Promoter sequences necessary for high-level expression of the plasmid-associated ampC β-lactamase gene bla_MIR-1

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 β-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.