Characterization and nucleotide sequence of a Klebsiella oxytoca cryptic plasmid encoding a CMY-type beta-lactamase: confirmation that the plasmid-mediated cephamycinase originated from the Citrobacter freundii AmpC beta-lactamase

Abstract

Plasmid pTKH11, originally obtained by electroporation of a Klebsiella oxytoca plasmid preparation into Escherichia coli XAC, expressed a high level of an AmpC-like beta-lactamase. The enzyme, designated CMY-5, conferred resistance to extended-spectrum beta-lactams in E. coli; nevertheless, the phenotype was cryptic in the K. oxytoca donor. Determination of the complete nucleotide sequence of pTKH11 revealed that the 8,193-bp plasmid encoded seven open reading frames, including that for the CMY-5 beta-lactamase (blaCMY-5). The blaCMY-5 product was similar to the plasmidic CMY-2 beta-lactamase of K. pneumoniae and the chromosomal AmpC of Citrobacter freundii, with 99.7 and 97.0% identities, respectively; there was a substitution of phenylalanine in CMY-5 for isoleucine 105 in CMY-2. blaCMY-5 was followed by the Blc and SugE genes of C. freundii, and this cluster exhibited a genetic organization identical to that of the ampC region on the chromosome of C. freundii; these results confirmed that C. freundii AmpC was the evolutionary origin of the plasmidic cephamycinases. In the K. oxytoca host, the copy number of pTKH11 was very low and the plasmid coexisted with plasmid pNBL63. Analysis of the replication regions of the two plasmids revealed 97% sequence similarity in the RNA I transcripts; this result implied that the two plasmids might be incompatible. Incompatibility of the two plasmids might explain the cryptic phenotype of blaCMY-5 in K. oxytoca through an exclusion effect on pTKH11 by resident plasmid pNBL63.

FIG. 1

Restriction maps of plasmids pKH11, pNBL63, and pTKH11. The restriction enzymes used in the construction of the maps are shown above the bars. The four fragments of pKH11 used as probes are indicated under the bars. The bars represent the DNA sequences of the plasmids. DNA fragments showing sequence similarities are indicated by hatched bars.

FIG. 2

Western blot of crude β-lactamase from an E. coli recipient, a K. oxytoca donor, and their transformants with AmpC β-lactamase-specific antisera. The AmpC β-lactamase-specific band is indicated by an arrow. Lane A, 50 μg of crude β-lactamase from K. oxytoca KH11; lane B, 50 μg of crude β-lactamase from E. coli SN03; lane C, 5 μg of crude β-lactamase from E. coli SN03/pNU6; lane D, molecular weight standards; lane E, 5 μg of crude β-lactamase from E. coli SN03/pTKH11; lane F, 0.5 μg of crude β-lactamase from E. coli SN03/pTKH11; lane G, 0.25 μg of crude β-lactamase from E. coli SN03/pTKH11; lane H, 0.125 μg of crude β-lactamase from E. coli SN03/pTKH11.

FIG. 3

Genetic organization of the genes identified on plasmid pTKH11. The direction of gene transcription is shown by the arrows. The DNA inserts in various recombinant plasmids are represented by lines of different lengths, and the significant restriction sites are indicated. The inverted repeat (IR) in the putative insertion element (IS) is shown, and mismatched nucleotides are indicated by bold letters.

FIG. 4

Multiple amino acid sequence alignments of the CMY-5 β-lactamase with K. pneumoniae CMY-2 and C. freundii AmpC (BLACF) β-lactamases. The amino acid stretches in bold letters are the active site (SVSK), the conserved triad (KTG), and the class C-typical motif (YXN). Amino acid substitutions are indicated with asterisks, and Phe-105 in CMY-5 and Leu-105 in CMY-2 are shown in bold letters.

FIG. 5

Nucleotide sequence of the 1,424-bp replication region of pNBL63. The sequence includes the 1,424-bp 3′ part of the 2,444-bp BglII fragment of pNBL63. The consensus sequences are underlined and labeled. The putative transcription start sites are indicated by bold letters. The putative start codons are underlined, the stop codons are designated with asterisks, and the gene designations are given below the deduced amino acids, which are specified by standard one-letter abbreviations.

FIG. 6

Bestfit comparison of the pNBL63 and pTKH11 RNA Is and RNA IIs. Identical nucleotides are indicated by vertical bars, and gaps are shown by periods.