Class iib microcins and methods of use thereof

Abstract

Provided herein are antimicrobial microcin peptides and genetically engineered microorganisms expressing the peptides, and compositions comprising the peptides and microorganisms for treating or reducing the risk of dysbiosis or bacterial infections in animals or plants, and further discloses methods of making and using such microorganisms.

Description

[0001] Class lib Microcins and Methods of Use Thereof

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Patent Application Serial Nos. 63 / 603.032, filed on November 27, 2023, and 63 / 684.595, filed on August 19, 2024. The entire contents of the foregoing are hereby incorporated by reference.

[0004] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with Government support under Grant No. AG075283 awarded by the National Institutes of Health, and Grant No. W81XWH2020013 awarded by the Department of Defense. The Government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] Provided herein are antimicrobial microcin peptides and genetically engineered microorganisms expressing the peptides, and compositions comprising the peptides and microorganisms for treating or reducing the risk of dysbiosis or bacterial infections in animals or plants, and further discloses methods of making and using such microorganisms.

[0008] BACKGROUND

[0009] A large body of theoretical and experimental work has shown that dynamics of microbiomes are shaped by the network of interbacterial interactions1 4These cooperative and competitive interactions are often achieved via the secretion of crossfeeding metabolites5, antimicrobial peptides6and bacterially produced small molecules78and are crucial to ecological properties including stability and ability to respond to external perturbations9 10. Among the competitive interactions, bacteriocin production is proposed to be a prominent mediator of microbiomes dynamics11and, specifically, several reports including ours have shown that a bacteriocins subclass, Class lib microcins, mediates Enterobacteriaceae dynamics in vivoiW.

[0010] SUMMARY

[0011] The results described herein challenge the prevailing notion that class lib microcin production is limited to Ec and Kp. Through comprehensive genomic analysis of publicly available bacterial genomes, coupled with heterologous overexpression, a set of previously undiscovered class lib microcins was discovered across Enterobacteriaceae species. The present findings not only expand the known repertoire of class Uh microcins but also hold significant implications for synthetic hybrid compounds. As shown herein, these newly identified class lib microcins exert remarkable inhibitory’ effects on ESKAPE pathogen species not affected by any of the currently known microcins. These microcins can be used as agents against a broader spectrum of pathogens, including those affecting humans and plants.

[0012] Thus provided herein are genetically engineered microorganisms capable of producing one or more microcins Bg E492, Bg X, Gq W, Ko H47, Ps G492, Ro Z, Ro H47, Ro 147. Ro X, Sf H47, Se G492, Se M, or Ec W, wherein the microorganism comprises: a microcin operon comprising microcin antimicrobial gene Bg E492A, Bg XA, Gq WA, Ko H47A, Ps G492A, Ro ZA, Ro H47A, Ro 147 A, Ro XA, Sf H47A, Se G492A, Se MA, or Ec WA.and preferably an immunity' gene Bg E492I, Bg XI, Gq WI, Ko H47I, Ps G492I, Ro ZI, Ro H47I, Ro 1471, Ro XI. Sf H47I, Se G492I, Se MI, or Ec WI, and genes for the production of the antimicrobial gene and immunity genes, optionally mchCDEF and mcmL, or mceCDGHIJ^ and a promoter for the microcin operon, w herein either or both of the microcin operon and the promoter are heterologous to the microorganism.

[0013] In some embodiments, the genetically engineered microorganism is a bacterium, optionally wherein the genetically engineered microorganism is Escherichia coli.

[0014] In some embodiments, the microcin operon further comprises microcin genes mchE and mchF.

[0015] In some embodiments, the promoter is a controllable promoter for the microcin operon that controls a level of expression of one or more of the microcin genes, thereby controlling the amount of microcin produced by the genetically engineered microorganism,

[0016] In some embodiments, the microorganism comprises a second microcin operon comprising microcin gene mchA and a second controllable promoter for the second microcin operon, wherein the second controllable promoter controls a level of expression of mchA, thereby controlling the amount of microcin produced by the genetically engineered microorganism. In some embodiments, the first or the second microcin operon, or both the first and the second microcin operons and the first or second controllable promoter, or both the first and the second controllable promoters are in the genome of the microorganism, or are in a vector.

[0017] Also provided herein are compositions for use in treating a bacterial infection, wherein the composition comprises the genetically engineered microorganisms described herein. In some embodiments, the composition is packaged in a capsule for intestinal delivery. In some embodiments, the bacterial infection is a gram-negative bacterial infection in an animal or plant.

[0018] Also provided herein are methods of treating or reducing risk of a bacterial infection or intestinal dysbiosis in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising a genetically engineered microorganism described herein, optionally wherein the bacterial infection is a gram-negative bacterial infection.

[0019] In some embodiments, the subject is a human and the composition is administered by endoscopy, enteroscopy, colonoscopy, a nasoduodenal catheter, enema, or by oral administration.

[0020] In some embodiments, the composition is orally administered, optionally in a capsule.

[0021] Additionally, provided herein are methods of treating a bacterial infection in a plant, the method comprising administering to the plant an effective amount of a composition comprising a genetically engineered microorganism as described herein. In some embodiments, the plant has an infection with Gibbsiella quercinecans, Gibbslella greigii, Rahnella victoriana. or Brenneria goodwinii. In some embodiments, the plant is a tree, optionally an oak tree, walnut tree, or autumn olive.

[0022] Further, provided herein are isolated microcin antimicrobial peptides comprising Bg E492A, Bg XA, Gq WA, Ko H47A, Ps G492A, Ro ZA, Ro H47A, Ro 147 A, Ro XA, Sf H47A, Se G492A, Se MA, or Ec WA. optionally wherein the peptide is modified. In some embodiments, the peptides are at least 80%. 85%. 90%. 95%, or 99% identical to an antimicrobial peptide described herein, or have at least 1 and up to 2, 3, 4, or 5 amino acid substitutions, e.g., conservative amino acid substitutions. Also provided herein are compositions comprising the isolated microcin antimicrobial peptides and a carrier, optionally a pharmaceutically or agriculturally acceptable carrier. In some embodiments, the composition is packaged in a capsule for intestinal delivery. Also provided are the compositions described herein for use in treating or reducing risk of a bacterial infection, optionally wherein the bacterial infection is a gram-negative bacterial infection in an animal or plant.

[0023] Additionally provided herein are methods of treating or reducing risk of a bacterial infection or intestinal dysbiosis in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition described herein, optionally wherein the bacterial infection is a gram-negative bacterial infection. In some embodiments, the subject is a human. In some embodiments, the composition is administered by endoscopy, enteroscopy, colonoscopy, a nasoduodenal catheter, enema, or by oral administration. In some embodiments, the composition is orally administered, optionally in a capsule.

[0024] Further, provided herein are methods of treating a bacterial infection in a plant, the method comprising administering to the plant an effective amount of a composition comprising a composition as described herein. In some embodiments, the plant has an infection with Gibbsiella quercinecans, Gibbsiella greigii, Rahnella victor iana. or Brenneria goodwinii. In some embodiments, the plant is a tree, optionally an oak tree, walnut tree, or autumn olive.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0026] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. DESCRIPTION OF DRAWINGS

[0027] FIGs. 1A-D: Novel class lib microcins are found in numerous Enterobacteriaceae genomes. (A) Sequence alignments of the newly identified mi crocin and immunity genes with the gene clusters of Ec CA46 and Kp RYC492 using Easyfig35. Antimicrobial (A) and immunity (I) genes in the center are represented by darker and lighter shades, respectively. X=mchX, I=mchl. B=mchB, E=mceE. L=mceL. M=mceM. (B.C) Phylogenetic trees of antimicrobial and corresponding immunity genes using codon-aligned nucleotide sequences with General Time Reversible model with discrete gamma distribution (GTR+G) and the Hasegawa-Kishino-Yano model with discrete gamma distribution (HKY+G), respectively. (D) MUSCLE30alignment of the amino acid sequence of the signal peptide sequence as well as the C-terminus of the antimicrobial peptides.

[0028] FIGs. 2A-C: Novel class lib microcins are effective inhibitors of Enterobacteriaceae and extend Gram-negative ESKAPE pathogens. (A) Heatmap summarizing the inhibitory’ potential of known and novel class lib microcins against a library of Enterobacteriaceae, Pseudomonadales, and Gram-positive bacteria, including multidrug-resistant isolates (red) as determined by static inhibition assays with live producing bacteria. *=acti vity determined through microcin purification and minimum inhibitory concentration assays13 16. (B) Relative minimum inhibitory concentrations for Se G492 against different bacterial species. Note that Se G492 is 256-times more potent against A. baumannii (BAA 1790) compared to K. pneumoniae (BAA 1705). (C) Static inhibition assays comparing Kp E492, Kp G492, Ec H47, Ec 147, Ec M, and Se G492 activity from single colony production against multidrugresistant A. baumannii (BAA 1790) and / 5aeruginosa (PA14). Note that iron-limited conditions (DP) are required for antimicrobial activity, confirming action of class lib microcins. L-ara=L-arabinose, DP=2,2-dipyridyl, scale bars: 1 cm.

[0029] FIG. 3: Comparison of class lib microcin gene clusters from Escherichia coli CA46 and Klebsiella pneumoniae RYC492. BLAST sequence comparison of gene clusters was created using Easyfig35. Antimicrobial and immunity genes of each cluster are represented by darker and lighter shades, respectively. Colored polygons represent functional microcins MccE492 (pink) and MccG492 (dark blue) from K. pneumoniae as well as MccH47 (light blue). MccI47 (red), and MccM (green) Prom E coll. X=mchX, I=mchl, B=mchB, E=mceE, L=mceL, M=mceM. Asterisks indicate truncated and non-functional microcin or immunity genes.

[0030] FIGs. 4A-G: Comparison of class lib microcin gene cluster from newly identified genomes with Escherichia coli CA46 and Klebsiella pneumoniae RYC492. BLAST sequence comparison of gene clusters was created using Easyfig35. Antimicrobial and immunity genes of each cluster are represented by darker and lighter shades, respectively. Colored polygons represent novel microcins from the genome indicated on the left: MccE492 (pink), MccG492 (dark blue), MccEI47 (light blue), MccI47 (red), MccM (green), MccX (black), MccW (dark red), and MccZ (dark green). X=mchX, I=mchl, B=mchB, E=mceE. L=mceL, M=mceM. Asterisks indicate truncated and non-functional microcin or immunity genes.

[0031] FIGs. 5A-B: Region and domain prediction of (A) antimicrobial and (B) immunity peptides using SMART29.

[0032] FIG. 5C. Phylogeny of all microcins from the classes I, Ila, and lib. Phylogenetic tree of all known microcin genes using codon-aligned nucleotide sequences with General Time Reversible model with discrete gamma distribution (GTR+G). The respective signal peptides were removed to ensure alignment of the active regions with antimicrobial activity7. Previously knoyvn class lib microcins are highlighted in bold.

[0033] FIGs. 6A-G. Ec W is a novel class lib microcin found in E. coli. (A) Two homologs of the class lib microcin gene clusters were identified in E. coli LR134092. Sequence alignment with E. coli CA46 reveals very7high conservation of the nucleotide sequence with minimal sequence variability7. The genes of the glycosyltransferase (mcmL) and the enterobactin esterase (mcmK) are only present in one of the homologs (bottom). Note that the novel microcin Ec W is inserted at the microcin M position in reverse complement orientation without any sequence similarity to the known gene cluster. Alignment was created using Easyfig. (B) Phylogenetic tree of the antimicrobial peptides using codon-aligned nucleotide sequences with General Time Reversible model with discrete gamma distribution and empirical base frequencies (GTR+G+F). (C) Table summarizing the amino acid sequence similarity7between Ec W and Gq W. (D) Map of plasmid used for heterologous overexpression of microcin Ec W, where Ec W and its corresponding immunity peptide are coexpressed under control of a pBAD promoter. (E) Bar plot of zone of inhibition diameters against a I i bran- of Enterobacteriaceae species from static inhibition assays. (F) MUSCLE alignment of the amino acid sequence of all known class lib microcins. (G) Uncompressed phylogenetic tree of the antimicrobial peptides using codon-aligned nucleotide sequences with General Time Reversible model with discrete gamma distribution and empirical base frequencies (GTR+G+F).

[0034] FIGs. 7A-J. Exemplary Microcin sequences

[0035] FIGs. 8A-T. Exemplary sequences for operon genes.

[0036] 8A-J, DNA and protein sequences for MchCDEF and McmL (mcmA) from E.

