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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

Role of LC fatty acyl-CoA infabAexpressionin vitroandin vivo. (A) EMSA-based visualization for effects of medium and long chain acyl-CoA species on binding of FadR_ec to the fabAshe probe. (B) Effects of different long chain acyl-CoA species on binding of FadR_she to the fabAshe probe. In the binding reaction mixtures (10 µL in total), the FadR (~5 pmol) was incubated with 0.2 pmol of DIG-labeled fabAshe probe. When required, acyl-CoA (~50 pmol) was added as we recently described (Feng & Cronan, 2011b). The gel shift assays were conducted for more than three times using 7% native PAGE, and the representative result is given. The shifted fabAshe probe band is indicated with a triangle (A) or an arrow (B). Designations C9:0, nonanoyl-CoA; C10:0, decanoyl-CoA; C16:0, palmitoyl-CoA; C18:0, stearoyl-CoA; C18:1, oleoyl-CoA. Abbreviations: ec and she denote E. coli and Shewanella, respectively. Plus sign denotes addition of either FadR proteins or acyl-CoA species, whereas minus sign denotes no addition of either FadR protein or acyl-CoA species. (C) Transcription of fabAshe is activated by FadR in E. coli and repressed upon oleic acid supplementation. All the E. coli strains used here carried a single copy of fabAshe-lacZ transcriptional fusion which is integrated on chromosomes. Bacterial cultures in mid-log phase were collected for assaying the LacZ (β-gal) activity. The three strains used here included FYJ241 (WT), FYJ246 (ΔfadR), and FYJ247 (ΔfabR), respectively. As anticipated, transcription of fabA she is also negatively regulated by FabR in E. coli
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Fig6: Role of LC fatty acyl-CoA infabAexpressionin vitroandin vivo. (A) EMSA-based visualization for effects of medium and long chain acyl-CoA species on binding of FadR_ec to the fabAshe probe. (B) Effects of different long chain acyl-CoA species on binding of FadR_she to the fabAshe probe. In the binding reaction mixtures (10 µL in total), the FadR (~5 pmol) was incubated with 0.2 pmol of DIG-labeled fabAshe probe. When required, acyl-CoA (~50 pmol) was added as we recently described (Feng & Cronan, 2011b). The gel shift assays were conducted for more than three times using 7% native PAGE, and the representative result is given. The shifted fabAshe probe band is indicated with a triangle (A) or an arrow (B). Designations C9:0, nonanoyl-CoA; C10:0, decanoyl-CoA; C16:0, palmitoyl-CoA; C18:0, stearoyl-CoA; C18:1, oleoyl-CoA. Abbreviations: ec and she denote E. coli and Shewanella, respectively. Plus sign denotes addition of either FadR proteins or acyl-CoA species, whereas minus sign denotes no addition of either FadR protein or acyl-CoA species. (C) Transcription of fabAshe is activated by FadR in E. coli and repressed upon oleic acid supplementation. All the E. coli strains used here carried a single copy of fabAshe-lacZ transcriptional fusion which is integrated on chromosomes. Bacterial cultures in mid-log phase were collected for assaying the LacZ (β-gal) activity. The three strains used here included FYJ241 (WT), FYJ246 (ΔfadR), and FYJ247 (ΔfabR), respectively. As anticipated, transcription of fabA she is also negatively regulated by FabR in E. coli

Mentions: Given the fact that long-chain (but not short chain) fatty acyl-CoA species can antagonize the DNA-binding activity of FadR with origins of E. coli (Henry & Cronan, 1992, Cronan, 1997) and Vibrio (Iram & Cronan, 2005, Feng & Cronan, 2011b), it is of much interest to test the behaviors of theses ligands in the case of S. oneidensis FadR. Therefore, we tested six acyl-CoA species of different acyl chain lengths. The EMSA-based competition assays showed that medium-chain acyl-CoA (C9:0; C10:0) don’t interfere with the fabA_she binding to either FadR_ec (Fig. 6A) or FadR_she (Fig. 6B). In contrast, the long-chain acyl-CoA species (C16:0, C16:1, C18: 0 and C18:1) strongly impaired DNA binding (Fig. 6A and 6B). We believed that long-chain but not medium-chain acyl-CoA can specifically interact with FadR_she ligand-binding site and release FadR_she from the S. oneidensisfabA promoter. In summary, the in vitro data accumulated suggest that long-chain acyl-CoAs regulate the Shewanella fabA transcription via their interaction with the FadR protein.Figure 6


