neutral effect on bacterial fitness.

[0331] A library of ten second heterologous gene expression cassettes was designed (FIGURE 41), comprising (i) anifH promoter sensitive to oxygen and nitrogen levels (SEQ ID NO: 595 or 596) and (ii) a ribosome binding site (SEQ ID NO: 444, 448, or 449), both operably linked to DNA encoding the PhiC31 integrase protein (SEQ ID NO: 35), wherein the start codon of the PhiC31 integrase protein coding region was any of ATG, ATT, or ACG, as described in Sacerdot C. et al. Molecular Microbiology (1996) 21(2), 331-346. In a Kosakonia sacchari host strain comprising the first heterologous gene expression cassette above, each second heterologous gene expression cassette was integrated into the chromosome in single copy downstream of each host strain’s glmS gene via site-specific Tn7 transposon-mediated recombination. Strains comprising successfully integrated gene expression cassettes were selected via gentamicin resistance, and chromosomal cassette integration was validated by PCR using primers that anneal to junction of the chromosomal DNA and the inserted heterologous gene expression cassettes.

[0332] To test recombinase-mediated irreversible expression of the sfGFP reporter gene of interest induced under w / frogen-fixing conditions, the Kosakonia strains constructed above were streaked onto selective solid media and grown for 24 hours at 30°C. For each strain, 3 single colonies were each re-streaked onto selective solid media, grown at 30°C, resuspended in 1 mL of 1% potassium chloride, and inoculated to a final ODeoo of 0.3 into 4.5 mL of NFbHP with MEM and 20 mM glucose, with or without 20 mM ammonium chloride, in a gas chromatography (GC) vial. Samples were each sealed, incubated shaking at room temperature and 2% oxygen tension for three to four hours, and 2.8 mL of headspace gas was replaced with 2.2 mL of 100% acetylene. Samples were incubated for an additional three hours, and headspace gas was withdrawn and analyzed on a gas chromatograph.

[0333] Presence of ethylene (FIGURE 42) confirmed nitrogen fixing conditions because acetylene was only reduced to ethylene under conditions where the nitrogenase enzyme was active. Expression of the sfGFP gene of interest was only achieved by recombinase-mediated promoter inversion when environmental nitrogen concentration was low' (FIGURE 43). Additionally, expression of the sfGFP gene of interest was greatest in the Kosakonia strains comprising the BCD 22 vL ribosome binding site (SEQ ID NO: 449), operably linked to DNA encoding the PhiC31 integrase protein (SEQ ID NO: 35), wherein the start codon of the PhiC31 integrase protein coding region was ATG.Example 14. Engineering Bacteria Isolated from Agricultural Fields to Irreversibly Express a Reporter Gene via Recombinase in Response to Increasing Cell Density.

[0334] Agriculturally useful bacterial (AUB) strains of Enterobacter chinensis. Klebsiella variicola, Kosakonia sacchari, ar Rahnella aceris were isolated from maize roots harvested from commercial agricultural fields in the midwestem United States. A first heterologous gene expression cassette (FIGURE 36) was designed comprising: the PhiC31 integrase attB recombinase recognition site (SEQ ID NO: 43), the J231 19 constitutive promoter (SEQ ID NO: 594), the PhiC31 integrase attP recombinase recognition site (SEQ ID NO: 42), and a BCD 5 ribosome binding site (SEQ ID NO: 432) operably linked to a super-folder green fluorescent protein (sfGFP) coding sequence. This first heterologous gene expression cassette was chromosomally integrated into each of the agriculturally useful bacterial strains above via double homologous recombination at location in the chromosome with a neutral effect on bacterial fitness.

[0335] A library of three second heterologous gene expression cassettes was designed (FIGURE 44). comprising (i) the Pahll quorum sensing promoter sensitive to cell density (SEQ ID NO: 567) and (ii) the BCD 22 vL ribosome binding site (SEQ ID NO: 449), both operably linked to DNA encoding the PhiC31 integrase protein (SEQ ID NO: 35), wherein the start codon of the PhiC31 integrase protein coding region was any of ATG, ATT. or ACG, as described in Sacerdot C. et al. Molecular Microbiology (1996) 21(2), 331-346. Additionally, all three second heterologous gene expression cassettes comprised: (i) a Ptet promoter (SEQ ID NO: 32) and BCD 22 vS ribosome binding site (SEQ ID NO: 450), each operably linked to DNA encoding the quorum sensing regulator protein (QSRP) AhlR (SEQ ID NO: 550); (ii) a Pahll quorum sensing promoter (SEQ ID NO: 567) and BCD 8 ribosome binding site (SEQ ID NO: 435), each operably- linked to DNA encoding the quorum sensing synthase protein (QSSP) Ahll (SEQ ID NO: 559); and (iii) a pOmega2 synthetic promoter (SEQ ID NO: 597) and B0034 RBS v2 ribosome binding site (SEQ ID NO: 598), each operably linked to DNA encoding the reverse tet repressor protein rTetR (SEQ ID NO: 28). In each of the agriculturally useful bacterial (AUB) host strains of Enterobacter chinensis, Klebsiella variicola. Kosakonia sacchari, wARahnella aceris comprising the first heterologous gene expression cassette above, each second heterologous gene expression cassette was integrated into the chromosome in single copy downstream of each host strain’s glmS gene via site-specific Tn7 transposon-mediated recombination. Strains comprising successfully integrated gene expression cassettes were selected via gentamicin resistance, and chromosomalcassette integration was validated by PCR using primers that anneal to junction of the chromosomal DNA and the inserted heterologous gene expression cassettes.

[0336] To test recombinase-mediated irreversible expression of the sfGFP reporter gene of interest induced when bacteria exceed a threshold population density, the Enterobacter. Klebsiella, Kosakonia, anA Rahnella strains constructed above were streaked onto LB agar media, with antibiotics for selection, with or without 400 ng per mL of an anhydrotetracycline (aTc) quorum quenching compound (QQ). Cells were grown until visible colonies emerged and resuspended in 1 mL of 1% potassium chloride for analysis by flow cytometry essentially as described in EXAMPLE 3.

[0337] When the bacteria were contacted with the aT c quorum quenching compound, aT c bound the rTetR deactivator to form the aTc-rTetR complex. The aTc-rTetR complex bound the Ptet promoter, repressing the gene encoding the AhlR QSRP and inhibiting expression of the PhiC31 recombinase. When the aTc quorum quenching compound was absent, 76% to 86.97% of Enterobacter cells with PhiC31 integrase comprising an ATT or ATG start codon demonstrated sfGFP expression following PhiC31 -mediated promoter inversion (FIGURE 45), 93.73% to 94.72% of Klebsiella cells with PhiC31 integrase comprising an ATT or ATG start codon demonstrated sfGFP expression following PhiC31 -mediated promoter inversion (FIGURE 46), 68.81% to 84.31% of Kosakonia cells with PhiC31 integrase comprising an ATT or ATG start codon demonstrated sfGFP expression following PhiC31 -mediated promoter inversion (FIGURE 47), and 77.06% to 83.7% of Rahnella cells with PhiC31 integrase comprising an ATT or ATG start codon demonstrated sfGFP expression following PhiC31 -mediated promoter inversion(FIGURE 48)Example 15. Irreversibly Inducing Reporter Gene Expression in Bacteria Isolated from an Agricultural Field in Response to Increasing Cell Density in an Agricultural Field Soil Substrate.

[0338] The Kosakonia sacchari (ATG start codon driving PhiC31 expression) and Rahnella aceris (ATG or ATT start codon driving PhiC31 expression) strains of EXAMPLE 14 irreversibly activated GFP expression on com roots following an increase in cell density in agricultural field soil used as a growth medium. Topsoil was collected from within 10 inches of the surface from American commercial maize fields in the state of Iowa (“Field Soil”) and underwent soil chemistry analysis. 2 x 10” Deepot cells (Stuewe & Sons, Inc.) were each filled with 300 g of Field Soil from a single location that had been titrated to 100 ppm potassium and 100 ppm nitrate. After 24 hours, surface sterilized com seeds were sown at a depth of 2 inches and inoculated with(i) 4.0 x 107cells of ?, sacchari (ATG-PhiC31), (ii) 2.6 x 107cells of A. aceris (ATG-PhiC31), or (iii) 3.8 x 107cells of . aceris (ATT-PhiC31), as confirmed by viable CFU counts. After inoculation, plants were incubated in a growth room set with the following parameters: light intensity 250 pmol / m2 / s at 7 inches from the floor; 1 :8 light cycles with 30 minute sunrise / sunset dimming; 50-55% humidity. Plants were sampled at 7 days (“1 week”), 14 days (“2 weeks”), 21 days (“3 weeks”) and 28 days (“4 weeks”) post inoculation. For each sampling point, five plant replicates were destructively uprooted and loose field soil was discarded. The roots were then excised from the shoot at the cotyledon and vortexed in phosphate-buffered saline with 10 mM ammonium chloride for 1 hour to create a bacterial cell suspension. This fraction was defined in these experiments as the rhizosphere. To isolate bacterial cells for analysis of the GFP reporter, plant and soil debris remaining in the rhizosphere suspension were selectively cleared by centrifugation at low speed, and one hundred microlitre aliquots of the supernatant containing the bacterial cells were analyzed by flow cytometry.

