Patent Publication Number: US-2022218787-A1

Title: Genetically engineered microorganisms and methods of use

Description:
CLAIM OF PRIORITY 
     This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/843,149, filed May 3, 2019 and U.S. Provisional Patent Application Ser. No. 62/942,537, filed Dec. 2, 2019. The entire contents of the foregoing applications are hereby incorporated by reference. 
    
    
     FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made with Government support under Grant Nos. NSF 1458347 awarded by National Science Foundation and NIH AI112985 awarded by the National Institutes of Health. The Government has certain rights to the invention. 
    
    
     TECHNICAL FIELD 
     This disclosure relates to genetically engineered microorganisms and methods of use. 
     BACKGROUND 
     Medical complications related to drug-resistant bacteria (bacteria that cannot be treated with currently available antibiotics), including those from members of the family Enterobacteriaceae, are a major issue in modern healthcare due to the increased morbidity, mortality, length of hospitalization and related healthcare costs. The CDC estimates that every year more than two million people acquire multi-drug-resistant bacterial infections, which result in over 23,000 directly-related deaths and several more lethal outcomes from associated complications. A 2014 report from the World Health Organization (WHO) found that all WHO surveyed regions are characterized by high-rates of multi-drug resistant microorganisms, which are responsible for common health care facility and community acquired urinary tract infections (UTIs), pneumonias and blood stream infections. Of particular gravity is the fact that WHO&#39;s surveillance data are showing that more than 50% of  Klebsiella  species-related infections in all WHO regions are resistant to third generation Cephalosporin, with a significant portion (&gt;20%) also showing concurrent resistance to its only alternative, Carbapenem. 
     A recent report has estimated that the economic burden for society due to Carbapenem-Resistant Enterobacteriaceae (CRE) infections, which include CR  Klebsiella pneumoniae  (CRKp), CR  Klebsiella oxytoca  (CRKo), CR  Enterobacter cloaceae  (CREc), and Enterobacter aerogenes (CREa), ranges from $37,000 to $83,000 per infection. When considering an infection incidence range of 2.93-15 per 100,000 people in the USA (i.e. 9,418-48,213 infections), CRE infections are estimated to cost society anything in the order of $1-$2 billion every year and to cause the loss of up to 45,261 quality-adjusted life years. Thus, there is pressing need to develop novel therapeutics that selectively kill pathogenic bacteria, reduce infection rates (and duration of infection), and curb the emergence of new drug-resistance mechanisms. 
     SUMMARY 
     The present disclosure provides compositions of engineered microorganisms to produce microcin I47 and methods of treating bacterial infections, e.g., gram-negative bacterial infections, and dysbiosis. The disclosure further provides genetically engineered microorganisms that include a microcin operon and a controllable promoter for the microcin operon. In particular, the microcin operon includes mciI, mciA, mchC, mchD, mchE, and mchF genes. The controllable promoter controls a level of expression of the one or more microcin genes, thereby controlling the amount of microcin produced by the genetically engineered microorganism. Either or both of the microcin operon and the controllable promoter are heterologous to the microorganism. 
     In one aspect, the disclosure provides genetically engineered microorganisms capable of producing microcin I47, wherein the microorganism include a microcin operon, and a first controllable promoter for the microcin operon, wherein the microcin operon comprises microcin genes mciI, mciA, mchC, and mchD, wherein the first controllable promoter controls a level of expression of at least the one of the microcin genes, thereby controlling the amount of microcin produced by the genetically engineered microorganism, and wherein either or both of the microcin operon and the first controllable promoter are heterologous to the microorganism. 
     In some embodiments, the genetically engineered microorganism is a bacterium. In some embodiments, the genetically engineered microorganism is  Escherichia coli.    
     In some embodiments, the microorganism further 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 second microcin operon further comprises microcin gene mchS4, microcin gebe mchS1, or both. 
     In some embodiments, the controllable promoter is a pJ23119 promoter. 
     In some embodiments, the first microcin operon further comprises microcin genes mchE and mchF. 
     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. 
     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 a vector. 
     In some embodiments, the composition includes any one of the genetically engineered microorganisms described herein. 
     In some embodiments, the composition is packaged in a capsule for intestinal delivery. 
     In another aspect, the disclosure provides methods of treating intestinal dysbiosis, the methods including identifying a subject as having intestinal dysbiosis; and administering to the subject a therapeutically effective amount of a composition comprising any one of the genetically engineered microorganism described herein. 
     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. In some embodiments, the composition is orally administered, optionally in a capsule. 
     In another aspect, the disclosure features methods of treating a bacterial infection, the methods including identifying a subject as having a bacterial infection; and administering to the subject a therapeutically effective amount of a composition comprising any one of the genetically engineered microorganisms described herein. 
     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. In some embodiments, the composition is orally administered, optionally in a capsule. In some embodiments, the bacterial infection is a gram-negative bacterial infection. In some embodiments, the bacterial infection is carbapenem-resistant Enterobacteriaceae infection, campylobacter infection,  E. coli  infection,  Salmonella  infection,  Shigella  infection and/or  Yersinia  infection. 
     Also provided herein are methods of reducing a risk of a bacterial infection, the method comprising: identifying a subject as having a risk of a bacterial infection; and administering to the subject a composition comprising any one of the genetically engineered microorganisms described herein. 
     In some embodiments, the subject is being administered one or more antibiotics. 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. In some embodiments, the composition is orally administered, optionally in a capsule. 
     This disclosure provides new  E. coli  single-strain probiotics that produce microcin MccI47. As described herein, in vitro experiments using both heterologous I47 production from a probiotic or purified I47 show that microcin I47 is especially capable in killing CR  K. pneumoniae.  The disclosure further provides plasmid-based systems capable of producing microcin I47 and provides the use of mature (post-translationally modified) microcin I47 delivered from a probiotic or in its purified form as new antibiotic to kill bacteria, e.g.,  Klebsiella  species with a specific focus on drug-resistant  Klebsiella  species. Moreover, I47 is able to kill with different efficacy other Enterobacteriaceae including  Escherichia coli, Salmonella typhimurium, Salmonella typhi,  and  Shigella flexneri  as well as drug resistant strains of these species. Based on the results described herein, the overproduction vectors described herein result in strong signals of decolonization, which greatly opens the opportunities for engineered biotherapeutics aimed at eradication of multidrug resistant (MDR) enteric bacteria. 
     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. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 
     Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. 
    
    
     
       DESCRIPTION OF DRAWINGS 
       The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. 
         FIG. 1  is a diagram of select  E. coli  mch gene clusters. Genes sharing the same shade in the same strain are involved in similar function (e.g. mchEF-secretion). Genes sharing the same shade across different strains, and with different names indicate homologs (e.g. mchA-mcmL). Black coding regions indicate transposable elements. Microcins listed within parentheses denote presence of corresponding gene for microcin structural protein (mchB, mciA, mcmA). 
         FIG. 2  is a diagram depicting the design of the six vectors used for MccH47 and MccI47 production. The first variable region inducibly expresses mchXIB or mciIA, and the second variable region express either mchA, mchAS4, or mchAS4S1. mchCDEFA are essential genes for MccH47 and MccI47 production and post-translational modification, while mchS4S1 are non-essential. mchCDEF retain their natural expression regulatory features, while mchXIB and mciIA are regulated by P BAD  and mchAS4S1 are expressed from the strong constitutive promoter, P J23119 . All vectors are built into the high-copy pUC19, ColE1 origin backbone. 
         FIGS. 3A-3H  are representations of assay plates showing results of static inhibitory assays utilizing stabs of  E. coli  NEB10β transformed with 1) pBBAD-H47, 2) pS4BAD-H47, 3) pJBAD-H47, 4) pBBAD-I47, 5) pS4BAD-I47, 6) pJBAD-I47, and 7) pUC19. Target overlays include A.)  E. coli  DH5α, B.)  E. coli  BAA-196, C.)  S. typhimurium  29630 pUC19, D.)  S. typhimurium  BAA-190 E.)  K. oxytoca  700324 pUC19, F.)  K. oxytoca  51983, G.)  K. pneumoniae  BAA-1705, H.)  K. pneumoniae  BAA-2524. Target strains B, D, F, G, and H are classified as multidrug resistant. 
         FIGS. 4A-4D  are representations of assay plates showing results of static inhibitory assays utilizing stabs of  E. coli  NEB10β transformed with pBBAD-I47 against (A)  E. coli  DH5alpha, (B)  S. typhimurium,  (C)  K. oxytoca  and (D) Carbapenem-resistant  K. pneumoniae.  (Right) Plasmid overexpressing genes for I47 production and for its post-translational modification and export. 
     
