Regulation of Class A β-Lactamase CzoA by CzoR and IscR in S44.

A genomic analysis of S44 revealed a gene that encodes a LysR family transcriptional regulator (here named , czo for cefazolin) located upstream of a putative class A β-lactamase encoding gene (here named ). A putative DNA-binding motif of the Fe-S cluster assembly regulator IscR was identified in the - intergenic region. Real-time RT-PCR and fusion expression assays indicated that transcription of and were induced by multiple β-lactams. CzoA expressed in was shown to contribute to susceptibility to a wide range of β-lactams judged from minimum inhibitory concentrations. enzymatic assays showed that CzoA hydrolyzed seven β-lactams, including benzylpenicillin, ampicillin, cefalexin, cefazolin, cefuroxime, ceftriaxone, and cefepime. Deletion of either or increased susceptibility to cefalexin and cefazolin, while complemented strains restored their wild-type susceptibility levels. Electrophoretic mobility shift assays (EMSA) demonstrated that CzoR and IscR bind to different sites of the - intergenic region. Precise CzoR- and IscR-binding sites were confirmed via DNase I footprinting or short fragment EMSA. When cefalexin or cefazolin was added to cultures, deletion completely inhibited expression but did not affect transcription, while deletion decreased the expressions of both and These results reveal that CzoR positively affects the expression of with its own expression upregulated by IscR.