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Probing regulon of ArcA in Shewanella oneidensis MR-1 by integrated genomic analyses.

Gao H, Wang X, Yang ZK, Palzkill T, Zhou J - BMC Genomics (2008)

Bottom Line: To further determine genes regulated by the Arc system, an ArcA recognition weight matrix from DNA-binding data and bioinformatics analysis was generated and used to produce an ArcA sequence affinity map.By combining both techniques, we identified an ArcA regulon of at least 50 operons, of which only 6 were found to be directly controlled by ArcA in E. coli.These results indicate that the Arc system in S. oneidensis differs from that in E. coli substantially in terms of its physiological function and regulon while their binding motif are strikingly similar.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Environmental Genomics and Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019, USA. haichun@ou.edu

ABSTRACT

Background: The Arc two-component system is a global regulator controlling many genes involved in aerobic/anaerobic respiration and fermentative metabolism in Escherichia coli. Shewanella oneidensis MR-1 contains a gene encoding a putative ArcA homolog with ~81% amino acid sequence identity to the E. coli ArcA protein but not a full-length arcB gene.

Results: To understand the role of ArcA in S. oneidensis, an arcA deletion strain was constructed and subjected to both physiological characterization and microarray analysis. Compared to the wild-type MR-1, the mutant exhibited impaired aerobic growth and a defect in utilizing DMSO in the absence of O2. Microarray analyses on cells grown aerobically and anaerobically on fumarate revealed that expression of 1009 genes was significantly affected (p < 0.05) by the mutation. In contrast to E. coli ArcA, the protein appears to be dispensable in regulation of the TCA cycle in S. oneidensis. To further determine genes regulated by the Arc system, an ArcA recognition weight matrix from DNA-binding data and bioinformatics analysis was generated and used to produce an ArcA sequence affinity map. By combining both techniques, we identified an ArcA regulon of at least 50 operons, of which only 6 were found to be directly controlled by ArcA in E. coli.

Conclusion: These results indicate that the Arc system in S. oneidensis differs from that in E. coli substantially in terms of its physiological function and regulon while their binding motif are strikingly similar.

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ArcA(-P) Binding to selected promoters by EMSA. (A). Overproduced and purified recombinant S. oneidensis His6-ArcA from E. coli BL21 cells. (B). Interaction of so1661 promoter DNA with S. oneidensis His6-ArcA. The probe was prepared by PCR with SO1661-EMSA-F (33P end-labeled) and SO1661-EMSA-R primers (Table S4 in additional file 5). The EMS assay was performed with 2 nM 33P end-labeled probes and various amounts of ArcA (left panel) or ArcA-P proteins (right panel). The protein concentrations for lanes 1–9 are 0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 4.0, 4.0 μM, respectively. Non-specific competitor DNA, (2 μg poly dI·dC), was added (lane 8) and specific competitor (10 μM unlabeled SO1661 probe) was added (lane 9). (C). The binding assay was performed in the presence of 0, 1, or 2 μM ArcA-P and 2–5 nM radiolabeled promoter DNA 0.2 μg/μl poly(dI·dC) was used in all these binding reactions to block non-specific interactions. Promoter region of so0011 (gyrB) was included as negative control. The phosphorylation of the ArcA protein was done with carbamoyl phosphate.
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Figure 5: ArcA(-P) Binding to selected promoters by EMSA. (A). Overproduced and purified recombinant S. oneidensis His6-ArcA from E. coli BL21 cells. (B). Interaction of so1661 promoter DNA with S. oneidensis His6-ArcA. The probe was prepared by PCR with SO1661-EMSA-F (33P end-labeled) and SO1661-EMSA-R primers (Table S4 in additional file 5). The EMS assay was performed with 2 nM 33P end-labeled probes and various amounts of ArcA (left panel) or ArcA-P proteins (right panel). The protein concentrations for lanes 1–9 are 0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 4.0, 4.0 μM, respectively. Non-specific competitor DNA, (2 μg poly dI·dC), was added (lane 8) and specific competitor (10 μM unlabeled SO1661 probe) was added (lane 9). (C). The binding assay was performed in the presence of 0, 1, or 2 μM ArcA-P and 2–5 nM radiolabeled promoter DNA 0.2 μg/μl poly(dI·dC) was used in all these binding reactions to block non-specific interactions. Promoter region of so0011 (gyrB) was included as negative control. The phosphorylation of the ArcA protein was done with carbamoyl phosphate.

Mentions: Expression of the S. oneidensis ArcA protein was initiated by insertion of the arcA gene into the Gateway entry vector pDONR221 using a lambda recombinase cloning strategy [56]. The entry vector containing the arcA gene was then converted to a protein expression system by recombination with the Gateway destination vector pDEST17 which resulted in the attachment of an N-terminal His-tag for protein purification. The His-tagged ArcA protein was expressed in E. coli and purified from inclusion bodies (Fig. 5A).


