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Antimicrobial Agents and Chemotherapy, November 2004, p. 4177-4182, Vol. 48, No. 11
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.11.4177-4182.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Promoter Sequences Necessary for High-Level Expression of the Plasmid-Associated ampC ß-Lactamase Gene bla_MIR-1

Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology, Creighton University School of Medicine, Omaha, Nebraska

Received 9 February 2004/ Returned for modification 23 April 2004/ Accepted 26 June 2004

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 ß-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.