Identification and Properties of the Genes Encoding Microcin E492 and Its Immunity Protein

The gene coding for the immunity protein (mceB) and the structural gene of microcin E492 (mceA), a low-molecular-weight channel-forming bacteriocin produced by a strain of Klebsiella pneumoniae, have been characterized. The microcin gene codes for a precursor protein of either 99 or 103 amino acids. Protein sequencing of the N-terminal region of microcin E492 unequivocally identified this gene as the microcin structural gene and indicated that this microcin is synthesized as a precursor protein that is cleaved at either amino acid 15 or 19, at a site resembling the double-glycine motif. The gene encoding the 95-amino-acid immunity protein (mceB) was identified by cloning the DNA segment that encodes only this polypeptide into an expression vector and demonstrating the acquisition of immunity to microcin E492. As expected, the immunity protein was found to be associated with the inner membrane. Analysis of the DNA sequence indicates that these genes belong to the same family as microcin 24, and they do not share structural motifs with any other known channel-forming bacteriocin. The organization of the microcin- and immunity protein-encoding genes suggests that they are coordinately expressed.     

Antibiotic resistance in Salmonella enterica serovar Typhimurium exposed to microcin-producing Escherichia coli

Microcin 24 is an antimicrobial peptide secreted by uropathogenic Escherichia coli. Secretion of microcin 24 provides an antibacterial defense mechanism for E. coli. In a plasmid-based system using transformed Salmonella enterica, we found that resistance to microcin 24 could be seen in concert with a multiple-antibiotic resistance phenotype. This multidrug-resistant phenotype appeared when Salmonella was exposed to an E. coli strain expressing microcin 24. Therefore, it appears that multidrug-resistant Salmonella can arise as a result of an insult from other pathogenic bacteria.     

Antibiotic Resistance in Salmonella enterica Serovar Typhimurium Exposed to Microcin-Producing Escherichia coli

Microcin 24 is an antimicrobial peptide secreted by uropathogenic Escherichia coli. Secretion of microcin 24 provides an antibacterial defense mechanism for E. coli. In a plasmid-based system using transformed Salmonella enterica, we found that resistance to microcin 24 could be seen in concert with a multiple-antibiotic resistance phenotype. This multidrug-resistant phenotype appeared when Salmonella was exposed to an E. coli strain expressing microcin 24. Therefore, it appears that multidrug-resistant Salmonella can arise as a result of an insult from other pathogenic bacteria.     

Structure, organization and characterization of the gene cluster involved in the production of microcin E492, a channel-forming bacteriocin

Microcin E492 is a low-molecular-weight, channel-forming bacteriocin produced and excreted by Klebsiella pneumoniae RYC492. A 13 kb chromosomal DNA fragment from K. pneumoniae RYC492 was sequenced, and it was demonstrated by random Tn5 mutagenesis that most of this segment, which has at least 10 cistrons, is needed for the production of active microcin and its immunity protein. Genes mceG and mceH correspond to an ABC exporter and its accessory protein, respectively, and they are closely related to the colicin V ABC export system. The microcin E492 system also requires the product of gene mceF as an additional factor for export. Despite the fact that this bacteriocin lacks post-translational modifications, genes mceC, mceI and mceJ are needed for the production of active microcin. Genes mceC and mceI are homologous to a glycosyl transferase and acyltransferase, respectively, whereas mceJ has no known homologue. Mutants in these three genes secrete an inactive form of microcin, able to form ion channels in a phospholipidic bilayer, indicating that the mutation of these microcin genes does not alter the process of membrane insertion. On the other hand, microcin isolated from mutants in genes mceC and mceJ has a lethal effect when incubated with spheroplasts of sensitive cells, indicating that the microcin defects in these mutants are likely to alter receptor recognition at the outer membrane. A model for synthesis and export is proposed as well as a novel maturation pathway that would involve conformational changes to explain the production of active microcin E492.     

