JAC Advance Access originally published online on January 16, 2004
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Journal of Antimicrobial Chemotherapy (2004) 53, 197-202
© 2004 The British Society for Antimicrobial Chemotherapy
Role of the ‘cre/blr-tag’ DNA sequence in regulation of gene expression by the Aeromonas hydrophila ß-lactamase regulator, BlrA
Bristol Centre for Antimicrobial Research and Evaluation, Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK
Received 5 August 2003; returned 14 October 2003; revised 7 November 2003; accepted 17 November 2003
Objectives: To further understand the mechanisms used to regulate expression of the blr regulon of Aeromonas hydrophila T429125, including three unlinked ß-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 ß-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-ß-lactamase genes being co-ordinately regulated with a normally co-resident ß-lactamase gene, and the first direct evidence for a role of the cre/blr-tag sequence in the regulation of transcription by BlrA.
Keywords: blr regulon, cre regulon, CreBC, BlrD, cre/blr-tag
* Corresponding author. Tel: +44-117-9287897; Fax: +44-117-9287896; E-mail: Matthewb.Avison{at}bris.ac.uk
Present address. Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanuloke 65000, Thailand.
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