    
    
     DETAILED DESCRIPTION 
     Members of drug-resistant Enterobacteriaceae spp. include opportunistic pathogens (e.g.,  Salmonella  spp.) are among the leading causes of morbidity and mortality worldwide. Overgrowth of these bacteria is considered a hallmark of intestinal dysbiosis. Some gut commensals produce microcins, small antimicrobial peptides, that inhibit growth of select pathogens. As described herein, select gut commensals can be genetically altered and used to effectively treat pathogenic bacteria infections and/or to limit the growth of pathogenic bacteria. 
     Delivery of rationally-designed combinations of gastrointestinal commensals has the benefit of ensuring CRE decolonization via a number of concurring mechanisms including competition for nutrient and space, production of antimicrobial molecules and immune-system stimulation. However, the cost of large-scale production of these consortia linearly scales with the number of employed species (1-2 months per strain based on work from our industrial partners), thus making the generation of consortia of dozens of strains a big and time-consuming endeavor. Recent work has shown that addition of single strains of microcinogenic intestinal residents (i.e. bacteria capable of secreting small antimicrobial peptides) can lead to the killing of pathogenic Gram-negative Enterobacteriaceae, and therefore could be used as novel live biotherapeutics. However, because native microcin production is performed by strains with unknown mammalian gut colonization capability, and is dependent on the conditions experienced in the intestine (e.g., iron limitation), this phenomenon is difficult to control and thus exploit for therapies. 
     We have built new prototypes of  E. coli  single-strain probiotics that produce a previously minimally-characterized microcin I47. Performing in vitro experiments using both heterologous I47 production from a probiotic or I47, purified for the first time by us, we observed that microcin I47 is especially capable in killing CR  K. pneumoniae,  suggesting that we have identified a novel molecule for the killing of this deadly pathogen. 
     The disclosure provides plasmid-based systems capable of producing microcin I47. The disclosure also provides the use of mature (post-translationally modified) microcin I47 delivered from a probiotic or in its purified form as new antibiotic to kill bacteria, e.g.,  Klebsiella  species with a specific focus on drug-resistant  Klebsiella  species. Our data show that I47 can also kill with different efficacy other Enterobacteriaceae including  Escherichia coli, Salmonella typhimurium, Salmonella typhi,  and  Shigella flexneri.  Additionally, this disclosure provides genetically engineered probiotics capable of conditionally producing microcin I47. 
     Microcins 
     Microcins are low-molecular-weight antimicrobial peptides secreted by members of the Enterobacteriaceae family. They include, e.g., Class I microcins, Class IIa microcins, Class IIb microcins, and Class IIc microcins. Class I microcins have molecular masses &lt;5 kDa, are post-translationally modified, and bind to a spectrum of targets. Class IIb microcins are relatively large (˜5-10 kDa) polypeptides and feature a C-terminal siderophore post-translational modification. Class IIb microcins include, e.g., Microcin H47 (MccH47), MccE492, MccM, MccG492 and MccI47. 
     MccI47 
     Microcin I47 is a bactericidal antibiotic. Due to its size, it shares with other microcins the ability to pass through cellophane membranes. Microcin I47 has been reported to be produced by the MccH47 genetic system and detected in iron deprivation conditions (Azpiroz et al., 2011,  PLOS ONE  6(10):e26179; Poey et al., 2006,  Antimicrob Agents Chemother  50(4):1411-8). 
     Production and purification of microcin I47 can be conducted by any suitable method known in the art. In some embodiments, microcin I47 can be purified using an amylose resin column eluted with maltose. For example, cultures of  E. coli  producing microcin I47, e.g.,  E. coli  NEB10β pHMT-I47, are 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 β-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. 
     Cultures can then be thawed in cold water, sonicated, and the crude lysate is passed through a resin column, e.g., an amylose resin (New England Biolabs, Ipswich, Mass.) 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). 
     The eluent can be concentrated, for example, using MilliporeSigma (Burlington, Mass.) 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, Mass.), 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, Del.). Ni-NTA slurry can be pelleted by centrifugation and the supernatant can be removed by pipetting. 
     Genetic System for Producing MccI47 and MccH47 
     The genes required for production of MccI47 and MccH47 are clustered in a 10-kb DNA segment located in the  E. coli  chromosome and include the genes: mchA, mchB, mchC, mchD, mchE, mchF, mchI, mchX, mciA (formerly known as mchS2), mciI (formerly known as mchS3),mchS1, and mchS4. Three genes, mchA, mchC, and mchD, are devoted to mature microcin synthesis; whereas mciA and mciI encode for the precursor and the corresponding immunity peptide of MccI47 and mchB and mchI for the precursor and the corresponding immunity peptide of MccH47. Two further genes, mchE and mchF, are required for the secretion of the antibiotic into the extracellular medium. 
     Production of class IIb 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 H 47,  Antimicrobial agents and chemotherapy  54.1:288-297, which is incorporated herein by reference in its entirety. The complexity of the MccI47 antibiotic system parallels that of other microcin systems, such as those of microcins B17 and C7. MccI47 maturation, in which mchA, mchC, and mchD gene products are known to be necessary, is believed to endow the antibiotic molecule with the ability to enter cells. 
     
       
         
           
               
            
               
                 mchA gene sequence 
               
               
                 (SEQ ID NO: 1) 
               
               
                 ATGCGAAAACGTATTCTTTTTATTGGCCCACCGCTGTACGGTTTGTTATA 
               
               
                   
               
               
                 CCCATTGATTTCTCTGGCTCAGGCCTTTCGTGTAATCGGACATGATGTAG 
               
               
                   
               
               
                 TAATTAGTAGTGCTGGCAAATTCGCGAATAAAGCAGCAGAAGCTGGACTG 
               
               
                   
               
               
                 GTTGTTTTTGATGCAGTTCCAGGTTTAGATTCAGAGGCTGGATATCGCCA 
               
               
                   
               
               
                 TCAGGAAGAGTTGAGGAAAAAAAGTAATATTATTGGTCATTTCTCTTTTT 
               
               
                   
               
               
                 TTAGCGATGAAATGGCAGATAACCTCATCGATTTTGCAGGAAAATGGAGG 
               
               
                   
               
               
                 CCAGATTTAATAGTCTATCCCCCGCTTGGTCCGGCAGGCCCATTGGTTGC 
               
               
                   
               
               
                 TGCTAAATATAGAATTCCTTCAGTGATGCTGGCTGTTGGATTCGCGCATA 
               
               
                   
               
               
                 CATCTGCCCATATTCAGATGTTAAACCGTTCTTTAAGCAATGCTTACAGG 
               
               
                   
               
               
                 CGGCATGGAGTCAGCGGTCCACTATGTGATTTAGCATGGATTGATGTTGC 
               
               
                   
               
               
                 TCCCCCAAGTATGAGCATTCTTAAAAATGCTGAAGAACCGGTTATCTCAA 
               
               
                   
               
               
                 TGAGATATATTCCTTATAACGGAGGTGCTGTAAAGGAAACATGGTGGGAC 
               
               
                   
               
               
                 AGGGATTCTGATCGAAAACGTTTACTCATCAGCCTTGGCACTGTAAAACC 
               
               
                   
               
               
                 AATGGTTGATGGTCTGGAGCTGATTTCATGGGTTATGGATTCTGCAAATG 
               
               
                   
               
               
                 AAGTTGATGCTGATATCATTTTGCAACTTGCAATAAATGCTCGTACTGGA 
               
               
                   
               
               
                 TTACGAAAACTACCATCAAATGTACGTCTGGTTGACTGGATACCTATGGG 
               
               
                   
               
               
                 TGTATTCCTTAATGGAGCTGATGGATTTATTCATCATGGTGGCGCAGGTA 
               
               
                   
               
               
                 ATACCCTGACAGCGTTGTATAGTGGGATACCACAGATTGTGTTTGGCGAA 
               
               
                   
               
               
                 GGTGCAGATCGCTCTGTTAATGCAGAAATTGTTGCGATGCGTGGGTGTGG 
               
               
                   
               
               
                 GATTATTCCGGACAAGCATGGACTGACCAGTGATTTGGTAAATCGCCTGC 
               
               
                   
               
               
                 TTTATGATGATTCACTACGCTTCTGTTCAGATCAGGTAGCCGCTGAAATG 
               
               
                   
               
               
                 GCTGAACAACCCAGTCCTGCAGAGATCGCAGAGGTTTTGATGAGAAAATT 
               
               
                   
               
               
                 AAAAAACAACGGGAAATAA. 
               