[0339] Because tested strains comprised both the GFP reporter cassette and carried a constitutively expressed LSSmScarlett reporter protein, flow cytometry' events corresponding to bacterial cells could be gated based on LSSmScarlett fluorescence above the background (here defined as LSSmScarlehT), as validated by Haskett etal. (doi: 10.3389 / fmicb.2021.690439). The percentage of the LSSmScarlett+ population activated for GFP expression at each time point was quantified by establishing GFP and “+” threshold fluorescence values for the bacteria.

[0340] As shown in FIGURE 48, engineered K. sacchari demonstrated over 30% to over 50% activation with a 2 week delay field soil while engineered R. aceris demonstrated from over 25% to over 75% (FIGURE 49) activation with a 2 week delay field soil, depending on the start codon of the PhiC31 integrase coding sequence.Example 16. Optimizing a Dual Promoter Control Element in Bacteria Isolated from an Agricultural Field for Ammonia Production in Response to Decreasing Phosphate Concentration.

[0341] In agriculturally useful bacterial (AUB) strains of Enterobacter chinensis, Klebsiella variicola. Kosakonia sacchari, and Rahnella aceris isolated from maize roots, the native glnA gene encoding glutamine synthetase was deleted, and one of each of the constructs of FIGURE 50 was integrated into the chromosome in single copy dow nstream of each host strain’s glmS gene via site-specific Tn7 transposon-mediated recombination. In the constructs of FIGURE 50, the attB SSRRS is given by SEQ ID NO: 43, the attP SSRRS is given by SEQ ID NO: 42, the BCD 22 RBS is given by SEQ ID NO: 449, the Azospirillum brasilense glnA sequence encodes aprotein of the amino acid sequence of SEQ ID NO: 229, and all promoter sequences beginning with “J” are given by SEQ ID NO: 44, 576, i, vi-xv, and xvi. Successfully integrated gene expression cassettes were selected via gentamicin resistance, and chromosomal cassette integration was validated by PCR using primers that anneal to junction of the chromosomal DNA and the inserted heterologous gene expression cassettes.

[0342] To determine the fold-reduction in glutamine synthetase expression that would inhibit bacterial growth, each of the strains constructed above was streaked on LB agar with selective antibiotics and incubated overnight at 30 degrees centigrade. Cells were resuspended in 200 uL of 1% potassium chloride solution, inoculated in triplicate to ODeoo 0.001 in 200 uL NFb + 20mM glucose + lOmM ammonium chloride, and incubated shaking at 30 degrees centigrade for 48 hours with frequent ODeoo measurements to build a growth curve (FIGURES 51-53).

[0343] To determine the fold-reduction in glutamine synthetase expression necessary for ammonia release, each of the strains constructed above was streaked on LB agar with selective antibiotics and incubated at 30 degrees centigrade until colonies appeared. Cells were resuspended in 1 mL of 1% potassium chloride solution and inoculated into a gas chromatography vial in triplicate to ODeoo 0.3 in a final volume of 5 mL NFbHP minimal medium + 20mM glucose + 20mM fructose. Open samples were incubated shaking at room temperature and 2% oxygen tension for 0.5 - 1 hour, sealed, and incubated shaking (200 rpm) for another 24 hours (at room temperature for Rahnella strains but at 30 degrees centigrade for Kosakonia and Klebsiella strains). ODsoo was measured, and ammonia concentration was quantified according to manufacturer instructions using the Sigma- Aldrich Ammonia Assay Kit (FIGURES 54-56).

[0344] Only the strongest J23100 promoter permitted cell growth equivalent to wild-type for Kosakonia sacchari (FIGURE 51), Klebsiella variicola (FIGURE 52), and Rahnella aceris (FIGURE 53). Promoters known to decrease expression as little as 14% (J23102) and as much as 94% (J23117) caused a fitness defect in all strains when operably linked to the glnA gene. For Kosakonia sacchari (FIGURE 54) and Rahnella aceris (FIGURE 56), ammonia release was maximized w hen the glnA gene w as operably linked to a promoter known to decrease expression between 14% and 53%. If glnA expression was reduced further, ammonia was not released by Kosakonia and Rahnella strains. Without being limited by theory, a moderate decrease in glnA expression may have had the potential for ammonia release without triggering such a large fitness defect as to cause cell metabolism to stall altogether.Example 17. Engineered Ammonia Production and Release in Response to Decreasing Phosphate Concentration in Bacteria Isolated from an Agricultural Field.

[0345] Essentially as described in EXAMPLE 16, strains of Kosakonia sacchari and Rahnella aceris comprising the heterologous gene expression cassettes of FIGURE 57 were constructed and assessed for ammonia release.

[0346] In Kosakonia sacchari, ammonia release induced by low phosphate concentration was maximized when the first heterologous gene expression cassette comprised a PhiC31 attB (SEQ ID NO: 43) and attP (SEQ ID NO: 42) SSRRS in an inverted configuration, flanking a control element, wherein the control element comprised a first strong J23100 constitutive promoter (SEQ ID NO: 609) directed opposite a second weak J23106 constitutive promoter (SEQ ID NO: 613). In this first heterologous gene expression cassette, the first strong J23100 promoter was operably linked to a BCD 22 vL RBS (SEQ ID NO: 449). which was operably linked to a gene encoding the Azospirillum brasilense glnA gene, prior to inversion of the control element by the PhiC31 integrase. The maximal ammonia release Kosakonia sacchari strain also comprised a second heterologous gene expression cassette comprising a phosphate-sensitive PstS promoter operably linked to a BCD 22 vL RBS. which was operably linked to a gene encoding the PhiC31 integrase protein (SEQ ID NO: 41) with an ATT start codon (FIGURE 58).

[0347] In Rahnella aceris, ammonia release induced by low phosphate concentration was maximized in two strains. Both comprised a first heterologous gene expression cassette comprising a PhiC31 attB (SEQ ID NO: 43) and attP (SEQ ID NO: 42) SSRRS in an inverted configuration, flanking a control element, wherein the control element comprised a first strong J23100 constitutive promoter (SEQ ID NO: 609) directed opposite a second weak constitutive promoter. In this first heterologous gene expression cassette, the first strong J23100 promoter was operably linked to a BCD 22 vL RBS (SEQ ID NO: 449), which was operably linked to a gene encoding the Azospirillum brasilense glnA gene, prior to inversion of the control element by the PhiC31 integrase. The maximal ammonia release Rahnella aceris strains also comprised a second heterologous gene expression cassette comprising a phosphate-sensitive PstS promoter operably linked to a gene encoding the PhiC31 integrase protein (SEQ ID NO: 41) with an ATT start codon. In one ammonia-maximizing Rahnella aceris strain, the weak promoter of the control element was the J23106 constitutive promoter (SEQ ID NO: 613), and a BCD 12 RBS (SEQ ID NO: 439) was operably linked to the gene encoding the PhiC31 integrase protein. In the second ammonia-maximizing Rahnella aceris strain, the weak promoter of the control element was the J23102 constitutive promoter (SEQ ID NO: 611), and a BCD 22 vL RBS (SEQ ID NO: 449) was operably linked to the gene encoding the PhiC31 integrase protein (FIGURE 59).

Claims

WHAT IS CLAIMED IS:

1. A method of providing at least one agriculturally relevant compound to a plant comprising placing at least one genetically engineered bacterium into a plant growth medium, wherein the genetically engineered bacterium is an agriculturally useful bacterium comprising:(i) a first heterologous gene expression cassette comprising at least one DNA molecule encoding a protein or RNA sequence of interest, wherein the DNA molecule is operably linked to at least one DNA molecule comprising a site-specific recombinase recognition sequence (SSRRS) and wherein the first heterologous gene expression cassette lacks an irreversible insertion, excision, or inversion of a DNA molecule in the first heterologous gene expression cassette; and(ii) a second heterologous gene expression cassette comprising at least one DNA molecule encoding at least one recombinase that can catalyze site-specific recombination at said SSRRS, wherein the DNA molecule encoding said recombinase is operably linked to a promoter activated by a change of at least one condition in the plant growth medium, and wherein the site-specific recombination induces an irreversible insertion, excision, or inversion of: (a) a DNA molecule comprising a control element in, from, at, or near the DNA molecule encoding the protein or RNA sequence of interest in the first heterologous gene expression cassette; or (b) a DNA molecule encoding the protein or RNA sequence of interest in the first heterologous gene expression cassette.

2. A method of providing at least one agriculturally relevant compound to a plant comprising placing at least one genetically engineered bacterium into a plant growth medium, wherein the genetically engineered bacterium is an agriculturally useful bacterium comprising:(i) a first part of a first heterologous gene expression cassette comprising a DNA molecule encoding a control element, protein or RNA sequence of interest, or a part thereof, wherein the first part of the first heterologous gene expression cassette has reduced or no function and wherein the DNA molecule is operably linked to at least one site-specific recombinase recognition sequence (SSRRS); and a second part of the first heterologous gene expression cassette comprising the control element, the protein or RNA sequence of interest, or part thereof absent from the first part of the first heterologous gene expression cassette; and(ii) a second heterologous gene expression cassette comprising at least one nucleic acid sequence encoding at least one recombinase that can catalyze site-specific recombination at said SSRRS, wherein the DNA molecule encoding said recombinase is operably linked to a promoter activated by a change of at least one condition in the plant grow th medium, and wherein the sitespecific recombination induces an irreversible insertion, excision, or inversion of DNA whichresults in the operable linkage of the first and second part of the first heterologous gene expression cassette to provide a functional first heterologous gene expression cassette.

