Abstract
The Escherichia colifadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other Ɣ–proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensisfabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensisfabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of ShewanellafabA expression by β-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid synthesis in the marine bacteria Shewanella genus.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0172-2) contains supplementary material, which is available to authorized users.
Highlights
Current knowledge on the regulation of fatty acid metabolism is mostly from studies with Escherichia coli (E. coli)
The data reported here defined that the Shewanella FadR homologue is a functional regulator with the involvement of fatty acid metabolism
We experimentally proved the proposal by Rodionov et al (Rodionov et al, 2011) that fad regulon is contracted in Shewanella
Summary
Current knowledge on the regulation of fatty acid metabolism is mostly from studies with Escherichia coli (E. coli). The accumulated crystal structures of FadR alone and its complex with DNA/acyl-CoA defined clearly the structural basis for FadR-mediated regulatory mechanism in the context of lipid metabolism (van Aalten et al, 2001, van Aalten et al, 2000, Xu et al, 2001). The mechanism by which LCFA induces fad expression lies in the fact that the binding of LCFA acyl-CoA to FadR protein results in the alteration of protein configuration, which in turn triggers the loss of its DNA binding ability. It still remains unclear why the unexpected functional diversity exists amongst the FadR regulatory proteins (Iram & Cronan, 2005). Unlike the scenario seen with its closely-relative V. cholerae, the FadR homologue from the other marine bacterium Shewanella, is quite similar to that of the paradigm organism E. coli
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