Role of the 'cre/blr-tag' DNA sequence in regulation of gene expression by the Aeromonas hydrophila beta-lactamase regulator, BlrA.
OBJECTIVE
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.
Avison
Matthew B
MB
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. Matthewb.Avison@bris.ac.uk
Niumsup
Pannika
P
Nurmahomed
Kurshid
K
Walsh
Timothy R
TR
Bennett
Peter M
PM
D016980:Aeromonas hydrophila / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D007202:Indicators and Reagents; D005810:Multigene Family / Q000235:genetics; D046915:Penicillin-Binding Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016324:Sequence Tagged Sites; D014158:Transcription, Genetic; D001618:beta-Lactamases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; C104885:BlrA protein, Aeromonas jandaei; D002352:Carrier Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D046915:Penicillin-Binding Proteins; C487761:ampH protein, E coli; D001618:beta-Lactamases; C108449:oxacillinase