3. The method of claim 2, wherein the first part and the second part of the first heterologous gene expression cassette are located on separate DNA molecules which are not covalently linked.

4. The method of claim 2, wherein the first part and the second part of the first heterologous gene expression cassette are located on a single DNA molecule.

5. The method of claim 1. wherein the DNA molecule encoding the recombinase comprises an ATG. ACG, or ATT translation initiation codon which is operably linked to the open reading frame of the DNA molecule encoding the recombinase.

6. The method of claim 1, wherein the irreversible insertion, excision, or inversion of the DNA molecule comprising the control element or the DNA molecule encoding the protein or RNA sequence of interest reduces expression of the protein or RNA sequence of interest in comparison with the first heterologous gene expression cassette lacking the irreversible insertion, excision, or inversion of the DNA molecule.

7. The method of claim 6, wherein the irreversible insertion, excision, or inversion of the DNA molecule causes the expression of the protein or RNA sequence of interest to be reduced by 10% or 20% to 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% in companson to the first heterologous gene expression cassette of (i) lacking the irreversible insertion, excision, or inversion of a DNA molecule.

8. The method of claim 6, wherein the control element comprises a first promoter and a second promoter directed opposite one another, wherein the control element is flanked by two SSRRS in an inverted configuration, wherein the first promoter of the control element is operably linked to the DNA molecule encoding at least one protein or RNA sequence of interest prior to inversion by the site-specific recombinase, wherein transcription promoting-activity of the second promoter is at least 14%, 28%, 42%, or 53% less than the transcription promoting-activity of the first promoter, and wherein the second promoter of the control element is operably linked to theDNA molecule encoding the at least one protein or RNA sequence of interest upon inversion by the site-specific recombinase.

9. The method of claim 8, wherein:(i) the first promoter comprises a constitutive promoter, a native promoter of the native gene encoding the protein or RNA sequence of interest, or a promoter having activity equivalent to or greater than the native promoter; and / or(ii) the protein of interest comprises a GlnA, AmtB, GlnB, GlnK, GlnZ, NifL, and / or DraT protein and the agriculturally relevant compound is ammonia.

10. The method of claim 8. wherein:(i) the first promoter comprises a J23100 constitutive promoter given by SEQ ID NO: 609;(ii) the second promoter comprises a J23106 constitutive promoter given by SEQ ID NO: 613, J23102 constitutive promoter given by SEQ ID NO: 611, J23111 constitutive promoter given by SEQ ID NO:

614. or a J23104 constitutive promoter given by SEQ ID NO: 612;(iii) the protein of interest comprises a GlnA protein and the agriculturally relevant compound is ammonia; and(iv) the agriculturally useful bacterium is a member of the genus Azospirillum, Enterobacter, Klebsiella, Kosakonia, Paenibacillus, Phytobacter, Rahnella, Sphingomonas, or Variovorax, wherein the bacterium is optionally a member of the genus Azospirillum, Klebsiella, Kosakonia. or Rahnella.

11. The method of claim 6, wherein the nucleic acid molecule encoding the protein or RNA sequence of interest is flanked by two SSRRS in a direct configuration and wherein the sitespecific recombination induces excision of said nucleic acid molecule encoding a protein or RNA sequence of interest to reduce expression of the protein or RNA sequence of interest.

12. The method of claim 6, wherein the first heterologous gene expression cassette comprises a promoter flanked by two SSRRS in a direct configuration, wherein said promoter is operably linked to the DNA molecule encoding a protein or RNA sequence of interest, and wherein the site-specific recombination induces excision of said promoter to reduce expression of the protein or RNA sequence of interest.

13. The method of claim 11, wherein: (i) the SSRRS comprise attB and attP sites and / or wherein the DNA molecule comprises aglnA, amiB. glnB, segment o glnE encoding an adenylyl- removing domain of a glutamine synthetase adenylyltransferase, glnK, glnZ, nifL, and / or draT gene; and (ii) the agriculturally relevant compound is ammonia.

14. The method of claim 8, wherein: (i) the SSRRS comprise attB and attP sites and / or wherein the DNA molecule comprises aglnA, amtB. glnB. segment of glnE encoding an adenylyl- removing domain of a glutamine synthetase adenylyltransferase, glnK, glnZ, nifL, and / or draT gene; and (ii) the agriculturally relevant compound is ammonia.

15. The method of claim 6, wherein the first heterologous gene expression cassette comprises a promoter flanked by two SSRRS in an inverted configuration, wherein said promoter is operably linked to the DNA molecule encoding a protein or RNA sequence of interest, and wherein the site-specific recombination induces inversion of said promoter to uncouple the promoter from the DNA molecule and to reduce expression of the protein or RNA sequence of interest.

16. The method of claim 6, wherein the DNA molecule encoding the protein or RNA sequence of interest is flanked by two SSRRS in an inverted configuration, wherein the DNA molecule is operably linked to a promoter, and wherein the site-specific recombination induces inversion of said DNA molecule encoding the protein or RNA sequence of interest to uncouple the DNA molecule from the promoter, thereby reducing expression of the protein or RNA sequence of interest.

17. The method of claim 6, wherein the first heterologous gene expression cassette comprises a promoter which is: (i) inoperably linked to a terminator flanked by two SSRRS in an inverted configuration; and (ii) operably linked to a DNA molecule encoding the protein or RNA sequence of interest, and wherein the site-specific recombination induces inversion of said terminator to operably link the promoter to the terminator and to uncouple the DNA molecule from the promoter, thereby reducing expression of the protein or RNA sequence of interest.

18. The method of claim 6, wherein the DNA molecule encoding the protein or RNA sequence of interest comprises a first SSRRS comprising an in-frame open reading frame (ORF), a part of the DNA molecule encoding a part of the protein or RNA sequence of interest, and asecond SSRRS comprising an in-frame ORF, wherein the protein or RNA sequence is functional, and wherein the site-specific recombination induces excision of the part of the DNA molecule and one SSRRS to reduce activity of the protein or RNA sequence of interest.

19. The method of claim 6, wherein: (i) the DNA molecule encoding the protein or RNA sequence of interest comprises an inserted first SSRRS comprising an in-frame open reading frame (ORF) and wherein the protein or RNA sequence is functional; and (ii) the genetically engineered bacterium further comprises a blocking DNA covalently linked to at least one SSRRS; wherein the site-specific recombination induces insertion of the blocking DNA into the DNA molecule to reduce expression and / or activity of the protein or RNA sequence of interest.

20. The method of claim 6. wherein the protein of interest comprises a GlnA, AmtB. GlnB, GlnK, GlnZ, NifL, and / or DraT protein and the agriculturally relevant compound is ammonia.

21. The method of claim 1 , wherein the irreversible insertion, excision, or inversion of the DNA molecule comprising the control element or the DNA molecule encoding the protein or RNA sequence of interest increases expression of the protein or RNA sequence of interest in comparison to the first heterologous gene expression cassette lacking the irreversible insertion, excision, or inversion of the DNA molecule.

22. The method of claim 21, wherein the irreversible insertion, excision, or inversion of the DNA molecule causes the expression of the protein or RNA sequence of interest to be increased: (i) at least 2-fold, 5-fold, or at least 10-fold; or (ii) about 2-fold or 5-fold to about 15- fold, both in comparison to the first heterologous gene expression cassette lacking the irreversible insertion, excision, or inversion of a DNA molecule.

23. The method of claim 21, wherein the control element comprises a promoter, wherein the promoter is flanked by two SSRRS in an inverted configuration, and wherein the promoter is operably linked to the at least one protein or RNA sequence of interest upon inversion by the sitespecific recombinase.

24. The method of claim 21, wherein the control element comprises a first promoter and a second promoter directed opposite one another, wherein the control element is flanked by two SSRRS in an inverted configuration, wherein the first promoter of the control element is operablylinked to the DNA molecule encoding at least one protein or RNA sequence of interest prior to inversion by the site-specific recombinase, wherein transcription promoting-activity of the second promoter is at least 2-. 5-, or 10-fold greater than the transcription promoting-activity of the first promoter, and wherein the second promoter of the control element is operably linked to the at least one protein or RNA sequence of interest upon inversion by the site-specific recombinase.

25. The method of claim 24, wherein:(i) the first promoter comprises a constitutive promoter, a native promoter of the native gene encoding the protein or RNA sequence of interest, or a promoter having activity equivalent to or lower than the native promoter; and / or(ii) the protein of interest is a NifA protein, NtrC protein, GlnR protein, or modified GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity.

26. The method of claim 21, wherein the control element comprises a promoter, wherein the DNA molecule encoding the protein or RNA sequence of interest is flanked by two SSRRS in an inverted configuration, and wherein the DNA molecule encoding the protein or RNA sequence of interest is operably linked to the control element upon inversion by the site-specific recombinase.

27. The method of claim 21, wherein the control element comprises a terminator, wherein the terminator is flanked by two SSRRS in a direct configuration, wherein the terminator and two SSRRS are between the at least one DNA molecule encoding protein or RNA sequence of interest and a promoter in the first heterologous gene expression cassette, and wherein the DNA molecule encoding the protein or RNA sequence of interest is operably linked to the promoter in the first heterologous gene expression cassette upon excision of said terminator.

