Complete nucleotide sequence of Klebsiella pneumoniae multiresistance plasmid pJHCMW1

Abstract

The multiresistance plasmid pJHCMW1, harbored by a clinical Klebsiella pneumoniae strain isolated from a neonate with meningitis, was sequenced. A circular sequence of 11,354 bp was generated, of which 7,993 bp make up Tn1331, a transposon including the antibiotic resistance genes aac(6')-Ib, aadA1, bla(OXA-9), and bla(TEM-1). The gene aac(6')-Ib is included in a gene cassette, and both aadA1 and bla(OXA-9) are included in a single-gene cassette that may have arisen as a consequence of a recombination event involving two integrons. The pJHCMW1 plasmid replicates through a ColE1-like RNA-regulated mechanism, includes a functional oriT, and two loci with similarity to XerCD site-specific recombination target sites involved in plasmid stabilization by the resolution of multimers. One of these two loci, mwr, is active and has been the subject of previous studies, and the other, dxs, is not functional but binds the recombinase XerD with low affinity. Two additional open reading frames were identified, one with low similarity to two hypothetical membrane proteins from Mycobacterium tuberculosis and Mycobacterium leprae and the other with low similarity to psiB, a gene encoding a function that facilitates the establishment of the transferring plasmid in the recipient bacterial cell during the process of conjugation.

FIG. 1.

Genetic map of pJHCMW1. Genes or ORFs are shown with the starting and ending coordinates. The transposon Tn1331, the direct repeats, and the replication (Rep), mobilization, and recombination elements are indicated. The gray arrowhead in tnpR∗ indicates that this is a truncated gene (the N terminus of the encoded protein is absent [32]).

FIG. 2.

Comparison of structures of regions including aac(6′)-Ib, aadA1, and bla_OXA-9 from pJHCMW1 and aac(6′)-IId and aadA1 from A. baumannii. The black lines represent DNA fragments with high homology. The numbers are the base pair coordinates in the pJHCMW1 nucleotide sequence. The nucleotide sequence of the 59-be element following aadA1 is shown. The homology between A. baumannii and pJHCMW1 DNA ends at the end of the aadA1 structural gene and starts again at coordinate 9727 (final portion of the bla_OXA-9 59-be element). At the bottom of the diagram, the positions and lengths of the gene cassettes are shown.

FIG. 3.

Possible mechanism for the generation of the gene cassette containing aadA1 and bla_OXA-9. Two putative integrons, one containing bla_OXA-9 immediately following attI and one with a gene cassette containing aadA1 crossover (probably an illegitimate recombination event), generating the gene cassette, including both genes found in pJHCMW1. In the process, the sequence of the 59-be element located 3′ of bla_OXA-9 has been conserved, but the attI site and the 59-be element 3′ of aadA1 have been lost, leaving a sequence indicated as attI∗. The attI site is located adjacent to the intI gene in the 5′ conserved region and is where gene cassettes are inserted in integrase-mediated reactions (22). The nucleotide sequences of the 59-be elements are shown.

FIG. 4.

Alignment of the nucleotide sequences at the N terminus of aac(6′)-Ib from pJHCMW1 and aac(6′)-IId from A. baumannii (A. b.). The proteins encoded by these genes have 98% identity [comparison starts at the AAC(6′)-Ib Met at nucleotide 7389]. Identical nucleotides are indicated by the vertical lines between the two sequences. The sequence identical to that of bla_TEM-1 in the pJHCMW1 aac(6′)-Ib gene is boxed.

FIG. 5.

Comparison of Xer recombination sites and DNA-protein binding. (a) Alignment of the nucleotide sequences of the core recombination sites mwr, cer, psi, dif, and dxs. The XerC and XerD binding sites and the central regions (gray) are shown. (b) Oligonucleotides with the dif, mwr, and dxs nucleotide sequences were end labeled and incubated with (+) and without (−) the indicated proteins. The products of the binding reactions were separated by electrophoresis in an 8% polyacrylamide gel, and the bands were detected by autoradiography. The nature of the complexes for each signal is shown.