Probing regulon of ArcA in Shewanella oneidensis MR-1 by integrated genomic analyses.

Gao H, Wang X, Yang ZK, Palzkill T, Zhou J - BMC Genomics (2008)

ArcA(-P) Binding to selected promoters by EMSA. (A). Overproduced and purified recombinant S. oneidensis His6-ArcA from E. coli BL21 cells. (B). Interaction of so1661 promoter DNA with S. oneidensis His6-ArcA. The probe was prepared by PCR with SO1661-EMSA-F (33P end-labeled) and SO1661-EMSA-R primers (Table S4 in additional file 5). The EMS assay was performed with 2 nM 33P end-labeled probes and various amounts of ArcA (left panel) or ArcA-P proteins (right panel). The protein concentrations for lanes 1–9 are 0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 4.0, 4.0 μM, respectively. Non-specific competitor DNA, (2 μg poly dI·dC), was added (lane 8) and specific competitor (10 μM unlabeled SO1661 probe) was added (lane 9). (C). The binding assay was performed in the presence of 0, 1, or 2 μM ArcA-P and 2–5 nM radiolabeled promoter DNA 0.2 μg/μl poly(dI·dC) was used in all these binding reactions to block non-specific interactions. Promoter region of so0011 (gyrB) was included as negative control. The phosphorylation of the ArcA protein was done with carbamoyl phosphate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2262068&req=5

Figure 5: ArcA(-P) Binding to selected promoters by EMSA. (A). Overproduced and purified recombinant S. oneidensis His6-ArcA from E. coli BL21 cells. (B). Interaction of so1661 promoter DNA with S. oneidensis His6-ArcA. The probe was prepared by PCR with SO1661-EMSA-F (33P end-labeled) and SO1661-EMSA-R primers (Table S4 in additional file 5). The EMS assay was performed with 2 nM 33P end-labeled probes and various amounts of ArcA (left panel) or ArcA-P proteins (right panel). The protein concentrations for lanes 1–9 are 0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 4.0, 4.0 μM, respectively. Non-specific competitor DNA, (2 μg poly dI·dC), was added (lane 8) and specific competitor (10 μM unlabeled SO1661 probe) was added (lane 9). (C). The binding assay was performed in the presence of 0, 1, or 2 μM ArcA-P and 2–5 nM radiolabeled promoter DNA 0.2 μg/μl poly(dI·dC) was used in all these binding reactions to block non-specific interactions. Promoter region of so0011 (gyrB) was included as negative control. The phosphorylation of the ArcA protein was done with carbamoyl phosphate.
Mentions: Expression of the S. oneidensis ArcA protein was initiated by insertion of the arcA gene into the Gateway entry vector pDONR221 using a lambda recombinase cloning strategy [56]. The entry vector containing the arcA gene was then converted to a protein expression system by recombination with the Gateway destination vector pDEST17 which resulted in the attachment of an N-terminal His-tag for protein purification. The His-tagged ArcA protein was expressed in E. coli and purified from inclusion bodies (Fig. 5A).

Bottom Line: To further determine genes regulated by the Arc system, an ArcA recognition weight matrix from DNA-binding data and bioinformatics analysis was generated and used to produce an ArcA sequence affinity map.By combining both techniques, we identified an ArcA regulon of at least 50 operons, of which only 6 were found to be directly controlled by ArcA in E. coli.These results indicate that the Arc system in S. oneidensis differs from that in E. coli substantially in terms of its physiological function and regulon while their binding motif are strikingly similar.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Environmental Genomics and Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019, USA. haichun@ou.edu

ABSTRACT

Background: The Arc two-component system is a global regulator controlling many genes involved in aerobic/anaerobic respiration and fermentative metabolism in Escherichia coli. Shewanella oneidensis MR-1 contains a gene encoding a putative ArcA homolog with ~81% amino acid sequence identity to the E. coli ArcA protein but not a full-length arcB gene.

Results: To understand the role of ArcA in S. oneidensis, an arcA deletion strain was constructed and subjected to both physiological characterization and microarray analysis. Compared to the wild-type MR-1, the mutant exhibited impaired aerobic growth and a defect in utilizing DMSO in the absence of O2. Microarray analyses on cells grown aerobically and anaerobically on fumarate revealed that expression of 1009 genes was significantly affected (p < 0.05) by the mutation. In contrast to E. coli ArcA, the protein appears to be dispensable in regulation of the TCA cycle in S. oneidensis. To further determine genes regulated by the Arc system, an ArcA recognition weight matrix from DNA-binding data and bioinformatics analysis was generated and used to produce an ArcA sequence affinity map. By combining both techniques, we identified an ArcA regulon of at least 50 operons, of which only 6 were found to be directly controlled by ArcA in E. coli.

Conclusion: These results indicate that the Arc system in S. oneidensis differs from that in E. coli substantially in terms of its physiological function and regulon while their binding motif are strikingly similar.

Show MeSH
Related in: MedlinePlus