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

Zhang H, Zheng B, Gao R, Feng Y - Protein Cell (2015)

Bottom Line: 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 a chromosome fabA-lacZ transcriptional fusion in E. coli.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology & Parasitology, Zhejiang University School of Medicine, Hangzhou, 310058, China.

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 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. 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 a chromosome 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.

No MeSH data available.


Related in: MedlinePlus

SPR-based dynamic analyses for binding offabAtoShewanellaFadR. (A) SPR assay for interaction between fabA and FadR_she. (B) Measurement of the KD value for fabA-FadR_she
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Fig5: SPR-based dynamic analyses for binding offabAtoShewanellaFadR. (A) SPR assay for interaction between fabA and FadR_she. (B) Measurement of the KD value for fabA-FadR_she

Mentions: Through sequence comparison of the fabA and fabB promoter regions of E. coli, V. cholerae and S. oneidensis, we found that the cognate FadR-specific binding site in front of the fabA promoter regions of these three bacterial species are much more conservative (Fig. S1A), but that of fabB promoter region is not (Fig. S1B). This observation is generally consistent with the prediction by Rodionov and coworkers (Rodionov et al., 2011) that only fabA (not fabB) of Shewanella has a binding site for the FadR regulator. To further prove the function of this predicted site, termed fabA probe, we synthesized it using the approach of annealing the two complementary DNA strand. This DNA probe is digoxigenin-labeled DNA fragment of 56 bp that overlaps the candidate FadR_she binding site (Fig. S1A and Table 2). Gel shift assays confirmed that FadR_ec (Fig. 4A) and FadR_she (Fig. 4B) both can efficiently bind the S. oneidensisfabA promoter. In much similarity to the scenario seen with FadR_ec here (Fig. 4A), plus our former observations with FadR regulators of E. coli (Feng & Cronan, 2009b, Feng & Cronan, 2010) and V. cholerae (Feng & Cronan, 2011b), we also found that the fabA_she probe binds FadR_she protein in a dose-dependent manner (Fig. 4B). To preliminarily elucidate the kinetics of FadR_she/fabA_she interaction, we conducted surface plasom resonance (SPR)-based measurements. SPR results revealed that the binding affinity (KD) of fabA_she to FadR_she is roughly 436 nmol/L (Fig. 5A and 5B), and the binding mode is 2:1 (a dimeric FadR protein is bound to a target DNA fragment (not shown).Figure 4


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

Zhang H, Zheng B, Gao R, Feng Y - Protein Cell (2015)

SPR-based dynamic analyses for binding offabAtoShewanellaFadR. (A) SPR assay for interaction between fabA and FadR_she. (B) Measurement of the KD value for fabA-FadR_she
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4537474&req=5

Fig5: SPR-based dynamic analyses for binding offabAtoShewanellaFadR. (A) SPR assay for interaction between fabA and FadR_she. (B) Measurement of the KD value for fabA-FadR_she
Mentions: Through sequence comparison of the fabA and fabB promoter regions of E. coli, V. cholerae and S. oneidensis, we found that the cognate FadR-specific binding site in front of the fabA promoter regions of these three bacterial species are much more conservative (Fig. S1A), but that of fabB promoter region is not (Fig. S1B). This observation is generally consistent with the prediction by Rodionov and coworkers (Rodionov et al., 2011) that only fabA (not fabB) of Shewanella has a binding site for the FadR regulator. To further prove the function of this predicted site, termed fabA probe, we synthesized it using the approach of annealing the two complementary DNA strand. This DNA probe is digoxigenin-labeled DNA fragment of 56 bp that overlaps the candidate FadR_she binding site (Fig. S1A and Table 2). Gel shift assays confirmed that FadR_ec (Fig. 4A) and FadR_she (Fig. 4B) both can efficiently bind the S. oneidensisfabA promoter. In much similarity to the scenario seen with FadR_ec here (Fig. 4A), plus our former observations with FadR regulators of E. coli (Feng & Cronan, 2009b, Feng & Cronan, 2010) and V. cholerae (Feng & Cronan, 2011b), we also found that the fabA_she probe binds FadR_she protein in a dose-dependent manner (Fig. 4B). To preliminarily elucidate the kinetics of FadR_she/fabA_she interaction, we conducted surface plasom resonance (SPR)-based measurements. SPR results revealed that the binding affinity (KD) of fabA_she to FadR_she is roughly 436 nmol/L (Fig. 5A and 5B), and the binding mode is 2:1 (a dimeric FadR protein is bound to a target DNA fragment (not shown).Figure 4

Bottom Line: 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 a chromosome fabA-lacZ transcriptional fusion in E. coli.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology & Parasitology, Zhejiang University School of Medicine, Hangzhou, 310058, China.

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 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. 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 a chromosome 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.

No MeSH data available.


Related in: MedlinePlus