The IncF plasmid p1658/97 (c. 125 kb) from Escherichia coli isolates recovered during a clonal outbreak in a hospital in Warsaw, Poland, in 1997 contains the extended-spectrum β-lactamase (ESBL) gene bla(SHV-5), originated from the Klebsiella pneumoniae chromosome. A region containing the bla(SHV-5) gene is flanked by two IS26 copies and its copy number multiplies spontaneously within p1658/97 and RecA-deficient E. coli strains. Here, we demonstrate that the amplified IS26-bla(SHV-5) units were arranged in tandems, containing up to more than 10 units, which could raise ceftazidime MICs for host strains from 4 μg mL(-1) to more than 128 μg mL(-1). Successive deletions within p1658/97, located outside the amplifiable module and encompassing even as little as c. 15% of the plasmid, blocked the amplification. Moreover, the complementing re-introduction of the deleted fragments in trans did not restore the process. Similarly, insertions of a 1-kb DNA fragment into the amplicon inhibited its self-multiplication ability. The module was able to transmit into another IS26-containing plasmid by recombination. The results prompted us to speculate that local DNA structure, especially favorable in p1658/97, might have been responsible for the IS26-bla(SHV-5) multiplication ability.

The widespread prokaryotic toxin-antitoxin (TA) systems involve conditional interaction between two TA proteins. The interaction between the Epsilon and Zeta proteins, constituting the TA system of plasmid pSM19035 from Streptococcus pyogenes, was detected in vivo using a yeast two-hybrid system. As we showed using Saccharomyces cerevisiae, the Zeta toxin hybrid gene also exerts its toxic effects in a dose-dependent manner in eukaryotic cells. Analysis of mutant proteins in the two-hybrid system demonstrated that the N-terminal part of Zeta and the N-terminal region of Epsilon are involved in the interaction. The N-terminal region of the Zeta protein and its ATP/GTP binding motif were found to be responsible for the toxicity.

Escherichia coli isolates recovered from patients during a clonal outbreak in a Warsaw, Poland, hospital in 1997 produced different levels of an extended-spectrum beta-lactamase (ESBL) of the SHV type. The beta-lactamase hyperproduction correlated with the multiplication of ESBL gene copies within a plasmid. Here, we present the complete nucleotide sequence of plasmid p1658/97 carried by the isolates recovered during the outbreak. The plasmid is 125,491 bp and shows a mosaic structure in which all modules constituting the plasmid core are homologous to those found in plasmids F and R100 and are separated by segments of homology to other known regions (plasmid R64, Providencia rettgeri genomic island R391, Vibrio cholerae STX transposon, Klebsiella pneumoniae or E. coli chromosomes). Plasmid p1658/97 bears two replication systems, IncFII and IncFIB; we demonstrated that both are active in E. coli. The presence of an active partition system (sopABC locus) and two postsegregational killing systems (pemIK and hok/sok) indicates that the plasmid should be stably maintained in E. coli populations. The conjugative transfer is ensured by the operons of the tra and trb genes. We also demonstrate that the plasmidic segment undergoing amplification contains the bla(SHV-5) gene and is homologous to a 7.9-kb fragment of the K. pneumoniae chromosome. The amplicon displays the structure of a composite transposon of type I.

ABSTRACT Here we report the nucleotide sequence of pCTX-M3, a highly conjugative plasmid that is responsible for the extensive spread of the gene coding for the CTX-M-3 extended-spectrum β-lactamase in clinical populations of the family Enterobacteriaceae in Poland. The plasmid belongs to the IncL/M incompatibility group, is 89,468 bp in size, and carries 103 putative genes. Besides bla CTX-M-3 , it also bears the bla TEM-1 , aacC2 , and armA genes, as well as integronic aadA2 , dfrA12 , and sul1 , which altogether confer resistance to the majority of β-lactams and aminoglycosides and to trimethoprim-sulfamethoxazole. The conjugal transfer genes are organized in two blocks, tra and trb , separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Only bla CTX-M-3 , accompanied by an IS Ecp1 element, is placed separately, in a DNA fragment previously identified as a fragment of the Kluyvera ascorbata chromosome. On the basis of sequence analysis, we speculate that pCTX-M3 might have arisen from plasmid pEL60 from plant pathogen Erwinia amylovora by acquiring mobile elements with resistance genes. This suggests that plasmids of environmental bacterial strains could be the source of those plasmids now observed in bacteria pathogenic for humans.

