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

Abstract

The structure of Vibrio cholerae 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 Vibrio alginolyticus, a species closely-related to V. cholerae. Here, we show crystal structures of V. alginolyticus 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 E. coli 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 fabA, an important gene of unsaturated fatty acid (UFA) synthesis. The removal of fadR from V. cholerae attenuates fabA transcription and results in the unbalance of UFA/SFA incorporated into membrane phospholipids. Genetic complementation of the mutant version of fadR (Δ42, 136-177) lacking site 2 cannot restore the defective phenotypes of ΔfadR while the wild-type fadR 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 Vibrio FadR-mediated lipid regulation and its role in pathogenesis.

A single regulator NrtR controls bacterial NAD+ homeostasis via its acetylation

“…Wild-type MsNrtR and its point mutants were overexpressed in E. coli or M. smegmatis MC 2 155 (Magni et al, 2004). The expression of proteins in E. coli (BL21) was induced by the addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) (Gao et al, 2017). For protein purification, the cells were harvested by centrifugation and lysed by sonication.…”

“…For protein purification, the cells were harvested by centrifugation and lysed by sonication. The clarified lysate was loaded onto a Ni-nitrilotriacetic acid (Ni-NTA) column (Qiagen) and eluted with 150 mM imidazole (Gao et al, 2017). The protein preparation was further purified by gel filtration through a Superdex 75 10/300 column (GL, GE Healthcare) and the protein purity was judged with 12% SDS-PAGE.…”

Crystal structure and acetylation of BioQ suggests a novel regulatory switch for biotin biosynthesis in Mycobacterium smegmatis

“…The reaction system of gel shift (~20 μl) was maintained at room temperature for about half an hour (Tang et al, ; Zhang et al, ; ). Finally, the DNA/protein mixtures were separated with the native 7% PAGE at 80 V for 1.5 h, and the shifted DNA bands were visualized with ethidium bromide (EB) staining (Gao et al, ).…”

“…Polyclonal antiserum of MsBioQ was generated via the immunization of rabbit with the purified MsBioQ as we recently described with the Vibrio FadR regulatory protein (Gao et al, ). In brief, a female white New Zealand rabbit was immunized with MsBioQ for five times with 2‐week intervals for each injection.…”

Recent Progress in the Identification of Aptamers Against Bacterial Origins and Their Diagnostic Applications

“…It is resistant to salt and can thrive in oceans. It can cause infections in open wounds and ears in humans [ 153 ]. Zhao et al described a method of sensing this bacteria using magnetic beads [ 141 ].…”