               
                   
               
               
                 mchC gene sequence: 
               
               
                 (SEQ ID NO: 2) 
               
               
                 ATGAGTCATCAGTGTTCACTTTCTGAACTGAATGAAAACCTGGTGCCTTT 
               
               
                   
               
               
                 CACTGCCAGGCAGATCAAGTCCTCATTAATCTGGTGTGCAGAGGATGTCA 
               
               
                   
               
               
                 GAAATCCAGGCGAGCTGCAAAATGCCTGCAGTTATATTATCGATCCTGAC 
               
               
                   
               
               
                 AGTACGGCTTCTGCCAAAGTGTTCCATGCAGAGCGCTATGGTGGCAGTGG 
               
               
                   
               
               
                 TATTCAGCGTAATGGAGGTGGTGCACGTTGTGGGTTTGATGGTAACTACC 
               
               
                   
               
               
                 AGGTTAAAGGAATAGGAAGTAATCCGTTGGTTGGTGAAGGTACTGACGAA 
               
               
                   
               
               
                 CGTCATTCTAATGGTGCACTCGGCGCTGTTCATGCAATATATGAGGCTTT 
               
               
                   
               
               
                 GTGGGGAGAAGTACTGGCTCAAATATTACCTTATAGTGCTGTGCGGGTTC 
               
               
                   
               
               
                 GGGCGGTTTTACTTACAGATCTCTATACTGAAAAGGCATTTGAGCGCTCC 
               
               
                   
               
               
                 GGTATGAAATCACGAAGAGCCCTGTTGGTACGTGAGCCTGTTGTTCGCCC 
               
               
                   
               
               
                 GGCGCATTTTGAACGGGCACCATACTTCCAAGTAAAACCGGAGTATTCCA 
               
               
                   
               
               
                 GTCAGTTAATTCACGATGCCTGTCGGGTTAGATCTGTGATCCACAAGCTG 
               
               
                   
               
               
                 CCAGGATATCTACCTGTACCACCGGAAGAAATTGATGCTGAAGCACGAAC 
               
               
                   
               
               
                 TGATCCCCGGATTTATTGCATTGAGGGATTATGTGAACTGGCACGTCGTG 
               
               
                   
               
               
                 AGGCCTGGCAAATGGCATTTTGTCGAACACGTTTCCTGAGATTGACAACT 
               
               
                   
               
               
                 TCTCCTTCTAATATTGCAATGGATGGCAGATTAATGGATTTTAACGGACT 
               
               
                   
               
               
                 CAGTTGCTCGTTTCCGGGAGATTCCCCAGCTGATTTTGGGTATAAACTAA 
               
               
                   
               
               
                 GATTAGCTGAACTGGCAAAAGAACCGATGGTACTTATGCAAGGGCTGTCT 
               
               
                   
               
               
                 GATCTCTGCTTGTATATCGGAAAATATATGTTTGACCCTGACTTCACTCT 
               
               
                   
               
               
                 TGCAGCCCGTTTGAAGGTTGAGGAGATATTTCAGAAAACTTTTCATGAAG 
               
               
                   
               
               
                 CATGTTATTACTGTTATCTAGAACTGTTGGGTATTCCTGGAGAATTTATA 
               
               
                   
               
               
                 ACACAAAAAGAGATACCTGATATATTGAAACAACTGGTTAACAGTTTTGT 
               
               
                   
               
               
                 TGCATTACTCAATAAATACTGCGAGAAATCACATGCCCAAGATATTGTCA 
               
               
                   
               
               
                 ATCAGGATGGTTCACCATTGCAAAAGTTGGTTGTGACGCTAATCCATCAT 
               
               
                   
               
               
                 AGGCATAATCAAAAGCAGGCACTGAATAGTAGCATCAAGAATGATGTTTA 
               
               
                   
               
               
                 TTTCACCGTTGCACAACAGTGTTTTTCCCAGACTATCCACTGGCTGACGC 
               
               
                   
               
               
                 AAGGCAGTACCAGACGTCAGATAAATGCTTCATTACTCCTGAAAGAAATT 
               
               
                   
               
               
                 GAACATCATACCATGAAAAGGCTGCAACCCAGGGAAGAGCTGAGGAAAGA 
               
               
                   
               
               
                 GAATATGTGCGAAAAAATTGCCATCCTGCTGGATAATCATGGCGATGATC 
               
               
                   
               
               
                 CCCTTTTTTTACAAGAAGCAATTTCTGATATGAAAAATTTTATGCTTAAG 
               
               
                   
               
               
                 TTTTCCAGAGATGCATTTGGATATCTTGAACCGATAAGAAACACAGTGTA 
               
               
                   
               
               
                 A. 
               
               
                   
               
               
                 mchD gene sequence: 
               
               
                 (SEQ ID NO: 3) 
               
               
                 ATGTCTTATATAAGGGAAACCATCAGAGGAAAAGATGAATGGACTGTTTA 
               
               
                   
               
               
                 TGAACAGATAGGTTTTGCGGTCAGTTGTATGCTCTACAATCGTAATTACA 
               
               
                   
               
               
                 GTCTGTATCCGGTGTTAACCATTCAATACTGGACTGAATATGCGATACAG 
               
               
                   
               
               
                 CATAATCAGATTAAATTCCTGTTTGATTCACGAGGTTTTCCACTGGCGTA 
               
               
                   
               
               
                 TATAACCTGGGCATATCTTGAGGCTGATACGGAAGCGCGCCTGCTCAGGG 
               
               
                   
               
               
                 ATCCAGAATTCAGGTTGCATCCGTCTGAATGGAATGAAGATGGAAGGATC 
               
               
                   
               
               
                 TGGATCCTGGATTTCTGTTGTAAACCAGGCTTTGGTCGAAAAGTTATTGA 
               
               
                   
               
               
                 CTATCTCATACAGCTTCAGCCATGGGGGGAAGGAGAAGTACGATGGTTAA 
               
               
                   
               
               
                 GCAGGCGAAAGAAAATTGTGACATACATCCCTGAGCGGCTGCATAAAACG 
               
               
                   
               
               
                 TAG. 
               
            
           
         
       
     
     The mchB genes encodes the pre-microcin H47 peptide. Once the peptide product of the mchB gene has gone through modification and secretion steps, the pre-microcin H47 peptide becomes microcin H47. 
     
       
         
           
               
            
               
                 mchB gene sequence: 
               
               
                 (SEQ ID NO: 4) 
               
               
                 ATGCGAGAAATAACAGAATCACAGTTAAGATATATTTCCGGGGCG 
               
               
                   
               
               
                 GGAGGTGCGCCAGCGACTTCAGCTAATGCCGCAGGTGCTGCAGCTATTGT 
               
               
                   
               
               
                 TGGAGCTCTCGCCGGAATACCTGGTGGTCCACTTGGGGTTGTAGTTGGAG 
               
               
                   
               
               
                 CCGTATCTGCCGGTTTGACAACAGCAATTGGCTCGACCGTGGGAAGTGGT 
               
               
                   
               
               
                 AGTGCCAGTTCTTCTGCTGGTGGCGGTAGCTAA. 
               
            
           
         
       
     
     The mchE and mchF genes encode secretion proteins, which are necessary for secretion out of the cell. In some embodiments, the secretion proteins encoded by mchE and mchF are required for export of the microcin, but are not required for the production of the microcin. 
     
       
         
           
               
            
               
                 mchE gene sequence: 
               
               
                 (SEQ ID NO: 5) 
               
               
                 TTGTTTCGTCAGGATGCTTTAGAAAACAGAAAAATGAAGTGGCAGGGACG 
               
               
                   
               
               
                 GGCAATATTACTTCCCGGAATACCACTATGGTTAATCATGCTGGGAAGCA 
               
               
                   
               
               
                 TTGTGTTTATTACGGCATTTCTGATGTTCATTATTGTTGGTACCTATAGC 
               
               
                   
               
               
                 CGCCGTGTTAATGTCAGTGGTGAGGTCACAACCTGGCCAAGAGCTGTCAA 
               
               
                   
               
               
                 TATATATTCAGGTGTACAGGGATTTGTTGTCAGGCAATTTGTTCATGAA 
               
               
                   
               
               
                 GGGCAGTTGATAAAAAAAGGGGATCCTGTTTATCTGATTGACATCAGTAA 
               
               
                   
               
               
                 AAGTACACGTAGTGGTATTGTCACTGATAATCATCGGCGGGATATAGAAA 
               
               
                   
               
               
                 ATCAGCTGGTTCGTGTGGACAACATTATTTCCCGTCTGGAAGAAAGTAAA 
               
               
                   
               
               
                 AAAATAACGTTAGATACCCTGGAAAAACAACGTCTGCAATACACAGATGC 
               
               
                   