High segregational stability of the streptococcal plasmid pSM19035 is achieved by the concerted action of systems involved in plasmid copy number control, multimer resolution, and postsegregational killing. In this study, we demonstrate the role of two genes, delta and omega, in plasmid stabilization by a partition mechanism. We show that these two genes can stabilize the native pSM19035 replicon as well as other theta- and sigma-type plasmids in Bacillus subtilis. In contrast to other known partition systems, in this case the two genes are transcribed separately; however, they are coregulated by the product of the parB-like gene omega. Analysis of mutants of the parA-like gene delta showed that the Walker A ATPase motif is necessary for plasmid stabilization. The ParB-like product of the omega gene binds to three regions containing repeated WATCACW heptamers, localized in the copS (regulation of plasmid copy number), delta, and omega promoter regions. We demonstrate that all three of these regions can cause partition-mediated incompatibility. Moreover, our data suggest that each of these could play the role of a centromere-like sequence. We conclude that delta and omega constitute a novel type of plasmid stabilization system.

pSM19035 of the pathogenic bacterium Streptococcus pyogenes is a low-copy-number plasmid carrying erythromycin resistance, stably maintained in a broad range of gram-positive bacteria. We show here that the omega-epsilon-zeta operon of this plasmid constitutes a novel proteic plasmid addiction system in which the epsilon and zeta genes encode an antitoxin and toxin, respectively, while omega plays an autoregulatory function. Expression of toxin Zeta is bactericidal for the gram-positive Bacillus subtilis and bacteriostatic for the gram-negative Escherichia coli. The toxic effects of zeta gene expression in both bacterial species are counteracted by proper expression of epsilon. The epsilon-zeta toxin-antitoxin cassette stabilizes plasmids in E. coli less efficiently than in B. subtilis.

from the Streptococcal Plasmid pSM19035 Genes ω and δ System Encoded by the

The postsegregational killing system of pSM19035 plasmid consists of the proteins Zeta and Epsilon, a toxin and an antidote, respectively. Zeta mutants were isolated with the use of Bacillus subtilis strain with the zeta gene under control of an inducible promoter integrated into the chromosome. Results of mutant analysis point to the amino terminal part of the Zeta protein as being responsible for the toxicity.

Plasmid pIPO2 is a cryptic, conjugative, broad-host-range plasmid isolated from the wheat rhizosphere. It efficiently self-transfers between alpha, beta and gamma Proteobacteria and has a mobilizing/retromobilizing capacity for IncQ plasmids. The complete nucleotide sequence of pIPO2 is presented on the basis of its mini-Tn5::luxABtet-tagged derivative, pIPO2T. The pIPO2 sequence is 39815 bp long and contains at least 43 complete ORFs. Apart from a suite of ORFs with unknown function, all of the genes carried on pIPO2 are predicted to be involved in plasmid replication, maintenance and conjugative transfer. The overall organization of these genes is different from previously described plasmids, but is similar to the genetic organization seen in pSB102, a conjugative plasmid recently isolated from the bacterial community of the alfalfa rhizosphere. The putative conjugative transfer region of pIPO2 covers 23 kb and contains the genes required for DNA processing (Dtr) and mating pair formation (Mpf). The organization of these transfer genes in pIPO2 is highly similar to the genetic organization seen in the environmental plasmid pSB102 and in pXF51 from the plant pathogen Xylella fastidiosa. Plasmids pSB102 and pXF51 have recently been proposed to form a new family of environmental broad-host-range plasmids. Here it is suggested that pIPO2 is a new member of this family. The proposed Mpf system of pIPO2 shares high amino acid sequence similarity with equivalent VirB proteins from the type IV secretion system of Brucella spp. Sequence information was used to design primers specific for the detection of pIPO2. Environmental DNA from a range of diverse habitats was screened by PCR with these primers. Consistently positive signals for the presence of pIPO2 were obtained from a range of soil-related habitats, including the rhizospheres of young wheat plants, of field-grown oats and of grass (all gramineous plants), as well as from the rhizosphere of tomato plants. These data add to the growing evidence that plasmids carry advantageous genes with as yet undefined functions in plant-associated communities.

The stable inheritance of bacterial plasmids is achieved by a number of different mechanisms. Among them are resolution of plasmid oligomers into monomers, active plasmid partitioning into dividing cells and selective killing of plasmid-free segregants. A special focus is given to the last mechanism. It involves a stable toxin and an unstable antidote. The antidotes neutralize their cognate toxins or prevent their synthesis. The different decay rates of the toxins and the antidotes underlie molecular mechanisms of toxin activation in plasmid-free cells. By eliminating of plasmid-free cells from the population of plasmid-bearing ones the toxin-antidote couples therefore act as plasmid addiction systems.