[0037] Coli H47 8K-T, DNA and protein sequences for mceCGHIJ from Kp

[0038] DETAILED DESCRIPTION

[0039] Interspecies interactions involving direct competition via bacteriocin production play a vital role in shaping ecological dynamics within microbial ecosystems. For instance, bacteriocins known as class lib microcins have been observed to affect the colonization of pathogenic Enterobacteriaceae species within hosts.

[0040] Class lib microcins are ribosomally synthesized bacteriocins between 5 kDa to

[0041] 10 kDa in size with activity against closely related strains or species13 17Unlike all other microcins, they carry a serine-rich C-terminal motif for a posttranslational modification with a siderophore, here an enterobactin or an enterobactin derivative, before they are secreted into the extracellular space18. Siderophores are iron-chelating molecules commonly employed by various bacteria to scavenge free iron to compete with other bacteria, particularly in resource-scarce environments such as the gastrointestinal tract15,16’19and are often associated with increased pathogenicity or virulence20 22. The iron chelating moiety of these post-translationally modified antimicrobial peptides is recognized by high-affinity receptors and functions as a ‘Trojan Horse’ key to susceptible bacteria as it triggers import into the periplasmic space, where the peptide inhibits the molecular target of susceptible bacteria19-23 25. Because of these features, delivery of class lib microcins by wildtype and engineered probiotics has been recently proposed as a strategy' to combat drug-resistant enteric bacteria12 13,16’26, which is in line with, a growing body of work from the past decade that explores siderophore conjugation, including with enterobactin. to specifically deliver antibiotics and other small molecules to drug-resistant Gram-negative pathogens (Page, Clinical Infectious Diseases [Internet], 2019 Nov 13;69(Supplement_7):S529-37; Negash et al.. Molecules [Internet]. 2019 Sep

[0042] 11 ;24( 18); Rayner et al., RSC Med Chem [Internet]. 2023 May 25 [cited 2024 Jul 6];14(5):800-22).

[0043] To date only five class lib microcins have been described and only four have been characterized in terms of their antimicrobial activity. Specifically, the class lib microcins MccE492 and MccG492 (uncharacterized) are solely present in Klebsiella pneumoniae (Kp), whereas MccH47 is specific for Escherichia coli (Ec)15. Additionally, truncated versions of mciA (MccI47) and mcmM (MccM) are present in Kp RYC492, whereas they are intact in the Ec CA46 genome15. Interestingly, while the genes encoding for microcins posttranslational modifications are highly conserved between Ec and Kp, suggesting a conserved pathway for mi crocin maturation, the toxin and corresponding immunity genes are significantly more variable (Figure 3).

[0044] After a comprehensive analysis of publicly available bacterial genomes, we describe twelve previously undiscovered class lib microcins. Among these findings, we identified three novel microcin clades, specifically MccW, MccX, and MccZ. Through heterologous expression, we showed antimicrobial activity for all but one of the new microcins and were the first to demonstrate activity for the known class lib microcin Kp G492. Hence, this research demonstrates that class lib microcin genes exhibit a higher prevalence in Enter obact er iaceae genomes than previously reported. As a result, their impact on ecological community dynamics in natural environments, including the growth of Pseudomonadales species, may be broader than previously thought. For antimicrobial activity testing, microcin and immunity genes were overexpressed recombinantly in our Ec-derived expression system optimized for microcin-MGE production. The common process of posttranslational modification with the siderophore consolidated the import mechanism of the hybrid microcins towards enterobactin, the most characteristic siderophore of the Enterobacteriaceae family. This allowed us to test the target-specific antimicrobial activity of the microcins irrespective of siderophore production in the native genomic background. However, it is important to note that class lib microcin activity is dependent on active import through siderophore receptors and consequently some of these microcins might display different activity spectrums when tested in their native genomic background of siderophore biosynthesis. Furthermore, static plate inhibition assays exhibit lower sensitivity7compared to purification approaches with quantitative minimum inhibitory concentration (MIC) assays. Thus, the activity spectrums of the hybrid microcins could encompass a wider range than what has been described in this study when tested as a purified product. However, historically the microcin literature proves that ideal approaches for purification and MIC testing can vary between the antimicrobials13,15716724 Remarkably, we were able to expand the origins of class lib microcins from the enteric bacteria Ec and Kp to other members of the Enterobacteriaceae family, including well-known phytopathogens36-38. Specifically, B. goodwinii and G. quercinecans are associated with Acute Oak Decline (AOD) and are frequently isolated together39, and the two strains containing microcin genes were isolated within the same research project. Notably, these bacteria grow synergistically40, while upregulating iron transporters during co-culture41. hinting at class lib microcin-related competition. We were able to show activity of the overexpressed hybrid microcins against human-derived enteric isolates, however, their native antimicrobial spectrum might have evolved to target more frequently encountered strains from the genus Brenneria or Gibbsiella. Further, we demonstrated activity of several class lib microcins against the three tree pathogen genera Brenneria, Gibbsiella, and Rahnella (e.g., G. quercinecans, G. greigii as well as Rahnella Victor iana)40A2A3. Thus, treatment with potent microcins, purified or produced in live bacteria, could present a viable option to target bacteria-caused plant diseases.

[0045] In health care settings the burden by gram-negative ESKAPE pathogens and multi drug-resistant Enterobacteriaceae weighs heavily on modem medicine and novel antimicrobials are needed to develop new treatment options44,45. In addition to enteric pathogens and pathobionts. bacteria outside of the Enterobacteriaceae family have also been shown to scavenge for and to import enterobactin. including P. aeruginosa and A. baumannii. (Moynie et al., Nature Communications 2019 10: 1. 2019 Aug 14; 10(1): 1—14; Subashchandrabose et al., mSphere. 2016 Feb 25; 1 (1)). Therefore, different siderophore conjugates could be a viable option to target these pathogens as well or to finetune the desired target range (Negash et al., Molecules [Internet]. 2019 Sep 11;24(18); Rayner et al., RSC Med Chem [Internet]. 2023 May 25 [cited 2024 Jul 6]; 14(5): 800-22). Antimicrobial peptides and particularly microcins are promising candidates for selective eradication of enteric pathogens and have been demonstrated to potently reduce pathogen colonization in vivo, when produced by a live probiotic12,13. Here we present the most comprehensive library of class lib microcins created so far, that is suited for heterologous expression and in vivo application for the development of novel live biotherapeutic products against drug-resistant enteric bacteria and gram-negative ESKAPE pathogens. Class lib Microcins

[0046] Table A provides a list of class lib microcins described herein, with antimicrobial peptides and their corresponding immunity genes. Exemplary sequences for each are provided below and in FIGs. 7A-H.

[0047] TABLE A. Microcins and Corresponding Immunity Genes

[0048] Provided herein are compositions comprising isolated microcin proteins, e.g., isolated anti-microbial proteins, e.g., comprising isolated anti-microbial proteins selected from Bg E492A, Bg XA, Gq WA, Ko H47A, Ps G492A, Ro ZA, Ro H47A, Ro 147 A. Ro XA, Sf H47A, Se G492A, Se MA. and Ec W.

[0049] The proteins can be produced using methods known in the art, e.g., chemically or enzymatically synthesized, or expressed recombinantly or using cell free systems, siderophore-conjugates. See, e.g., Garenne et al., Nature Reviews Methods Primers volume 1, Article number: 49 (2021)(cell-free protein synthesis of proteins); Wang et al., ACS Chemical Biology 2024 19 (9). 1953-1962 (fast-flow solid phase peptide synthesis coupled with chemoenzymatic chemistry); the proteins can be produced, e.g., as siderophore-conjugates, or conjugates with non-natural siderophore analogs optionally with cleavable linkers (see, e.g., Puja et al., Biomolecules 2023, 13(6), 959; Peukert et al., Chem. Sci., 2023, 14, 5490-5502; Zheng et al., Journal of the American Chemical Society 2012 134 (44), 18388-18400; Caradec et al., ACS Central Science 2023 9 (11), 2138-2149).

[0050] In some embodiments, the microcins can be purified using an amylose resin column eluted with maltose. For example, cultures of E. coll producing the microcin can be grown under antibiotic selection (e.g., ampicillin and / or chloramphenicol), and in iron-limiting conditions, e.g., via the addition of 0.2 mM 2’2-dipyridyl, and induced, e.g., with isopropyl 0- d-1 -thiogalactopyranoside (IPTG). Cultures are grown for an additional time, e.g., 4 to 10 hours, e.g., 5 to 7 hours, post-induction, then pelleted and frozen overnight, e.g., at -20 °C.

[0051] Cultures are then thawed in cold water, sonicated, and the crude lysate is passed through a resin column, e.g., an amylose resin (New England Biolabs. Ipswich. MA) column, to capture maltose-binding protein (MBP) fusion proteins, then finally eluted, e.g., with maltose. Elution is performed by adding the elution buffer (e.g., 200 mM NaCl, 20 mM Tris-HCl. 10 mM maltose; pH 7.5).

[0052] The eluent can be concentrated, for example, using Millipore Sigma (Burlington, MA) MWCO 10,000 filters. The concentrated MBP-MccI47 is then digested by an endopeptidase, such as the Tobacco etch virus nuclear-inclusion-a endopeptidase (TEV) (New' England Biolabs, Ipswich, MA), yielding a buffered solution of MccI47, TEV, and MBP. This solution can then be further purified, e.g., by subsequent rounds of resuspension with Ni-NTA agarose resin (Qiagen, Hilden, DE). Ni-NTA slurry can be pelleted by centrifugation and the supernatant can be removed by pipetting.

[0053] The proteins can include one or more chemical modifications, e.g., covalent attachment of polyethylene glycol (PEG) chains (PEGylation), glycosylation, acetylation, biotinylation (e g., of the amino terminus), amidation (e.g., of the carboxy terminus), derivatization with protecting / blocking groups, and / or linkage to a cellular ligand or other protein: fusion to another protein or peptide, e.g., to an immunoglobin Fc domain, albumin, XTEN. or carboxy -terminal peptide, or other polypeptide fusion approaches to make drugs with more desirable pharmacokinetic profiles. See, e.g., Werle and Bemkop-Schniirch, Amino Acids. 2006 Jun;30(4):351-67; Strohl, BioDrugs. 2015; 29(4): 215-239; Wang et al., Signal Transduction and Targeted Therapy 7: 48 (2022). The proteins can comprise a C-terminal siderophore post- translational modification, e.g., with an enterobactin or an enterobactin derivative. As noted above, the proteins can be siderophore-conjugates, or conjugates with nonnatural siderophore analogs optionally with cleavable linkers (see, e.g., Puja et al., Biomolecules 2023, 13(6), 959; Peukert et al., Chem. Sci., 2023, 14, 5490-5502; Zheng et al., Journal of the American Chemical Society 2012 134 (44), 18388-18400; Caradec et al., ACS Central Science 2023 9 (11), 2138-2149).

[0054] Compositions

[0055] The antimicrobial proteins or engineered microorganisms can be present in a composition comprising a buffer or other carrier for delivery to a tissue in a subject. For example, biodegradable polymeric microparticles or nanoparticles such as liposomes, micelles, polymer nanoparticles, and inorganic nanomaterials, which are ty pically in the range of 10-150 nm in size, can be used as carriers. See, e.g., Yu et al., J Control Release. 2016 Oct 28; 240: 24-37. The proteins can also be present in a gel. e.g., a hydrogel composition, e.g.. for topical applications. The hydrogels can be synthetic (e.g., polyethylene oxide (PEO), polyvinyl alcohol (PVA), polyacrylic acid (PAA), polypropylene fumarate-co-ethylene glycol (P (PF-coEG), PEO-PEG-PEO, etc.), natural (e.g., alginate, chitosan, collagen, gelatin, hyaluronic acid, gellan gum, polyhydroxybutyrate valerate, cellulose, fibrin, etc.), or a combination thereof; see. e.g.. De Angelis et al., Biomedicines. 2021 Sep; 9(9): 1235.

[0056] The compositions can also include a pharmaceutically acceptable carrier. The term ‘‘pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a subject. The term “pharmaceutically acceptable carrier,” as used herein, includes any and all solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants and the like, that may be used as media for a pharmaceutically acceptable substance.

[0057] See also US 11560543; US20220218787; US20230126514; WO2020227155; WO2023114477, all of which are incorporated by reference herein.