               
               
                 GTTTCGTCGCTCATCAGATATTATACAGCGTGCAGAGGAAGGGATAAAAA 
               
               
                   
               
               
                 TAATGAAAAACAATATGGAGAATTACAGAAACTATCAGGCAAAAGGGCT 
               
               
                   
               
               
                 GATTAATAAAGATCAGTTAACTAACCAGGTGGCATTATATTATCAGCAAC 
               
               
                   
               
               
                 AAAACAATCTTCTCAGCCTGAGCGGACAGAACGAACAGAATGCCCTGCAG 
               
               
                   
               
               
                 ATAACCACTCTGGAGAGTCAGATTCAGACTCAGGCTGCAGATTTTGATAA 
               
               
                   
               
               
                 CCGTATCTACCAGATGGAACTGCAACGGTACGAGTTACAGAAAGAACTGG 
               
               
                   
               
               
                 TTAACACTGATGTGGAGGGCGAAATTATTATCCGGGCGTTGACTGACGGG 
               
               
                   
               
               
                 AAAGTTGACTCCCTGAGTGTCACTGTCGGGCAAATGGTCAATACCGGAGA 
               
               
                   
               
               
                 CAGCCTTCTGCAGGTTATTCCTGAGAACATTGAAAACTATTATCTTATTC 
               
               
                   
               
               
                 TCTGGGTCCCAAATGATGCTGTTCCTTATATTTCGGCTGGTGACAAAGTG 
               
               
                   
               
               
                 AATATTCGTTATGAAGCCTTTCCGGCAGAAAAATTTGGGCAGTTCTCTGC 
               
               
                   
               
               
                 TACGGTTAAAACTATATCCAGGACTCCTGCGTCAACACAGGAAATGTTGA 
               
               
                   
               
               
                 CCTATAAGGGTGCACCACAGAATACGCCGGGCGCCTCTGTTCCCTGGTAT 
               
               
                   
               
               
                 AAAGTCATTGCGATGCCTGAAAAGCAGATTATCAGATATGACGAAAAATA 
               
               
                   
               
               
                 CCTCCCTCTGGAAAATGGAATGAAAGCCGAAAGTACACTATTTCTGGAAA 
               
               
                   
               
               
                 AAAGGCGTATTTACCAGTGGATGCTTTCTCCTTTCTATGACATGAAACAC 
               
               
                   
               
               
                 AGTGCAACAGGACCGCTCAATGACTAA. 
               
               
                   
               
               
                 mchF gene sequence: 
               
               
                 (SEQ ID NO: 6) 
               
               
                 ATGACTAACGGGAGTTTCAGACAAATTATAAATCAGCTTGATATGCGCTG 
               
               
                   
               
               
                 GCGACGTCGTGTTCCGGTTATTCATCAGACGGAGACCGCTGAATGTGGAC 
               
               
                   
               
               
                 TGGCCTGCCTGGCAATGATATGCGGTCATTTTGGTAAGAATATTGACCTG 
               
               
                   
               
               
                 ATATCTCTTCGCCGGAAGTTTAATCTCTCGGCCCGTGGAGCAAACCTTGC 
               
               
                   
               
               
                 AGGAATCAATGGAATAGCGGAGCAGCTGGGGATGGTCACCCGGGCTCTTT 
               
               
                   
               
               
                 CACTGGAGCTGGATGAACTTGGTGCCCTCAAAATGCCGTGTATTCTCCAC 
               
               
                   
               
               
                 TGGGATTTCAGTCACTTTGTCGTGCTGGTCAGCGTAAAGCGTAACCGTTA 
               
               
                   
               
               
                 TGTACTGCATGATCCGGCCAGAGGCAGAAGATATCTCGGTCGGGAGGAAA 
               
               
                   
               
               
                 TGAGCCGGTATTTTACGGGCATTGCACTTGAGGTCTGGCCTGGAAGTGAA 
               
               
                   
               
               
                 TTCCTGGCGGAAACCCAGCAGATCCGCATAAGTCTCCGTTCACTGATTAA 
               
               
                   
               
               
                 CAGTATTTACGGTATTAAAAGAACACTGGCGAAAATTTTCTGTCTGTCAG 
               
               
                   
               
               
                 TTGTAATTGAAGCAATCAATCTGGTAATGCCGGTGGGGACTCAGCTGGTT 
               
               
                   
               
               
                 ATGGATCATGCGATTCCGGCGGGGGACAGAGGGCTGCTGACGCTTATTTC 
               
               
                   
               
               
                 TGCTGGCCTGATGTTCTTTATATTGCTCAGGGCCGCGGTGAGTATGCTGC 
               
               
                   
               
               
                 GTGCATGGTCCTCACTGGTTATGAGCACGCTCATCAATATACAGTGGCAG 
               
               
                   
               
               
                 TCGGGTCTGTTTAACCATCTTCTCAGACTGCCGCTGGCGTTTTTTGAACG 
               
               
                   
               
               
                 CCGTAAATTAGGTGATATCCAGTCGCGTTTTGGCTCCCTTGACACTTTGA 
               
               
                   
               
               
                 GGGCCACCTTTACCACCTGTGTGGTTGGGGCAATCATGGACAGTATTATG 
               
               
                   
               
               
                 GTTGTGGGGGTTTTTGTGATGATGCTGTTATATGGAGGATATCTTACCTG 
               
               
                   
               
               
                 GATAGTGCTCGGTTTTACCATGGTTTACGTTCTTATTCGTCTGGTGACAT 
               
               
                   
               
               
                 ACGGCTATTACCGGCAAATATCGGAAGAAACTCTTGTCAGGGGGGCCCGG 
               
               
                   
               
               
                 GCCAGCTCCTATTTTATGGAAAGCCTGTATGGTATTGCCACGGTAAAAAT 
               
               
                   
               
               
                 CCAAGGTATGGCTGGGATCCGGGGAACACACTGGCTTAACCTGAAAATAG 
               
               
                   
               
               
                 ATGCGATCAATTCAGGTATTAAGTTAACCAAGATGGATTTGCTCTTCGGG 
               
               
                   
               
               
                 GGGATAAATACTTTTGTTGCCGCCTGTGATCAGGTGGCGATTTTATGGCT 
               
               
                   
               
               
                 GGGTGCAAGCCTTGTGATCGATAATCAGATGACAATAGGGATGTTTGTGG 
               
               
                   
               
               
                 CATTTGGTTCTTTTCGTGGGCAGTTTTCGGATCGGGTTGCTTCGCTGACC 
               
               
                   
               
               
                 AGTTTTCTTCTTCAACTGAGAATAATGAGTCTGCATAATGAGCGCATTGC 
               
               
                   
               
               
                 AGATATTGCACTACATGAAAAGGAAGAAAAGAAACCGGAAATTGAAATCG 
               
               
                   
               
               
                 TTGCTGACATGAGCCCGGTTTCACTGGAAACCACTGATTTAAGCTACCGG 
               
               
                   
               
               
                 TATGACAGCCAGTCAGCACAGGTATTCAGTGGTCTGAATTTGTCTGTGGC 
               
               
                   
               
               
                 TCCGGGAGAAAGTGTGGCTATAACTGGTGCCTCCGGTGCCGGAAAAACCA 
               
               
                   
               
               
                 CATTAATGAAAGTATTATGTGGACTGTTTGAACCAGATAGTGGAAAAGTA 
               
               
                   
               
               
                 CTGGTTAATGGCACGGATATACGTCAACTTGGAATAAATAATTATCACCG 
               
               
                   
               
               
                 TATGATAGCCTGTGTTATGCAGGACGACCGGCTATTTTCAGGATCAATTC 
               
               
                   
               
               
                 GTGAAAATATCTGTGGGTTTGCAGAAGAAACAGACGACGAATGGATGACA 
               
               
                   
               
               
                 GAATGTGCCAGAGCAAGTCATATTCATGATGTGATAATGAAAATGCCAAT 
               
               
                   
               
               
                 GGGGTATGAAACGTTAATAGGTGAACTGGGGGAAGGTCTTTCCGGCGGTC 
               
               
                   
               
               
                 AAAAACAGCGTATATTCATTGCCCGAGCTTTATACCGGAAACCTGGAATA 
               
               
                   
               
               
                 TTATTTATGGATGAGGCTACAAGTTCTCTTGATACAGAAAGTGAACGTTT 
               
               
                   
               
               
                 CGTGAATGCTGCCATAAAAAAAATGAATATCACCCGGGTGATTATTGCAC 
               
               
                   
               
               
                 ACAGAGAAACTACGTTGAGAACTGTTGACAGGATTATTTCTATTTAA. 
               