28. The method of claim 21, wherein the control element comprises a terminator, wherein the terminator is flanked by two SSRRS in an inverted configuration, wherein the terminator and two SSRRS are between the DNA molecule encoding at least one protein or RNA sequence of interest and a promoter in the first heterologous gene expression cassette, and wherein the DNA molecule encoding the protein or RNA sequence of interest is operably linked to the promoter in the first heterologous gene expression cassette upon inversion of said terminator.

29. The method of claim 2, wherein the first part of first heterologous gene expression cassette comprises the first part of the DNA molecule encoding the first part of the protein or RNA sequence of interest, wherein the SSRRS is located at the beginning of, within, or at the end of the first part of the DNA molecule, wherein the genetically engineered bacterium further comprises a third heterologous gene expression cassette comprising a site-specific recombinase recognition site and the second part of the DNA molecule encoding the second part of the protein or RNA of interest absent from the first heterologous gene expression cassette, and wherein the first and second part of the DNA molecule encoding the protein or RNA sequence of interest are operably linked to provide a functional protein or RNA sequence of interest upon insertion of the second part of the DNA molecule into the first heterologous gene expression cassette.

30. The method of claim 2. wherein the first part of the first heterologous gene expression cassette comprises the DNA molecule encoding the protein or RNA sequence of interest but lacks a control element, wherein the SSRRS is located at the beginning of, within, or at the end of the first part of the DNA molecule, wherein the genetically engineered bacterium further comprises a third heterologous gene expression cassette comprising a site-specific recombinase recognition site and a control element, and wherein the control element and the DNA molecule encoding the protein or RNA sequence of interest are operably linked to provide a functional first heterologous gene expression cassette upon insertion of the control element into the first heterologous gene expression cassette.

31. The method of claim 21, 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 agriculturally relevant compound 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 agriculturally relevant compound is ammonia;(c) a GlnR protein and the agriculturally relevant compound is ammonia;(d) a glutaminase enzyme and the agriculturally relevant compound is ammonia;(e) a protein product of a refactored nif or fix gene cluster and the agriculturally relevant compound 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 (cl), the tet repressor (TetR), the lac repressor (Lad), 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, lnB. glnK. glnZ, nifL, and / or draT gene and the agriculturally relevant compound is ammonia; or(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 agriculturally relevant compound is ammonia.

32. The method of claim 1 wherein:(i) the first heterologous gene expression cassette comprises a control element comprising a promoter flanked by two SSRRS oriented as inverted repeats, wherein the promoter is operably linked to a first DNA molecule encoding a first protein or RNA sequence of interest, wherein the promoter flanked by two SSRRS is uncoupled from a second DNA molecule encoding a second protein or RNA sequence of interest; and(ii) the site-specific recombination induces the inversion of the DNA molecule comprising the promoter to uncouple the promoter from the first DNA molecule, to operably link the promoter to the second DNA molecule, and to reduce expression of the first protein or RNA sequence of interest and increase expression of the second protein or RNA sequence of interest, both in comparison to the first heterologous gene expression cassette of (i) lacking the irreversible inversion of the DNA molecule.

33. The method of claim 2, wherein:(i) the first heterologous gene expression cassette comprises a control element comprising a promoter which is operably linked to a first DNA molecule encoding a first protein or RNA sequence of interest which is operably linked to an SSRRS, wherein the promoter is uncoupledfrom a second DNA molecule encoding a second protein or RNA sequence of interest which is operably linked to a terminator element and an SSRRS; and(ii) the site-specific recombination induces the insertion of the second DNA molecule encoding the second protein or RNA sequence of interest to uncouple the promoter from the first DNA molecule, to operably link the promoter to the second DNA molecule, and to reduce expression of the first protein or RNA sequence of interest and increase expression of the second protein or RNA sequence of interest, both in comparison to the first heterologous gene expression cassette of (i) lacking the irreversible inversion of the DNA molecule.

34. The method of claim 32, wherein:(a) the first protein of interest comprises a wild-type glutamine synthetase (GS) or variant thereof with improved catalytic activity in comparison to wild-type GS and the second protein of interest comprises a wild-type GS with reduced levels of expression in comparison to the wildtype GS or a GS variant with decreased catalytic activity in comparison to wild-type GS, and the agriculturally relevant compound is ammonia;(b) the first protein of interest comprises a GS adenylyltransferase protein and the second protein of interest comprises a GS adenylyltransferase protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, and the agriculturally relevant compound is ammonia; or(c) the first target gene is a nifL gene and the second target gene is a nifA gene, and the agriculturally relevant compound is ammonia.

35. The method of any of claims 1 to 32 wherein said change of at least one condition in the plant growth medium condition comprises:(a) growth of the genetically engineered bacterium to exceed a threshold population density in the plant grow th medium;(b) reduction of at least one fertilizer or plant nutrient in the soil, 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;(c) growth of a plant in the plant grow th media;(d) change in soil temperature;(e) change in motility of the bacterium;(f) change in light levels in the soil;(g) change in oxygen concentration in the soil;(h) change in concentration of bioavailable nitrogen in the soil;(i) change in pH in the soil;(j) change in overall solute concentration (osmotic pressure) in the soil;(k) change in phytohormone or plant signaling molecule concentrations;(l) change in an amino acid concentration;(m) change in a sugar concentration; and / or(n) a change in plant or microbial metabolite concentrations, optionally wherein the plant or microbial metabolite is naringenin, quercetin, luteolin, apigenin, octopine, nopaline, or Scyllo- inosamine.

36. The method of any one of claims 1 to 32. wherein said promoter activated by a change of at least one plant growth medium condition comprises:(i) a phosphate-sensitive promoter;(ii) a nitrogen-sensitive promoter;(iii) a quorum sensing promoter;(iv) a promoter which is induced or repressed by decreased oxygen levels;(v) a promoter which is induced or repressed by increased oxygen levels;(vi) a promoter which is induced by decreased oxygen and decreased nitrogen levels, optionally wherein the promoter is a nifH promoter;(vii) a promoter which is induced or repressed by increased or decreased potassium levels;(viii) a promoter which is induced by decreased carbon levels, optionally wherein the promoter is a Pfic promoter;(ix) a promoter that is induced by increased nitrate levels, optionally wherein the promoter is an oxygen insensitive PnarG promoter;(x) a promoter that is induced by increased nitrate levels, optionally wherein the promoter is a narK or narX promoter;(xi) a promoter which is upregulated by an increase in the concentration of naringenin; and / or(xii) a promoter or system comprising a promoter set forth in Table 5, Table 6, or Table 7.

37. The method of claim 36, wherein the promoter which is induced by decreased oxygen and decreased nitrogen levels is a NifH promoter.

38. The method of claim 37, wherein the NifH promoter comprises a DNA molecule having at least 75%, 80%, 85%, 90%, 95%, 98%. or 99% sequence identity to SEQ ID NO: 595 or 596.

39. The method of claim 36, wherein the genetically engineered bacterium comprises the phosphate-sensitive promoter which is operably linked to the recombinase and wherein:(a) the threshold concentration of phosphate in the plant growth medium which activates expression of the recombinase is about 0 pM to about 50 pM;(b) the phosphate-sensitive promoter and / or a segment of the 5’ untranslated region (UTR) which is operably linked to the phosphate-sensitive promoter comprises at least one copy of an operably linked Pho box, wherein the Pho box comprises the sequence of SEQ ID NO: 525-529, 234-396. or 397 and sequences with at least 95% identity to any one of SEQ ID NO: 234-397, optionally wherein the Pho box comprises SEQ ID NO: 525-529, 376, 377, 383-396, or 397;(c) the phosphate-sensitive promoter and / or a segment of the 5 ’ UTR comprises a promoter and / or a segment of a 5’UTR of a phoA, phoX. phy, pstS gene, a variant thereof, or a combination thereof; and / or(d) the phosphate-sensitive promoter and / or the segment of the 5’ UTR comprises a promoter of a gene encoding:(i) a PstS protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 524, 399, 401, 403. 405, 407, 409, 415, 416, 513, 523. and 524 or having an identity of at least 76%, 80%, 85%, 90%, 95%, 98%, or 99% with any one of SEQ ID NO: 399, 401, 403, 405, 407, 409, 415, 416, 513, or 524;(ii) a PhoX protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 408, 410, 411, 412, and 413 or having an identity of at least 80% with any one of SEQ ID NO: 408, 410. 411, 412, and 413;(iii) a Phy protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 414, 57-138, and 139 or having an identity of at least 80% with any one of SEQ ID NO: 414, 57-138, or 139; and / or(iv) a PhoA protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 398, 400, 402, 404, and 406 or having an identity of at least 80% with any one of SEQ ID NO: 398, 400, 402, 404, or 406.

