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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

The working model proposed forfadregulon and its regulation inShewanellagenus. Multiple sequence alignments (A) and sequence logo (C) of the known palindromes recognized by E. coli FadR. Sequence analyses (B) and sequence logo (D) of the predicted FadR-binding sites of Shewanella. The alignment of DNA sequences was carried out using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html), and the output was given via processing with the program ESPript 2.2. (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) (Feng & Cronan, 2011b). Identical residues are in white letters with red background, similar residues are in the form with mixture of red/black letters, and the varied residues are in black letters. Sequence logo of the FadR-binding sites was generated using the program of WebLogo (http://weblogo.berkeley.edu/logo.cgi). The sequences of the known E. coli FadR sites were sampled from E. coli K-12 MG1655 (http://regprecise.lbl.gov/RegPrecise/regulon.jsp?regulon_id=10286), and the putative Shewanella FadR sites were collected from S. oneidensis MR-1(http://regprecise.lbl.gov/RegPrecise/regulon.jsp?regulon_id=5431). (E) In the absence of a long chain acyl-CoA, E. coli FadR of E. coli and S. oneidensis represses the fad regulon genes, whereas it activates transcription of fabA (and/or fabB) with critical roles in the unsaturated fatty acid synthetic pathway. (F) Binding of long chain acyl-CoA species leads to the release of FadR protein from its operator sites. The fad members of Shewanella are in red except that fabA is highlighted in green. Such kind of FadR-DNA dissociation increases fad regulon expression whereas reduces the expression of fabA (and/or fabB). The oval denotes FadR regulatory protein whereas the small open circle represents the acyl-CoA pool
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Fig1: The working model proposed forfadregulon and its regulation inShewanellagenus. Multiple sequence alignments (A) and sequence logo (C) of the known palindromes recognized by E. coli FadR. Sequence analyses (B) and sequence logo (D) of the predicted FadR-binding sites of Shewanella. The alignment of DNA sequences was carried out using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html), and the output was given via processing with the program ESPript 2.2. (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) (Feng & Cronan, 2011b). Identical residues are in white letters with red background, similar residues are in the form with mixture of red/black letters, and the varied residues are in black letters. Sequence logo of the FadR-binding sites was generated using the program of WebLogo (http://weblogo.berkeley.edu/logo.cgi). The sequences of the known E. coli FadR sites were sampled from E. coli K-12 MG1655 (http://regprecise.lbl.gov/RegPrecise/regulon.jsp?regulon_id=10286), and the putative Shewanella FadR sites were collected from S. oneidensis MR-1(http://regprecise.lbl.gov/RegPrecise/regulon.jsp?regulon_id=5431). (E) In the absence of a long chain acyl-CoA, E. coli FadR of E. coli and S. oneidensis represses the fad regulon genes, whereas it activates transcription of fabA (and/or fabB) with critical roles in the unsaturated fatty acid synthetic pathway. (F) Binding of long chain acyl-CoA species leads to the release of FadR protein from its operator sites. The fad members of Shewanella are in red except that fabA is highlighted in green. Such kind of FadR-DNA dissociation increases fad regulon expression whereas reduces the expression of fabA (and/or fabB). The oval denotes FadR regulatory protein whereas the small open circle represents the acyl-CoA pool

Mentions: In relative to E. coli that has no less than 12 fad members, those genes controlled by the fatty acid-responsive FadR regulator (Fig. 1A, 1C and 1E), the fad members seemed to be contracted in the cousin S. oneidensis in that only 4 well-known fad genes/operons (fadE, fadL, fadIJ and fabA) has the putative FadR-binding sites (Fig. 1B, 1D and 1F). Also, S. oneidensis possesses two more new FadR-regulated genes (SO_4761 encoding the GNAT family of Acetyltransferease and SO_0572 representing a possible Enoyl-CoA hydratase (EC 4.2.1.17)) (Fig. 1B and 1F), somewhat suggesting the expansion of limited fad members in this marine bacterium. However, this observation argues the possibility of gene horizontal transfer in that the GC contents of two genes SO_4761 (45.23%) and SO_0572 (46.84%) is similar to that of the whole genome (46%). Unlike E. coli that encodes only one FadL fatty acid transporter (Blattner et al., 1997), S. oneidensis has three FadL-like homologues (FadL-1 (SO_3099, 440 aa), FadL-2 (SO_3276, 311 aa) and FadL-3 (SO_4232, 437 aa)) (Heidelberg et al., 2002), only FadL-1 of which is directly regulated by FadR regulatory protein (Fig. 1B and 1F). This situation seems unusual, but not without any precedent. The similar scenario was observed in V. cholerae, the other marine bacterium since three FadL orthologues are distributed in its two chromosomes (Heidelberg et al., 2000), and only FadL-2 is regulated by FadR repressor (http://regprecise.lbl.gov/RegPrecise/regulon.jsp?regulon_id=16367). It is reasonable that three FadL transporters coupled with one FadD inner-membrane protein (3FadL-1FadD) constitute a more efficient system of fatty acid uptake than the prototypical version of 1FadL-1FadD in E. coli. Unlike the V. cholerae FadL-2 that has only one FadR-recognizable site not shown), S. oneidensis FadL-1 exhibits two tandem FadR-binding sites (Fig. 1B), similar to the scenario seen in E. coli FadL (Fig. 1A). Different from the E. colifadD that also carries two tandem FadR-specific palindromes, the fadD gene with origins of both S. oneidensis and V. cholerae seems not to be regulated by the FadR regulator in that the typical site is cryptic (Fig. 1B). In views of genomic evolutions, we anticipated that S. oneidensis has the relics of both E. coli and Vibrio in the context of fatty acid transporter system.Figure 1