Cloning and mapping of the genetic determinants for microcin C51 production and immunity

Microcin C51 is a small peptide antibiotic produced by Escherichia coli cells harbouring the 38 kb low copy number plasmid pC51, which codes for microcin production and immunity. The genetic determinants for microcin synthesis and immunity were cloned into the vectors pBR325, pUC19 and pACYC184. Physical and phenotypic analysis of deletion derivatives and mutant plasmids bearing insertions of transposon Tn5 showed that a DNA fragment of about 5 kb is required for microcin C51 synthesis and expression of complete immunity to microcin. Partial immunity can be provided by a 2 kb DNA fragment. Mutant plasmids were tested for their ability to complement Mic^– mutations. Results of these experiments indicate that at least three plasmid genes are required for microcin production. The host OmpR function is also necessary for microcin C51 synthesis.     

Cloning and mapping of the genetic determinants for microcin B17 production and immunity.

Plasmid pMccB17 (70 kilobases [kb]) codes for the production of microcin B17, a peptide that inhibits DNA synthesis, and for microcin B17 immunity. A BamHI-EcoRI fragment of 5.1 kb from pMccB17 was cloned into pBR322 in two steps. The resulting plasmid (pMM102) overproduced microcin B17 and expressed immunity against microcin. Mcc- and Mcc- Imm- mutants were isolated on plasmids pMccB17 and pMM102 by deleting various DNA fragments and by inserting different translocatable elements. Physical and phenotypic characterization of these mutants showed that a DNA region of 3.0 to 3.5 kb is required to produce microcin B17, whereas an adjacent region of about 1.0 kb is required to express microcin B17 immunity.     

YojI of Escherichia coli functions as a microcin J25 efflux pump

In the present study, we showed that yojI, an Escherichia coli open reading frame with an unknown function, mediates resistance to the peptide antibiotic microcin J25 when it is expressed from a multicopy vector. Disruption of the single chromosomal copy of yojI increased sensitivity of cells to microcin J25. The YojI protein was previously assumed to be an ATP-binding-cassette-type exporter on the basis of sequence similarities. We demonstrate that YojI is capable of pumping out microcin molecules. Thus, one obvious explanation for the protective effect against microcin J25 is that YojI action keeps the intracellular concentration of the peptide below a toxic level. The outer membrane protein TolC in addition to YojI is required for export of microcin J25 out of the cell. Microcin J25 is thus the first known substrate for YojI.     

The Leucine-Responsive Regulatory Protein, Lrp, Modulates Microcin J25 Intrinsic Resistance in Escherichia coli by Regulating Expression of the YojI Microcin Exporter

Many Escherichia coli K-12 strains display an intrinsic resistance to the peptide antibiotic microcin J25. In this study, we present results showing that the leucine-responsive regulatory protein, Lrp, is involved in this phenotype by acting as a positive regulator of YojI, a chromosomally encoded efflux pump which expels microcin out of cells. Exogenous leucine antagonizes the effect of Lrp, leading to a diminished expression of the pump and an increased susceptibility to microcin J25.     

The DNA replication inhibitor microcin B17 is a forty-three-amino-acid protein containing sixty percent glycine

Microcin B17 is a low-molecular-weight protein that inhibits DNA replication in a number of enteric bacteria. It is produced by bacterial strains which harbor a 70-kilobase plasmid called pMccB17. Four plasmid genes (named mcbABCD) are required for its production. The product of the mcbA gene was identified by labelling minicells. The mcbA gene product was slightly larger when a mutation in any of the other three production genes was present. This indicates that these genes are involved in processing the primary mcbA product to yield the active molecule. The mcbA gene product predicted from the nucleotide sequence has 69 amino acids including 28 glycine residues. Microcin B17 was extracted from the cells by boiling in 100 mM acetic acid, 1 mM EDTA, and purified to homogeneity in a single step by high-performance liquid chromatography through a C18 column. The N-terminal amino acid sequence and amino acid composition demonstrated that mcbA is the structural gene for microcin B17. The active molecule is a processed product lacking the first 26 N-terminal residues. The 43 remaining residues include 26 glycines. While microcin B17 is an exported protein, the cleaved N-terminal peptide does not have the characteristic properties of a "signal sequence", which suggests that it is secreted by a mechanism different from that used by most secreted proteins of E. coli.     

