Characterization and nucleotide sequence of CARB-6, a new carbenicillin-hydrolyzing beta-lactamase from Vibrio cholerae

Abstract

A clinical strain of Vibrio cholerae non-O1 non-O139 isolated in France produced a new beta-lactamase with a pI of 5.35. The purified enzyme, with a molecular mass of 33,000 Da, was characterized. Its kinetic constants show it to be a carbenicillin-hydrolyzing enzyme comparable to the five previously reported CARB beta-lactamases and to SAR-1, another carbenicillin-hydrolyzing beta-lactamase that has a pI of 4.9 and that is produced by a V. cholerae strain from Tanzania. This beta-lactamase is designated CARB-6, and the gene for CARB-6 could not be transferred to Escherichia coli K-12 by conjugation. The nucleotide sequence of the structural gene was determined by direct sequencing of PCR-generated fragments from plasmid DNA with four pairs of primers covering the whole sequence of the reference CARB-3 gene. The gene encodes a 288-amino-acid protein that shares 94% homology with the CARB-1, CARB-2, and CARB-3 enzymes, 93% homology with the Proteus mirabilis N29 enzyme, and 86.5% homology with the CARB-4 enzyme. The sequence of CARB-6 differs from those of CARB-3, CARB-2, CARB-1, N29, and CARB-4 at 15, 16, 17, 19, and 37 amino acid positions, respectively. All these mutations are located in the C-terminal region of the sequence and at the surface of the molecule, according to the crystal structure of the Staphylococcus aureus PC-1 beta-lactamase.

FIG. 1

Sequencing strategy for CARB-6 β-lactamase gene. The CARB-6 gene is represented by the black box. The arrows indicate the oligonucleotide positions.

FIG. 2

Gene sequence and deduced amino acid sequence of the V. cholerae β-lactamase (CARB-6). The deduced amino acid sequence is designated by the one-letter code. The active site, STFK, is boxed, and the differences relative to CARB-1 to CARB-3 are underlined.

FIG. 3

Multiple sequence alignment of the amino acid sequences of CARB-1, CARB-2, CARB-3, CARB-4, P. mirabilis N29, P. mirabilis GN79, and CARB-6 β-lactamases. The shadowed boxes (I to VII) correspond to amino acid boxes conserved in all penicillin-recognizing enzymes, as identified by Joris et al. (14). Alpha-helix and beta-barrel motifs are indicated from the PC-1 crystal structure (4, 12). Asterisks indicate the conserved residues specific for class A β-lactamases. Amino acid changes are written as black letters in white boxes. Sequences are numbered as described by Ambler (2).