title: Promoter sequences necessary for high-level expression of the plasmid-associated ampC beta-lactamase gene blaMIR-1.
abstract: Little is known about mechanisms involved in high-level expression of plasmid-associated ampC genes. The sequence for bla(MIR-1) has been elucidated, and the gene is not inducible. Although the sequence for the promoter (prA) that drives expression of Enterobacter cloacae chromosomal ampC is present upstream of bla(MIR-1), high-level expression from bla(MIR-1) is directed from a hybrid promoter (prB) located further upstream of prA. The purpose of this study was to determine the influence of each promoter on bla(MIR-1) expression and beta-lactam resistance. RNA expression by deletion clones with both promoters was measured and compared to that by clones in which -35 and/or -10 elements of prA and/or prB were altered. Primer extension revealed two start sites for bla(MIR-1) transcription. Expression of bla(MIR-1) in clones with both promoters was 171-fold higher than that in clones carrying only prA. In addition, bla(MIR-1) expression from prA increased 11-fold in the presence of the prB -10 element compared to expression driven from prA alone. Ceftazidime and cefotaxime MICs increased 42- and 64-fold, respectively, for the clone expressing bla(MIR-1) from both promoters compared to expression from prA alone. The upstream promoter prB of bla(MIR-1) is solely responsible for high-level expression required for cefotaxime and ceftazidime resistance. These data suggest that resistance to extended-spectrum cephalosporins mediated by noninducible plasmid-associated ampC genes requires the formation of novel promoter elements that are capable of increasing ampC expression.
authors.affiliation: Center for Research in Anti-Infectives and Biotechnology, Department of Microbiology and Immunology, Creighton University School of Medicine, 2500 California Pl., Omaha, NE 68178, USA.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Reisbig
authors.forename: Nancy D
authors.initials: ND
authors.lastname: Hanson
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017931:DNA Primers; D016972:Enterobacter cloacae / Q000201:enzymology / Q000235:genetics; D008826:Microbial Sensitivity Tests; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics*; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D017931:DNA Primers; D012329:RNA, Bacterial; D047090:beta-Lactams; D001618:beta-Lactamases; C494140:beta-lactamase MIR-1, Enterobacter cloacae