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.     

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.     

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.     

Plasmid genes required for microcin B17 production.

The production of the antibiotic substance microcin B17 (Mcc) is determined by a 3.5-kilobase DNA fragment from plasmid pMccB17. Several Mcc- mutations on plasmid pMccB17 were obtained by both transposon insertion and nitrosoguanidine mutagenesis. Plasmids carrying these mutations were tested for their ability to complement Mcc- insertion or deletion mutations on pMM102 (pMM102 is a pBR322 derivative carrying the region encoding microcin B17). Results from these experiments indicate that at least four plasmid genes are required for microcin production.     

Physical characterization of plasmids determining synthesis of a microcin which inhibits methionine synthesis in Escherichia coli.

Plasmid deoxyribonucleic acid (DNA) isolated from each of three antibiotic-resistant clinical strains of Escherichia coli producing the same microcin showed multiple bands upon agarose gel electrophoresis. Transformants selected either for microcin resistance or ampicillin resistance yielded plasmid DNA corresponding in size to only one of the multiple bands. Plasmids, isolated from all three hosts, which determined microcin resistance and microcin production measured about 4 megadaltons by sucrose density, restriction enzyme, and contour length analyses; cleavage of the DNAs by each of eight restriction enzymes showed the same response, and DNA-DNA hybridization indicated complete homology. The antibiotic resistance plasmids of the three host strains were uniformly larger, were of different sizes, and showed different restriction enzyme cleavage patterns. One of these R plasmids (pCP106) also determined the synthesis of the same microcin, and DNA-DNA hybridization studies indicated an approximate 2.4-megadalton homology with the 4-megadalton microcin plasmid pCP101. The microcin plasmids were present at approximately 20 copies per genome equivalent and were nonconjugative, whereas the R plasmids had a copy number of about 1, were conjugative, and could mobilize the microcin plasmid. Microcin plasmid pCP101 showed replication properties similar to those of a number of small multicopy plasmids such as ColE1.     

Cloning of the determinants for microcin D93 production and analysis of three different D-type microcin plasmids

Three different microcin plasmids coding for D-type microcins were analyzed. Two of the plasmids (pMccD93 and pCP101) were small, multicopy plasmids and were closely related. The third plasmid (pCP106) was a conjugative, antibiotic multiresistance plasmid. Although plasmids pCP101 and pCP106 were previously classified as A-type microcin plasmids, we have determined that they are, in fact, D type. Furthermore, the determinants for microcin D93 production were cloned from plasmid pMccD93, and a DNA probe for the region implicated in the synthesis of microcin was obtained. This probe hybridized to plasmid C from Escherichia coli strain V517, indicating that this plasmid might be involved in the synthesis of a D-type microcin. The characteristics of replication of plasmid pCP106 were analyzed and appeared to be similar to those of ColEl plasmids, although pCP106 is a conjugative single-copy plasmid.     

Microcin H47, a chromosome-encoded microcin antibiotic of Escherichia coli.

Microcin H47 (MccH47) is a novel microcin antibiotic produced by a natural Escherichia coli isolate. In contrast to all the other colicins and microcins examined to date, which are plasmid encoded, the genes for MccH47 synthesis and immunity are located on the chromosome. These genetic determinants were cloned and shown to extend over a continuous DNA region of ca. 10 kb.     

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.     

Evidence that colicin X is microcin B17.

The DNA replication inhibitor microcin B17 is a peptide antibiotic produced by Escherichia coli strains carrying plasmid pMccB17. Here we present evidence that antibiotic activities previously named colicin X are probably identical to microcin B17. Our results include comparison of the conditions of production of the antibiotics, their mode of action, cross-immunity of producer strains, and cross-resistance of resistant mutants. Plasmids encoding colicin X have been identified and shown to have a region of significant homology with the microcin B17-producing region of pMccB17 DNA.     

Microcin B17 blocks DNA replication and induces the SOS system in Escherichia coli

Microcin B17 is a novel peptide antibiotic of low Mr (about 4000) produced by Escherichia coli strains carrying plasmid pMccB17. The action of this microcin in sensitive cells is essentially irreversible, follows single-hit kinetics, and leads to an abrupt arrest of DNA replication and, consequently, to the induction of the SOS response. RecA- and RecBC- strains are hypersensitive to microcin B17. Strains producing a non-cleavable SOS repressor (lexAl mutant) are also more sensitive than wild-type, whereas strains carrying a mutation which causes constitutive expression of the SOS response (spr-55) are less sensitive to microcin. Microcin B17 does not induce the SOS response in cells which do not have an active replication fork. The results suggest that the mode of action of this microcin is different from all other well-characterized microcins and colicins, and from other antibiotics which inhibit DNA replication.     

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.     