40. The method of claim 39, wherein the phosphate-sensitive promoter and / or the segment of the 5’ UTR comprises:i. a pstS promoter comprising a DNA sequence selected from the group consisting of SEQ ID NO: 517, 417. 418, 420, 421, 422, 423, 424, 425, 428, 515, and 518; ii. a phoX promoter comprising the DNA sequence of SEQ ID NO: 426; iii. a phy promoter comprising the DNA sequence of SEQ ID NO: 427; iv. a phoA promoter comprising the DNA sequence of SEQ ID NO: 419; and / or v. a Pliar53 promoter comprising the DNA sequence of SEQ ID NO: 459; vi. a variant of the promoters under i. to v. having an identity of at least 76%, 80%, 90%, 95%, 98%, or 99% with any of SEQ ID NO: 515, 417, 418-428, 459, 517, or 518, wherein said variant comprises at least one Pho box and / or retains at least one Pho box present in each promoter and wherein said variant promoters are activated by a decrease in phosphate concentration to about 0 pM to about 1 pM. 2 pM, 5 pM. 7 pM, 10 pM, 20 pM, 30 pM, 40 pM, or 50 pM.

41. The method of claim 39, wherein the bacterial pstS gene encodes a protein having at least 76%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 513 and comprises the polypeptide of SEQ ID NO: 514.

42. The method of claim 39, wherein the phosphate-sensitive promoter comprises a sequence having at least 95% sequence identity to SEQ ID NO: 517.

43. The method of claim 36, wherein the genetically engineered bacterium comprising the quorum sensing promoter which is operably linked to the recombinase further comprises:(a) one or more heterologous gene expression cassette(s) comprising at least one control element which is operably linked to at least one DNA molecule encoding a first quorum sensing synthase protein (QSSP) able to synthesize a quorum sensing signal molecule Q (QSSM Q) and / or at least one DNA molecule encoding a first quorum sensing regulator protein (QSRP) that can bind said QSSM Q, wherein the first quorum sensing promoter can be 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(b) 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 recombinase at a population density of the genetically engineered bacterium which exceeds the threshold population density when the genetically engineered bacterium (GEB) is: (i) contactedwith a quorum quenching compound QQ; or (ii) exposed to a temperature above a threshold temperature.

44. The method of claim 43, 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.

45. The method of claim 44, 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.

46. The genetically engineered bacterium of claim 45, wherein:(i) the AHL acylase enz me 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: 537, 538. 539, or 540, 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 identity7to SEQ ID NO: 541, 542, or 543, 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:

549. 550, 551, 552, or 553, respectively; or(iv) the AHL lactonase enzyme comprises a metallo-beta-lactamase protein, optionally wherein the metallo-beta-lactamase protein is a Gel, AttM, AidC, AiiB, or AiiA protein and optionally wherein the Gel, AttM, AidC, AiiB, or AiiA protein has at least 75% sequence identity to SEQ ID NO: 544, 545, 546, 547, or 548, respectively.

47. The method of claim 43, wherein the deactivator comprises an inducible transcription factor which:(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.

48. The method of claim 43, 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-(P-Ketocaproyl)-L-homoserine lactone and the first QSSP comprises an Ahll protein; or(iv) the QSSM Q molecule comprises N-(P-Ketocaproyl)-L-homoserine lactone and the first QSRP comprises an AhlR protein.

49. The method of claim 43, 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.

50. The method of claim 43, wherein: i. the first QSSP comprises an Ahll protein, the first QSRP comprises an AhlR protein, and the first quorum sensing promoter comprises an ahll promoter; ii. the first QSSP comprises an CinI protein, the first QSRP comprises a CinR protein, and the first quorum sensing promoter comprises a cinl promoter; iii. the first QSSP comprises an Ceil protein, the first QSRP comprises a CciR protein, and the first quorum sensing promoter comprises a ceil promoter; iv. the first QSSP comprises an Cvil protein, the first QSRP comprises a CviR protein, and the first quorum sensing promoter comprises a evil promoter; v. the first QSSP comprises an Esal 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 Esal protein, the first QSRP comprises a EsaR protein, and the first quorum sensing promoter comprises an esal promoter; vii. the first QSSP comprises a LasI protein, the first QSRP comprises a LasR protein, and the first quorum sensing promoter comprises a Iasi or lasB promoter;viii. the first QSSP comprises an LuxI protein, the first QSRP comprises a LuxR protein, and the first quorum sensing promoter comprises a luxl promoter; or ix. the first QSSP comprises an Tral protein, the first QSRP comprises a TraR protein, and the first quorum sensing promoter comprises a tral promoter.

51. The method of claim 50, wherein:(i) the Ahll protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 565;(ii) the Ceil protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 566;(iii) the CinI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 567;(iv) the CinI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 568;(v) the Cvil protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 569;(vi) the Esal protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 570;(vii) the LasI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 593;(viii) the Luxl protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 571; or(ix) the Tral protein comprises a protein having at least 75% sequence identity7to SEQ ID NO: 572.

52. The method of claim 43, 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.

53. The method of claim 43, 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.

54. The method of claim 43, wherein the first QSRP comprises an AhlR, CciR, CinR, CviR, EsaR, LasR, LuxR, or TraR QSRP.

55. The method of claim 54, 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: 556, 557, 558 or 559, 560, 561, or 562, 592, 563. or 564, respectively.

56. The method of any one of claims 1 to 32, wherein the AUB is a diazotrophic and / or phosphate-solubilizing bacterium.

57. The method of any one of claims 1 to 32, wherein the AUB is a member of the genus Azospirillum. Enter obacter. Klebsiella, Kosakonia, Rahnella, Paenibacillus, Phytobacter, Rahnella, Sphingomonas, or Variovorax, wherein the bacterium is optionally a member of the genus Azospirillum, Klebsiella, Kosakonia, or Rahnella.

58. The method of any one of claims 1 to 32, wherein the AUB is selected from the taxonomic class of Gammaproteobacteria.

59. The method of claim 58, wherein the Gammaproteobacteria is a Kosakonia sp., Enterobacter sp., Klebsiella sp., Rahnella sp., Pseudomonas sp., or Acinetobacter sp.

60. The method of any one claims 1 to 32, wherein the recombinase 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: (i) attB and attP sites; or (ii) attL or attR sites; wherein the (i) attB and attP sites; or (ii) attL or attR sites recognized respectively by the PhiC31, IntS, IntM, IntG, YdcL, or Sxt / R39 integrase.

61. The method of claim 60, wherein the recombinase is a phage PhiC31 serine integrase having at least 90%, 95% . or 99% sequence identity’ to SEQ ID NO: 41 and the SSRRS comprise an attP of SEQ ID NO: 42 and an attB recombinase recognition site of SEQ ID NO: 43, or functional equivalents thereof recognized by the phage PhiC31 serine integrase.

62. The method of any one of claims 1 to 32, wherein: (i) the recombinase is a yeast flippase (FLP) recombinase and the SSRRS are FRT sites: or (ii) the recombinase is a Cre- recombinase and the SSRRS are loxP sites.

63. The method of any one of claims 1 to 32, 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.

64. The method of any one of claims 1 to 32, 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.

65. A genetically engineered bacterium comprising:(i) a first heterologous gene expression cassette comprising at least one DNA molecule encoding a protein or RNA sequence of interest, wherein the DNA molecule is operably linked to at least one DNA molecule comprising a site-specific recombinase recognition sequence (SSRRS) and wherein the first heterologous gene expression cassette lacks an irreversible insertion, excision, or inversion of a DNA molecule in the first heterologous gene expression cassette; and(ii) a second heterologous gene expression cassette comprising at least one DNA molecule encoding at least one recombinase that can catalyze site-specific recombination at said SSRRS, wherein the DNA molecule encoding said recombinase is operably linked to a promoter activated by a change of at least one condition in the plant growth medium, and wherein the site-specific recombination can induce an irreversible insertion, excision, or inversion of: (a) a DNA molecule comprising a control element in. from, at, or near the DNA molecule encoding the protein or RNAsequence of interest in the first heterologous gene expression cassette; or (b) a DNA molecule encoding the protein or RNA sequence of interest in the first heterologous gene expression cassette; wherein the genetically engineered bacterium is an agriculturally useful bacterium.

66. A genetically engineered bacterium comprising:(i) a first part of a first heterologous gene expression cassette comprising a DNA molecule encoding a control element, protein or RNA sequence of interest, or a part thereof, wherein the first part of the heterologous gene expression cassette has reduced or no function and wherein the DNA molecule is operably linked to at least one site-specific recombinase recognition sequence (SSRRS); and a second part of the first heterologous gene expression cassette comprising the control element, the protein or RNA of interest, or part of absent from the first part of the first heterologous gene expression cassette; and(ii) a second heterologous gene expression cassette comprising at least one DNA molecule encoding at least one recombinase that can catalyze site-specific recombination at said SSRRS, wherein the DNA molecule encoding said recombinase is operably linked to a promoter activated by a change of at least one condition in the plant growth medium, and wherein the site-specific recombination induces an irreversible insertion, excision, or inversion of DNA which results in the operable linkage of the first and second part of the first heterologous gene expression cassette to provide a functional first heterologous gene expression cassette, wherein the genetically engineered bacterium is an agriculturally useful bacterium.

67. The genetically engineered bacterium of claim 66, wherein the first part and the second part of the first heterologous gene expression cassette are located on separate DNA molecules which are not covalently linked.

68. The genetically engineered bacterium of claim 66, wherein the first part and the second part of the first heterologous gene expression cassette are located on a single DNA molecule.

67. The genetically engineered bacterium of claim 65, wherein the DNA molecule encoding the recombinase comprises an ATG, ACG, or ATT translation initiation codon which is operably linked to the open reading frame of the DNA molecule encoding the recombinase.

