Klebsiella pneumoniae Multiresistance Plasmid pMET1: Similarity with the Yersinia pestis Plasmid pCRY and Integrative Conjugative Elements

Dissemination of antimicrobial resistance genes has become an important public health and biodefense threat. Plasmids are important contributors to the rapid acquisition of antibiotic resistance by pathogenic bacteria.The nucleotide sequence of the Klebsiella pneumoniae multiresistance plasmid pMET1 comprises 41,723 bp and includes Tn1331.2, a transposon that carries the bla(TEM-1) gene and a perfect duplication of a 3-kbp region including the aac(6')-Ib, aadA1, and bla(OXA-9) genes. The replication region of pMET1 has been identified. Replication is independent of DNA polymerase I, and the replication region is highly related to that of the cryptic Yersinia pestis 91001 plasmid pCRY. The potential partition region has the general organization known as the parFG locus. The self-transmissible pMET1 plasmid includes a type IV secretion system consisting of proteins that make up the mating pair formation complex (Mpf) and the DNA transfer (Dtr) system. The Mpf is highly related to those in the plasmid pCRY, the mobilizable high-pathogenicity island from E. coli ECOR31 (HPI(ECOR31)), which has been proposed to be an integrative conjugative element (ICE) progenitor of high-pathogenicity islands in other Enterobacteriaceae including Yersinia species, and ICE(Kp1), an ICE found in a K. pneumoniae strain causing primary liver abscess. The Dtr MobB and MobC proteins are highly related to those of pCRY, but the endonuclease is related to that of plasmid pK245 and has no significant homology with the protein of similar function in pCRY. The region upstream of mobB includes the putative oriT and shares 90% identity with the same region in the HPI(ECOR31).The comparative analyses of pMET1 with pCRY, HPI(ECOR31), and ICE(Kp1 )show a very active rate of genetic exchanges between Enterobacteriaceae including Yersinia species, which represents a high public health and biodefense threat due to transfer of multiple resistance genes to pathogenic Yersinia strains

Genetic structures located upstream of <i>parF</i> and <i>parG.</i>

<p>A. The direct repeats within the pMET1 putative <i>parH</i>-like locus are shown in red. The diagram also shows the −35 and −10 sequences, as well as the inverted repeats (arrows). The inverted repeat within the putative <i>parH</i> locus is shown in blue. The beginning of the ParF amino acid sequence including the deviant Walker motif A and motif A' are shown. B. Logo plot <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Crooks1" target="_blank">[60]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Schneider1" target="_blank">[61]</a> of a multiple alignment of the direct repeats shown in red.</p

Genetic map of pMET1 and comparison to plasmid pCRY and chromosomal elements.

<p>A. The genetic maps of pMET1 and pCRY are compared showing the homologous regions. The arrows indicate genes locations and orientation. Genes with different functions are shown with different colors and if the genes in the different structures shown are homologus they are represented with the same colors. Yellow: mobilization; green: replication and partition; red: antibiotic resistance; purple: virB/pilX-like; blue: transposition; grey: unknown. Since pCRY is smaller than pMET1, to represent it in circular form a dotted line was added to fill the gap. Solid line represents non-homologous DNA. B. Comparison of a pMET1 region with chromosomal HPIs or ICEs is shown using a linearized version of the plasmid. The HPIs shown are those from <i>E. coli</i> ECOR31 (HPI_ECOR31) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Schubert1" target="_blank">[43]</a>, <i>K. pneumoniae</i> NTUH-K2044 (ICE_Kp1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Lin1" target="_blank">[44]</a>, and <i>Y. pestis</i> KIM (HPI_Yp)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Schubert1" target="_blank">[43]</a>. The diagram shows the HP core regions, which are not at scale and are represented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Schubert1" target="_blank">[43]</a>, and the RB-HPIs. The sequence described in this manuscript has been deposited in GenBank, accession number is EU383016.</p

Replication region of pMET1.

<p>A. The bar shows a genetic map of the pMET1 replication region and the GC content plot generated using a window size of 100 bp on top. Recombinant clones were obtained by inserting the indicated fragments into pCR2.1 or ligated to the pUC4K <i>aph</i> cassette. The ability to be maintained in <i>E. coli</i> C2110 (a <i>polA</i> mutant) of the recombinant plasmids made using pCR2.1 as vector is indicated to the right by a + or − sign. The ability to generate kanamycin resistant colonies in <i>E. coli</i> TOP10 of the indicated fragments when ligated to the <i>aph</i> cassette from pUC4K is also represented by a + or − sign. B. BLASTP comparison of the amino acid sequences of the putative RepA proteins from pMET1 and pCRY.</p