RESEARCH ARTICLE

Binding of Shewanella FadR to the fabA fatty acid biosynthetic gene: implications for contraction of the fad regulon

  • Huimin Zhang 1 ,
  • Beiwen Zheng 1,2 ,
  • Rongsui Gao 1 ,
  • Youjun Feng , 1,2
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  • 1. Department of Medical Microbiology & Parasitology, Zhejiang University School of Medicine, Hangzhou 310058, China
  • 2. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University School of Medicine, Hangzhou 310058, China

Received date: 22 Apr 2015

Accepted date: 06 May 2015

Published date: 11 Sep 2015

Copyright

2014 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

The Escherichia coli fadR 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 γ–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 DNAbinding 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 crosslinking 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. oneidensis fabA 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. oneidensis fabA was fused to a LacZ reporter gene to engineer achromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of Shewanella fabA 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.

Cite this article

Huimin Zhang , Beiwen Zheng , Rongsui Gao , Youjun Feng . Binding of Shewanella FadR to the fabA fatty acid biosynthetic gene: implications for contraction of the fad regulon[J]. Protein & Cell, 2015 , 6(9) : 667 -679 . DOI: 10.1007/s13238-015-0172-2

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