            
           
         
       
     
     The mchI gene encodes for the MccH47 immunity protein. 
     
       
         
           
               
            
               
                 mchI gene sequence: 
               
               
                 (SEQ ID NO: 7) 
               
               
                 ATGAGTTATAAAAAACTGTACCAATTGACGGCTATATTTAGTTTACCTCT 
               
               
                   
               
               
                 TACTATCTTATTGGTTTCACTTTCATCCCTTCGGATTGTTGGCGAAGGGA 
               
               
                   
               
               
                 ATTCTTATGTTGACGTTTTTCTAAGCTTTATAATATTTCTTGGTTTTATT 
               
               
                   
               
               
                 GAGCTGATTCATGGGATTCGAAAGATTTTGGTCTGGTCAGGCTGGAAAAA 
               
               
                   
               
               
                 CGGAAGTTAA. 
               
               
                   
               
               
                 mchX gene sequence: 
               
               
                 (SEQ ID NO: 8) 
               
               
                 ATGGAATTTGCTACAAACAGGGTTACTGTAAATGACAGTCGGTCAGCACT 
               
               
                   
               
               
                 GTCATCAACTTTGCTGTTGTCTTTGATCATGAGCGCCACTCTACTGGAAT 
               
               
                   
               
               
                 ATTCTTTATCGATGACCTGA. 
               
               
                   
               
               
                 mchS1 gene sequence: 
               
               
                 (SEQ ID NO: 9) 
               
               
                 ATGAAAAACTATCTTTTCCAGACTCCCGAAGATATTTGTGTACAGTTAAA 
               
               
                   
               
               
                 AAAAATGACACATCCTGTCACAATAAGAACAACAGATATTGCTAATTTCT 
               
               
                   
               
               
                 GGCACTATCTTGAGTCAGCAACTCTTCCGGTGATCACAAAAAGCACCACT 
               
               
                   
               
               
                 ACAGAAAATCGGGAGGTTACATTTCTGTGGCGCTCAGAGAAAGCAGTGCA 
               
               
                   
               
               
                 AGGCGTATATCTTCGCCTGAATCGTGTTACAGATAAAAAAGATGTCAAAA 
               
               
                   
               
               
                 AAGGACTAATGACTCATATCCCTTCGACAGATATCTGGATGCTGACACTG 
               
               
                   
               
               
                 GTGTTACCAGCTTCATATCGGGGCTCATACTCATTTATAGAAATTCCCAC 
               
               
                   
               
               
                 AGATATGACACAAAAAGACATATTTCAACTAGGAAGTCGCTTCTCTCCAT 
               
               
                   
               
               
                 TACCCGGTAAATCTGATCCATTTAACAAAACAGCAGAAATAAATATACGA 
               
               
                   
               
               
                 GGATTCGGAGAATCAGTCCTTTCTCTTGATATGGCTCCTGAACAAAAGGA 
               
               
                   
               
               
                 ATGGGATGATACTTCCCATAAATGTACAGGTATTCTTTCAACATTACATT 
               
               
                   
               
               
                 CCTTTGTTGCAGGATATCAACGCCGGATTCGTTTATATTTTCCCCAGAAT 
               
               
                   
               
               
                 CCAACATCAGTACCTCTTGGATTACTTGTGTTACCTGATGCTGAAATATG 
               
               
                   
               
               
                 GTTTGACCGGATGGATATTACCCGGGCATTAGATATGGCCATTACCACTG 
               
               
                   
               
               
                 GTCATATTGCGCCAATGGCAATTATGGGGATAGACAATATTAATGAATCT 
               
               
                   
               
               
                 GATCGTATGAATATACTGGGAGGCAATAAAGAACTTATCTTTGATATAGC 
               
               
                   
               
               
                 GGAAAATCTGATACCCCAGTTATACAGAGACTACCCGAATATCGTATGGG 
               
               
                   
               
               
                 CTGGTCGTTCTAATACTATACTGGCCGGTCAGAGCCTCGGTGGAGTGACA 
               
               
                   
               
               
                 GCACTGATGGCAGCTATATATGCGTCGACAACATTTGGTACAATCATTAG 
               
               
                   
               
               
                 CCACTCACCTTCAATGTGGTGGAACCCTGACCAGGGCAGCCCGATTTTGT 
               
               
                   
               
               
                 TTACTGAGAATGATATCTCCTGGGTAAGTGAGCAGATACTTTCAGCGCCT 
               
               
                   
               
               
                 CCGAAAGATGTAAATATCCAACTTGGAGTCGGTTCTTTAGAAGGTACAAC 
               
               
                   
               
               
                 CGTCTCACATGTTCAGCGGTTGCATCAGTCGTTAATCGCAGCAGGTTTGG 
               
               
                   
               
               
                 AAAGTAACCTCACTGTCTATGCCGGTGGTCATGATTATGCCTGGTGGCGC 
               
               
                   
               
               
                 GGAGCAATTATTGATGCATTAGCAAATTATAATTGCAGGAAGATATCAGA 
               
               
                   
               
               
                 TAATAACTTTGTGTAA. 
               
               
                   
               
               
                 mchS4 gene sequence: 
               
               
                 (SEQ ID NO: 10) 
               
               
                 ATGAATTGTGATAATAATCACAGAAATGAAGAATTCATTGTTACCTTTGA 
               
               
                   
               
               
                 TAAAGGCAACAAGCAAGACAATTCAAGACGAAAACACGATAATTTTCCTA 
               
               
                   
               
               
                 TAGAGGTAGAATCCTCCGTAGAGCTGGAGACACACTGTATCACAAATAAT 
               
               
                   
               
               
                 AAGTCGGCTTCCGGTATAGTAACACATGACTATGATGCCGATTATATTTG 
               
               
                   
               
               
                 TGGTTGTGGTGAAATTATGTGTCCTGGTTGCGGTCATGACCTATAA. 
               
            
           
         
       
     
     The mciA (formerly known as mchS2) gene encodes the pre-microcin I47 peptide. Once the peptide product of the mciA gene has gone through modification and secretion steps, the pre-microcin I47 peptide becomes microcin I47. 
     
       
         
           
               
            
               
                 (SEQ ID NO: 11) 
               
               
                 ATGAGAGAAATATCAGATAACATGCTTGATTCCGTGAAAGGAGGGAT 
               
               
                   
               
               
                 GAATCTTAATGGATTACCTGCTTCTACTAATGTAATAGATCTACGTGGA 
               
               
                   
               
               
                 AAAGATATGGGAACATATATTGATGCTAATGGAGCATGCTGGGCTCCGGA 
               
               
                   
               
               
                 TACTCCATCCATCATCATGTATCCGGGGGGAAGTGGACCTTCTTATAGTA 
               
               
                   
               
               
                 TGAGTAGTTCCACATCCAGTGCAAACAGCGGCAGTTAA 
               
            
           
         
       
     
     The mciI (formerly known as mchS3) gene encodes for the MccI47 immunity protein. 
     
       
         
           
               
            
               
                 mciI gene sequence: 
               
               
                 (SEQ ID NO: 12) 
               
               
                 ATGTATCTTACGAAAAAGATTATAATAAGTATGATGTTTATATTACCATC 
               
               
                   
               
               
                 TGCTGCATTTTCATCAGATCCACCTCCCCTTCAACAATCGTTAGAAAAAA 
               
               
                   
               
               
                 CAACCTATTTTTCTATAGGTATGAATGGGTTTATAGGCTATCAGAGCGAA 
               
               
                   
               
               
                 GGGGAAAAATTATACACACACATTCTTACATTAGATAATCCCGAAGAGAT 
               
               
                   
               
               
                 ATTTAAAAATATAATAAAAAATAGAAAGTCAACTAAGGAGTCTAAAATTT 
               
               
                   
               
               
                 ATGCTGCTTGTGGGCTATATTATTTAAACGTAGAAAATATAGAGTCATTG 
               
               
                   
               
               
                 TTTAATGAAAATGATAAACAAGAATATGTGTCTGTCTTAAGAGGGGATAT 
               
               
                   
               
               
                 TTTAACAAAAATAAAACTGAATGATATTCTGAATTCTGTGATAATAAATG 
               
               
                   
               
               
                 GTTGCAACACCAAATTAATATCTGAACATAAATGA. 
               