Transcription initiation of the copy-number control and better-than-random segregation genes of the broad-host-range and low-copy-number plasmid pSM19035 are subjected to repression by the autoregulated pSM19035-encoded omega product in Bacillus subtilis cells. The promoters of the copS (Pcop1 and Pcop2), delta (Pdelta), and omega (Pomega) genes have been mapped. These promoters are embedded in a set of either seven copies of a 7-bp direct repeat or in a block consisting of two 7-bp direct repeats and one 7-bp inverted repeat; the blocks are present either two or three times. The cooperative binding of omega protein to the repeats on the Pcop1, Pcop2, Pdelta, and Pomega promoters represses transcription initiation by a mechanism that does not exclude sigma(A)RNAP from the promoters. These results indicate that omega protein regulates plasmid maintenance by controlling the copy number on the one hand and by regulating the amount of proteins required for better-than-random segregation on the other hand.

In all cytosine‐C5‐DNA‐methyltransferases (MTases) from prokaryotes and eukaryotes, remarkably conserved amino acid sequence elements responsible for general enzymatic functions are arranged in the same canonical order. In addition, one variable region, which includes the target‐recognizing domain(s) (TRDs) characteristic for each enzyme, has been localized in one region between the same blocks of these conserved elements. This conservation in the order of conserved and variable sequences suggests stringent structural constraints in the primary structure to obtain the correct folding of the enzymes. Here we report the characterization of a new type of a multispecific MTase, M.(φ)BssHII, which is expressed as two isoforms. Isoform I is an entirely novel type of MTase which has, in addition to the TRDs at the conventional location, one TRD located at a non‐canonical position at its N‐terminus. Isoform II is represented by the same MTase, but without the N‐terminal TRD. The N‐terminal TRD provides HaeII methylation specificity to isoform I. The TRD is fully functional when engineered into either the conventional variable region of M.(φ)BssHII or the related monospecific M.φ3TII MTase. The implications of this structural plasticity with respect to the evolution of MTases are discussed.

The Lactococcus lactis gene encoding trimethoprim resistance has been cloned and expressed in Escherichia coli and Bacillus subtilis. Several lines of evidence indicate that the cloned gene encodes dihydrofolate reductase (DHFR). (i) It fully complements the fol ''null'' mutation in E. coli. (ii) Nucleotide sequencing of the cloned fragment revealed the presence of one open reading frame encoding a protein that shares homology with the family of bacterial DHFR enzymes. (iii) Overexpression of this open reading frame in E. coli resulted in the appearance in cell extracts of a protein of the expected size as well as in a dramatic increase of DHFR activity. In cell extracts, the DHFR activity was not inhibited by low trimethoprim concentration. By Northern (RNA) blotting and primer extension analyses, the size and the start point of the dhfr transcript, respectively, have been determined. Results of these experiments indicate that in L. lactis the dhfr gene represents part of a larger transcription unit.

The gene organization of the broad-host-range low-copy-number pSM19035-derived plasmid pDB101 is presented. Analysis of the 19202-bp sequence revealed thirteen different open reading frames (orfs). Nine of these orfs (repS-orf-orfβ-orfγ-orfδ-orfε-orfζ,-erm2-erm1 have been previously identified [Ceglowski et al., Gene 136 (1993) 1–12]. The extraordinarily long inverted repeated sequence, which includes orfα-orfβ-orfγ-orfδ-orfε-orfζ, comprises 76% of the pDB101 molecule. The gene order in pDB101 is repS-orfα-orfβ-orf|γ-orfδ-orfε-orfζ-erm2-erm1-orfζ-orfε- orfδ-orfγ-orfβ-orfα-orfη-orf θ-orf1-copS. The organization of genes of the orfη-orfγ region resembles the organization of genes in the orfA-orfI region of pAMβ1. Except for Orf1, bands of radioactive proteins corresponding to the molecular mass of the deduced reading frames (26.7, 14.3 and 10.3 kDa) were detected using the T7 promoter-expression system. The orf1 encoded a product (deduced molecular mass 28.3 kDa) which shows anomalous electrophoretical mobility corresponding to 60 kDa. The copS- and orf1-encoded proteins share homology to plasmid copy number control systems and Gram + cocci surface proteins, respectively. The orfη and orfθ encode proteins with unknown activity.

We determined the effect of various Bacillus subtilis dna(Ts) mutations on pSM19035 replication. At non-permissive temperature, plasmid replication depends on the dnaB, dnaC, dnaG and dnaI initiation replication products, but does not require the dnaA function. DNA elongation is performed by DNA polymerase III, since replication is blocked at non-permissive temperature in dna(Ts) mutants impaired in different subunits of the enzyme. Single-stranded plasmid replication intermediates, corresponding to the leading strand, accumulate in the dnaD strain. On the basis of electron microscopic analysis of replication intermediates, we confirmed that the plasmid replicates unidirectionally by a theta mechanism.