CHARACTERIZATION OF HAPR, A POSITIVE REGULATOR OF THE VIBRIO-CHOLERAEHA PROTEASE GENE HAP, AND ITS IDENTIFICATION AS A FUNCTIONAL HOMOLOG OF THE VIBRIO-HARVEYI LUXR GENE

The Vibrio cholerae HA/protease gene (hap) promoter is inactive in Esc herichia coli. We cloned and sequenced the 0.7 kb hap promoter fragmen t 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 activati on of the hap promoter in E. coli, Expression from the hap promoter on ly occurred late in the growth phase, A single complete open reading f rame (ORF) designated HapR was identified on a 1.7 kb DNA fragment tha t was required for activation, Allelic replacements showed that hapR w as also essential for hap expression in V. cholerae, In El Tor, but no t 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 posi tive regulator of the lux operon that has no previously identified hom ologues, The amino-terminal domain (residues 21-68) showed significant homology to the TetR family of helix-turn-helix DNA-binding domains a nd was 95% identical to the same domain of LuxR, HapR and LuxR activat ed both the hap and the lux promoters at near wild-type levels, despit e only limited homology in the promoter sequences (46% identity with 1 2 gaps over 420 bp). DNA sequences and ORFs 5' (but not 3') of the hap R and luxR loci were homologous, suggesting a common origin for these loci, and hapR-hybridizing sequences were found in other vibrios, We c onclude that HapR is absolutely required for hap expression and that H apR and LuxR form a new family of transcriptional activator proteins.