[0058] Food Compositions

[0059] The present disclosure also provides a food composition comprising the genetically engineered microorganisms. In some embodiments, the food composition comprises carbohydrates such as, but not limited to, starches such as are contained in rice flour, flour, tapioca flour, tapioca starch, and whole wheat flour, modified starches or mixtures thereof.

[0060] In some embodiments, the compositions including the genetically engineered microorganisms are in the form of a liquid, and thus can be used as a beverage. In some embodiments, the beverage composition comprising the genetically engineered microorganisms is naturally sweetened. Suitable natural sweeteners include, but are not limited to, sugars and sugar sources such as sucrose, lactose, glucose, fructose, maltose, galactose, com syrup (including high fructose com syrup), sugar alcohols, maltodextrins, high maltose com syrup, starch, glycerin, brown sugar and mixtures thereof. In some embodiments, fiber, inulin, or prebiotics or probiotics are included.

[0061] In some embodiments, the food or beverage compositions include milk or milk-derived product, e g., yogurt. In some embodiments, a stabilizer may be combined with the milk-derived product. Combining a stabilizer with the milk- derived product may thicken the milk-derived product. In some embodiments, a stabilizer can be combined with the milk-derived product following completion of microorganism culture. The stabilizer can be selected from, as examples, gums, salts, emulsifiers, and their mixtures. Gums can be selected from, as examples, locust bean gum, xanthan gum, guar gum, gum arabic, and carageenan. In some embodiments, salts include, but are not limited to, sodium chloride and potassium chloride.

[0062] In some embodiments, the genetically engineered microorganisms can be administered to a subject with alkaline phosphatase. These methods involve administering to the subject a composition including the genetically engineered microorganisms and an amount of an alkaline phosphatase effective to increase the number of commensal bacteria in the gastrointestinal tract, wherein alkaline phosphatase decreases the number of pathogenic bacteria in the gastrointestinal tract, or increases the number of commensal bacteria and decreases the number of pathogenic bacteria in the gastrointestinal tract, thereby modulating gastrointestinal tract flora levels in the subject. The alkaline phosphatase composition, and the methods of use is described in WO 2010 / 025267, which is incorporated by reference in its entirety.

[0063] Genetically Engineered Microorganisms and Vectors

[0064] Many microorganisms can be genetically engineered to treat bacterial infection as described herein. In some embodiments, a bacterium is used. In some embodiments, the bacterium is E. coll (e.g., E. coli Nissle 1917 or E. coli NGF-19), Bifidobacteria (e.g., B. animalis. B. breve. B. lactis, B. longum, or B. infantis), Lactobacillus (e.g., L. acidophilus, L. reuteri, L. bulgaricus, L. lactis. L. casei, L. rhamnosus, L. plantarum, L. paracasei, or L. delbreuckii / bulgaricus), Saccharomyces boulardii, Saccharomyces cerevisiae, Streptococcus thermophiles, or Bacillus subtilis. One useful E. coli strain is Nissle 1917 (EcN). E. coll Nissle 1917 is a Gram-negative species that is easily cultured, easily genetically manipulated, able to colonize a human host, and easy to use for human probiotic applications. EcN is the active component of Mutaflor® (Ardeypharm GmbH, Herdecke, Germany), a microbial probiotic drug that is marketed and used in several countries. Clinical trials have shown EcN to be effective for maintaining remission of ulcerative colitis (UC), for stimulation of the of the immune system in premature infants, for treatment of infectious GI diseases, for the relief of constipation, and also for treatment of irritable bowel syndrome in some patients.

[0065] In some embodiments, useful microorganisms that can be used in the methods disclosed herein include bacteria for making yogurt, e.g., Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophiles .

[0066] A vector or a set of genes (e.g., an operon) as described herein can be introduced into a microorganism to generate a genetically engineered microorganism by known molecular biology, microbiology, and recombinant DNA techniques. These techniques are familiar to one of skilled in the art and are explained fully in the literature. See, e.g., Molecular Cloning: A Laboratory Manual (Michael R. Green, Joseph Sambrook, Fourth Edition, 2012); Oligonucleotide Synthesis: Methods and Applications (Methods in Molecular Biology) (Piet Herdewijn, 2004); Nucleic Acid Hybridization (M. L. M. Andersen, 1999); Short Protocols in Molecular Biology (Ausubel et al., 1990), each of which is incorporated herein by reference in its entirety. This disclosure provides various vectors comprising microcin genes and controllable promoters (e.g., inducible promoters). In some embodiments, the vector is a plasmid (e.g., pBR322, pLJV3, pJPMcH47, pttrMcH47, and pEX2000).

[0067] The vector or organism can include genes for various mi crocins, e.g., Class I microcins, Class Ila microcins, Class lib microcins, and / or Class lie microcins. In some embodiments, the vector can include a set of genes for a Class Ila microcin as desenbed herein, e.g., Bg E492, Bg X. Gq W. Ko H47, Ps G492, Ro Z. Ro H47, Ro 147, Ro X, Sf H47, Se G492, Se M, and Ec W.

[0068] In some embodiments, the vector or organism includes a set of genes (e.g., an operon) for microcin Bg E492. Bg X, Gq W, Ko H47, Ps G492. Ro Z, Ro H47. Ro 147, Ro X, Sf H47. Se G492. Se M, or Ec W, e.g.. antimicrobial gene Bg E492A. Bg XA, Gq WA, Ko H47A, Ps G492A, Ro ZA, Ro H47A, Ro 147 A, Ro XA, Sf H47A, Se G492A, Se MA, or Ec WA, and optionally an immunity gene Bg E492I, Bg XL Gq WI, Ko H47I, Ps G492I, Ro ZI, Ro H47I, Ro 1471, Ro XI, Sf H47I, Se G492I, Se MI, or Ec WI, and including genes required to express a functional mi crocin, e.g., a microcin that can inhibit the growth of other bacteria.

[0069] For example, in some embodimtes, to produce a microcin in a cell in the classical way with postranslational modification of a siderophore, the methods include expressing in the cell at least the following:

[0070] 1. Antimicrobial (A) gene;

[0071] 2. Immunity (I) gene; and

[0072] 3. mchCD to attach a siderophore.

[0073] In some embodiments, the minimum gene required to produce the toxin is the antimicrobial gene (A). The immunity gene (I) prevents a cell expressing the antimicrobial gene from self-killing during production. However, in some embodiments a cell-free system is used, in which only the antimicrobial gene is used because there is no cell to protect. In some embodiments, mchCD are included, to attach a siderophore to the antimicrobial to create an antimicrobial posttranslationally modified with a siderophore. Other tags or modifications that could be added biochemically. mchEF are part of the microcin cluster and are for export, specific to the signal peptides for each peptide. However, one could use different signal peptides to secrete mi crocins into the extracellular environment, e.g., when using other engineered probiotics, that are not closely related to E. coli (e g., Lactobacilli or yeast).

[0074] Genes for production of the immunity and antimicrobial proteins described herein are clustered in a 10-kb DNA segment located in the E. coli chromosome and include the genes mchA. mchB, mchC, mchl). mchE, mchF, mchl, and mchX. The mchA, mchC, and mchD genes are devoted to mature microcin synthesis; mchE and mchF are required for the secretion of the antibiotic into the extracellular medium. Sequences of the precursor and the corresponding immunity peptides, and the precursor and the corresponding immunity peptides, of Bg E492, Bg X, Gq W, Ko H47, Ps G492, Ro Z, Ro H47, Ro 147, Ro X, Sf H47, Se G492, Se M, and Ec W, are provided herein, as are sequences encoding the peptides. In some embodiments, genes used for postranslational modification and export are:

[0075] For Ec CA46: mchCDEF and mcmL. mcmL is referred to differently in different E. coli genomes and, for example, refers to mchA in the E. coli H47 genome.

[0076] For Kp: mceCGHIJ. mcmK (mchSl) in Ec or mceD in Kp are an enterobactin esterase and introduce some diversity of the siderophore but are not essential for function or activity. In some exemplary expression systems, the genes from Ecoli H47 (mchACDEF) are used. However, mature microcin production can also be obtained with the genes from Kp mceCGHIJ or some of the homologs from the other species (e.g., as described in FIGs. 7A-H).

[0077] Production of class lib microcins is a process involving three main steps: synthesis of the precursor peptide, subsequent maturation of the molecule, and its final secretion. These microcin genes are described, e.g., in Vassiliadis et al. (2010) Isolation and characterization of two members of the siderophore-microcin family, microcins M and H47, Antimicrobial agents and chemotherapy 54. 1: 288-297, which is incorporated herein by reference in its entirety.

[0078] In some embodiments, the set of genes includes one, tw o, three, four, five, six, seven, or eight genes that are selected from the group consisting of mchC, mchD, mchE, mchF. and mcmL (e.g., from / ■ / . coli H47), or mceC, mceD. mceG, mceH, mcel, and mceJ(e.g., from Kp); see exemplary sequences in FIGs. 8A-T; in some embodiments, homologs thereof, e.g., from the organisms in which the new A / I genes were found can also be used. In some embodiments, these genes can be located within one operon. Thus, in some embodiments, the operon includes mchC, mchD, mchE. mchF, and mcmL for Ec-like microcins, or mceC, mceD, mceG, mceH, mcel, and mceJ for Kp-like microcins. Exemplary sequences for each of these genes are provided herein. In some embodiments, all of the genes in the vector or operon are from the same organism; alternatively, the genes can include immunity and / or antimicrobial genes from one organism, and the remaining genes in the vector can be from one or more other organisms. For example, the vector can include mchCDEF and mcmL from Ec CA46 and an immunity and / or antimicrobial gene described herein for an Ec-like microcins, e.g., Gq W, Ko H47, Ro Z, Ro H47, Ro 147, Ro X, Sf H47, Se M. or Ec W: or mceCDGHIJ from Kp RYC492 for X / i-like microcins, e.g., Bg E492. Bg X, Ps G492, and Se G492.

[0079] In some embodiments, the set of genes or the operon is under the control of a controllable promoter. As used herein, the term “controllable promoter” refers to a promoter of which the initiation of transcription is controllable. For example, the initiation of transcription of a controllable promoter can be induced by a ligand, such as tetracycline, arabinose, galactose, isopropyl 0-D-1 -thiogalactopyranoside (IPTG). or allolactose. In some embodiments, the controllable promoter is rha / 7?.4 / ) or Pttr.

[0080] High levels of microcins may be harmful to a subject, thus, according to the present disclosure, mechanisms can be used to control the transcription of the genes or the operon. and thus control the level of microcins. The transcription of the microcin genes can be controlled by a controllable promoter. Some exemplary controllable promoters include, but are not limited to, Pttr promoter or pBAD promoter. The pBAD promoter is found in bacteria and was originally part of the arabinose operon that regulates transcription of araB. araA. and araD. Transcription initiation at the pBAD promoter occurs in the presence of high arabinose and low glucose concentrations. Upon arabinose binding to AraC, the N-terminal arm of AraC is released from its DNA binding domain via a “light switch” mechanism. This allows AraC to dimerize and bind the II and 12 operators. The AraC-arabinose dimer at this site contributes to activation of the pBAD promoter.

[0081] Additionally, cyclic AMP receptor protein (CAP) binds to two CAP binding sites upstream of the II and 12 operators and helps activate the pBAD promoter. In the presence of both high arabinose and high glucose concentrations however, low cAMP levels prevent CAP from activating the pBAD promoter. In the absence of arabinose. AraC dimerizes while bound to the 02 and II operator sites, looping the DNA. The looping prevents binding of CAP and RNA polymerase. Thus, without arabinose, the pBAD promoters are repressed by AraC. A detailed description of pBAD promoter can be found, e.g., in Schleif, FEMS Microbiol Rev. 2010 Sep;34(5): 779-96, which is incorporated by reference in its entirety. An exemplary pBAD promoter sequence is as follows:

[0082] CCACAATTCAGCAAATTGTGAACATCATCACGTTCATCTTTCCCTGGTTGCC AATGGCCCATTTTCCTGTCAGTAACGAGAAGGTCGCGTATTCAGGCGCTTT TTAGACTGGTCGTAATGAA. In some embodiments, the controllable promoter is Pttr and is activated in the presence of tetrathionate as the inducing agent. The vector can also include genes that are required to determine the level of tetrathionate. Thus, the vector can include one, two, three, four or five genes that are selected from the group consisting of ttrA, ttrB, ttrC, ttrS, and ttrR. In some embodiments, the vector includes ttrS and ttrR.