Construction and characterization of mutations at codon 751 of the Escherichia coli gyrB gene that confer resistance to the antimicrobial peptide microcin B17 and alter the activity of DNA gyrase

Microcin B17 is a peptide antibiotic that inhibits DNA replication in Escherichia coli by targeting DNA gyrase. Previously, two independently isolated microcin B17-resistant mutants were shown to harbor the same gyrB point mutation that results in the replacement of tryptophan 751 by arginine in the GyrB polypeptide. We used site-directed mutagenesis to construct mutants in which tryptophan 751 was deleted or replaced by other amino acids. These mutants exhibit altered DNA gyrase activity and different levels of resistance to microcin B17.     

A novel plasmid vector allowing positive selection for cloned fragments

Abstract The use of plasmid pMM102 as a positive selection cloning vector is described. This plasmid is derived from pBR322 and contains the DNA encoding microcin B17 production and immunity. RecA⁻ Escherichia coli K12 cells containing this plasmid are unable to grow in minimal medium. Inactivation of any of the 4 genes required for microcin production allows the bacterial host to produce colonies. This property has been used to clone DNA inserts in the unique sites for restriction endonucleases Bgl II, Sac I, Sac II and Sma I in pMM102. DNA fragments with asymmetrical termini of many different kinds can also be cloned. We have also identified a fragment in the wild-type plasmid pMccB17 that suppresses the pMM102-induced growth inhibition.     

The antibiotic microcin B17 is a DNA gyrase poison: characterisation of the mode of inhibition

Microcin B17 is a 3.1-kDa bactericidal peptide; the putative target of this antibiotic is DNA gyrase. Microcin B17 has no detectable effect on gyrase-catalysed DNA supercoiling or relaxation activities in vitro and is unable to stabilise DNA cleavage in the absence of nucleotides. However, in the presence of ATP, or the non-hydrolysable analogue 5'-adenylyl beta,gamma-imidodiphosphate, microcin B17 stabilises a gyrase-dependent DNA cleavage complex in a manner reminiscent of quinolones, Ca(2+), or the bacterial toxin CcdB. The pattern of DNA cleavage produced by gyrase in the presence of microcin B17 is different from that produced by quinolones and more closely resembles Ca(2+)-mediated cleavage. Several gyrase mutants, including well-known quinolone-resistant mutants, are cross resistant to microcin-induced DNA cleavage. We suggest that microcin exerts its effects through a mechanism that has similarities to those of both the bacterial toxin CcdB and the quinolone antibacterial agents.     

Gene ompR and regulation of microcin 17 and colicin e2 syntheses.

The production of microcin 17 is controlled by plasmid pRYC17. Chromosomal mutants unable to produce a normal amount of microcin were isolated in Escherichia coli. One of the mutations maps in the ompR locus, indicating that an active OmpR product is required for the synthesis of microcin 17. The same conclusion was obtained for the synthesis of colicin E2. Therefore, two new functions of the regulatory gene ompR have been revealed.     

The production in vivo of microcin E492 with antibacterial activity depends on salmochelin and EntF

Microcin E492 is a channel-forming bacteriocin that is found in two forms, namely, a posttranslationally modified form obtained by the covalent linkage of salmochelin-like molecules to serine 84 and an unmodified form. The production of modified microcin E492 requires the synthesis of enterochelin, which is subsequently glycosylated by MceC and converted into salmochelin. mceC mutants produced inactive microcin E492, and this phenotype was reversed either by complementation with iroB from Salmonella enterica or by the addition of exogenous salmochelin. Cyclic salmochelin uptake by Escherichia coli occurred mainly through the outer membrane catecholate siderophore receptor Fiu. The production of inactive microcin E492 by mutants in entB and entC was reverted by the addition of the end product of the respective mutated pathway (2,3-dihydroxybenzoic acid and enterochelin/salmochelin, respectively), while mutants in entF did not produce active microcin E492 in the presence of enterochelin or salmochelin. The EntF adenylation domain was the only domain required for this microcin E492 maturation step. Inactivation of the enzymatic activity of this domain by site-directed mutagenesis did not prevent the synthesis of active microcin E492 in the presence of salmochelin, indicating that the adenylation activity is not essential for the function of EntF at this stage of microcin E492 maturation.     

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.     

Cloning and expression in Escherichia coli of genetic determinants for production of and immunity to microcin E492 from Klebsiella pneumoniae.

Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae RYC492. The genetic determinants for microcin synthesis and immunity were cloned in Escherichia coli VCS257 into the cosmid vector pHC79, starting from total DNA of K. pneumoniae RYC492. The microcin E492 expressed in E. coli had the same properties as that of K. pneumoniae, i.e., the same molecular weight, the ability to form ionic channels in planar phospholipid bilayers, and essentially identical biological properties. Microcin E492 expression in E. coli, like that in K. pneumoniae, was mainly in the exponential phase of growth, declining in the stationary phase. The immunity determinant was subcloned into the same vector, and its expression was found to disappear in the stationary phase. This phenomenon is not dependent on rpoS, the stationary-phase sigma factor.     

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.