Structural and Functional Characterization of the FadR Regulatory Protein from Vibrio alginolyticus

Abstract

The structure of <i>Vibrio cholerae</i> FadR (VcFadR) complexed with the ligand oleoyl-CoA suggests an additional ligand-binding site. However, the fatty acid metabolism and its regulation is poorly addressed in <i>Vibrio alginolyticus</i>, a species closely-related to <i>V. cholerae</i>. Here, we show crystal structures of <i>V. alginolyticus</i> FadR (ValFadR) alone and its complex with the palmitoyl-CoA, a long-chain fatty acyl ligand different from the oleoyl-CoA occupied by VcFadR. Structural comparison indicates that both VcFadR and ValFadR consistently have an additional ligand-binding site (called site 2), which leads to more dramatic conformational-change of DNA-binding domain than that of the <i>E. coli</i> FadR (EcFadR). Isothermal titration calorimetry (ITC) analyses defines that the ligand-binding pattern of ValFadR (2:1) is distinct from that of EcFadR (1:1). Together with surface plasmon resonance (SPR), electrophoresis mobility shift assay (EMSA) demonstrates that ValFadR binds <i>fabA</i>, an important gene of unsaturated fatty acid (UFA) synthesis. The removal of <i>fadR</i> from <i>V. cholerae</i> attenuates <i>fabA</i> transcription and results in the unbalance of UFA/SFA incorporated into membrane phospholipids. Genetic complementation of the mutant version of <i>fadR</i> (Δ42, 136-177) lacking site 2 cannot restore the defective phenotypes of Δ<i>fadR</i> while the wild-type <i>fadR</i> gene and addition of exogenous oleate can restore them. Mice experiments reveals that VcFadR and its site 2 have roles in bacterial colonizing. Together, the results might represent an additional example that illustrates the <i>Vibrio</i> FadR-mediated lipid regulation and its role in pathogenesis.