[0083] In some embodiments, ttrA, ttrC, and ttrB are located within one operon. In some embodiments, this operon further includes mchB. mchC. mchD. mchE, mchF, mchX and mchl. In some embodiments, this operon is under the control of Pttr.

[0084] In some embodiments, the tetrathionate promoter (Pttr) is located immediately upstream of the mchXlB genes (mchX, mchl, mchB), and encoding them on a single transcript based on activation of the ttr promoter. The mchA can controlled by a constitutive promoter (e.g., J23119). Exemplary sequences include the following:

[0085] Pttr promoter sequence: CCCAATATCCCTGTCAATTATGTTGTTTTAGATCAACAACAAGCCGGGTATG TGGTTAACCACAATAGAGCGCACCCCGCCTCGATTTTTACACTGTAAATCAT CGACATTTTTTATTCATTACACATGAACCAACATCGTGACAAATGTTTCATT GTTGGCA.

[0086] J23110 promoter sequence: TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAG (SEQ ID NO: 154#).

[0087] This disclosure further provides genetically engineered microorganisms comprising the vectors as described herein. In some embodiments, the vector are integrated into the genome of the microorganism, e.g., by recombinant DNA techniques. Thus, in one aspect, this disclosure provides an engineered strain of EcN harboring a plasmid-based system carrying mchAXIBCDEF and ttrRSBCA. capable of producing MccH47 in response to environmental tetrathionate, resulting in the abi 1 i ty to inhibit and out-compete Salmonella.

[0088] In some embodiments, the vector or the set of genes is integrated into a bacterial or other microbial genome.

[0089] Methods of Treating Bacterial Infections in Animals and Plants

[0090] The compositions and methods as described herein can be used, e.g., to treat a gram-negative bacterial infection, e.g., an infection with a bacterial pathogen as described herein; the methods include administering one or a combination of the antimicrobials described herein, e.g., as isolated peptides (e.g., in a carrier) or as genetically engineered organisms (e.g., genetically engineered microorganisms) that secrete one or a combination of two or more of the antimicrobials. In some embodiments, the infection is an infection in a wound, e.g., a surgical or accidental wound, e.g., an Acinetobacter baumannii infection in a wound, as iron scavenging through siderophores is critical in the pathogenesis of wound infections (see, e.g., Fleming et al., J Trauma Acute Care Surg. 2017 Mar; 82(3): 557-565). Therefore, the class lib microcins can be used in wound infection treatments as well.

[0091] In animals, the antimicrobial proteins and engineered microorganisms expressing the antimicrobial proteins described herein can be used inhibit various bacteria, e.g.. gram-negative bacteria, including ESKAPE pathogens (six highly virulent and often antibiotic-resistant bacterial pathogens including: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii. Pseudomonas aeruginosa, and Enterobacter spp). As used herein, the term “gramnegative bacterium” refers to a bacterium that do not retain the cry stal violet stain used in the Gram staining method of bacterial differentiation. Gram-negative bacteria include, e.g., proteobacteria, cocci, bacilli, etc. The proteobacteria are a major group of gram-negative bacteria, including Escherichia coli (E. coll). Salmonella, Shigella, and other Enterobacteriaceae , Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella etc. Gram-negative bacteria also include, e.g., the cyanobacteria, spirochaetes, green sulfur, and green non-sulfur bacteria. Medically relevant gram-negative cocci include, e.g., Neisseria gonorrhoeae. Neisseria meningitidis, and Moraxella catarrhalis, Haemophilus influenzae. Medically relevant gram-negative bacilli include a multitude of species. Some of them cause primarily respiratory problems (Klebsiella pneumoniae, Legionella pneumophila. Pseudomonas aeruginosa), primarily urinary problems (Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens), and primarily gastrointestinal problems (Helicobacter pylori, Salmonella enteritidis, Salmonella Typhi). Gram-negative bacteria associated with hospital-acquired infections include, e.g., Acinetobacter baumannii. which causes bacteremia, secondary meningitis, and ventilator-associated pneumonia in hospital intensive-care units.

[0092] In some embodiments, the bacterial infection is carbapenem-resistant Enterobacteriaceae infection, Klebsiella oxytoca infection, Klebsiella pneumoniae infection, Campylobacter infection, extended spectrum Enterobacteriaceae (e.g., E. coll, Salmonella, Shigella and Yersinia) infection. In some embodiments, the infection is with extended spectrum beta-lactamase (ESBL)-producing organisms. In some embodiments, the subject has an infection with an ESBL-producing Enterobacteriaceae .

[0093] The methods described in the present disclosure are effective for treating bacterial infection in a variety of subjects including mammals, e.g.. humans and veterinary subjects, such as laboratory animals, e.g., mice, rats, rabbits, or monkeys, or domesticated and farm animals, e.g., cats, dogs, goats, sheep, pigs, cows, horses, and birds, e.g., chickens and turkeys.

[0094] Healthcare providers can identify subjects in need of treatment for bacterial infection using their experience and judgment, which can be based on subjective (e.g., based on the healthcare provider’s opinion) or objective (e.g., measurable by a test or diagnostic method) information. As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

[0095] The present disclosure provides methods of inhibiting or reducing the risk of bacterial infections and for treating bacterial infections. As used herein, the term “reducing the risk” refers to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of, or susceptible to, developing a disorder or condition.

[0096] In some embodiments, the genetically engineered microorganisms or a combination thereof, or composition comprising one antimicrobial protein as described herein or a combination thereof, can be administered to a subject in combination with some other know n treatments for bacterial infection. For example, the genetically engineered microorganisms or a combination thereof, or composition comprising one antimicrobial protein as described herein or a combination thereof, can be used in combination with an antibiotic therapy, such as metronidazole, vancomycin, bacitracin, and / or teicoplatin. In some embodiments, the genetically engineered microorganisms are administered to the subject after the subject have received an antibiotic therapy. In some embodiments, the genetically engineered microorganisms are administered to the subject before the subject has received an antibiotic therapy. In other embodiments, the genetically engineered microorganisms are administered to the subject when the subject is under an antibiotic therapy.

[0097] Table B provides a list of exemplary target pathogens and application sites for the methods described herein.

[0098] Table B. Exemplary targets and application sites

[0099]

[0100] Methods of Treating Dysbiosis

[0101] The compositions and the methods as described herein can be used to treat and / or reduce the risk of dysbiosis and its associated diseases.

[0102] Dysbiosis is a term for a microbial imbalance or maladaptation on or inside the body. As used herein, the term “intestinal dysbiosis” refers to microbial imbalance in intestines. Dysbiosis is most commonly reported as a condition in the gastrointestinal tract, particularly during small intestinal bacterial overgrowth (SIBO) or small intestinal fungal overgrowth (SIFO). It has been reported to be associated with various diseases, such as periodontal disease, inflammatory bowel disease, chronic fatigue syndrome, obesity, cancer, bacterial vaginosis, and colitis.

[0103] The methods described in the present disclosure are effective for treating dysbiosis in a variety’ of subjects including humans and animals, such as laboratory animals, e.g., mice, rats, rabbits, or monkeys, or domesticated and farm animals, e.g., cats, dogs, goats, sheep, pigs, cows, horses, and birds, e g., chickens and turkeys.

[0104] Healthcare providers can identify subjects in need of treatment for dysbiosis using their experience and judgment, which can be based on subjective (e.g., based on the healthcare provider’s opinion) or objective (e.g., measurable by a test or diagnostic method) information. As used herein, the terms “treat.” treating,” “treatment.” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated. The present disclosure provides methods of inhibiting or reducing the risk of dysbiosis and for treating dysbiosis. As used herein, the term “reducing the risk” refers to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of, or susceptible to, developing a disorder or condition. For example, subjects who are entering, living in, or leaving a group housing facility such as a nursing home, rehabilitation facility, or hospital are at risk of developing bacterial dysbiosis.

[0105] In some embodiments, the genetically engineered microorganisms can be administered to a subject with some other known treatments for dysbiosis.

[0106] Methods of Administration in Animal Subjects

[0107] The therapeutic methods disclosed herein (including prophylactic treatments) generally include administration of a therapeutically effective amount of a composition comprising the antimicrobials, e.g.. in compositions or genetically engineered microorganisms, to a subject in need thereof. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom of bacterial infection and / or dysbiosis. Determination of those subjects who are “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a health care provider.

[0108] A subject is effectively treated when a clinically beneficial result ensues. This may mean, for example, a resolution of the symptoms associated with bacterial infection and / or dysbiosis, a decrease in the severity of the symptoms associated with bacterial infection and / or dysbiosis, or a slowing of the progression of symptoms associated with bacterial infection and / or dysbiosis.

[0109] Compositions comprising the genetically engineered microorganisms can be administered to a subject through many different routes, e.g., by endoscopy, byenteroscopy, by colonoscopy, by a nasoduodenal catheter, by enema, or by oral administration. In the case of oral administration, the composition can be delivered in a capsule or pill form, e.g., for intestinal delivery. In some embodiments, the composition is in a capsule form, e.g., packaged in gelatin capsules. Dosage

[0110] The compositions can be formulated in a unit dosage form, each dosage containing, for example, from about 0.005 mg to about 2000 mg of the genetically engineered microorganisms. The dosage scheduling can be approximately once per week, twice per week, three times per week, or four times per week. In some embodiments, the compositions can be administered to a subject every day. every’ other day, every three days, every four days, even' five days, every six days, or once per week. A person skilled in the art can refine the dosage scheduling as needed.

[0111] The phrase “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. When referring to these pre-formulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms.

[0112] The compositions can be formulated in a unit dosage form, each dosage containing, for example, from about 0.1 mg to about 50 mg, from about 0.1 mg to about 40 mg, from about 0.1 mg to about 20 mg, from about 0. 1 mg to about 10 mg, from about 0.2 mg to about 20 mg, from about 0.3 mg to about 15 mg, from about 0.4 mg to about 10 mg, from about 0.5 mg to about 1 mg; from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 0.5 mg to about 30 mg, from about 0.5 mg to about 20 mg, from about 0.5 mg to about 10 mg, from about 0.5 mg to about 5 mg; from about 1 mg from to about 50 mg, from about 1 mg to about 30 mg, from about 1 mg to about 20 mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg; from about 5 mg to about 50 mg, from about 5 mg to about 20 mg, from about 5 mg to about 10 mg; from about 10 mg to about 100 mg, from about 20 mg to about 200 mg, from about 30 mg to about 150 mg, from about 40 mg to about 100 mg, from about 50 mg to about 100 mg of the genetically engineered microorganisms.

[0113] Methods of Treating Plant Diseases

[0114] The antimicrobial proteins and engineered microorganisms expressing the antimicrobial proteins described herein can be used inhibit vanous plant pathogens, including tree pathogens Gibbsiellct quercinecans, Gibbsiella greigii^ Rahnella vicloriana. and Brenneria goodwinii, all of which are associated with tree decline. Thus the methods described herein can be used to treat infections, e.g., bacterial cankers, in trees and tree decline, e.g., acute oak decline (see, e.g., Denman et al.. The ISME Journal volume 12, pages386-399 (2018); Brady et al., Curr Res Microb Sci. 2021 Dec 20:3:100102; Brady et al., Syst Appl Microbiol. 2014 Sep;37(6):417-22) and walnut bacterial canker or walnut decline (Hajialigol et al., Sci Rep. 2023 Jul 12;13(1): 11286; Allahverdipour et al.. New Dis Rep. 2020;41: 12-12) and canker in Russian olive trees (Basavand et al., 3 Biotech. 2021 Jun; 11(6): 286).

[0115] Thus provided herein are methods and compositions for treating a bacterial disease in a plant, the method comprising administering to the plant an effective amount of an engineered microorganism or antimicrobial peptide as described herein, e.g., as a spray or gel, to and / or around the infected tissue (e.g., injected into and / or around a canker), or systemically, e.g., by injection into or near the roots or root flare. In some embodiments, the engineered microorganisms or antimicrobial peptides are present in a composition that comprises one or more other agriculturally acceptable items, such as dispersants, surfactants and / or humectants, inert components, thickeners, bactericides, resistance inductors, biopesticides, fungicides, foliage fertilizers, hormones, and the like, e.g., as described in US20200060277. The methods can also be used to reduce the risk of developing a bacterial infection in a susceptible tree or plant, e.g., a tree or plant that is growing near an infected organism.

