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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

Characterization ofS. oneidensisFadR protein. (A) Sequence analyses of three different FadR homologues. The multiple alignments of FadR protein sequences were performed using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html), and the resultant output was processed by program ESPript 2.2 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi), generating the final BLAST photography (Feng & Cronan, 2011b). Identical residues are in white letters with red background, similar residues are in black letters in yellow background, the varied residues are in grey letters, and gaps are denoted with dots. In light of the structural architecture of E. coli FadR protein (PDB:1E2X) (van Aalten et al., 2000), the protein secondary structure was illustrated in cartoon (on top) (Zhang et al., 2014), α: alpha-helix; β: beta-sheet; T: Turn; η: coil. The seven known DNA-binding sites (R35, T44, R45, T46, T47, R49 and 65H) are highlighted with black triangles (Xu et al., 2001), the three known ligand-binding sites are shown with grey triangles (216G, 219S and 223W) (van Aalten et al., 2001), and the newly-proposed amino acids with indirect role for FadR-DNA interaction are highlighted with blue arrows (W60, F74 and W75) (Zhang et al., 2014). The extra 40-aa (138–177) longer region of V. cholerae FadR was underlined in blue. The FadR sequences are separately sampled from E. coli K12 (Accession no.: CAA30881), V. cholerae (Vibrio cholerae) (Accession no.: AAO37924) and S. oneidensis (Accession no.: NP_718457). (B) Gel exclusion chromatographic profile of the recombinant S. oneidensis FadR protein run on a Superdex 75 column (GE Healthcare). The expected peak of the target FadR was eluted at the position of 10.5 mL (highlighted with an arrow). The inset gel is the 15% SDS-PAGE photography of the collected S. oneidensis FadR protein sample. The mass of the monomeric S. oneidensis FadR is estimated to be ∼27 kDa. Abbreviations: M, protein marker; OD280, optical density at 280 nm; mAu, milli-absorbance units. The ruler on the top was given to describe the elution pattern of the standard proteins (Pharmacia). The standards used here included Ferritin (∼440 kDa), Aldolase (153 kDa), Bovine serum albumin (∼67 kDa), Ovalbumin (∼44 kDa) and ribonuclease (∼13.7 kDa), respectively. (C) Chemical cross-linking analyses for the purified S. oneidensis FadR protein. The level of EGS chemical cross-linker was illustrated with a triangle varies from 0, 0.1, 0.2, 0.5, 1.0, 1.5, to 2.0 µmol/L. (D) MS determination of the recombinant S. oneidensis FadR protein. The matched amino acid residues that exhibited 69% coverage to the native S. oneidensis FadR are given bold and underlined type
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Fig2: Characterization ofS. oneidensisFadR protein. (A) Sequence analyses of three different FadR homologues. The multiple alignments of FadR protein sequences were performed using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html), and the resultant output was processed by program ESPript 2.2 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi), generating the final BLAST photography (Feng & Cronan, 2011b). Identical residues are in white letters with red background, similar residues are in black letters in yellow background, the varied residues are in grey letters, and gaps are denoted with dots. In light of the structural architecture of E. coli FadR protein (PDB:1E2X) (van Aalten et al., 2000), the protein secondary structure was illustrated in cartoon (on top) (Zhang et al., 2014), α: alpha-helix; β: beta-sheet; T: Turn; η: coil. The seven known DNA-binding sites (R35, T44, R45, T46, T47, R49 and 65H) are highlighted with black triangles (Xu et al., 2001), the three known ligand-binding sites are shown with grey triangles (216G, 219S and 223W) (van Aalten et al., 2001), and the newly-proposed amino acids with indirect role for FadR-DNA interaction are highlighted with blue arrows (W60, F74 and W75) (Zhang et al., 2014). The extra 40-aa (138–177) longer region of V. cholerae FadR was underlined in blue. The FadR sequences are separately sampled from E. coli K12 (Accession no.: CAA30881), V. cholerae (Vibrio cholerae) (Accession no.: AAO37924) and S. oneidensis (Accession no.: NP_718457). (B) Gel exclusion chromatographic profile of the recombinant S. oneidensis FadR protein run on a Superdex 75 column (GE Healthcare). The expected peak of the target FadR was eluted at the position of 10.5 mL (highlighted with an arrow). The inset gel is the 15% SDS-PAGE photography of the collected S. oneidensis FadR protein sample. The mass of the monomeric S. oneidensis FadR is estimated to be ∼27 kDa. Abbreviations: M, protein marker; OD280, optical density at 280 nm; mAu, milli-absorbance units. The ruler on the top was given to describe the elution pattern of the standard proteins (Pharmacia). The standards used here included Ferritin (∼440 kDa), Aldolase (153 kDa), Bovine serum albumin (∼67 kDa), Ovalbumin (∼44 kDa) and ribonuclease (∼13.7 kDa), respectively. (C) Chemical cross-linking analyses for the purified S. oneidensis FadR protein. The level of EGS chemical cross-linker was illustrated with a triangle varies from 0, 0.1, 0.2, 0.5, 1.0, 1.5, to 2.0 µmol/L. (D) MS determination of the recombinant S. oneidensis FadR protein. The matched amino acid residues that exhibited 69% coverage to the native S. oneidensis FadR are given bold and underlined type

