Role of the 'cre/blr-tag' DNA sequence in regulation of gene expression by the Aeromonas hydrophila β-lactamase regulator, BlrA

Objectives: To further understand the mechanisms used to regulate expression of the blr regulon of Aeromonas hydrophila T429125, including three unlinked beta-lactamase genes, ampH, cepH and imiH, and to examine the role of the 'cre/blr-tag' DNA sequence (TTCAC) in transcriptional control exerted by the two-component system, BlrAB. Methods: Genes linked to blrAB-ampH were cloned using standard methods; gene expression was measured by RT-PCR or beta-lactamase assays; transcription start sites were determined by reversed-transcript analysis; cepH promoter probe reporter constructs including cre/blr-tag deletions were generated by PCR; and BlrA was overexpressed in Escherichia coli using the pBAD plasmid. Results: The blrD gene, encoding a putative inner membrane protein, was found to be located downstream of blrAB-ampH. RT-PCR analysis showed that blrD is part of the A. hydrophila blr regulon, and transcript start-point determinations revealed that blr-regulon promoters (including that of blrD) are preceded by at least one cre/blr-tag. Targeted deletion of the 16 bp cepH cre/blr-tag dimer blocked BlrA-induced overproduction of cepH in E. coli. Conclusions: This is the first report of non-beta-lactamase genes being co-ordinately regulated with a normally co-resident beta-lactamase gene, and the first direct evidence for a role of the cre/blr-tag sequence in the regulation of transcription by BlrA.