[0116] Kits

[0117] The present disclosure also provides kits comprising the antimicrobial proteins and / or genetically engineered microorganisms described herein. In some embodiments, the kit includes a sterile container which contains a therapeutic or prophylactic composition comprising the antimicrobial proteins and / or genetically engineered microorganisms. Such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0118] The kit can also include instructions, e.g., information about the use of the composition for treating a bacterial infection. The kit can further contain precautions; warnings; indications; counter-indications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0119] Exemplary Sequences

[0120] In some embodiments, the sequence of a microcin or immunity protein, or a nucleic acid encoding a microcin or immunity protein, used in a composition or method described herein is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical, optionally 100% identical, to a sequence set forth herein. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0121] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using the default parameters, e.g.. a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0122] Exemplary Microcin Antimicrobial Protein Sequences

[0123] These sequences include a signal peptide (secretion tag) represented by the first 15 amino acids, which are cleaved off during secretion. Consequently, for intracellular production of microcins during purification, the signal peptide would be removed (bold italic font), optionally retaining or adding an initial methionine (ATG / start). In some embodiments, other signal peptides are used in place of the ones shown below; e.g., in cells other than E. coli. >Se_MA

[0124] 47 / ?A7 / / 7.A'£'LA'Y7S’G / lGDGDGVGRDIATALGGLAGGFVAGAVGAAAGGVAG

[0125] GAVYDFASTHTVNPAMSPSGLGGYLGGYYGSSSSAVNSGS

[0126] >Se_G492A

[0127] / WM / / / 7WEE / \STTG71DRGDSAVTGAVAGGTAGGAAGGWAGAELGAELGSL

[0128] AGPIGTFVGFLGGAAIGAFGGAYIYDSFSSPSNGS

[0129] >Ec_WA

[0130] Af / ?A7777)£'T / 1E / / / G / 1GLDKDASVAIGTAAGGFLGKATKIPGADIAGSAIGGYI

[0131] GGVIADGSNRTITIP SNGINYNPGIGLGSFNPNYNGGLLNS GFNS STS S S SS GS

[0132] Exemplary Microcin Antimicrobial Nucleic Acid Sequences

[0133] >Ec_H47A

[0134] ATGCGAGAAATAACAGAATCACAGTTAAGATATATTTCCGGGGCGGGAGGT

[0135] GCGCCAGCGACTTCAGCTAATGCTGCAGGTGCTGCAGCTATTGTTGGAGC

[0136] TCTCGCCGGAATACCTGGTGGTCCACTTGGGGTTGTAGTTGGAGCCGTATC

[0137] TGCCGGTTTGACAACAGCAATTGGCTCGACCGTGGGAAGTGGTAGTGCCA

[0138] GTTCTTCTGCTGGTGGCGGTAGCTAA

[0139] >Ec_I47A atgagagaaatatcagataacatgcttgattccgtgaaaggagggatgaatctaatggattacctggfictactaatgtaa tagatctacgtggaaaagatatgggaacatatattgatgctaatggagcatgctgggctccggatactccatccatcatcatgt atccggggggaagtggaccctctatagtatgagtagtccacatccagtgcaaacagcggcagtaa

[0140] >Ec_MA

[0141] ATGAGAAAACTATCTGAAAATGAAATAAAACAAATATCTGGAGGTGACGGG

[0142] AATGACGGGCAGGCAGAATTAATTGCTATTGGTTCACTTGCAGGTACGTTT

[0143] ATTAGCCCGGGATTTGGTTCTATTGCAGGGGCTTATATAGGTGATAAAGTA

[0144] CATTCATGGGCAACGACTGCGACGGTTAGTCCCTCCATGTCTCCCTCAGGT

[0145] ATAGGATTATCATCCCAGTTTGGATCCGGCAGAGGTACATCAAGTGCCTC

[0146] TTCGTCTGCGGGGAGTGGAAGTTAA >Kp_E492A atgagagaaattagtcaaaaggacttaaatctgctttggtgcaggagagaccgatccaaatactcaactctaaacgac ctggaaataatatggcatggggtgctgctctggcgctcctggcggataggatcagcagcttgggggccgcgggaggt gcattacaaactgtagggcaaggattaattgaccatggtcctgtaaatgtccccatccctgtactcatcgggccaagctggaa tggtagcggtagtggtataacagcgcaacatccagttccggtagtggtagttaa

[0147] >Kp_G492A

[0148] ATGAGAGCACTGACAGAAAATGATTTTTTTGCAGTATCCGGCGCAGATCGA

[0149] GGTGATGCAGCAGTGGCTGGAGCGGTAGCAGGAGGTACAGCAGGTGCAG

[0150] CCGCAGGGGGATGGGCCGGAGCACAAATGGGGGCTACAGTTGGGAGTTT

[0151] GGCCGGACCTGTCGGAACTGTTGTCGGGTTTGTTGCAGGTGCTGCGGCAG GTCGCTATGGTGGTGCTTTTATCTATGATTCCTTTAGTTCACCATCAAATTC TTCTTCCAGCGGAAGTTAA

[0152] >Bg_XA atgcgagaaataagtcatgtcgagttatctgcaatatcaggtgccggtgaattagtatggagagctgccggagtgtg ctggaggagcggttgctggagcgatgacaggggctgctgctgctggattaggtgccggacctggagctgttctcggtgga actgctgccggggctgctatgtaactggccacttggttcaatcattttttggtggaagtaacacgagcagtagttcaagtagc accagttclgctgccaglagtgccagltctgctgccagtaatggtagltaa

[0153] >Bg_E492A

[0154] A TGA GA GCGTTAAA TGTTTA TGAAA TAAAAA TGGCTTCA GCTGCA GGA ACT

[0155] CCTGGTATCGCCCCTGGCAATGCTCAGATAATTAAAGATGTGGCCATCGA

[0156] TGCTGGGATTGGTGCAGCGTTTACTCCAGGAGCTCCATTGATTGGCGCTGG

[0157] ACTGGGGGCTGCAGGCAGTGTAATCCATGGTGCTATCAACCATGGGCCTG

[0158] TATGCGTCCCTATTCCCGTCATGATCGGACCGACTTGGAATGGCAGTGGC GGCGGTAACTCTGTTAGCGTTAGCGCTGCTGCTGCCAACGCTCTGGCTGCT GCCAGGGATGGTAGTTAA

[0159] >Gq_WA

[0160] ATGAGAGAGCTATCTATGTTAGACATAACATTGGCAAAAGGTGGATATGA

[0161] CGAGGTGAAACAATTGCCGGGGCTGTAGGCGCTGCTGTCGGTGCAGAAAT

[0162] AGGAGGGCCTGTAGGAGGAGTTATTGTTACGATAGTAGGAGCTGAACTCT ATAACAGCATTAACGAAGGTTGGACGATGGCGCCAGGGGTTTCACAAGGT GTAGGCTCTTATAACCCAAATTATAATGGCTCATTAGTTAGCCCTGGATAT TCATCATCATGGGGTGGTTCGTGGGGAGGAGCGACTTCAAGTGCTGGTGG

[0163] TGGTAGCTAA

[0164] >Ko_H47A

[0165] A TGCGA GAA TTAA CGCAA TA TCAAA TGGAAAA TGTA GCTGGTTCCGGAGGT GCTCCTGCAACTGTAGCAAATGCTGCCGGTGTAGGCATTATTATTGGTTCC CTCGGTGCAATTGCCACAGGTGTTGCGGCAGGAACTCCCGTTGGGCCTGT TGGTATGGTGATTGGTGGCATATTGGGTGGGTTAGGTGTTGCAGTAGGTTC AGCTATAGGTAGCTCGGGAGGTGGTTCTTAA

[0166] >Ps_G492A atgagagcactttcagcaaacgattatgtcggtctatggcgcg^aic^c^c^ai^cagc^aiccggt^ccaGac cggagcaacgacaggcgctgctgctggtgcctgggctggtgcccagctgggcgcagaaggcgggagtcttgccggac cgatggtgccgtgtcggtttggcgtaggtgctgcggtcggtgctatggtggtgcggctatctgtgattcatttagctctca tctgattcagcatcaagcggtagctaa

[0167] >Ro_H47A atgcgagaattaacacaagatcaaatagaaaatgtagctggtgcc^a^^cYccc^caac^a^caaa^clacc ggtgtaggtattattattggctctctggtgcaatggcgcaggtattgcggcaggaactcccgttgggcctgctggtatgata atggtggtatattgggggggttaggtgttgcagtaggttcagctacaggtagctcgggagctggaggaggttcttaa

[0168] >Ro_I47A

[0169] ATGAGAGAGTTGGATGTTAAGATGTTGGCTTTTGCCAAAGGTGGAATGATT CTGGATGGAAGGCCTGAGTCATCTAATGTAGTAGATCTTCGAGGAAATGA TCGGGGGTCTTATATTGATGCTAATGGTTCTTGTTGGGCTCCGGGCACCTC ATCTATTGTTATGTATCCTAGTGGGAGTTATTCTTCATTCAATTGGGGAGG CTCAAGTTCTACATCCAGTGCTGGGGGGGGGAGTTAA

[0170] >Ro_XA a / ^« / fflccffla / a«^Z^a / accgaac / ^a§ / a^a / « / cagg / ^c / ggtgagtcagtatggtgaactgctggtgtgtgt tggaggcgctgtcgctggtgcaatgggaggtgtgttataggtagatgggagcaggttcaggagctatagctggcgcaac gagttattgctgatggttcaaataggacaataacaataccatcaaacggtattaactataatccgggtataggacttggaagttt taatccaaactacaatggaggttattaaactcaggattaactcatcaacatcctctctagtagtggaagtga

