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

J Antimicrob Chemother. 2004 Feb;53(2):197-202. doi: 10.1093/jac/dkh077. Epub 2004 Jan 16.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aeromonas hydrophila / enzymology
  • Aeromonas hydrophila / genetics*
  • Bacterial Proteins / genetics*
  • Carrier Proteins / genetics
  • Chemical Phenomena
  • Chemistry, Physical
  • Cloning, Molecular
  • Culture Media
  • DNA, Bacterial / genetics*
  • Escherichia coli / genetics
  • Escherichia coli Proteins*
  • Gene Expression Regulation, Bacterial / genetics*
  • Indicators and Reagents
  • Multigene Family / genetics
  • Penicillin-Binding Proteins*
  • Promoter Regions, Genetic / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Tagged Sites*
  • Transcription, Genetic
  • beta-Lactamases / biosynthesis
  • beta-Lactamases / chemistry
  • beta-Lactamases / genetics*

Substances

  • Bacterial Proteins
  • BlrA protein, Aeromonas jandaei
  • Carrier Proteins
  • Culture Media
  • DNA, Bacterial
  • Escherichia coli Proteins
  • Indicators and Reagents
  • Penicillin-Binding Proteins
  • ampH protein, E coli
  • beta-Lactamases
  • oxacillinase