            
           
         
       
     
     Vectors 
     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., pBBAD, pS4BAD, pJBAD, pBR322, pLJV3, and pEX2000). 
     The vector can include genes for various microcins, e.g., Class I microcins, Class IIa microcins, Class IIb microcins, and/or Class IIc microcins. In some embodiments, the vector can include a set of genes for a Class IIa microcin (e.g., MccH47, MccE492, MccM, MccG492, and MccI47). In some embodiments, the vector can include a set of genes for MccI47. 
     In some embodiments, the vector includes a set of genes for MccI47. These genes are required to express a functional MccI47 that can inhibit the growth of other bacteria. In some embodiments, the set of genes includes one, two, three, four, five, six, seven, or eight genes that are selected from the group consisting of mchA, mchB, mchC, mchD, mchE, mchF, mchX, mchI, mchS1, mchS4, mciI and mciA. In some embodiments, the set of genes includes mchA, mchC, and mchD. In some embodiments, the set of genes includes mchA, mchC, mchD, mchE, and mchF (pBBAD). In some embodiments, the set of genes includes mchA, mchC, mchD, mchE, mchF, and mchS4 (pS4BAD). In some embodiments, the set of genes includes mchA, mchC, mchD, mchE, mchF, mchS1, and mchS4 (pJBAD). 
     In some embodiments, the set of genes includes mchA, mchC, mchD, mchE, mchF, mciI, and mciA (pBBAD-I47). In some embodiments, the set of genes includes mchA, mchC, mchD, mchE, mchF, mchS4, mciI, and mciA (pS4BAD-I47). In some embodiments, the set of genes includes mchA, mchC, mchD, mchE, mchF, mchS1, mchS4, mciI, and mciA (pJBAD-I47). In some embodiments, the minimum genes required for the production of active, mature microcin I47 are mciIA and mchACD. In some embodiments, the set of genes includes mchA, mchC, mchD, mciI and mciA. 
     In some embodiments, the vector for the production of active, mature microcin I47 is constructed by combining a plasmid expressing mciI and mciA, a plasmid expressing mchC, mchD, mchE, and mchF, and a plasmid expressing mchA. In some embodiments, the vector for the production of active, mature microcin I47 is constructed by combining a plasmid expressing mciI and mciA, a plasmid expressing mchC, mchD, mchE, and mchF, and a plasmid expressing mchA, and mchS4. In some embodiments, the vector for the production of active, mature microcin I47 is constructed by combining a plasmid expressing mciI and mciA, a plasmid expressing mchC, mchD, mchE, and mchF, and a plasmid expressing mchA, mchS1 and mchS4. In some embodiments, the plasmids are combined into a single backbone vector, e.g., pUC19. 
     In some embodiments, the vector includes a set of genes for MccH47. These genes are required to express a functional MccH47 that can inhibit the growth of other bacteria. In some embodiments, the set of genes includes mchA, mchB, mchC, mchD, mchI, ad mchX. In some embodiments, the set of genes includes mchA, mchB, mchC, mchD, mchE, mchF, mchI, and mchX (pBBAD-H47). In some embodiments, the set of genes includes mchA, mchB, mchC, mchD, mchE, mchF, mchI, mchS4, and mchX (pS4BAD-H47). In some embodiments, the set of genes includes mchA, mchB, mchC, mchD, mchE, mchF, mchI, mchS1, mchS4, and mchX (pJBAD-H47). In some embodiments, the minimum genes required for the production of active, mature microcin H47 are mchXIB and mchACD. In some embodiments, the set of genes includes mchA, mchB, mchC, mchD, mchI, ad mchX. 
     In some embodiments, one or more genes in a set of genes are located within one operon. In some embodiments, the set of genes are located within more than one operons. Thus, in some embodiments, the operon includes one, two, three, four, five, six, seven, eight, or nine, or ten genes. 
     In some embodiments, the vector for the production of active, mature microcin H47 is constructed by combining a plasmid expressing mchX, mchI and mchB, a plasmid expressing mchC, mchD, mchE, and mchF, and a plasmid expressing mchA. In some embodiments, the vector for the production of active, mature microcin H47 is constructed by combining a plasmid expressing mchX, mchI and mchB, a plasmid expressing mchC, mchD, mchE, and mchF, and a plasmid expressing mchA, and mchS4. In some embodiments, the vector for the production of active, mature microcin H47 is constructed by combining a plasmid expressing mchX, mchI and mchB, a plasmid expressing mchC, mchD, mchE, and mchF, and a plasmid expressing mchA, mchS1 and mchS4. In some embodiments, the plasmids are combined into a single backbone vector, e.g., pUC19. 
     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 β-D-1-thiogalactopyranoside (IPTG), allolactose, etc. In some embodiments, the controllable promoter is rhaPBAD or Pttr. 
     High levels of microcins may be harmful to a subject, thus, according to the present disclosure, mechanisms can be introduced to the genetically engineered microorganisms 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 I1 and I2 operators. The AraC-arabinose dimer at this site contributes to activation of the pBAD promoter. Additionally, cyclic AMP receptor protein (CAP) binds to two CAP binding sites upstream of the I1 and I2 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 O2 and I1 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 R. AraC protein, regulation of the L-arabinose operon in  Escherichia coli,  and the light switch mechanism of AraC action. FEMS Microbiol. Rev., (2010) 1-18, which is incorporated by reference in its entirety. 
     
       
         
           
               
            
               
                 pBAD promoter sequence: 
               
               
                 (SEQ ID NO: 13) 
               
               
                 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. 
     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 mchI. In some embodiments, this operon is under the control of Pttr. 
     In some embodiments, the tetrathionate promoter (Pttr) is located immediately upstream of the mchXIB genes (mchX, mchI, 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). 
     
       
         
           
               
            
               
                 Pttr promoter sequence: 
               
               
                 (SEQ ID NO: 14) 
               
               
                 CCCAATATCCCTGTCAATTATGTTGTTTTAGATCAACAACAAGCCGGGTATG 
               
               
                   
               
               
                 TGGTTAACCACAATAGAGCGCACCCCGCCTCGATTTTTACACTGTAAATCAT 
               
               
                   
               
               
                 CGACATTTTTTATTCATTACACATGAACCAACATCGTGACAAATGTTTCATT 
               
               
                   
               
               
                 GTTGGCA. 
               
               
                   
               
               
                 J23119 promoter sequence: 
               
               
                 (SEQ ID NO: 15) 
               
               
                 TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAG. 
               
            
           
         
       