Mentions: An earlier study (Iram & Cronan, 2005) has found that the FadR lipid metabolism regulator of V. cholerae has an unusual insert of 40 residues. Our results (submitted) plus Shi’s observations (Shi et al., 2015) revealed an unexpected contribution of this unique inserting sequence in constituting an extra-ligand binding motif for FadR regulatory protein. The second ligand-binding site confers its excellent ability in fatty acid sensing. Given the fact that both V. cholerae and S. oneidensis are closely-related marine bacteria that shared a similar ecological niche with poor availability of fatty acids, we initially anticipated that this insert might be an indicator or relic for such kind of unparalleled regulation by FadR (Fig. 1). In fact, it is not this case. Multiple sequence alignments of three FadR proteins (FadR_ec for E. coli, FadR_vc for V. cholerae, and FadR_she for S. oneidensis) showed that: 1) the N-terminal DNA-binding motifs are very conserved featuring a full set of all the known residues critical for DNA binding; 2) the C-terminal ligand-interacting domains are appreciably diversified; and 3) the so-called insert of 40 residues (138–177 aa) is only present in FadR_vc (Fig. 2A). Considered the fact combined with atypical features seemed in fatty acid transport system, we favored the anticipation that Shewanella somewhat retains the evolutional relic that are partially observed with E. coli and Vibrio, respectively.Figure 2


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)

