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

A clinical strain of Vibrio cholerae non-Or non-O139 isolated in France pro duced a new beta-lactamase with a pI of 5.35. The purified enzyme, with a m olecular mass of 33,000 Da, was characterized. Its kinetic constants show i t to be a carbenicillin-hydrolyzing enzyme comparable to the five previousl y reported CARB beta-lactamases and to SAR-1, another carbenicillin-hydroly zing beta-lactamase that has a pi of 4.9 and that is produced by a V. chole rae 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 conju gation. The nucleotide sequence of the structural gene was determined by di rect sequencing of PCR-generated fragments from plasmid DNA with four pairs of primers covering the whole sequence of the reference CARB-3 gene. The g ene encodes a 288-amino-acid protein that shares 94% homology with the CARB -1, CARB-2, and CARB-3 enzymes, 93% homolog with the Proteus mirabilis N29 enzyme, and 86.5% homology with the CARB-4 enzyme. The sequence of CARB-6 d iffers 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 loc ated in the C-terminal region of the sequence and at the surface of the mol ecule, according to the crystal structure of the Staphylococcus aureus PC-1 beta-lactamase.