     
     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 mchACDEF, mciI, mciA, and ttr RSBCA, capable of producing MccI47 in response to environmental tetrathionate, resulting in the ability to inhibit and out-compete  Salmonella.  In another 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 ability to inhibit and out-compete  Salmonella.    
     Genetically Engineered Microorganisms 
     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. coli  (e.g.,  E. coli  Nissle 1917 or  E. coli  NGF-19). One useful  E. coli  strain is Nissle 1917 (EcN).  E. coli  Nissle 1917 is a Gram-negative species, which 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. 
     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.    
     A vector or a set of genes as described herein can be introduced into a microorganism, e.g., a bacterium, such as,  E. coli,  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. 
     In some embodiments, the vector or the set of genes is integrated into the bacterial or other microbial genome. 
     Examples of bacterial species that can be used as engineered microorganisms include, but are not limited to:  Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumonia, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella typhi, Serratia marcescens, Shigella flexneri,  and  Staphylococcus aureus.    
     Methods of Treating Bacterial Infection 
     MccI47 has been shown to be active to inhibit various bacteria, e.g., gram-negative bacteria. As used herein, the term “gram-negative bacterium” refers to a bacterium that do not retain the crystal 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. coli ),  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 cause bacteremia, secondary meningitis, and ventilator-associated pneumonia in hospital intensive-care units. 
     In some embodiments, the composition and the methods as described herein can be used to treat gram-negative bacterial infection. 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. coli, Salmonella, Shigella  and  Yersinia ) infection. 
     The methods described in the present disclosure are effective for treating bacterial infection 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. 
     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&#39;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 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. 
     In some embodiments, the genetically engineered microorganisms can be administered to a subject with some other known treatments for bacterial infection. For example, the genetically engineered microorganisms 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. 
     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. 
     Methods of Treating Dysbiosis 
     The compositions and the methods as described herein can be used to treat and/or reduce the risk of dysbiosis and its associated diseases. 
     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. 
     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. 
     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&#39;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. 
     In some embodiments, the genetically engineered microorganisms can be administered to a subject with some other known treatments for dysbiosis. 
     Methods of Administration 
     The therapeutic methods disclosed herein (including prophylactic treatments) generally include administration of a therapeutically effective amount of a composition comprising the 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. 
     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. 
     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. 
     Compositions comprising the genetically engineered microorganisms can be administered to a subject through many different routes, e.g., by endoscopy, by enteroscopy, 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. 
     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. 
     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, corn syrup (including high fructose corn syrup), sugar alcohols, maltodextrins, high maltose corn syrup, starch, glycerin, brown sugar and mixtures thereof. 
     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. 
     Dosage 
     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, every five days, every six days, or once per week. A person skilled in the art can refine the dosage scheduling as needed. 
     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. 
     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. 
     Kits 
     The present disclosure also provides kits of the genetically engineered microorganisms. In some embodiments, the kit includes a sterile container which contains a therapeutic or prophylactic composition having the 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. 
     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. 
     EXAMPLES 
     The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. 
     Example 1: Construction and Analysis of Plasmid-Based Systems for the Production of Microcin H47 or Microcin I47 
     As outlined in the introduction, the mch gene cluster of  E. coli  strain H47 contains twelve genes: mchABCDEFIXS1S4 and mciAI. Known or proposed functions were provided for all twelve. Notably, other  E. coli  strains containing parts, or all, of the mch cluster also occasionally contain three additional genes, mcmAIM. Strains of  E. coli  housing all twelve mch cluster genes, in addition to the mcmAIM genes include strains  E. coli  strain CA46 and  E. coli  strain CA58. Interestingly,  E. coli  strain Nissle (EcN) and a uropathogenic strain  E. coli  CFT073 lack mchS1S2S3, but have mcmAIM, while  E. coli  H47 has truncated versions of mcmAIM. As mentioned, mciAI were previously referred to as mchS2S3 and have been renamed, comprising the gene pair responsible for microcin I47 (MccI47) production and immunity. mcmAI are the corresponding gene pair encoding the peptide and immunity protein for microcin M (MccM), the third and final known member of the  E. coli -derived Class IIb microcins. An overview of the known mch gene clusters is represented in  FIG. 1 . 
     The focus of the current study is two-fold. First, MccI47 inhibitory action against select Enterobacteriaceae representatives was determined. Second, inhibitory activity from three distinct vector backbones were compared to best determine candidate mch genes to design vectors for eradication of enteric targets during an in vivo challenge study, specifically focused on the relevancy of mchS1 and mchS4. 
     The roles of MchS4 and MchS1 are known to be optional in the originally characterized host strain  E. coli  H47, but their functions could possibly affect inhibitory activity in terms of both potency and spectrum. First, MchS4 has no known homologs, with the Basic Local Alignment Search Tool (BLAST) returning results of &gt;50% amino acid identity from other hypothetical proteins (seven, in total) exclusively from  E. coli.  However, in  E. coli  strain K-12, the presence of mchS4 led to elevated iron-chelating activity, allowing for enterobactin production even in iron-rich environments, while  E. coli  K-12 strains without mchS4 lack this capability 22 . This result demonstrates that MchS4 likely plays some role in enterobactin biosynthetic pathway regulation, as the two main pathways leading to enterobactin biosynthesis in  E. coli  are multi-enzymatic (shikimate and non-ribosomal peptide synthesis (NRPS)). Maturation of class IIb microcins is dependent upon linkage to catecholate siderophores, and therefore it is reasonable to presume that inclusion of mchS4 could lead to elevated levels of inhibitory activity by MccH47 and MccI47. 
     Unlike MchS4, MchS1 has numerous known homologs, all belonging to the Fes superfamily of enterochelin esterases (e.g. IroD, Fes), present in  E. coli  as well as other known siderophore producers (e.g.  Klebsiella, Salmonella, Serratia ). While enzymatic kinetics of MchS1 have not been specifically elucidated, Fes homologs have been purified to homogeneity and demonstrated to hydrolyze both enterobactin and the enterobactin ferric complex in vitro, though the former was more effectively hydrolyzed. Fes/MchS1 activity is represented in  FIGS. 3A-3H . Interestingly, comparative studies of Fes, IroD, and IroE demonstrated that both IroD and IroE hydrolyze ferric enterobactin more effectively than their non-ferric counterparts, potentially justifying the presence of both Fes and IroD/IroE. It is unknown whether MchS1 hydrolyzes the ferric or non-ferric enterobactin more efficiently, though it has been demonstrated that expression of MchS1 in  E. coli  K-12 led to reduced iron chelating activity, implying degradation of non-ferric enterobactin prior to secretion taking place. Previous studies have purified MccM and MccE492 containing the mixture of a structural microcin peptide linked to DHBS 3 , DHBS 2 , and DHBS 1  and tested their inhibitory activity, but individual compounds of the mixture have not been tested and directly compared, to date. This collectively leads to the hypothesis that the presence of MchS1 may lead to altered inhibitory effect against variable targets, due to an alteration of DHBS 3 :DHBS2:DHBS 1 . 
     Siderophore scavenging Enterobacteriaceae often contain multiple siderophore receptors, which have been shown to have variable binding and transport efficiencies for different siderophores, and knockout mutants have variable susceptibility to different class IIb microcins. This phenomenon of apparent redundancy increases fitness in  P. aeruginosa,  allowing strains to switch between highly efficient, but metabolically costly siderophores and less efficient, less metabolically expensive siderophores. Considering this, altering investment of catecholate siderophores in a microcin producing strain towards DHBS 3  or DHBS 1  may have variable effect against any targets that tend to preferentially uptake more costly or less costly siderophores. Careful separation of compounds using advanced chromatography techniques may be the best method for testing this hypothesis, however, as a simple test of principle for this study, it will be assumed that strains with MchS1 ( FIG. 2 ) likely produce a MccH47/MccI47 mixture more strongly skewed towards MchB-DHBS 1 /MciA-DHBS 1 , as compared with strains that lack MchS1 ( FIG. 2 ). 
     Materials and Methods 
     The following materials and methods were used in the following examples. 
     Microbial Strains, Media, and Growth Conditions 
     Strains used in this study include  Escherichia coli  strain NEB10β (New England Biolabs. Ipswich, Mass.) and the strains listed utilized in  FIGS. 3A-3H , all of which were purchased from ATCC, except for  E. coli  DH5α (New England Biolabs. Ipswich, Mass.). Plasmid constructs developed in this work were transformed by electroporation into  E. coli  NEB10β cells. All media and additional reagents listed in this study were purchased from Sigma Aldrich, St. Louis, Mo., unless otherwise indicated. 
     Vectors pBBAD-H47, pS4BAD-H47, pJBAD-H47, pBBAD-I47, pS4BAD-I47, pJBAD-I47 were constructed using standard methods for Gibson Assembly 47 , and the Gibson Assembly Master Mix (New England Biolabs. Ipswich, Mass.). Construction of the six vectors of this study were assembled in the same manner, utilizing the source vectors, pUC19 (New England Biolabs. Ipswich, Mass.), pTARA, pEX2000 (mchS23) 20 , and pPP2000 (mchAXIBCDEF). 
     Solid Media Inhibition Assays 
     Inhibition assays in solid media were designed and carried out based on the methods as described in Delgado et al. (2005) YojI of  Escherichia coli  functions as a microcin J25 efflux pump.  J. Bacteriol.  187, 3465-3470, which is incorporated by reference in its entirety. Briefly, select bacterial strains were grown overnight on LB agar plates, individual colonies were selected and used to inoculate 3 mL of LB broth, and after overnight growth 1 μL of liquid culture was then used to create an agar stab in solid media and incubated at 37° C., either aerobically or anaerobically, for 24 hours. Post incubation, cells were inactivated with chloroform and UV. Molten 3% agar was then added to an overnight culture of susceptible cells to a final concentration of 0.75%, and then 3 mL of the mixture was overlaid on top of the inactivated agar stab plates and allowed to solidify. After incubation of plates in aerobic conditions overnight at 37° C. ImageJ software was utilized to quantify the area corresponding to the inhibition halo. 
     Purification 
     Briefly, cultures of  E. coli  NEB10β pHMT-I47 were grown in under antibiotic selection (ampicillin and chloramphenicol), and in iron-limiting conditions via the addition of 0.2 mM 2′2-dipyridyl, and induced with IPTG. Cultures were grown for an additional 5-7 hours post-induction, then pelleted and frozen overnight at −20° C. Cultures were then thawed in cold water, sonicated, and the crude lysate was passed through an amylose resin (New England Biolabs, Ipswich, Mass) column to capture the MBP fusion proteins, then finally eluted with maltose. 
     Elution was performed by adding the elution buffer (200 mM NaCl, 20 mM Tris-HCl, 10 mM maltose; pH 7.5). The eluent was concentrated using MilliporeSigma (Burlington, Mass.) MWCO 10,000 filters. 
     The concentrated MBP-MccI47 was digested by Tobacco etch virus nuclear-inclusion-a endopeptidase (TEV) (New England Biolabs, Ipswich, Mass.), yielding a buffered solution of MccI47, TEV, and MBP. This solution was then further purified by subsequent rounds of resuspension with Ni-NTA agarose resin (Qiagen, Hilden, Del.). Ni-NTA slurry was pelleted by centrifugation and the supernatant was carefully removed by pipetting. 
     Results 
     One objective of this research is to generate vectors and strains capable of producing microcins to eradicate multi-drug resistant (MDR) Enterobacteriaceae. The most obvious mode of delivery would be to orally provide the antibiotic peptide in a purified form. While this benefits from being a conventional standard and potentially more accepted by an end consumer, it is perhaps more plausible that the microcin will be more effectively delivered via a live engineered probiotic strain, directly to the site of need (gastrointestinal tract). However, before progressing towards transformation of these strains with a particular plasmid capable of microcin production, the best subset of genes of the mch cluster in order to most effectively inhibit select targets should be determined. This was performed by testing their activity in the common lab strain,  E. coli  strain NEB10β. 
     A series of twelve vectors were designed and constructed using a combination of PCR and Gibson Assembly, and were built as three different backbone sets, producing each of the three microcins in isolation, as well as a fourth set that expressed all three microcins from a single operon. The three different plasmid backbones were termed pBBAD, pS4BAD, and pJBAD, where pBBAD vectors contain the essential and recommended genes for microcin production: mchACDEF pS4BAD vectors are pBBAD with the addition of mchS4, and pJBAD vectors are pBBAD with the addition of mchS4 and mchS1. However, while all twelve vectors were constructed and tested, only the data pertaining the MccH47 and MccI47 are within the scope of this report, and therefore any data regarding MccM and MccIMHo is summarized in  FIGS. 4A-4D . A schematic demonstrating the modular vector design of the six L-arabinose inducible MccH47/MccI47 production vectors can be seen in  FIG. 2 . 
     The P BAD  promoter chosen and utilized for expression of the genes that code for the structural peptide and immunity protein is currently in use in clinical trials with the engineered EcN to treat phenylketonuria. The EcN can also include an fnr operator region to prevent heterologous expression in aerobic environments, providing an additional safety measure. 
     Strains of  E. coli  NEB10β were transformed with the six vectors discussed above and demonstrated in  FIG. 3.2 , and a seventh was transformed with pUC19 to serve as a negative control. Numbers were assigned to the strains based on the vector harbored: 1.) pBBAD-H47, 2.) pS4BAD-H47, 3.) pJBAD-H47, 4.) pBBAD-I47, 5.) pS4BAD-I47, 6.) pJBAD-I47, and 7.) pUC19. Static inhibition assays were performed by stabbing each strain individually into LB agar supplemented with 0.2 mM 2,2′-dipyridyl, 0.4% L-arabinose, and 100 μg/mL ampicillin, following methods described previously, and allowed to incubate overnight prior to inactivation by chloroform and UV. In this manner, the inhibitory activity of all six vectors can be evaluated against an array of target strains, where each target strain requires a single Petri dish, as can be seen in  FIGS. 3A-3H . Because plates were supplemented with 100 μg/mL ampicillin to select for retention of microcin production vectors, strains lacking known mechanisms for ampicillin resistance were transformed with pUC19 ( E. coli  DH5α,  S. typhimurium  29630, and  K. oxytoca  700324). All other strains utilized in  FIGS. 3A-3H  are MDR organisms with resistance to beta-lactam antibiotics, although it is immediately apparent that ESBL- E. coli  BAA-196 ( FIG. 3B ) was not resistant to ampicillin at the concentration these assays were performed, yet the interesting dynamics of this experiment justifies its retention as a part of  FIGS. 3A-3H  and its discussion in detail, below. Strain seven behaved as expected for a negative control and did not exhibit antimicrobial activity in all cases. 
     For all assays of  FIGS. 3A-3H , strains harboring pBBAD-I47 (shown as strain 4 in the figures) appear to have greater activity against susceptible targets (A, B, C, G, H) than strains harboring either pS4BAD-I47 (shown as strain 5 in the figures) or pJBAD-I47 (shown as strain 6 in the figures). This contrasts MccH47 producing strains, where activity levels are most commonly strongest in pS4BAD-H47 (shown as strain 2 in the figures), followed by pBBAD-H47 (shown as strain 1 in the figures), and pJBAD-H47 with visible activity only against  E. coli  DH5αpUC19 (A),  E. coli  BAA-196 (B), and  S. typhimurium  26930 (C). 
     Additionally, purifications of MccI47 were obtained and tested against an array of bacterial targets. The minimum inhibitory concentrations (MICs) for MccI47 are shown in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Minimum Inhibitory Concentrations (MICs) of bacterial strains producing MccI47 
               