68. The genetically engineered bacterium of claim 65, wherein the irreversible insertion, excision, or inversion of the DNA molecule comprising the control element or the DNA moleculeencoding the protein or RNA sequence of interest reduces expression of the protein or RNA sequence of interest in comparison with the first heterologous gene expression cassette lacking the irreversible insertion, excision, or inversion of the DNA molecule.

69. The genetically engineered bacterium of claim 68, wherein the irreversible insertion, excision, or inversion of the DNA molecule causes the expression of the protein or RNA sequence of interest to be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%. 80%. 90%, 95%, 98%, or 99% in comparison to the first heterologous gene expression cassette of (i) lacking the irreversible insertion, excision, or inversion of a DNA molecule.

70. The genetically engineered bacterium of claim 68, wherein the control element comprises a first promoter and a second promoter directed opposite one another, wherein the control element is flanked by two SSRRS in an inverted configuration, wherein the first promoter of the control element is operably linked to the DNA molecule encoding at least one protein or RNA sequence of interest prior to inversion by the site-specific recombinase, wherein transcription promoting-activity of the second promoter is at least 14%, 28%, 42%, or 53% less than the transcription promoting-activity of the first promoter, and wherein the second promoter of the control element is operably linked to the DNA molecule encoding the at least one protein or RNA sequence of interest upon inversion by the site-specific recombinase.

71. The genetically engineered bacterium of claim 70, wherein:(i) the first promoter comprises a constitutive promoter, a native promoter of the native gene encoding the protein or RNA sequence of interest, or a promoter having activity equivalent to or greater than the native promoter; and / or(ii) the protein of interest comprises a GlnA, AmtB, GlnB. GlnK, GlnZ, NifL, and / or DraT protein and the agriculturally relevant compound is ammonia.

72. The genetically engineered bacterium of claim 70, wherein:(i) the first promoter comprises a J23100 constitutive promoter given by SEQ ID NO: 609;;(ii) the second promoter comprises a J23106 constitutive promoter given by SEQ ID NO: 613, J23102 constitutive promoter given by SEQ ID NO: 611, J23111 constitutive promoter given by SEQ ID NO: 614, or a J23104 constitutive promoter given by SEQ ID NO: 612;(iii) the protein of interest comprises a GlnA protein and the agriculturally relevant compound is ammonia; and(iv) the agriculturally useful bacterium is a member of the genus Azospirillum. Enlerobacler, Klebsiella, Kosakonia, Rahnella, Paenibacillus. Phylobacler, Rahnella, Sphingomonas. or Variovorax, wherein the bacterium is optionally a member of the genus Azospirillum, Klebsiella, Kosakonia, or Rahnella.

73. The genetically engineered bacterium of claim 68, wherein the nucleic acid molecule encoding the protein or RNA sequence of interest is flanked by two SSRRS in a direct configuration and wherein the site-specific recombination can induce excision of said nucleic acid molecule encoding a protein or RNA sequence of interest to reduce expression of the protein or RNA sequence of interest.

74. The genetically engineered bacterium of claim 68. wherein the first heterologous gene expression cassette comprises a promoter flanked by two SSRRS in a direct configuration, wherein said promoter is operably linked to the DNA molecule encoding a protein or RNA sequence of interest, and wherein the site-specific recombination can induce excision of said promoter to reduce expression of the protein or RNA sequence of interest.

75. The genetically engineered bacterium of claim 73, wherein: (i) the SSRRS comprise attB and attP sites and / or wherein the DNA molecule comprises a lnA. amtB, glnB, segment of glnE encoding an adenylyl-removing domain of a glutamine synthetase adenylyltransferase, glnK, glnZ, nifL, and / or draT gene; and (ii) the agriculturally relevant compound is ammonia.

76. The genetically engineered bacterium of claim 73, wherein: (i) the SSRRS comprise attB and attP sites and / or wherein the DNA molecule comprises a glnA, amlB. glnB, segment of glnE encoding an adenylyl-removing domain of a glutamine synthetase adenylyltransferase, glnK, glnZ, nifL, and / or draT gene; and (ii) the agriculturally relevant compound is ammonia.

77. The genetically engineered bacterium of claim 68, wherein the first heterologous gene expression cassette comprises a promoter flanked by two SSRRS in an inverted configuration, wherein said promoter is operably linked to the DNA molecule encoding a protein or RNA sequence of interest, and wherein the site-specific recombination can induce inversion of said promoter to uncouple the promoter from the DNA molecule and to reduce expression of the protein or RNA sequence of interest.

78. The genetically engineered bacterium of claim 68, wherein the DNA molecule encoding the protein or RNA sequence of interest is flanked by two SSRRS in an inverted configuration, wherein the DNA molecule is operably linked to a promoter, and wherein the sitespecific recombination can induce inversion of said DNA molecule encoding the protein or RNA sequence of interest to uncouple the DNA molecule from the promoter, thereby reducing expression of the protein or RNA sequence of interest.

79. The genetically engineered bacterium of claim 68, wherein the first heterologous gene expression cassette comprises a promoter which is: (i) inoperably linked to a terminator flanked by two SSRRS in an inverted configuration; and (ii) operably linked to a DNA molecule encoding the protein or RNA sequence of interest, and wherein the site-specific recombination can induce inversion of said terminator to operably link the promoter to the terminator and to uncouple the DNA molecule from the promoter, thereby reducing expression of the protein or RNA sequence of interest.

80. The genetically engineered bacterium of claim 68, wherein the DNA molecule encoding the protein or RNA sequence of interest comprises a first SSRRS comprising an inframe open reading frame (ORF), a part of the DNA molecule encoding a part of the protein or RNA sequence of interest, and a second SSRRS comprising an in-frame ORF, wherein the protein or RNA sequence is functional, and wherein the site-specific recombination can induce excision of the part of the DNA molecule and one SSRRS to reduce activity of the protein or RNA sequence of interest.

81. The genetically engineered bacterium of claim 68. wherein: (i) the DNA molecule encoding the protein or RNA sequence of interest comprises an inserted first SSRRS comprising an in-frame open reading frame (ORF) and wherein the protein or RNA sequence is functional; and (ii) the genetically engineered bacterium further comprises a blocking DNA covalently linked to at least one SSRRS; wherein the site-specific recombination can induce insertion of the blocking DNA into the DNA molecule to reduce expression and / or activity of the protein or RNA sequence of interest.

82. The genetically engineered bacterium of claim 68, wherein the protein of interest comprises a GlnA. AmtB, GlnB, GlnK. GlnZ, NifL, and / or DraT protein and the agriculturally relevant compound is ammonia.

83. The genetically engineered bacterium of claim 65, wherein the irreversible insertion, excision, or inversion of the DNA molecule comprising the control element or the DNA molecule encoding the protein or RNA sequence of interest increases expression of the protein or RNA sequence of interest in comparison to the first heterologous gene expression cassette lacking the irreversible insertion, excision, or inversion of the DNA molecule.

84. The genetically engineered bacterium of claim 83, wherein the irreversible insertion, excision, or inversion of the DNA molecule causes the expression of the protein or RNA sequence of interest to be increased: (i) at least 2-fold, 5-fold ,or at least 10-fold; or (ii) about 2-fold or 5- fold to about 15-fold, both in comparison to the first heterologous gene expression cassette lacking the irreversible insertion, excision, or inversion of a DNA molecule.

85. The genetically engineered bacterium of claim 83, wherein the control element comprises a promoter, wherein the promoter is flanked by two SSRRS in an inverted configuration, and wherein the promoter is operably linked to the at least one protein or RNA sequence of interest upon inversion by the site-specific recombinase.

86. The genetically engineered bacterium of claim 83, wherein the control element comprises a first promoter and a second promoter directed opposite one another, wherein the control element is flanked by two SSRRS in an inverted configuration, wherein the first promoter of the control element is operably linked to the DNA molecule encoding at least one protein or RNA sequence of interest prior to inversion by the site-specific recombinase, wherein transcription promoting-activity of the second promoter is at least 2-, 5-, or 10-fold greater than the transcription promoting-activity of the first promoter, and wherein the second promoter of the control element is operably linked to the at least one protein or RNA sequence of interest upon inversion by the site-specific recombinase.

87. The genetically engineered bacterium of claim 86, wherein:(i) the first promoter comprises a constitutive promoter, a native promoter of the native gene encoding the protein or RNA sequence of interest, or a promoter having activity equivalent to or lower than the native promoter; and / or(ii) the protein of interest is a NifA protein, NtrC protein, GlnR protein, or modified GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity.

88. The genetically engineered bacterium of claim 83, wherein the control element comprises a promoter, wherein the DNA molecule encoding the protein or RNA sequence of interest is flanked by two SSRRS in an inverted configuration, and wherein the DNA molecule encoding the protein or RNA sequence of interest is operably linked to the control element upon inversion by the site-specific recombinase.

89. The genetically engineered bacterium of claim 83, wherein the control element comprises a terminator, wherein the terminator is flanked by two SSRRS in a direct configuration, wherein the terminator and two SSRRS are between the at least one DNA molecule encoding protein or RNA sequence of interest and a promoter in the first heterologous gene expression cassette, and wherein the DNA molecule encoding the protein or RNA sequence of interest is operably linked to the promoter in the first heterologous gene expression cassette upon excision of said terminator.

