Molecular and genetic analysis of the Bacteroides uniformis cephalosporinase gene, cblA, encoding the species-specific β-lactamase

Abstract

The gene, cblA, encoding the species-specific, clavulanate-susceptible, endogenous cephalosporinase was cloned from Bacteroides uniformis WAL-7088. The nucleotide sequence was determined, and the cblA structural gene was found to be 891 nucleotides, with a 48% G+C composition, which is similar to that of the B. uniformis genome. The cblA open reading frame encoded an Ambler class A β-lactamase polypeptide precursor of 296 amino acid residues with a predicted molecular weight of 33,450. A β-lactamase-deficient B. uniformis mutant with increased β-lactam susceptibility was constructed by insertional inactivation of the chromosomal gene. This mutant was complemented by plasmids bearing the cblA gene, and the resulting strains were resistant to cephaloridine and had a β-lactamase that comigrated with the parental β-lactamase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (30,500 Da) and in isoelectric focusing gels (pI 4.6), confirming a role for this β-lactamase in resistance.