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)

The working model proposed forfadregulon and its regulation inShewanellagenus. Multiple sequence alignments (A) and sequence logo (C) of the known palindromes recognized by E. coli FadR. Sequence analyses (B) and sequence logo (D) of the predicted FadR-binding sites of Shewanella. The alignment of DNA sequences was carried out using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html), and the output was given via processing with the program ESPript 2.2. (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) (Feng & Cronan, 2011b). Identical residues are in white letters with red background, similar residues are in the form with mixture of red/black letters, and the varied residues are in black letters. Sequence logo of the FadR-binding sites was generated using the program of WebLogo (http://weblogo.berkeley.edu/logo.cgi). The sequences of the known E. coli FadR sites were sampled from E. coli K-12 MG1655 (http://regprecise.lbl.gov/RegPrecise/regulon.jsp?regulon_id=10286), and the putative Shewanella FadR sites were collected from S. oneidensis MR-1(http://regprecise.lbl.gov/RegPrecise/regulon.jsp?regulon_id=5431). (E) In the absence of a long chain acyl-CoA, E. coli FadR of E. coli and S. oneidensis represses the fad regulon genes, whereas it activates transcription of fabA (and/or fabB) with critical roles in the unsaturated fatty acid synthetic pathway. (F) Binding of long chain acyl-CoA species leads to the release of FadR protein from its operator sites. The fad members of Shewanella are in red except that fabA is highlighted in green. Such kind of FadR-DNA dissociation increases fad regulon expression whereas reduces the expression of fabA (and/or fabB). The oval denotes FadR regulatory protein whereas the small open circle represents the acyl-CoA pool
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: The working model proposed forfadregulon and its regulation inShewanellagenus. Multiple sequence alignments (A) and sequence logo (C) of the known palindromes recognized by E. coli FadR. Sequence analyses (B) and sequence logo (D) of the predicted FadR-binding sites of Shewanella. The alignment of DNA sequences was carried out using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html), and the output was given via processing with the program ESPript 2.2. (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) (Feng & Cronan, 2011b). Identical residues are in white letters with red background, similar residues are in the form with mixture of red/black letters, and the varied residues are in black letters. Sequence logo of the FadR-binding sites was generated using the program of WebLogo (http://weblogo.berkeley.edu/logo.cgi). The sequences of the known E. coli FadR sites were sampled from E. coli K-12 MG1655 (http://regprecise.lbl.gov/RegPrecise/regulon.jsp?regulon_id=10286), and the putative Shewanella FadR sites were collected from S. oneidensis MR-1(http://regprecise.lbl.gov/RegPrecise/regulon.jsp?regulon_id=5431). (E) In the absence of a long chain acyl-CoA, E. coli FadR of E. coli and S. oneidensis represses the fad regulon genes, whereas it activates transcription of fabA (and/or fabB) with critical roles in the unsaturated fatty acid synthetic pathway. (F) Binding of long chain acyl-CoA species leads to the release of FadR protein from its operator sites. The fad members of Shewanella are in red except that fabA is highlighted in green. Such kind of FadR-DNA dissociation increases fad regulon expression whereas reduces the expression of fabA (and/or fabB). The oval denotes FadR regulatory protein whereas the small open circle represents the acyl-CoA pool
Mentions: In relative to E. coli that has no less than 12 fad members, those genes controlled by the fatty acid-responsive FadR regulator (Fig. 1A, 1C and 1E), the fad members seemed to be contracted in the cousin S. oneidensis in that only 4 well-known fad genes/operons (fadE, fadL, fadIJ and fabA) has the putative FadR-binding sites (Fig. 1B, 1D and 1F). Also, S. oneidensis possesses two more new FadR-regulated genes (SO_4761 encoding the GNAT family of Acetyltransferease and SO_0572 representing a possible Enoyl-CoA hydratase (EC 4.2.1.17)) (Fig. 1B and 1F), somewhat suggesting the expansion of limited fad members in this marine bacterium. However, this observation argues the possibility of gene horizontal transfer in that the GC contents of two genes SO_4761 (45.23%) and SO_0572 (46.84%) is similar to that of the whole genome (46%). Unlike E. coli that encodes only one FadL fatty acid transporter (Blattner et al., 1997), S. oneidensis has three FadL-like homologues (FadL-1 (SO_3099, 440 aa), FadL-2 (SO_3276, 311 aa) and FadL-3 (SO_4232, 437 aa)) (Heidelberg et al., 2002), only FadL-1 of which is directly regulated by FadR regulatory protein (Fig. 1B and 1F). This situation seems unusual, but not without any precedent. The similar scenario was observed in V. cholerae, the other marine bacterium since three FadL orthologues are distributed in its two chromosomes (Heidelberg et al., 2000), and only FadL-2 is regulated by FadR repressor (http://regprecise.lbl.gov/RegPrecise/regulon.jsp?regulon_id=16367). It is reasonable that three FadL transporters coupled with one FadD inner-membrane protein (3FadL-1FadD) constitute a more efficient system of fatty acid uptake than the prototypical version of 1FadL-1FadD in E. coli. Unlike the V. cholerae FadL-2 that has only one FadR-recognizable site not shown), S. oneidensis FadL-1 exhibits two tandem FadR-binding sites (Fig. 1B), similar to the scenario seen in E. coli FadL (Fig. 1A). Different from the E. colifadD that also carries two tandem FadR-specific palindromes, the fadD gene with origins of both S. oneidensis and V. cholerae seems not to be regulated by the FadR regulator in that the typical site is cryptic (Fig. 1B). In views of genomic evolutions, we anticipated that S. oneidensis has the relics of both E. coli and Vibrio in the context of fatty acid transporter system.Figure 1

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