90. The genetically engineered bacterium of claim 83, wherein the control element comprises a terminator, wherein the terminator is flanked by two SSRRS in an inverted configuration, wherein the terminator and two SSRRS are between the DNA molecule encoding at least one protein or RNA sequence of interest and a promoter in the first heterologous gene expression cassette, and wherein the DNA molecule encoding the protein or RNA sequence of interest is operably linked to the promoter in the first heterologous gene expression cassette upon inversion of said terminator.

91. The genetically engineered bacterium of claim 66, wherein the first part of first heterologous gene expression cassette comprises the first part of the DNA molecule encoding the first part of the protein or RNA sequence of interest, wherein the SSRRS is located at the beginning of, within, or at the end of the first part of the DNA molecule, wherein the genetically engineered bacterium further comprises a third heterologous gene expression cassette comprising a sitespecific recombinase recognition site and the second part of the DNA molecule encoding the second part of the protein or RNA of interest absent from the first heterologous gene expression cassette, and wherein the first and second part of the DNA molecule encoding the protein or RNA sequence of interest are operably linked to provide a functional protein or RNA sequence of interest upon insertion of the second part of the DNA molecule into the first heterologous gene expression cassette.

92. The genetically engineered bacterium of claim 66, wherein the first part of the first heterologous gene expression cassette comprises the DNA molecule encoding the protein or RNA sequence of interest but lacks a control element, wherein the SSRRS is located at the beginning of, within, or at the end of the first part of the DNA molecule, wherein the genetically engineered bacterium further comprises a third heterologous gene expression cassette comprising a sitespecific recombinase recognition site and a control element, and wherein the control element and the DNA molecule encoding the protein or RNA sequence of interest are operably linked to provide a functional first heterologous gene expression cassette upon insertion of the control element into the first heterologous gene expression cassette.

93. The genetically engineered bacterium of claim 83, 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 agriculturally relevant compound 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-ty pe or refactored nif or fix gene cluster and the agriculturally relevant compound is ammonia;(c) a GlnR protein and the agriculturally relevant compound is ammonia;(d) a glutaminase enzyme and the agriculturally relevant compound is ammonia;(e) a protein product of a refactored nif or fix gene cluster and the agriculturally relevant compound 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 (cl), 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 optionally7wherein the first target gene(s) is / are a glnA, amtB, glnB, glnK, glnZ, nifL, and / or draT gene and the agriculturally relevant compound is ammonia; or(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 aglnA, amlH. glnH. glnK, glnZ, nifL, and / or draT gene and the agriculturally relevant compound is ammonia.

94. The genetically engineered bacterium of claim 65, wherein:(i) the first heterologous gene expression cassette comprises a control element comprising a promoter flanked by two SSRRS oriented as inverted repeats, wherein the promoter is operably linked to a first DNA molecule encoding a first protein or RNA sequence of interest, wherein the promoter flanked by two SSRRS is uncoupled from a second DNA molecule encoding a second protein or RNA sequence of interest; and(ii) the site-specific recombination can induce the inversion of the DNA molecule comprising the promoter to uncouple the promoter from the first DNA molecule, to operably link the promoter to the second DNA molecule, and to reduce expression of the first protein or RNA sequence of interest and increase expression of the second protein or RNA sequence of interest, both in comparison to the first heterologous gene expression cassette of (i) lacking the irreversible inversion of the DNA molecule.

95. The genetically engineered bacterium of claim 66, wherein:(i) the first heterologous gene expression cassette comprises a control element comprising a promoter which is operably linked to a first DNA molecule encoding a first protein or RNA sequence of interest which is operably linked to an SSRRS, wherein the promoter is uncoupled from a second DNA molecule encoding a second protein or RNA sequence of interest which is operably linked to a terminator element and an SSRRS; and(ii) the site-specific recombination can induce the insertion of the second DNA molecule encoding the second protein or RNA sequence of interest to uncouple the promoter from the first DNA molecule, to operably link the promoter to the second DNA molecule, and to reduce expression of the first protein or RNA sequence of interest and increase expression of the second protein or RNA sequence of interest, both in comparison to the first heterologous gene expression cassette of (i) lacking the irreversible inversion of the DNA molecule.

96. The genetically engineered bacterium of claim 94, wherein:(a) the first protein of interest comprises a wild-type glutamine synthetase (GS) or variant thereof with improved catalytic activity in comparison to wild-type GS and the second protein of interest comprises a wild-type GS with reduced levels of expression in comparison to the wildtype GS or a GS variant with decreased catalytic activity in comparison to wild-type GS, and the agriculturally relevant compound is ammonia:(b) the first protein of interest comprises a GS adenylyltransferase protein and the second protein of interest comprises a GS adenylyltransferase protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, and the agriculturally relevant compound is ammonia; or(c) the first target gene is a nifL gene and the second target gene is a nifA gene, and the agriculturally relevant compound is ammonia.

97. The genetically engineered bacterium of any of claims 65 to 96, wherein said change of at least one condition in the plant growth medium condition comprises:(a) growth of the genetically engineered bacterium to exceed a threshold population density in the plant growth medium;(b) reduction of at least one fertilizer or plant nutrient in the soil, 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;(c) growth of a plant in the plant growth media;(d) change in soil temperature;(e) change in motility of the bacterium;(f) change in light levels in the soil(g) change in oxygen concentration in the soil(h) change in concentration of bioavailable nitrogen in the soil;(i) change in pH in the soil(j) change in overall solute concentration (osmotic pressure) in the soil;(k) change in phytohormone or plant signaling molecule concentrations;(l) change in an amino acid concentration;(m) change in a sugar concentration; and / or(n) a change in plant or microbial metabolite concentrations, optionally wherein the plant or microbial metabolite is naringenin, quercetin, luteolin, apigenin, octopine, nopaline, or Scyllo- inosamine.

98. The genetically engineered bacterium of any one of claims 65 to 96, wherein said promoter activated by a change of at least one plant growth medium condition comprises:(i) a phosphate-sensitive promoter;(ii) a nitrogen -sensitive promoter;(iii) a quorum sensing promoter;(iv) a promoter which is induced or repressed by decreased oxygen levels;(v) a promoter which is induced or repressed by increased oxygen levels;(vi) a promoter which is induced by decreased oxygen and decreased nitrogen levels, optionally wherein the promoter is a nifH promoter;(vii) a promoter which is induced or repressed by increased or decreased potassium levels;(viii) a promoter which is induced by decreased carbon levels, optionally wherein the promoter is a Pfic promoter;(ix) a promoter that is induced by increased nitrate levels, optionally wherein the promoter is an oxygen insensitive PnarG promoter;(x) a promoter that is induced by increased nitrate levels, optionally wherein the promoter is a narK or narX promoter;(xi) a promoter which is upregulated by an increase in the concentration of naringenin; and / or(xii) a promoter or system comprising a promoter set forth in Table 5, Table 6, or Table 7.

99. The genetically engineered bacterium of claim 98, wherein the promoter which is induced by decreased oxygen and decreased nitrogen levels is a NifH promoter.

100. The genetically engineered bacterium of claim 99, wherein the NifH promoter comprises a DNA molecule having at least 75%, 80%, 85%, 90%, 95%. 98%. or 99% sequence identity to SEQ ID NO: 595 or 596.

101. The genetically engineered bacterium of claim 98, w herein the genetically engineered bacterium comprises the phosphate-sensitive promoter which is operably linked to the recombinase and wherein:(a) the threshold concentration of phosphate in the plant grow th medium which activates expression of the recombinase is about 0 pM to about 50 pM;(b) the phosphate-sensitive promoter and / or a segment of the 5?untranslated region (UTR) which is operably linked to the phosphate-sensitive promoter comprises at least one copy of an operably linked Pho box, wherein the Pho box comprises the sequence of SEQ ID NO: 525-529, 234-396, or 397 and sequences with at least 95% identity to any one of SEQ ID NO: 234-397, optionally wherein the Pho box comprises SEQ ID NO: 525-529. 376, 377. 383-396. or 397;(c) the phosphate-sensitive promoter and / or a segment of the 5 ' UTR comprises a promoter and / or a segment of a 5’UTR of a phoA, phoX, phy, pstS gene, a variant thereof, or a combination thereof; and / or(d) the phosphate-sensitive promoter and / or the segment of the 5' UTR comprises a promoter of a gene encoding:(i) a PstS protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 524, 399, 401, 403, 405, 407, 409, 415, 416, 513, 523, and 524 or having an identity of at least 76%, 80%, 85%, 90%, 95%, 98%, or 99% with any one of SEQ ID NO: 399, 401, 403, 405, 407. 409, 415, 416, 513, or 524;(ii) a PhoX protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 408, 410, 411, 412, and 413 or having an identity of at least 80% with any one of SEQ ID NO: 408, 410, 411, 412, and 413;(iii) a Phy protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 414, 57-138, and 139 or having an identity of at least 80% with any one of SEQ ID NO: 414, 57-138, or 139; and / or(iv) a PhoA protein comprising a protein sequence selected from the group consisting of SEQ ID NO: 398, 400, 402, 404, and 406 or having an identity of at least 80% with any one of SEQ ID NO: 398, 400. 402, 404, or 406.