[0171] Exemplary Immunity Protein Sequences

[0172] >Ec_H47I

[0173] MSYKKLSQLTAIFSLPITILLVSLSSLRIVGEGNSYVDVFLSFIIFLGFIELIHGIRR

[0174] ILVWSGWKNGS

[0175] >Ec_I47I

[0176] MYLTKKIIISMMFILPSAAFSSDPPPLQQSLEKTTYFSIGMNGFIGYQSEGEKLY

[0177] THILTLDNPEEIFKNIIKNRKSTKESKIYAACGLYYLNVENIESLFNENDKQEYV

[0178] SVLRGDILTKIKLNDILNSVIINGCNTKLISEHK

[0179] >Ec_MI

[0180] MGEVKKDIKITVIAFVINYLFFYIPVSLYLSYYYGYNFFNLYMFFLSLVVTFLS

[0181] LWLNVNFYFFTNLIAKVLK

[0182] >Kp_E492I

[0183] MTLLSFGFSPVFFSVMAFCIISRSKFYPQRTRNKVIVLILLTFFICFLYPLTKVYL

[0184] VGSYGIFDKFYLFCFISTLIAIAINVVILTINGAKNERN

[0185] >Kp_G492I

[0186] MIFLYLDKIPLFILGIGLLTSFALPGSSALDSPKFLCIYSSTILAGISFIYQVFRHG

[0187] TNTEFFLAMLITVSFVVMLPVIKMHFAY

[0188] >Bg_XI

[0189] MLCCPFLVFLMMSAEGVMRIFILARYWVLFFIVFYFFIYIGVWLFDGVFNYGR

[0190] TAMLSF VFS SFF ALFMAKLRS SPKK

[0191] >Bg_E492I

[0192] MITIINFYFLPAIFSVMAFLSLSSFLKKRNALLKIIISFAVACLAVFVYFFTHSVF

[0193] DGVSITQALFSFGFNDFIASFLICILNTVILWVLINLK >Gq_WI

[0194] MKFTLYGLMDMKTTCALLTFFFNKSSLIYFLLSCLLAVQGDLDGHTTEVLLIS

[0195] CFFGLIQMLINFKKITKPKQS

[0196] >Ko_H47I

[0197] MDHKTKRGWIDKAAVVLTVPLTIIFVSFSSLQVIGKGNSFIDVFLACMVYLGL

[0198] FNLFRVLKKAFYFFVK

[0199] >Ps_G492I

[0200] MAFLSSDKTPFLIIGVGLIFSGTLPGGPTLLSPKFLCIYFFVFLAVVFYIWLAISS

[0201] GEKSRIILACVISALSLVLLPMIKIHFSK

[0202] >Ro_H47I

[0203] MGHNTKLGWIDKAAVVLTVPLTIIFVSFSSLQIIGKGNSFIDVFLACMVCLGFF

[0204] NFFKVLKTALYFFIK

[0205] >Ro_I47I

[0206] MRLSKKIVMVILFLCAHSVYAHDILSEQKRLEKINYYSVGMNGFAGKESEGE

[0207] YLYRLILQKDNAEEIFMNIAKNDHSTNESKLYAACALRALGVGNINEIFNQSR

[0208] DKDVVVLTGDVLRRVSFKDKLSAIIQHGCD

[0209] >Ro_XI

[0210] MRKMKLLMYWFLFFIVFYGCrVIGVYVCGEVFKPSRIAMLAFIFSSLFTVLLLR

[0211] KRVRLDM

[0212] >Ro_ZI

[0213] MLDRLINFEISFLFFSNEKINILITAIVTIFLMFSLYLLPDKEWLVFIVGALIIWER

[0214] ILLLKIKKMQSLVKDNS

[0215] >Sf_H47I

[0216] MTNEASFSWVNKLAAILSFPLTVIFVSFSSLKVVGEGNSLVDIFLSFVIYIGFLG

[0217] LIRLTRKFLIWFS >Se_MI

[0218] MKTKKRNLTDFLISLFINALHYIPISLYNHFIVNLDFLSLHTLYLSIFIALINNLIG NSHYSIYDIIKRN

[0219] >Se_G492I

[0220] MFLLPFDKLYLFILGIGFIISLSFPSTSFVNSPKFLCIYLAVILSSTYSLLMAVRH GVGNEIITTIIIGVSFIILLPVIKIYFS

[0221] >Ec_WI

[0222] MKLFFYFSLFFIIQLVNSDLDTLTLYLLIKKIILSLLASVLFLFVLSFFNKKRK

[0223] Exemplary Immunity Nucleic Acid Sequences

[0224] >Ec_H47I

[0225] ATGAGTTATAAAAAACTGTCCCAATTGACGGCTATATTCAGTTTACCTATT

[0226] ACTATCTTATTGGTTTCACTTTCATCCCTTCGGATTGTTGGCGAAGGGAAT TCTTATGTTGACGTTTTTCTAAGCTTTATAATATTTCTTGGTTTTATTGAGC TGATTCATGGGATTCGAAGGATTCTGGTCTGGTCAGGCTGGAAAAACGGA AGTTAA

[0227] >Ec 1471 atgtatcttacgaaaaagattataataagtatgatgtttatattaccatctgctgcattttcatcagatccacctccccttcaacaat cgttagaaaaaacaacctatttttctataggtatgaatgggttataggctatcagagcgaaggggaaaaattatacacacaca ttctacattagataatcccgaagagatattaaaaatataataaaaaatagaaagtcaactaaggagtctaaaatttatgctgct tgtgggctatatatttaaacgtagaaaatatagagtcattgttaatgaaaatgataaacaagaatatgtgtctgtctaagagg ggatatttaacaaaaataaaactgaatgatatctgaattctgtgataataaatggtgcaacaccaaattaatatctgaacata aatga

[0228] >Ec_MI

[0229] ATGGGGGAGGTTAAGAAGGATATAAAAATAACAGTGATTGCTTTTGTTAT CAATTATCTGTTCTTTTATATTCCGGTGTCATTATATCTTAGTTATTACTAT GGATATAATTTTTTTAATCTATATATGTTTTTTTTATCACTTGTAGTTACAT TTTTATCGTTGTGGTTAAACGTGAATTTTTACTTCTTCACAAATCTTATAGC GAAGGTGTTGAAATGA >Kp_E492I atgacattacttcattggattttctcctgtttctttcagtcatggcgtctgtatcattcacgtagtaaattctatccgcagaga acgcgaaacaaagtattgttctgattttactaacttttttatttgttttttatatccattaacaaaagtgtatctggtgggaagttac ggtatatttgacaaattctacctcttttgctttatttctacgttaattgcaatagcaattaacgtagtgatacttacaataaatggagc taagaatgagagaaattag

[0230] >Kp_G492I

[0231] ATGATTTTTCTCTATTTAGACAAAATACCCTTATTTATATTAGGTATTGGTC

[0232] TTCTTACTTCATTTGCACTTCCTGGCAGTTCCGCTTTAGACTCGCCAAAATT

[0233] TCTTTGTATTTATTCCAGCACTATCCTTGCTGGTATTTCATTCATCTATCAG GTGTTTCGTCATGGAACAAATACAGAGTTTTTTTTAGCGATGCTGATAACT GTTTCTTTTGTTGTTATGCTCCCTGTTATCAAAATGCATTTTGCTTATTAA

[0234] >Bg_XI atgtatgttgtccattcctgtgtttaatgatgtctgctgagggggtatgagaatattatacttgctaggtatgggtttgtt ttattgtgttttatttttttatatatattggagtatggttgtttgatggtgtatttaattatgggcgaacagcgatgctgtcatttgtatttt ctagttctttgctttatttatggcgaagctcgtagttcgcctaaaaagtag

[0235] >Bg E492I

[0236] ATGATTACGATTATTAATTTTTATTTTCTACCTGCGATTTTTAGTGTAATGG

[0237] CCTTTTTGTCATTAAGTTCTTTTTTAAAAAAAAGAAATGCTTTGTTGAAAA

[0238] TTATAATTTCTTTTGCAGTGGCTTGCCTTGCTGTGTTTGTATACTTCTTTAC

[0239] TCATTCAGTTTTTGATGGAGTTTCAATTACACAGGCGTTGTTTTCATTTGGC TTTAACGATTTTATTGCTTCTTTTTTAATTTGTATTTTAAACACGGTTATTTT GTGGGTTTTAATTAATTTGAAATAA

[0240] >Gq_WI

[0241] ATGAAGTTTACTTTATATGGATTAATGGACATGAAAACAACCTGTGCTTTA

[0242] TTAACATTTTTCTTTAATAAATCAAGTTTAATCTATTTCTTATTGTCCTGTT

[0243] TACTCGCGGTACAGGGCGATTTGGATGGTCATACAACTGAGGTCTTATTG ATAAGCTGTTTTTTTGGATTGATTCAAATGCTAATCAACTTCAAAAAAATA ACCAAACCCAAGCAATCATAA >Ko_H47I

[0244] ATGGATCATAAGACTAAACGGGGATGGATAGATAAAGCTGCTGTTGTTCT

[0245] AACCGTTCCTCTAACAATTATCTTTGTTTCTTTTTCCTCTTTACAAGTTATC

[0246] GGTAAAGGTAATTCATTTATTGATGTTTTCCTGGCCTGCATGGTTTATTTG

[0247] GGTTTGTTTAATTTATTCAGGGTGCTCAAAAAGGCATTTTATTTCTTTGTCA AGTGA

[0248] >Ps_G492I atggcatttctgtcttcagacaaaacccctttttaatattggggtaggtctcattttttccggtacatgcctggtggaccaactc tgcttcgccaaagtttgtgcatttacttttgtttttctggccgtgtctttatatatggtagctatcagtagtggagaaaaga gcagaataatactggcttgtgtaatagcgcactgtcgttagtttgttacccatgataaaaatacatttagcaagtga

[0249] >Ro_H47I atgggtcataatactaaactgggatggatagataaagctgcagtgtctaaccgtcctctaacaatatcttgttctttcct cttacaaatatcggtaaaggaaattcattatgacgtttctagcctgtatggttgttgggtttttaatttctcaaggtgct caaaacggcactttatttctttatcaaataa

[0250] >Ro_I47I

[0251] ATGAGATTAAGTAAAAAAATTGTGATGGTGATATTGTTTTTATGCGCTCAT

[0252] TCGGTTTACGCACATGATATATTGTCTGAGCAGAAACGTTTGGAAAAAAT

[0253] AAATTATTATTCTGTCGGGATGAATGGTTTTGCTGGGAAAGAAAGTGAGG

[0254] GGGAGTATTTATATAGATTGATACTTCAAAAAGATAATGCAGAAGAAATA

[0255] TTTATGAATATTGCAAAAAACGATCATTCAACAAATGAATCAAAGCTTTA

[0256] TGCTGCATGCGCACTTCGTGCTCTAGGTGTAGGAAACATAAACGAGATCT

[0257] TTAATCAGTCTCGGGATAAAGATGTCGTAGTTTTAACTGGTGACGTACTAA

[0258] GGAGGGTCAGTTTTAAAGATAAGCTATCCGCAATTATACAGCATGGTTGC GATTGA

[0259] >Ro_XI atgcgtaaaatgaagttgtaatgtatggtttttgtttttcattgtttctatggtgtatctatatcggagtgtatgttgtggtgagg tctttaaaccttcccgtatagctatgctagcgttcattttttctagtctattacagtactctataagaaaaagagtacgtttggat atgtag >Ro_ZI

[0260] ATGCTGGATAGATTAATTAACTTTGAGATAAGCTTTCTATTTTTCTCAAAT

[0261] GAAAAAATCAACATTCTAATAACAGCTATAGTAACCATTTTTTTAATGTTT

[0262] TCATTATATCTTTTACCAGATAAGGAGTGGCTGGTATTCATCGTTGGGGCG

[0263] TTGATTATATGGGAGAGGATTCTTTTGCTGAAGATAAAAAAAATGCAGTC

[0264] TCTGGTGAAAGATAACAGCTGA

[0265] >Sf_H47I atgactaatgaggcaagtttctcgtgggtaaataaactagctgccatacttagctttccgctaacagtcatctttgtatcattttca tcactaaaagttgttggagaaggcaattcacttgtcgacattttcttatcctttgttatttacattggatttcttggtctaatcaggct gacgaggaagtttttgatttggttttcttag

[0266] >Se_MI

[0267] ATGAAAACAAAAAAAAGAAACCTTACAGATTTTTTAATTTCCTTATTTATT

[0268] AATGCATTAATAATATACATTCCAATATCTTTATATAATCATTTCATTGTTA

[0269] ATCTTGATTTTTTATCATTGCACACTCTCTATTTATCTATATTCATAGCACT

[0270] AATAAACAATCTAATCGGAAACAGCCACTACTCTATTTACGATATAATAA AGAGGAACTAA

[0271] >Se_G492I atgtttcttttaccttttgataaattatatttattcatattagggattggatttataatttctctttcttttcccagtacatccttcgtgaact ctccaaaatttttatgtatttacctggctgtgattttatccagtacatactctttgttgatggccgttcgccatggagtgggaaacg aaataatcacaaccattattataggtgtctcatttataatattattgccagtaattaaaatatactttagttag

[0272] >Ec_WI atgaaactatttttttatttctcgttgttctttataatacaacttgtaaattcagacttagacacattaaccctttatcttctcataaaga aaataattctttcgttacttgcgagtgtactgtttttatttgttttatctttctttaataaaaagaggaaataa

[0273] EXAMPLES

[0274] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Example 1. Novel class lib microcins show activity against gram-negative ESKAPE and plant pathogens.

[0275] Methods

[0276] The following methods and materials were used in Example 1 herein.

[0277] Bioinformatic microcin identification

[0278] We developed a pipeline that by leveraging the Basic Local Alignment Search Tool (BLAST27) enabled us to mine publicly available genome databases for novel, previously undescribed class lib microcins. We included mchCDEF and mcmL for Ec as well as mceCDGHIJ for Kp for posttranslational modification and export, expecting more reliable hits for longer and functionally conserved proteins in close proximity to class lib microcin and immunity genes. Thus, we first ran tblastn21against RefSeq28, to screen for all genes related to biosynthesis pathways, known microcin genes, as well as immunity gene sequences exhibiting homology7to the microcin gene clusters found in Ec C A46 and Kp RY C492. Homology to the microcin gene clusters were guided by BLAST parameters sseqid (Genome ID), pident (percentage of identical positions), sstart (start of alignment in Genome), and send (end of alignment position in Genome). Resulting hits were concatenated by genome ID and assessed for their proximity to one another in the genome. These gene clusters should, at best, contain all the known genes required for toxin maturation, including mchCDEF and mcmlfi’fi In addition to genomic hits to the known microcins, small ORFs of 50 to 150 amino acids in size close to the biosynthesis genes were screened and annotated manually using the criteria described below and their domains were predicted using SMART29The ORFs were meticulously examined and assessed against established class lib microcin criteria known from Ec H47, Ec 147, Ec M, Kp E492, and Kp G492: (i) a serine-rich C-terminus culminating in a final serine, (ii) the presence of fewer than one cysteine residue, (iii) a signal peptide within the initial 15 amino acids ending with GG or GA, and (iv) close proximity (<200 bp) to an ORF featuring a predicted transmembrane domain, typically encoding an immunity peptide. The identified genes were included in the pipeline's input to expand the scope of gene detection. We repeated this process iteratively through the pipeline until no additional genes were added to the output. Subsequently, blastp was used to assess microcin similarity shown in Table 1 and Table 2. below. Phylogenetic analyses

[0279] For the native full length coding sequence of the mi crocin and immunity genes a codon-based sequence alignment was generated using the MUSCLE algorithm30. For phylogeny of all microcins, the nucleotide sequences without the respective signal peptides were codon-aligned. Subsequently, we determined the best fit substitution models for maximum likelihood phylogenetic analyses, resulting in the General Time Reversible model with discrete gamma distribution (GTR+G) and the Hasegawa- Kishino-Yano model with discrete gamma distribution (HKY+G), respectively. A bootstrap test with 1000 replicates for maximum likelihood and random seed was conducted for all trees. Alignment, model testing, and tree building was performed in MEGA1131. antiSMASH analyses

[0280] To test if similar results of class lib microcin identification could be obtained with automated bioinformatic tools, we ran antiSMASH 7.0(34), a widely used tool for microbial genome mining and biosynthetic gene cluster detection. As input, we utilized the seven genomes from the newly identified class lib microcins: (i) Bg CP014137, (ii) Gq CP014136, (iii) Ko CP033844, (iv) Ps CP034363, (v) Ro CP008886, (vi) Se CP030220, (vii) Sf CP033055. As a positive control for the well- established microcins Kp E492 and Kp G492 as well as Ec H47 and Ec M we used the accession numbers CP127839 (Kp RYC492) and CP148105 (Ec Nissle 1917), respectively. Notably, using the “loose” setting, in none of the cases a class lib microcin biosynthesis gene cluster was detected, nor were any microcin genes identified. This was the case for both, the novel microcins and the original, well- annotated, microcins.