            
           
           
               
               
               
               
            
               
                 Bacterial species 
                 Strain 
                 Origin 
                 MIC (μg/ml) 
               
               
                   
               
               
                   Acinetobacter baumannii * 
                 BAA-1790 
                 Human clinical isolate (sputum) 
                 ND (&gt;138.75) 
               
               
                   Enterobacter cloacae * 
                 BAA-2341 
                 Human clinical isolate 
                 137.38 
               
               
                 
                   Escherichia coli 
                 
                 25922 
                 Human clinical isolate 
                 1.58 
               
               
                   Escherichia coli * 
                 BAA-196 
                 Human clinical isolate 
                 4.43 
               
               
                 
                   Escherichia coli 
                 
                 DH5α 
                 Laboratory 
                 2.26 
               
               
                   Klebsiella oxytoca * 
                 51983 
                 Human clinical isolate (blood) 
                 ND (&gt;197.25) 
               
               
                 
                   Klebsiella oxytoca 
                 
                 700324 
                 Human isolate (bioMérieux, Inc) 
                 ND (&gt;197.25) 
               
               
                   Klebsiella pneumoniae * 
                 BAA-1705 
                 Human clinical isolate (urine) 
                 36.13 
               
               
                   Klebsiella pneumoniae * 
                 BAA-2146 
                 Human clinical isolate 
                 29.44 
               
               
                   Klebsiella pneumoniae * 
                 BAA-2342 
                 Human clinical isolate 
                 16.39 
               
               
                   Klebsiella pneumoniae * 
                 BAA-2524 
                 Human clinical isolate 
                 14.72 
               
               
                 
                   Proteus mirabilis 
                 
                 29906 
                 Human clinical isolate (urogenital) 
                 ND (&gt;197.25) 
               
               
                 
                   Pseudomonas aeruginosa 
                 
                 PA14 
                 Human clinical isolate 
                 ND (&gt;138.75) 
               
               
                 
                   Salmonella Typhimurium 
                 
                 19585 
                 Derived from LT2 (natural source) 
                 7.96 
               
               
                 
                   Salmonella Typhimurium 
                 
                 29630 
                 Derived from LT2 (natural source) 
                 6.31 
               
               
                   Salmonella Typhimurium * 
                 BAA-190 
                 Human clinical isolate 
                 12.63 
               
               
                 
                   Salmonella Typhi 
                 
                 700931 
                 Derived from TY2 (Human isolate) 
                 8.67 
               
               
                 
                   Serratia marcescens 
                 
                 DB11 
                 Derived from DB10 ( Drosophila ) 
                 107.00 
               
               
                 
                   Shigella flexneri 
                 
                 2457T 
                 Human clinical isolate 
                 0.42 
               
               
                 
                   Shigella flexneri 
                 
                 M90T 
                 Human clinical isolate 
                 0.42 
               
               
                 
                   Staphylococcus aureus 
                 
                 27661 
                 Human isolate 
                 ND (&gt;197.25) 
               
               
                   
               
               
                 *multidrug-resistant isolate; 
               
               
                 ND = not determined, as MIC is higher than denoted concentration 
               
            
           
         
       
     
     Reported values are the median of at least three individual assays with each strain listed in Table 1. All tested strains of  K. pneumoniae, Salmonella, Shigella,  and  E. coli,  including many multi-drug resistant clinical isolates, were strongly inhibited by MccI47. One strain each of  Serratia marcescens  and  Enterobacter cloacae  were also inhibited. No inhibitory activity against  Acinetobacter baumannii, K. oxytoca, Proteus mirabilis, Pseudomonas aeruginosa,  or  Staphylococcus aureus  was detected with the used concentrations. This data represents the first demonstration of the inhibitory capability of purified MccI47. 
     Notably, MccI47 has potent activity against both MDR  K. pneumoniae  targets. In addition, it is likely that enterobactins function antagonistically and compete for receptor sites on target organisms. It may also be concluded that Class IIb microcins do not act additively, but rather compete for entry sites on target organisms, and therefore act antagonistically when supplied in conjunction. 
     Other Embodiments 
     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.