Characterization of hapR, a positive regulator of the Vibrio cholerae HA/protease gene hap, and its identification as a functional homologue of the Vibrio harveyi luxR gene

Abstract

The Vibrio cholerae HA/protease gene (hap) promoter is inactive in Escherichia coli. We cloned and sequenced the 0.7kb hap promoter fragment from strain 3083-2 and showed that hap is located immediately 3' of ompW, encoding a minor outer membrane protein. A clone from a genomic library of strain 3083-2 was isolated, which was required for activation of the hap promoter in E. coli. Expression from the hap promoter only occurred late in the growth phase. A single complete open reading frame (ORF) designated HapR was identified on a 1.7 kb DNA fragment that was required for activation. Allelic replacements showed that hapR was also essential for hap expression in V. cholerae. In El Tor, but not in classical biotypes of V. cholerae, hapR mutations also produced a rugose colonial phenotype. HapR was shown to encode a 203-amino-acid polypeptide with 71% identity to LuxR of V. harveyi, an essential positive regulator of the lux operon that has no previously identified homologues. The amino-terminal domain (residues 21-68) showed significant homology to the TetR family of helix-turn-helix DNA-binding domains and was 95% identical to the same domain of LuxR. HapR and LuxR activated both the hap and the lux promoters at near wild-type levels, despite only limited homology in the promoter sequences (46% identity with 12 gaps over 420bp). DNA sequences and ORFs 5' (but not 3') of the hapR and luxR loci were homologous, suggesting a common origin for these loci, and hapR-hybridizing sequences were found in other vibrios. We conclude that HapR is absolutely required for hap expression and that HapR and LuxR form a new family of transcriptional activator proteins.