102. The genetically engineered bacterium of claim 101, wherein the phosphate-sensitive promoter and / or the segment of the 5’ UTR comprises: i. a pstS promoter comprising a DNA sequence selected from the group consisting of SEQ ID NO: 517, 417, 418, 420, 421, 422, 423, 424, 425, 428, 515, and 518; ii. a phoX promoter comprising the DNA sequence of SEQ ID NO: 426; iii. a phy promoter comprising the DNA sequence of SEQ ID NO: 427; iv. a phoA promoter comprising the DNA sequence of SEQ ID NO: 419; and / or v. a Pliar53 promoter comprising the DNA sequence of SEQ ID NO: 459;vi. a variant of the promoters under i. to v. having an identity of at least 76%, 80%, 90%, 95%, 98%, or 99% with any of SEQ ID NO:

515. 417, 418-428, 459, 517, or 518, wherein said variant comprises at least one Pho box and / or retains at least one Pho box present in each promoter and wherein said variant promoters are activated by a decrease in phosphate concentration to about 0 pM to about 1 pM, 2 pM, 5 pM, 7 pM, 10 pM, 20 pM, 30 pM, 40 pM, or 50 pM.

103. The genetically engineered bacterium of claim 101, wherein the bacterial pstS gene encodes a protein having at least 76%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 513 and comprises the polypeptide of SEQ ID NO: 514.

104. The genetically engineered bacterium of claim 101, wherein the phosphate-sensitive promoter comprises a sequence having at least 95% sequence identity' to SEQ ID NO: 517.

105. The genetically engineered bacterium of claim 98, wherein the genetically engineered bacterium comprising the quorum sensing promoter which is operably linked to the recombinase further comprises:(a) one or more heterologous gene expression cassette(s) comprising at least one control element which is operably linked to at least one DNA molecule encoding a first quorum sensing synthase protein (QSSP) able to synthesize a quorum sensing signal molecule Q (QSSM Q) and / or at least one DNA molecule encoding a first quorum sensing regulator protein (QSRP) that can bind said QSSM Q, wherein the first quorum sensing promoter can be 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(b) 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 recombinase 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.

106. The genetically engineered bacterium of claim 105, wherein the deactivator comprises an enzyme which catalyzes the degradation of the quorum sensing signal molecule Qand wherein the expression of the enzy me is induced by the addition of the quorum quenching compound QQ or the increase in temperature.

107. The genetically engineered bacterium of claim 106, 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 enzy me, an AHL lactonase enzyme, or an AHL metallo-beta-lactamase enzyme.

108. The genetically engineered bacterium of claim 107, wherein:(i) the AHL acylase enzy me 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: 537, 538. 539, or 540, 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: 541, 542, or 543, 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:

549. 550, 551, 552, or 553, respectively; or(iv) the AHL lactonase enzyme comprises a metallo-beta-lactamase protein, optionally wherein the metallo-beta-lactamase protein is a Gel, AttM, AidC, AiiB, or AiiA protein and optionally wherein the Gel, AttM, AidC, AiiB, or AiiA protein has at least 75% sequence identity’ to SEQ ID NO: 544, 545, 546, 547, or 548, respectively.109.The genetically engineered bacterium of claim 105, wherein the deactivator comprises an inducible transcription factor which:(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.

110. The genetically engineered bacterium of claim 105, 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-(P-Ketocaproyl)-L-homoserine lactone and the first QSSP comprises an Ahll protein; or(iv) the QSSM Q molecule comprises N-(P-Ketocaproyl)-L-homoserine lactone and the first QSRP comprises an AhlR protein.

111. The genetically engineered bacterium of claim 105, 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.

112. The genetically engineered bacterium of claim 105, wherein: i. the first QSSP comprises an Ahll protein, the first QSRP comprises an AhlR protein, and the first quorum sensing promoter comprises an ahll promoter; ii. the first QSSP comprises an CinI protein, the first QSRP comprises a CinR protein, and the first quorum sensing promoter comprises a cinl promoter; iii. the first QSSP comprises an Ceil protein, the first QSRP comprises a CciR protein, and the first quorum sensing promoter comprises a ceil promoter; iv. the first QSSP comprises an Cvil protein, the first QSRP comprises a CviR protein, and the first quorum sensing promoter comprises a evil promoter; v. the first QSSP comprises an Esal 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 Esal protein, the first QSRP comprises a EsaR protein, and the first quorum sensing promoter comprises an esal promoter; vii. the first QSSP comprises a LasI protein, the first QSRP comprises aLasR protein, and the first quorum sensing promoter comprises a Iasi or lasB promoter; viii. the first QSSP comprises an LuxI protein, the first QSRP comprises a LuxR protein, and the first quorum sensing promoter comprises a luxl promoter; or ix. the first QSSP comprises an Tral protein, the first QSRP comprises a TraR protein, and the first quorum sensing promoter comprises a tral promoter.

113. The genetically engineered bacterium of claim 112, wherein:(i) the Ahll protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 565;(ii) the Ceil protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 566;(iii) the CinI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 567;(iv) the CinI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 568;(v) the Cvil protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 569;(vi) the Esal protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 570;(vii) the LasI protein comprises a protein having at least 75% sequence identity to SEQ ID NO: 593;(viii) the LuxI protein comprises a protein having at least 75% sequence identity’ to SEQ ID NO: 571; or(ix) the Tral protein comprises a protein having at least 75% sequence identity’ to SEQ ID NO: 572.

114. The genetically engineered bacterium of claim 105, 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.

115. The genetically engineered bacterium of claim 105, 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.

116. The genetically engineered bacterium of claim 105, wherein the first QSRP comprises an AhlR, CciR, CinR, CviR, EsaR, LasR, LuxR, or TraR QSRP.

117. The genetically engineered bacterium of claim 116, 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:

556. 557, 558 or 559. 560, 561. or 562. 592, 563. or 564, respectively.

118. The genetically engineered bacterium of any one of claims 65 to 94, wherein the AUB is a diazotrophic and / or phosphate-solubilizing bacterium.

119. The genetically engineered bacterium of any one of claims 65 to 94, wherein the AUB is a member of the genus Azospirillum, Enterobacter, Klebsiella, Kosakonia, Rahnella, Paenibacillus, Phytobacter, Rahnella, Sphingomonas, or Variovorax. wherein the bacterium is optionally a member of the genus Azospirillum, Klebsiella, Kosakonia, or Rahnella.

120. The genetically engineered bacterium of any one of claims 65 to 94, wherein the AUB is selected from the taxonomic class of Gammaproteobacteria.

121. The genetically engineered bacterium of claim 120, wherein the Gammaproteobacteria is a Kosakonia sp., Enterobacter sp., Klebsiella sp., Rahnella sp.. Pseudomonas sp., or Acinetobacter sp.,122. The genetically engineered bacterium of any one claims 65 to 94, wherein the recombinase 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: (i) attB and attP sites; or (ii) attL or attR sites; wherein the (i) attB and attP sites; or (ii) attL or attR sites recognized respectively by the PhiC31. IntS, IntM, IntG, YdcL, or Sxt / R39 integrase.

123. The genetically engineered bacterium of claim 122, wherein the recombinase is a phage PhiC31 serine integrase having at least 90%, 95%, or 99% sequence identity to SEQ ID NO: 41 and the SSRRS comprise an attP of SEQ ID NO: 42 and an attB recombinase recognition site of SEQ ID NO: 43, or functional equivalents thereof recognized by the phage PhiC31 serine integrase.

124. The genetically engineered bacterium of any one of claims 65 to 96, wherein: (i) the recombinase is a yeast flippase (FLP) recombinase and the SSRRS are FRT sites; or (ii) the recombinase is a Cre-recombinase and the SSRRS are loxP sites.

125. A method of producing a bacterial culture comprising:(i) growing the genetically engineered bacterium of any one of claims 65 to 96 either:(a) in contact with the quorum quenching compound QQ;(b) in exposure to a temperature above the threshold temperature;(c) or under conditions where expression or activity of the recombinase is suppressed; and(ii) harv esting the bacterial culture.

126. An agricultural system comprising(i) a plant grow th medium;(ii) at least one plant of an agronomically relevant plant species rooted in said plant growth medium; and(iii) the genetically engineered bacterium of any one of claims 65 to 96.

127. The system of claim 126, wherein the plant is an alfalfa, apple, banana, barley, bean, buckwheat, cabbage, cassava, chili, clover, coffee, com, 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.

128. The system of claim 127, wherein the plant growth medium comprises soil and / or water, optionally wherein the soil and / or w ater is non-axenic.

129. The system of claim 127, wherein the vegetative propagule comprises a cutting, tuber, or stolon.

130. A composition comprising the genetically engineered bacterium of any one of claims 65 to 96 and an agriculturally acceptable carrier.

131. The composition of claim 130, 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.

132. The composition of claim 130, wherein the composition is in a solid form, optionally wherein the solid form comprises a wettable powder, granules, a gel, pellets, or microencapsulated particles.

133. The composition of claim 130, 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.

134. A plant part or plant propagule which is at least partially coated, imbibed, or mixed with the composition of claim 130.

135. The plant part of claim 134. wherein the part is a leaf, stem, root, or seed.

136. The plant propagule of claim 134, wherein the propagule comprises a cutting, tuber, or stolon.

137. Use of the plant part or plant propagule of claim 134 to grow a crop.

138. The use of claim 137, wherein fertilizer input is reduced in comparison to a crop grown from a plant part or plant propagule which has not been treated.