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

TY - JOUR

T1 - Promoter sequences necessary for high-level expression of the plasmid-associated ampC β-lactamase gene blaMIR-1

AU - Reisbig, Mark

AU - Hanson, Nancy D.

PY - 2004/11

Y1 - 2004/11

N2 - Little is known about mechanisms involved in high-level expression of plasmid-associated ampC genes. The sequence for blaMIR-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 blaMIR-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 blaMIR-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 blaMIR-1 transcription. Expression of blaMIR-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 blaMIR-1 from both promoters compared to expression from prA alone. The upstream promoter prB of blaMIR-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.

AB - Little is known about mechanisms involved in high-level expression of plasmid-associated ampC genes. The sequence for blaMIR-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 blaMIR-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 blaMIR-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 blaMIR-1 transcription. Expression of blaMIR-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 blaMIR-1 from both promoters compared to expression from prA alone. The upstream promoter prB of blaMIR-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.

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U2 - 10.1128/AAC.48.11.4177-4182.2004

DO - 10.1128/AAC.48.11.4177-4182.2004

M3 - Article

C2 - 15504838

AN - SCOPUS:7244248673

VL - 48

SP - 4177

EP - 4182

JO - Antimicrobial Agents and Chemotherapy

JF - Antimicrobial Agents and Chemotherapy

SN - 0066-4804

IS - 11

ER -