title: Insertional activation of cepA leads to high-level beta-lactamase expression in Bacteroides fragilis clinical isolates.
abstract: Bacteroides fragilis is an important opportunistic pathogen of humans and is resistant to many drugs commonly used to treat anaerobic infections, including beta-lactams. A strain set comprised of B. fragilis isolates producing either low or high levels of the endogenous cephalosporinase activity, CepA, has been described previously (M. B. Rogers, A. C. Parker, and C. J. Smith, Antimicrob. Agents Chemother. 37:2391-2400, 1993). Clones containing cepA genes from each of seven representative strains were isolated, and the DNA sequences were determined. Nucleotide sequence comparisons revealed that there were few differences between the cepA coding sequences of the low- and high-activity strains. The cepA coding sequences were cloned into an expression vector, pFD340, and analyzed in a B. fragilis 638 cepA mutant. The results of beta-lactamase assays and ampicillin MICs showed that there was no significant difference in the enzymatic activity of structural genes from the high- or low-activity strains. Comparison of sequences upstream of the cepA coding region revealed that 50 bp prior to the translation start codon, the sequence for high-activity strains change dramatically. This region of the high-activity strains shared extensive homology with IS21, suggesting that an insertion was responsible for the increased expression of cepA in these isolates. Northern (RNA) blot analysis of total RNA by using cepA-specific DNA probes supported the idea that differential cepA expression in low- and high-activity strains was controlled at the level of transcription. However, the insertion did not alter the cepA transcription start site, which occurred 27 bp upstream of the ATG translation start codon in both expression classes. Possible mechanisms of cepA activation are discussed.
authors.affiliation: Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27858-4354.
authors.forename: M B
authors.initials: MB
authors.lastname: Rogers
authors.forename: T K
authors.initials: TK
authors.lastname: Bennett
authors.forename: C M
authors.initials: CM
authors.lastname: Payne
authors.forename: C J
authors.initials: CJ
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D001441:Bacteroides fragilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002510:Cephalosporinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D004251:DNA Transposable Elements; D012329:RNA, Bacterial; D012333:RNA, Messenger; D002510:Cephalosporinase