Amyloid formation modulates the biological activity of a bacterial protein

The aggregation of proteins into amyloid fibrils is the hallmark feature of a group of late-onset degenerative diseases including Alzheimer, Parkinson, and prion diseases. We report here that microcin E492, a peptide naturally produced by Klebsiella pneumoniae that kills bacteria by forming pores in the cytoplasmic membrane, assembles in vitro into amyloid-like fibrils. The fibrils have the same structural, morphological, tinctorial, and biochemical properties as the aggregates observed in the disease conditions. In addition, we found that amyloid formation also occurs in vivo where it is associated with a loss of toxicity of the protein. The finding that microcin E492 naturally exists both as functional toxic pores and as harmless fibrils suggests that protein aggregation into amyloid fibrils is an evolutionarily conserved property of proteins that can be successfully employed by bacteria to fulfill specific physiological needs.     

Microcin R51 and a plasmid determining its synthesis

A new microcin produced by an Citrobacter R51 strain has been detected. This antibiotic has been shown to inhibit the growth of a number of Gram-negative as well as Gram-positive strains of bacteria on the minimal medium plates. The properties of partially purified microcin were characterized. Constitutive synthesis of microcin is determined by a conjugative plasmid. The genes of microcin synthesis and immunity were cloned on a plasmid and plasmid vehicles. A physical map of the 12 kb fragment coding for the production of microcin R51 and immunity to this antibiotic is presented.     

Phenomic and genomic approaches to studying the inhibition of multiresistant Salmonella enterica by microcin J25

In livestock production, antibiotics are used to promote animal growth, control infections and thereby increase profitability. This practice has led to the emergence of multiresistant bacteria such as Salmonella, of which some serovars are disseminated in the environment. The objective of this study is to evaluate microcin J25 as an inhibitor of Salmonella enterica serovars of various origins including human, livestock and food. Among the 116 isolates tested, 37 (31.8%) were found resistant to at least one antibiotic, and 28 were multiresistant with 19 expressing the penta-resistant phenotype ACSSuT. Microcin J25 inhibited all isolates, with minimal inhibitory concentration values ranging from 0.06 μg/ml (28.4 nM) to 400 μg/ml (189 μM). Interestingly, no cross-resistance was found between microcin J25 and antibiotics. Multiple sequence alignments of genes encoding for the different proteins involved in the recognition and transport of microcin J25 showed that only ferric-hydroxamate uptake is an essential determinant for susceptibility of S. enterica to microcin J25. Examination of Salmonella strains exposed to microcin J25 by transmission electronic microscopy showed for the first-time involvement of a pore formation mechanism. Microcin J25 was a strong inhibitor of several multiresistant isolates of Salmonella and may have a great potential as an alternative to antibiotics.     

Microcin H47 system: an Escherichia coli small genomic island with novel features

Genomic islands are DNA regions containing variable genetic information related to secondary metabolism. Frequently, they have the ability to excise from and integrate into replicons through site-specific recombination. Thus, they are usually flanked by short direct repeats that act as attachment sites, and contain genes for an integrase and an excisionase which carry out the genetic exchange. These mobility events would be at the basis of the horizontal transfer of genomic islands among bacteria.Microcin H47 is a ribosomally-synthesized antibacterial peptide that belongs to the group of chromosome-encoded microcins. The 13 kb-genetic system responsible for its production resides in the chromosome of the Escherichia coli H47 strain and is flanked by extensive and imperfect direct repeats. In this work, both excision and integration of the microcin H47 system were experimentally detected. The analyses were mainly performed in E. coli K12 cells carrying the microcin system cloned in a multicopy plasmid. As expected of a site-specific recombination event, the genetic exchange also occurred in a context deficient for homologous recombination. The DNA sequence of the attachment sites resulting from excision were hybrid between the sequences of the direct repeats. Unexpectedly, different hybrid attachment sites appeared which resulted from recombination in four segments of identity between the direct repeats. Genes encoding the trans-acting proteins responsible for the site-specific recombination were shown to be absent in the microcin H47 system. Therefore, they should be provided by the remaining genetic context, not only in the H47 strain but also in E. coli K12 cells, where both excision and integration occurred. Moreover, a survey of the attachment sites in data banks revealed that they are widely spread among E. coli strains. It is concluded that the microcin system is a small island -H47 genomic island- that would employ a parasitic strategy for its mobility.     

