CLONING AND CHARACTERIZATION OF THE ENDOGENOUS CEPHALOSPORINASE GENE,CEPA, FROM BACTEROIDES-FRAGILIS REVEALS A NEW SUBGROUP OF AMBLER CLASS-A BETA-LACTAMASES

Bacteroides fragilis CS30 is a clinical isolate resistant to high conc entrations of benzylpenicillin and cephaloridine but not to cephamycin or penem antibiotics. Beta-Lactam resistance is mediated by a chromos omally encoded cephalosporinase produced at a high level. The gene enc oding this beta-lactamase was cloned from genomic libraries constructe d in Escherichia coli and then mated with B. fragilis 638 for identifi cation of ampicillin-resistant (Ap(r)) strains. Ap(r) transconjugants contained a nitrocefin-reactive protein with the physical and enzymati c properties of the original CS30 isolate. The beta-lactamase gene (ce pA) was localized by deletion analysis and subcloned, and its nucleoti de sequence was determined. The 903-bp cepA open reading frame encoded a 300-amino-acid precursor protein (predicted molecular mass, 34,070 Da). A beta-lactamase-deficient mutant strain of B. fragilis 638 was c onstructed by insertional inactivation with the cepA gene of CS30, dem onstrating strict functional homology between these chromosomal beta-l actamase genes. An extensive comparison of the CepA protein sequence b y alignment with other beta-lactamases revealed the strict conservatio n of at least four elements common to Ambler class A. A further compar ison of the CepA protein sequence with protein sequences of beta-lacta mases from two other Bacteroides species indicated that they constitut e their own distinct subgroup of class A beta-lactamases.