[0281] Plasmids and heterologous microcin expression

[0282] ORFs of identified microcin and immunity genes were codon optimized for frequent Ec codon usage without creating repetitive sequences and synthesized by Integrated DNA Technologies (Coralville, IA) with 18 bp of native 5’ upstream sequence and 20 bp of native 3’ downstream sequence, respectively. Using Gibson Assembly32, the genes were cloned into our previously established Ec class lib microcin expression system that results in mature class lib microcins posttranslationally modified with a monoglycosylated enterobactin (MGE)13,16.

[0283] Briefly, the antimicrobial and the immunity genes are co-expressed under the control of an arabinose-inducible pBad / araC promoter in a high copy plasmid with a pUC- derived origin of replication. All assemblies were verified using whole plasmid sequencing.

[0284] Static inhibition assays

[0285] Cultures of strains with confirmed plasmid assemblies were spread in LB agar plates containing 100 pg / ml ampicillin. In addition to a pUC19 control without microcin expression, single colonies for each microcin were picked with a sterile pipet tip and all placed into the same solid LB agar plate containing 100 pg / ml ampicillin for plasmid retention, 0.2 mM 2,2-dipyridyl to create iron limited conditions during the growth phase, and 0.4% L-arabinose for induction of gene expression. Plates were incubated at 37°C for up to 72 h, before they were overlaid with the target bacterial isolates. Note that testing all microcin-expressing stains on the same plate allowed us to confidently assess differential inhi bi tory activity7betw een all 17 tested microcins. For the overlay, the microcin-producing bacteria in the stabs were inactivated using chloroform vapors and ten minutes under ultraviolet light. Then, target bacteria were diluted 1 :2000 from overnight culture in LB media containing 100 pg / ml ampicillin and 0.2 mM 2,2-dipyridyl. Ec and S.flexneri strains were diluted 1:200 to acquire dense bacterial lawns. Finally, 0.5 ml of molten agar was added to the liquid media and the resulting soft agar was spread on the plate with the inactivated bacteria and incubated for 16 h at 37°C. The pUC19 control strain was unable to create any zone of inhibition against any of the tested target bacteria.

[0286] Relative MIC dilution factors

[0287] For enrichment of microcin Se G492, an MBP-microcin fusion protein was expressed from pHMT-SeG492 in A. coll BL21 cells as previously described(13.17). Harvested cells were resuspended in column buffer (200 mM NaCl, 20 mM Tris-HCl, pH 7.5), lysed by sonication, and passed through a high flow amylose resin (New England Biolabs, Ipswich, MA) as recommended by the manufacturer. The protein was eluted with 10 mM maltose, cleaved with Tobacco etch virus (TEV) protease, and further processed as previously reported(13.17). The relative MIC assays were conducted using sterile 96-well round bottom microplates. The plates were prepared as follows: the first row7contained 20 pl of 2x LB with 0.4 mM 2.2’ -di pyridyl and 20 pl of Se G492 containing solution in amylose resin elution buffer (200 mM NaCl, 20 mM Tris-HCl, 10 mM maltose, pH 7.5). All other wells were filled with 20 pl of lx LB. 0.2 rnM 2,2 ’-dipyridyl, and 0.5x amylose resin elution buffer, and a two-fold serial dilution was performed across the plate. The target bacteria were grown overnight in LB at 200 rpm and 37°C and were added to a final dilution of 10‘4into the wells. The plates were then incubated in the dark at 37°C with gentle agitation. Relative MICs were determined as the lowest concentration at which no grow th was observed after 24 hours. All reported values represent the median of at least three biological replicates.

[0288] Results

[0289] With the hypothesis that class lib microcin production is a common trait among Enter obacleriaceae. it was believed that the genes encoding for the antimicrobial and immunity would exhibit a high degree of dissimilarity to already known peptides as target specificity may result in accelerated adaptive coevolution33. Therefore, in addition to the known microcin and immunity genes from MccE492, MccG492, MccH47, MccI47, and MccM, in our informatic approach we included the genes that are necessary for mature class lib microcin biosynthesis, extending our search to longer sequences for more reliable BLAST results27. Moreover, we hypothesized that the amino acid sequences of genes responsible for posttranslational modification and microcin export would be less prone to evolutionary' changes, thereby maintaining the functional integrity of the gene cluster15’34. We then assessed their proximity in the respective genome location, because microcin genes are ty pically flanked by genes essential for toxin maturation15. Further, we manually assessed and annotated small open reading frames upstream and downstream of the maturation genes, allowing us to also identify novel class lib microcins without significant sequence similarity to the known antimicrobials, enabling the discovery of compounds with new' molecular targets or modes of action (see Methods).

[0290] Our informatics-driven analysis identified twelve promising class lib microcin candidates from seven gene clusters with high similarity to Ec CA46 and Kp RY C492 in seven species across the Enterobacteriaceae family (FIG. 1A, FIGs. 4A-G, FIGs. 7A-H): (i) Brenneria goodwinii (Bg; 2; GenBank: CP014137.1). (ii) Gibbsiella quercinecans (Gq; 1; CP014I36.1), (iii) Klebsiella oxytoca (Ko; 1; CP033844.1), (iv) Pantoea sp. (Ps; 1; CP034363.1), (v) Raoultella ornithinolytica (Ro; 4; CP008886.1), (vi) Salmonella enterica (Se; 2; CP030220. 1), (vii) Serratia fonticola (Sf. 1; CP033055.1), (viii) E. coli NCTC 10444 (LR134092.1). Although it has traditionally been a defining characteristic of class lib microcins that all required genes are encoded within the chromosome12, the gene cluster we discovered for Se is situated on a 159 kbp plasmid. Phylogenetic sequence analysis of both the antimicrobial and immunity peptide genes revealed the presence of eight different clades represented in both trees, respectively (FIGs. 1B,C). Regarding the well- established class lib microcins MccH47, MccI47, MccM, MccG492, and MccE492, we identified novel members for each group, supported by nucleotide sequence similarity, amino acid identity, the closest blastp match, and domain predictions (Table 1, FIGs. 5A-B). It is important to note that application of established tools for secondary' metabolite identification (e.g., antiSMASH 7.0) to these genomes did not yield identification of any of the old or novel microcins providing support of the relevance of this approach.

[0291] In order to then ensure that these novel microcins are unique and not part of any other microcin class, we performed phylogenetic analysis for all known microcin genes from the classes I, Ila, and lib and show distinct clustering for all neyvly described sequences (FIG. 5C). In light of this discovery, we propose a new nomenclature for class lib microcins that includes the species initials in which they were identified (e.g., Ec, Kp), the closest relative already characterized class lib microcin (G492, E492. H47, 147 or M), as well as the identifiers ‘A’ for antimicrobial or ‘I’ for immunity gene.

[0292] Based on this, the novel G492 relative found in Salmonella enterica will be called Se G492 with the antimicrobial peptide identified as Se G492A and the immunity peptide identified as Se G492I. It is worth highlighting that in the case of the G492 group, all its members have the immunity gene located downstream of the antimicrobial gene, whereas for the other clades, this arrangement is reversed. In addition to uncovering eight novel variants of the five previously characterized microcins, we have identified four additional microcins through manual curation of ORFs in proximity to the microcin maturation genes. These novel microcins, which we name microcin W (MccW), microcin X (MccX), and microcin Z (MccZ). seem to belong to three entirely new clades based on nucleotide similarity (FIGs. 1B,C). The two members of the microcin X group, found in B. goodwinii (Bg X) and R. ornithinolytica (Ro X), only show significant similarity betyveen one another, but not to any of the other antimicrobial or immunity peptides. This holds true for the nucleotide similarity as well as amino acid identity and the closest blastp hits (FIGs. 1B,C,D Table 1, Table 2). Similarly, MccW from Gibbsiella quercinecans (Gq W) does not show any sequence similarity to either the known or novel antimicrobial or immunity’ peptides in terms of sequence similarity, amino acid identity, the respective blastp hits, or phylogenetic localization (FIGs. 1B,C, Table 1, Table 2). Lastly, MccZ from R. ornithinolytica (Ro Z) shows insignificant similarity with Ec MA (inc mA) for the antimicrobial, whereas the immunity peptide does not have any match among the known or the novel microcins (FIG. 1C, Table 1, Table 2). Crucially, the identification of MccX, and MccZ within the same gene clusters as representatives of the E492 (Bg E492), H47 (Ro H47), and 147 (Ro 147) groups strongly implies that they are functional components of a microcin gene cluster.

[0293] To test the newly identified microcins for antimicrobial activity, we used our previously established Ac overexpression system13,16. All antimicrobial and immunity peptides were codon optimized, synthesized, and cloned into an inducible high copy vector (see Methods). Thus, we extracted the novel microcins out of their native genomic context of siderophore biosynthesis and transferred them into a heterologous expression background optimized for microcin-monoglycosylated enterobactin (MGE) linkage. This allowed us to create hybrid compounds that could be efficiently tested for antimicrobial activity in an E. coli background. Through static plate inhibition assays involving live-producing cells13,16,26, we successfully validated the robust antimicrobial activity of eleven out of the twelve newly discovered microcins (FIG. 2A). Notably, antimicrobial activity was only observed in iron-depleted media (FIGs. 2B,C). The hybrid microcins exhibited a range of specificities, with some inhibiting targets narrowly (e.g, Rs G492AI), while others exert a broader effect against multiple bacteria (e.g., Se G492AI). Moreover, this study also provides the first evidence of inhibitory activity' by Kp G492, a microcin whose existence and function have only been proposed in the scientific literature based on genetic sequence15.

[0294] To date class lib microcins have been only shown to be very selective and only active against different species within the Enterobacteriaceae family13,13,16,26. While the activity for the novel microcins varies, we here report, for the first time, antimicrobial activity outside of the Enterobacteriaceae family utilizing hybrid antimicrobial peptides. Specifically, we found that microcins Ps G492 and Se G492 have activity against gram-negative multi drug-resistant ESKAPE pathogens with both being capable of inhibiting Acinetobacter baumannii (BAA 1790), and with microcin Se G492 alone also showing activity against Pseudomonas aeruginosa (PA14) (FIGs. 2A,C). Specifically, compared to K. pneumoniae (BAA 1705) Se G492 is 256-times more effective against A. baumannii (BAA 1790), 128-times more effective against E. coli (BAA 196), and 8-times more effective against P. aeruginosa (PA14) (FIG. 2B).

[0295] Table 1: Blastp results and closest matches to the known class lib microcins MccE492, MccG492, MccH47, MccI47, or MccM.

[0296] Table 2: Blastp results and closest matches to the known or novel class lib microcins.

[0297] *, no significant match found. Example 2. Ec W: A novel class lib microcin from Escherichia coli.

[0298] Materials and Methods

[0299] The following materials and methods were used in Example 2.

