Transcriptional Repression of the VC2105 Protein by Vibrio FadR Suggests that It Is a New Auxiliary Member of the fad Regulon

Recently, our group along with others reported that theVibrio FadR regulatory protein is unusual in that, unlike the prototypicalfadR product ofEscherichia coli , which has only one ligand-binding site,Vibrio FadR has two ligand-binding sites and represents a new mechanism for fatty acid sensing. The promoter region of thevc2105 gene, encoding a putative thioesterase, was mapped, and a putative FadR-binding site (AA CTG GTA AGA GCA CTT) was proposed. Different versions of the FadR regulatory proteins were prepared and purified to homogeneity. Both electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) determined the direct interaction of thevc2105 gene with FadR proteins of various origins. Further, EMSAs illustrated that the addition of long-chain acyl-coenzyme A (CoA) species efficiently dissociates thevc2105 promoter from the FadR regulator. The expression level of theVibrio cholerae vc2105 gene was elevated 2- to 3-fold in afadR null mutant strain, validating that FadR is a repressor for thevc2105 gene. The β-galactosidase activity of avc2105-lacZ transcriptional fusion was increased over 2-fold upon supplementation of growth medium with oleic acid. Unlike thefadD gene, a member of theVibrio fad regulon, the VC2105 protein played no role in bacterial growth and virulence-associated gene expression ofctxAB (cholera toxin A/B) andtcpA (toxin coregulated pilus A). Given that the transcriptional regulation ofvc2105 fits the criteria for fatty acid degradation (fad ) genes, we suggested that it is a new member of theVibrio fad regulon.IMPORTANCE TheVibrio FadR regulator is unusual in that it has two ligand-binding sites. Different versions of the FadR regulatory proteins were prepared and characterizedin vitro andin vivo . An auxiliaryfad gene (vc2105 ) fromVibrio was proposed that encodes a putative thioesterase and has a predicted FadR-binding site (AAC TGG TA A GAG CAC TT). The function of this putative binding site was proved using both EMSA and SPR. Furtherin vitro andin vivo experiments revealed that theVibrio FadR is a repressor for thevc2105 gene. UnlikefadD , a member of theVibrio fad regulon, VC2105 played no role in bacterial growth and expression of the two virulence-associated genes (ctxAB andtcpA ). Therefore, since transcriptional regulation ofvc2105 fits the criteria forfad genes, it seems likely thatvc2105 acts as a new auxiliary member of theVibrio fad regulon.