Molecular characterization of a DNA fragment carrying the basic replicon of pTUC100, the natural plasmid encoding the peptide antibiotic microcin J25 system

The Escherichia coli plasmid pTUC100 encodes production of, and immunity to, the peptide antibiotic microcin J25. In the present study, an approximately 8-kb fragment immediately adjacent to the previously sequenced microcin region was isolated and its DNA sequence was determined. The main features of the newly characterized region are: (i) a basic replicon which is almost identical to that of the RepFIIA plasmid R100; (ii) two ORFs with 96% identity to two ORFs of unknown function on pO157, a large plasmid harbored by enterohemorragic E. coli, and a large ORF which does not show significant homology to any other reported nucleotide or protein sequence; and (iii) two intact insertion sequences, IS1294 and IS1. Sequence analysis, as well as that of the G+C content of both the 8-kb fragment and the previously sequenced microcin locus, lead us to propose that plasmid pTUC100 is a composite structure assembled from DNA elements from various sources.     

Escherichia coli outer membrane protein TolC is involved in production of the peptide antibiotic microcin J25

A Tn5 insertion in tolC eliminated microcin J25 production. The mutation had little effect on the expression of the microcin structural gene and presumably acted by blocking microcin secretion. The tolC mutants carrying multiple copies of the microcin genes were less immune to the microcin. TolC is thus likely a component of a microcin export complex containing the McjD immunity protein, an ABC exporter.     

Mapping of plasmid genes responsible for microcin R51 synthesis and immunity to it]

Microcin R51 is plasmid-determined low-molecular-weight peptide antibiotic produced by Escherichia coli. The spectrum of its action includes many different species of gram negative and some gram positive bacteria. Microcinogenic strains are immune to the action of the microcin they synthesize. As shown earlier, genes responsible for MccR51 production and immunity are located in a continuous 11.1 kb DNA fragment. These genes were cloned in pUC19 and pACYC184 plasmid vectors. Deletion derivatives and Tn5 insertion mutant plasmids which determined no microcin synthesis and immunity were obtained. Analysis of clones' phenotypes and physical mapping of mutant plasmids demonstrated that the 5 kb DNA fragment was indispensable for microcin production. The region of about 4.6 kb confers complete immunity of the producing strains, while partial immunity is provided by 1.8-1.9 kb DNA fragment.     

Cloning and mapping of the genetic determinants for microcin C7 production and immunity.

Microcin C7, a peptide antibiotic inhibitor of protein synthesis, is produced by Escherichia coli K-12 strains that carry the 43-kilobase low-copy-number plasmid pMccC7. Microcin C7 production and immunity determinants of this plasmid have been cloned into the vectors pBR322 and pACYC184. The resulting plasmids overproduce microcin C7 and express immunity against the microcin. Mcc- and Mcc- Imm- mutants have been isolated on recombinant plasmids by inserting transposable elements. Physical and phenotypic characterization of these mutants shows that a DNA region of 5 kilobases is required to produce microcin C7, and that two small regions located inside the producing region are also required to express immunity. Analysis of plasmids carrying mcc-lacZ gene fusions indicates that all microcin DNA is transcribed in the same direction. The results suggest that a structure like a polycistronic operon is responsible for microcin C7 production and immunity.     

The activity of microcin E492 from Klebsiella pneumoniae is regulated by a microcin antagonist

Abstract Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae . Different growth conditions of the producer strain affect microcin activity. The production of a microcin antagonist is responsible for the changes in microcin activity. The microcin antagonist is induced when cells are iron-deprived, resulting in a low microcin activity. The microcin antagonist was purified using a procedure developed for the isolation of a catechol-type siderophore, and its activity was titrated using purified microcin. The inhibitory effect of the microcin antagonist is not observed when this compound is forming a complex with iron. The same inhibitory effect on microcin activity was obtained using purified enterochelin from Escherichia coli . The microcin antagonist was identified as enterochelin through thin-layer chromatography.