[0300] Bioinformatic identification ofEc W

[0301] To identify additional novel class lib microcins, we leveraged BLAST to mine publicly available genome databases as described above. Briefly, since we expected higher variability for the antimicrobials themselves due to target specificity, we leveraged the genes of the class lib gene cluster mchCDEF and mcmL for E. coli and mceCDGHIJ for Klebsiella pneumoniae, which might be more conserved to retain functionality. Resulting hits were merged by genome ID and assessed for their proximity to one another in the genome. In the instance of E. coll NCTC 10444, we noticed inconsistent results regarding compactness of the gene cluster since the high conservation of the two homologs resulted in random hits in two parts of the genome. Once the two homologous gene clusters were identified, they were aligned to E. coll CA46 (FIG. 6A) using Easyfig35. Manual curation and annotation of the genes in each homolog led to the identification of Ec W. whereas the antimicrobial itself shows similarity the recently described class lib microcin Gq W.

[0302] Then, blastp was used to assess microcin similarity to other microcins as shown in Tables 3 and 4, below.

[0303] Phylogenetic analysis

[0304] The phylogenetic analysis was conducted with MEGA1131. Using the MUSCLE algorithm30a codon-based sequence alignment was generated for all class lib microcins and the best fit substitution models for maximum likelihood phylogenetic analyses was determined. Employing the General Time Reversible model with discrete gamma distribution and empirical base frequencies (GTR+G+F) a bootstrap test with 1000 replicates for maximum likelihood and random seed was conducted.

[0305] Plasmids and heterologous class lib microcin expression

[0306] The sequence of the of the putative microcin and immunity peptide with 18 bp of native 5’ upstream sequence and 20 bp of native 3’ downstream sequence was synthesized by Integrated DNA Technologies (Coralville, TA). The genes were cloned into our previously established Ec class lib microcin expression system using Gibson Assembly32under the control of an arabinose-inducible pBad / araC promoter. The assembly was confirmed using whole plasmid sequencing through Plasmidsaurus, Inc. (Eugene, OR) and the DNA file is attached as supplementary material.

[0307] Static inhibition assays

[0308] Static inhibition assays were performed as previously described on several occasions. Briefly, Ec W- producing E. coli NEB10P bacteria harboring plasmid pBBAD-EcW were stabbed into a solid agar plate containing 100 pg / ml ampicillin for plasmid retention, 0.2 mM 2,2-dipyridyl to create iron-limited conditions during the growth phase, and 0.4% L-arabinose for induction of gene expression. The plates were incubated at 37°C for up to 48 h, before the microcin-producing bacteria were inactivated using chloroform vapors and UV light. Finally, they were overlaid with an agar film containing the target bacterial isolates and incubated for 16 h at 37°C. E. coli NEB 1 op bacteria transformed with pUC19 served as the negative control in this assay. Note that the base media contained ampicillin for plasmid retention, whereas not all tested bacterial isolates are ampicillin resistant, however, grow th of the mi crocin-producers for 48 h at 37°C allows for clearance of the antibiotic in the agar by beta-lactamases. Multiple stabs on a single plate ensure clearance of the whole plate, while also providing multiple replicates for zone of inhibition measurements. Each target bacteria was tested at least times independently (n=3).

[0309] Results

[0310] Identifying novel antimicrobial compounds presents a great opportunity to expand existing antimicrobial libraries to target MDR pathogens in various environments. Mining of public databases as described above and elsewhere46has uncovered numerous novel compounds that had yet to be tested and characterized. As described above in Example 1, we overexpressed and functionally tested twelve novel class lib microcins from seven different Enterobacteriaceae species Brenneria goodwinii, Gibbsiella quercinecans , Klebsiella oxytoca, Pantoea sp., Raoultella ornithinolytica, Salmonella enterica, and Serratia fonticola. Reanalyzing our data, we came across E. coli strain NCTC 10444 (accession no. NZ_LR134092) that to our knowledge is the first known bacterial strain to harbor two homologs of the class lib biosynthesis gene cluster (FIG. 6A, FIG. 7H shows one of the clusters). NCTC 10444 has been isolated as a pathogen from a calf in 1948 and sequenced by the Wellcome Sanger Institute (Hixton, England). Interestingly, while the tw o homologs of the cluster are highly conserved and almost identical in nucleotide sequence, there are two major differences to be noted: The first cluster (genome location: complement [2143418..2152585]) is flanked by a transposase and lacks the glycosyltransferase (mcmL) and the enterobactin esterase (mcmK) but harbors all three to-date known class lib microcins from E. coli, microcin H47, microcin 147, and microcin M. It is noteworthy, that at least mcmL is required to produce mature class lib microcins, as it glycosylates the respective siderophore, which is then covalently linked to the microcin precursor. Strikingly, mcmL and mcmK are present in the second homolog of the cluster (genome location 1916105.. 1928399), however, in the position of microcin M (between mchF and mcmM), there is no nucleotide resemblance to the clusters usually known to E. coli (i.e. E. coli CA46) (FIG. 6A). Automatic annotation identified a 107 amino acid (aa) ‘uncharacterised protein’ in reverse complement orientation in this region (locus tag: NCTC10444 03266; complement 1926778.. 1927101]) with clear resemblance of a class lib microcin composing of a serine-rich C-terminus ending on a final serine, no cysteines that could form disulfide bonds, and a 15 aa signal peptide that ends on a GA (FIGs. 6E- F)(see Example 1). Through manual curation of the sequence, we were able to identify a putative immunity peptide directly upstream of this microcin with just a single nucleotide separating the two open reading frames. As characteristic for all other class lib microcins, this putative immunity peptide contains a predicted transmembrane region using SMART29. Sequence alignment and phylogenic analysis of all class lib microcins reveals that this novel microcin closest associates with our previously described Gq W from G. quercinecans , an oak tree pathogen (FIG. 6B)37. Notably, blastp search using Gq W returns low e-values for the protein alignment and reasonable protein identity for this new microcin and vice versa (FIG. 6C), whereas Gq W previously did not have any significant resemblance to any of the class lib microcins. Thus, we propose to name this novel class lib microcin from the E. coli strain NCTC 10444 Ec W, which is now the fourth known class lib microcin produced by the genus Escherichia and the longest one identified so far with 107 aa.

[0311] For functional characterization, we synthesized the nucleotide sequence encoding for the immunity and antimicrobial peptide and cloned it into our established class lib microcin overexpression system (FIG. 6D)13’16’26. Through static inhibition assays employing Ec W-producing E. coli and an overlay of the respective target bacteria, we show that Ec W has potent activity against B. alni, B. rubifaciens, B. goodwinii. E. coli. G. quercinecans. G. greigii, K. aerogenes, K. pneumoniae, S. Typhimurium, and Shigella flexneri including clinical isolates, while did not observe activity against Acinetobacter baumannii, Enterobacter cloacae, K. oxytoca, Proteus mirabilis, Pseudomonas aeruginosa, S. marcescens, or Staphylococcus aureus. As reported for previous class lib microcins, we saw consistency across all tested strains of a species irrespective of the clade or antibiotic resistance, where the activity spectrum appears to be species-dependent13,16’26. Lastly, we report the zone of inhibition diameter for the tested strains with the largest clearance for clinically relevant E. coli strains BAA 196 and EHEC (EDL933). Notably, one of the highest activities is also observed against G. quercinecans, the Enterobacteriaceae relative and tree pathogen that encodes for Gq W.

[0312]

[0313]

[0314] Table 4: Blastp results and closest matches to the known or novel class lib microcins. Asterisk indicates no significant match found. References

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[0357] OTHER EMBODIMENTS

[0358] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A genetically engineered microorganism capable of producing one or more microcins Bg E492, Bg X, Gq W, Ko H47, Ps G492, Ro Z, Ro H47, Ro 147, Ro X, Sf H47, Se G492, Se M, or Ec W, wherein the microorganism comprises: a microcin operon comprising microcin antimicrobial gene Bg E492A, Bg XA, Gq WA, Ko H47A, Ps G492A, Ro ZA, Ro H47A, Ro 147 A, Ro XA, Sf H47A, Se G492A, Se MA, or Ec WA, and preferably an immunity gene Bg E492I, Bg XI, Gq WI, Ko H47I, Ps G492I, Ro ZI, Ro H47I, Ro 1471, Ro XI. Sf H47I, Se G492I, Se MI, or Ec WI, and genes for the production of the antimicrobial gene and immunity genes, optionally mchCDEF and mcmL, or mceCDGHIJ^ and a promoter for the microcin operon, wherein either or both of the microcin operon and the promoter are heterologous to the microorganism.

2. The genetically engineered microorganism of claim 1 , wherein the genetically engineered microorganism is a bacterium, optionally wherein the genetically engineered microorganism is Escherichia coli.

3. The genetically engineered microorganism of any one of claims 1 or 2, wherein the microcin operon further comprises microcin genes mchE and mchF.

4. The genetically engineered microorganism of claim 1 , wherein the promoter is a controllable promoter for the microcin operon that controls a level of expression of one or more of the microcin genes, thereby controlling the amount of microcin produced by the genetically engineered microorganism,5. The genetically engineered microorganism of claim 4, wherein the microorganism comprises a second microcin operon comprising microcin gene mchA and a second controllable promoter for the second microcin operon, wherein the second controllable promoter controls a level of expression of mchA, thereby controlling the amount of microcin produced by the genetically engineered microorganism.

6. The genetically engineered microorganism of any one of claims 1 to 5, wherein the first or the second microcin operon. or both the first and the second microcin operons and the first or second controllable promoter, or both the first and the second controllable promoters are in the genome of the microorganism, or are in a vector.

7. A composition for use in treating a bacterial infection, wherein the composition comprises the genetically engineered microorganism of any one of claims 1 to 6.

8. The composition of claim 7, wherein the composition is packaged in a capsule for intestinal deliver}'.

9. The composition of claim 7 or 8. wherein the bacterial infection is a gramnegative bacterial infection in an animal or plant.

10. A method of treating or reducing risk of a bacterial infection or intestinal dysbiosis in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising the genetically engineered microorganism of any one of claims 1 to 9, optionally wherein the bacterial infection is a gram-negative bacterial infection.

11. The method of claim 10, wherein the subject is a human and the composition is administered by endoscopy, enteroscopy, colonoscopy, anasoduodenal catheter, enema, or by oral administration.

12. The method of claim 11, wherein the composition is orally administered, optionally in a capsule.

13. A method of treating a bacterial infection in a plant, the method comprising administering to the plant an effective amount of a composition comprising the genetically engineered microorganism of any one of claims 1 to 9.

14. The method of claim 13, wherein the plant has an infection with Gibbsiella quercinecans, Gibbsiella greigii, Rahnella victoriana. or Brenneria goodwinii.

15. The method of claim 13 or 14, wherein the plant is a tree, optionally an oak tree, walnut tree, or autumn olive.

16. An isolated microcin antimicrobial peptide comprising Bg E492A, Bg XA, Gq WA, Ko H47A, Ps G492A, Ro ZA, Ro H47A, Ro 147 A, Ro XA, Sf H47A, Se G492A, Se MA, or Ec WA, optionally wherein the peptide is modified.

17. A composition comprising the isolated microcin antimicrobial peptide of claim 16, and a carrier, optionally a pharmaceutically or agriculturally acceptable carrier.

18. The composition of claim 17, wherein the composition is packaged in a capsule for intestinal delivery.

19. The composition of claim 17 or 18 for use in treating or reducing risk of a bacterial infection, optionally wherein the bacterial infection is a gram-negative bacterial infection in an animal or plant.

20. A method of treating or reducing risk of a bacterial infection or intestinal dysbiosis in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of any one of claims 17 to 19, optionally wherein the bacterial infection is a gram-negative bacterial infection.

21. The method of claim 20, wherein the subject is a human and the composition is administered by endoscopy, enteroscopy, colonoscopy, a nasoduodenal catheter, enema, or by oral administration.

22. The method of claim 21, wherein the composition is orally administered, optionally in a capsule.

23. A method of treating a bacterial infection in a plant, the method comprising administering to the plant an effective amount of a composition comprising the composition of any one of claims 17 to 19.

24. The method of claim 23, wherein the plant has an infection with a pathogen of genera Gibbsiella. Rahnella. or Brenneria, optionally Gibbsiella quercinecans, Gibbsiella greigii, Rahnella vicioriana. ox Brenneria goodwinii.

25. The method of claim 23 or 24, wherein the plant is a tree, optionally an oak tree, walnut tree, or autumn olive.

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