Characterization ofS. oneidensisFadR protein. (A) Sequence analyses of three different FadR homologues. The multiple alignments of FadR protein sequences were performed using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html), and the resultant output was processed by program ESPript 2.2 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi), generating the final BLAST photography (Feng & Cronan, 2011b). Identical residues are in white letters with red background, similar residues are in black letters in yellow background, the varied residues are in grey letters, and gaps are denoted with dots. In light of the structural architecture of E. coli FadR protein (PDB:1E2X) (van Aalten et al., 2000), the protein secondary structure was illustrated in cartoon (on top) (Zhang et al., 2014), α: alpha-helix; β: beta-sheet; T: Turn; η: coil. The seven known DNA-binding sites (R35, T44, R45, T46, T47, R49 and 65H) are highlighted with black triangles (Xu et al., 2001), the three known ligand-binding sites are shown with grey triangles (216G, 219S and 223W) (van Aalten et al., 2001), and the newly-proposed amino acids with indirect role for FadR-DNA interaction are highlighted with blue arrows (W60, F74 and W75) (Zhang et al., 2014). The extra 40-aa (138–177) longer region of V. cholerae FadR was underlined in blue. The FadR sequences are separately sampled from E. coli K12 (Accession no.: CAA30881), V. cholerae (Vibrio cholerae) (Accession no.: AAO37924) and S. oneidensis (Accession no.: NP_718457). (B) Gel exclusion chromatographic profile of the recombinant S. oneidensis FadR protein run on a Superdex 75 column (GE Healthcare). The expected peak of the target FadR was eluted at the position of 10.5 mL (highlighted with an arrow). The inset gel is the 15% SDS-PAGE photography of the collected S. oneidensis FadR protein sample. The mass of the monomeric S. oneidensis FadR is estimated to be ∼27 kDa. Abbreviations: M, protein marker; OD280, optical density at 280 nm; mAu, milli-absorbance units. The ruler on the top was given to describe the elution pattern of the standard proteins (Pharmacia). The standards used here included Ferritin (∼440 kDa), Aldolase (153 kDa), Bovine serum albumin (∼67 kDa), Ovalbumin (∼44 kDa) and ribonuclease (∼13.7 kDa), respectively. (C) Chemical cross-linking analyses for the purified S. oneidensis FadR protein. The level of EGS chemical cross-linker was illustrated with a triangle varies from 0, 0.1, 0.2, 0.5, 1.0, 1.5, to 2.0 µmol/L. (D) MS determination of the recombinant S. oneidensis FadR protein. The matched amino acid residues that exhibited 69% coverage to the native S. oneidensis FadR are given bold and underlined type
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Fig2: Characterization ofS. oneidensisFadR protein. (A) Sequence analyses of three different FadR homologues. The multiple alignments of FadR protein sequences were performed using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html), and the resultant output was processed by program ESPript 2.2 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi), generating the final BLAST photography (Feng & Cronan, 2011b). Identical residues are in white letters with red background, similar residues are in black letters in yellow background, the varied residues are in grey letters, and gaps are denoted with dots. In light of the structural architecture of E. coli FadR protein (PDB:1E2X) (van Aalten et al., 2000), the protein secondary structure was illustrated in cartoon (on top) (Zhang et al., 2014), α: alpha-helix; β: beta-sheet; T: Turn; η: coil. The seven known DNA-binding sites (R35, T44, R45, T46, T47, R49 and 65H) are highlighted with black triangles (Xu et al., 2001), the three known ligand-binding sites are shown with grey triangles (216G, 219S and 223W) (van Aalten et al., 2001), and the newly-proposed amino acids with indirect role for FadR-DNA interaction are highlighted with blue arrows (W60, F74 and W75) (Zhang et al., 2014). The extra 40-aa (138–177) longer region of V. cholerae FadR was underlined in blue. The FadR sequences are separately sampled from E. coli K12 (Accession no.: CAA30881), V. cholerae (Vibrio cholerae) (Accession no.: AAO37924) and S. oneidensis (Accession no.: NP_718457). (B) Gel exclusion chromatographic profile of the recombinant S. oneidensis FadR protein run on a Superdex 75 column (GE Healthcare). The expected peak of the target FadR was eluted at the position of 10.5 mL (highlighted with an arrow). The inset gel is the 15% SDS-PAGE photography of the collected S. oneidensis FadR protein sample. The mass of the monomeric S. oneidensis FadR is estimated to be ∼27 kDa. Abbreviations: M, protein marker; OD280, optical density at 280 nm; mAu, milli-absorbance units. The ruler on the top was given to describe the elution pattern of the standard proteins (Pharmacia). The standards used here included Ferritin (∼440 kDa), Aldolase (153 kDa), Bovine serum albumin (∼67 kDa), Ovalbumin (∼44 kDa) and ribonuclease (∼13.7 kDa), respectively. (C) Chemical cross-linking analyses for the purified S. oneidensis FadR protein. The level of EGS chemical cross-linker was illustrated with a triangle varies from 0, 0.1, 0.2, 0.5, 1.0, 1.5, to 2.0 µmol/L. (D) MS determination of the recombinant S. oneidensis FadR protein. The matched amino acid residues that exhibited 69% coverage to the native S. oneidensis FadR are given bold and underlined type
Mentions: An earlier study (Iram & Cronan, 2005) has found that the FadR lipid metabolism regulator of V. cholerae has an unusual insert of 40 residues. Our results (submitted) plus Shi’s observations (Shi et al., 2015) revealed an unexpected contribution of this unique inserting sequence in constituting an extra-ligand binding motif for FadR regulatory protein. The second ligand-binding site confers its excellent ability in fatty acid sensing. Given the fact that both V. cholerae and S. oneidensis are closely-related marine bacteria that shared a similar ecological niche with poor availability of fatty acids, we initially anticipated that this insert might be an indicator or relic for such kind of unparalleled regulation by FadR (Fig. 1). In fact, it is not this case. Multiple sequence alignments of three FadR proteins (FadR_ec for E. coli, FadR_vc for V. cholerae, and FadR_she for S. oneidensis) showed that: 1) the N-terminal DNA-binding motifs are very conserved featuring a full set of all the known residues critical for DNA binding; 2) the C-terminal ligand-interacting domains are appreciably diversified; and 3) the so-called insert of 40 residues (138–177 aa) is only present in FadR_vc (Fig. 2A). Considered the fact combined with atypical features seemed in fatty acid transport system, we favored the anticipation that Shewanella somewhat retains the evolutional relic that are partially observed with E. coli and Vibrio, respectively.Figure 2

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