Limits...
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

ShewanellaFadR protein is functionally-exchangeable to the paradigmE. coliversion. (A) EMSA-based evidence for binding of E. coli fabA promoter to FadR protein of three origins. (B) EMSA analyses for crosstalk of E. coli fabA promoter with three kinds of bacterial FadR proteins. A representative photography was given here, which were from no less than three independent EMSA experiments (7% native PAGE). Three versions of FadR protein here are FadR_ec, FadR_vc and FadR_she, respectively. In gel shift assays, the FadR protein (5 pmol) is incubated with DIG-labeled fabAec (or fabBec) probe (0.2 pmol). Note: An unexpected but interesting scenario “super-shift” is consistently observed in our trials with V. cholerae FadR
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4537474&req=5

Fig3: ShewanellaFadR protein is functionally-exchangeable to the paradigmE. coliversion. (A) EMSA-based evidence for binding of E. coli fabA promoter to FadR protein of three origins. (B) EMSA analyses for crosstalk of E. coli fabA promoter with three kinds of bacterial FadR proteins. A representative photography was given here, which were from no less than three independent EMSA experiments (7% native PAGE). Three versions of FadR protein here are FadR_ec, FadR_vc and FadR_she, respectively. In gel shift assays, the FadR protein (5 pmol) is incubated with DIG-labeled fabAec (or fabBec) probe (0.2 pmol). Note: An unexpected but interesting scenario “super-shift” is consistently observed in our trials with V. cholerae FadR

Mentions: Gel shift assay was performed to detect the binding ability of FadR_ec, FadR_vc and FadR_she to the cognate DNA binding sites. As expected, EMSA-based experiments showed that the E. coli FadR protein (as the positive control) binds well to its own promoters of both fabA (Fig. 3A) and fabB (Fig. 3B) promoters. The fact that the FadR_vc protein gives consistently the super-shift bands for both fabA and fabB probes in the gel shift assays, is mostly attributed to the essence of its easy-forming the protein multimer (Fig. 3). Of note, FadR_she exhibited an excellent ability of interacting with the fabA (Fig. 3A) and fabB (Fig. 3B) with the origin of E. coli. It seemed likely that the FadR proteins of E. coli and S. oneidensis are functionally-equivalent (and/or exchangeable). It is not surprise since the FadR/FabR orthologue from other marine bacterium, Vibrio, also followed this rule (Feng & Cronan, 2011b, Feng & Cronan, 2011a). To our knowledge, the cases of similar functional exchange of transcriptional regulators can be extended to BioR, the other GntR-type regulators implicated into the metabolism of biotin, a sulfur-containing fatty acid (Feng et al., 2013a, Feng et al., 2013b). Thereby, it makes sense that the atypical regulation by FadR in UFA synthesis of Shewanella is due to the cryptic site in front of fabB (Fig. 1), not FadR_she (Fig. 3).Figure 3


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)

ShewanellaFadR protein is functionally-exchangeable to the paradigmE. coliversion. (A) EMSA-based evidence for binding of E. coli fabA promoter to FadR protein of three origins. (B) EMSA analyses for crosstalk of E. coli fabA promoter with three kinds of bacterial FadR proteins. A representative photography was given here, which were from no less than three independent EMSA experiments (7% native PAGE). Three versions of FadR protein here are FadR_ec, FadR_vc and FadR_she, respectively. In gel shift assays, the FadR protein (5 pmol) is incubated with DIG-labeled fabAec (or fabBec) probe (0.2 pmol). Note: An unexpected but interesting scenario “super-shift” is consistently observed in our trials with V. cholerae FadR
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4537474&req=5

Fig3: ShewanellaFadR protein is functionally-exchangeable to the paradigmE. coliversion. (A) EMSA-based evidence for binding of E. coli fabA promoter to FadR protein of three origins. (B) EMSA analyses for crosstalk of E. coli fabA promoter with three kinds of bacterial FadR proteins. A representative photography was given here, which were from no less than three independent EMSA experiments (7% native PAGE). Three versions of FadR protein here are FadR_ec, FadR_vc and FadR_she, respectively. In gel shift assays, the FadR protein (5 pmol) is incubated with DIG-labeled fabAec (or fabBec) probe (0.2 pmol). Note: An unexpected but interesting scenario “super-shift” is consistently observed in our trials with V. cholerae FadR
Mentions: Gel shift assay was performed to detect the binding ability of FadR_ec, FadR_vc and FadR_she to the cognate DNA binding sites. As expected, EMSA-based experiments showed that the E. coli FadR protein (as the positive control) binds well to its own promoters of both fabA (Fig. 3A) and fabB (Fig. 3B) promoters. The fact that the FadR_vc protein gives consistently the super-shift bands for both fabA and fabB probes in the gel shift assays, is mostly attributed to the essence of its easy-forming the protein multimer (Fig. 3). Of note, FadR_she exhibited an excellent ability of interacting with the fabA (Fig. 3A) and fabB (Fig. 3B) with the origin of E. coli. It seemed likely that the FadR proteins of E. coli and S. oneidensis are functionally-equivalent (and/or exchangeable). It is not surprise since the FadR/FabR orthologue from other marine bacterium, Vibrio, also followed this rule (Feng & Cronan, 2011b, Feng & Cronan, 2011a). To our knowledge, the cases of similar functional exchange of transcriptional regulators can be extended to BioR, the other GntR-type regulators implicated into the metabolism of biotin, a sulfur-containing fatty acid (Feng et al., 2013a, Feng et al., 2013b). Thereby, it makes sense that the atypical regulation by FadR in UFA synthesis of Shewanella is due to the cryptic site in front of fabB (Fig. 1), not FadR_she (Fig. 3).Figure 3

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