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)

Role of LC fatty acyl-CoA infabAexpressionin vitroandin vivo. (A) EMSA-based visualization for effects of medium and long chain acyl-CoA species on binding of FadR_ec to the fabAshe probe. (B) Effects of different long chain acyl-CoA species on binding of FadR_she to the fabAshe probe. In the binding reaction mixtures (10 µL in total), the FadR (~5 pmol) was incubated with 0.2 pmol of DIG-labeled fabAshe probe. When required, acyl-CoA (~50 pmol) was added as we recently described (Feng & Cronan, 2011b). The gel shift assays were conducted for more than three times using 7% native PAGE, and the representative result is given. The shifted fabAshe probe band is indicated with a triangle (A) or an arrow (B). Designations C9:0, nonanoyl-CoA; C10:0, decanoyl-CoA; C16:0, palmitoyl-CoA; C18:0, stearoyl-CoA; C18:1, oleoyl-CoA. Abbreviations: ec and she denote E. coli and Shewanella, respectively. Plus sign denotes addition of either FadR proteins or acyl-CoA species, whereas minus sign denotes no addition of either FadR protein or acyl-CoA species. (C) Transcription of fabAshe is activated by FadR in E. coli and repressed upon oleic acid supplementation. All the E. coli strains used here carried a single copy of fabAshe-lacZ transcriptional fusion which is integrated on chromosomes. Bacterial cultures in mid-log phase were collected for assaying the LacZ (β-gal) activity. The three strains used here included FYJ241 (WT), FYJ246 (ΔfadR), and FYJ247 (ΔfabR), respectively. As anticipated, transcription of fabA she is also negatively regulated by FabR in E. coli
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig6: Role of LC fatty acyl-CoA infabAexpressionin vitroandin vivo. (A) EMSA-based visualization for effects of medium and long chain acyl-CoA species on binding of FadR_ec to the fabAshe probe. (B) Effects of different long chain acyl-CoA species on binding of FadR_she to the fabAshe probe. In the binding reaction mixtures (10 µL in total), the FadR (~5 pmol) was incubated with 0.2 pmol of DIG-labeled fabAshe probe. When required, acyl-CoA (~50 pmol) was added as we recently described (Feng & Cronan, 2011b). The gel shift assays were conducted for more than three times using 7% native PAGE, and the representative result is given. The shifted fabAshe probe band is indicated with a triangle (A) or an arrow (B). Designations C9:0, nonanoyl-CoA; C10:0, decanoyl-CoA; C16:0, palmitoyl-CoA; C18:0, stearoyl-CoA; C18:1, oleoyl-CoA. Abbreviations: ec and she denote E. coli and Shewanella, respectively. Plus sign denotes addition of either FadR proteins or acyl-CoA species, whereas minus sign denotes no addition of either FadR protein or acyl-CoA species. (C) Transcription of fabAshe is activated by FadR in E. coli and repressed upon oleic acid supplementation. All the E. coli strains used here carried a single copy of fabAshe-lacZ transcriptional fusion which is integrated on chromosomes. Bacterial cultures in mid-log phase were collected for assaying the LacZ (β-gal) activity. The three strains used here included FYJ241 (WT), FYJ246 (ΔfadR), and FYJ247 (ΔfabR), respectively. As anticipated, transcription of fabA she is also negatively regulated by FabR in E. coli
Mentions: Given the fact that long-chain (but not short chain) fatty acyl-CoA species can antagonize the DNA-binding activity of FadR with origins of E. coli (Henry & Cronan, 1992, Cronan, 1997) and Vibrio (Iram & Cronan, 2005, Feng & Cronan, 2011b), it is of much interest to test the behaviors of theses ligands in the case of S. oneidensis FadR. Therefore, we tested six acyl-CoA species of different acyl chain lengths. The EMSA-based competition assays showed that medium-chain acyl-CoA (C9:0; C10:0) don’t interfere with the fabA_she binding to either FadR_ec (Fig. 6A) or FadR_she (Fig. 6B). In contrast, the long-chain acyl-CoA species (C16:0, C16:1, C18: 0 and C18:1) strongly impaired DNA binding (Fig. 6A and 6B). We believed that long-chain but not medium-chain acyl-CoA can specifically interact with FadR_she ligand-binding site and release FadR_she from the S. oneidensisfabA promoter. In summary, the in vitro data accumulated suggest that long-chain acyl-CoAs regulate the Shewanella fabA transcription via their interaction with the FadR protein.Figure 6

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