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Multiple σEcfG and NepR Proteins Are Involved in the General Stress Response in Methylobacterium extorquens.

Francez-Charlot A, Frunzke J, Zingg J, Kaczmarczyk A, Vorholt JA - PLoS ONE (2016)

Bottom Line: We identify distinct levels of regulation for the different sigma factors, as well as two NepR paralogues that interact with PhyR.Our results suggest that in M. extorquens AM1, ecfG and nepR paralogues have diverged in order to assume new roles that might allow integration of positive and negative feedback loops in the regulatory system.Comparison of the core elements of the GSR regulatory network in Methylobacterium species provides evidence for high plasticity and rapid evolution of the GSR core network in this genus.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, ETH Zurich, Zurich, Switzerland.

ABSTRACT
In Alphaproteobacteria, the general stress response (GSR) is controlled by a conserved partner switch composed of the sigma factor σ(EcfG), its anti-sigma factor NepR and the anti-sigma factor antagonist PhyR. Many species possess paralogues of one or several components of the system, but their roles remain largely elusive. Among Alphaproteobacteria that have been genome-sequenced so far, the genus Methylobacterium possesses the largest number of σ(EcfG) proteins. Here, we analyzed the six σ(EcfG) paralogues of Methylobacterium extorquens AM1. We show that these sigma factors are not truly redundant, but instead exhibit major and minor contributions to stress resistance and GSR target gene expression. We identify distinct levels of regulation for the different sigma factors, as well as two NepR paralogues that interact with PhyR. Our results suggest that in M. extorquens AM1, ecfG and nepR paralogues have diverged in order to assume new roles that might allow integration of positive and negative feedback loops in the regulatory system. Comparison of the core elements of the GSR regulatory network in Methylobacterium species provides evidence for high plasticity and rapid evolution of the GSR core network in this genus.

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Characterization of NepR paralogues.A. Interactions between NepR paralogues and PhyR. Co-immunoprecipitation of C-terminal triple FLAG-tagged NepR paralogues. The control strain (WT) bears the empty plasmid pCM80. The input (I), flow-through (FT), last washing step (W) and elution (E) fractions were analyzed by Western blot using anti-PhyR (upper panel) or anti-FLAG (middle panel) antibodies, or stained with Coomassie Blue (lower panel). B. Stress sensitivity of strains overexpressing nepR paralogues. Methylglyoxal sensitivity of the wild-type strains overexpressing nepR (WT/nepR) or one of the putative nepR homologues (WT/1275, WT/2700, WT/735) from the mxaF promoter, or bearing the empty plasmid (WT/80). Data are displayed as means +/- SD of three independent biological replicates. C. Putative σEcfG-dependent promoter of META2_0735. The -35 and -10 boxes are highlighted in grey. D. Activity of the 735p::luxCDABE transcriptional fusion in response to ethanol or salt in the wild-type, Δ6 or ΔphyR strain. Values are given as means +/- SD of two independent biological replicates.
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pone.0152519.g006: Characterization of NepR paralogues.A. Interactions between NepR paralogues and PhyR. Co-immunoprecipitation of C-terminal triple FLAG-tagged NepR paralogues. The control strain (WT) bears the empty plasmid pCM80. The input (I), flow-through (FT), last washing step (W) and elution (E) fractions were analyzed by Western blot using anti-PhyR (upper panel) or anti-FLAG (middle panel) antibodies, or stained with Coomassie Blue (lower panel). B. Stress sensitivity of strains overexpressing nepR paralogues. Methylglyoxal sensitivity of the wild-type strains overexpressing nepR (WT/nepR) or one of the putative nepR homologues (WT/1275, WT/2700, WT/735) from the mxaF promoter, or bearing the empty plasmid (WT/80). Data are displayed as means +/- SD of three independent biological replicates. C. Putative σEcfG-dependent promoter of META2_0735. The -35 and -10 boxes are highlighted in grey. D. Activity of the 735p::luxCDABE transcriptional fusion in response to ethanol or salt in the wild-type, Δ6 or ΔphyR strain. Values are given as means +/- SD of two independent biological replicates.

Mentions: Because no interactions between the putative NepR homologues and the σEcfG proteins could be observed, we wondered whether they were involved in the PhyR cascade by other means. Most NepR residues known to interact with PhyR based on C. crescentus and S. melonis PhyR-NepR complexes are conserved in these proteins (S2C Fig), suggesting they can bind PhyR, and for two of them (MexAM1_META1_2700 and MexAM1_META2_0735), putative σEcfG-type promoters are found in the upstream region of their encoding genes, linking them to the GSR. Interactions between PhyR and NepR homologues were analyzed using co-immunoprecipitation experiments using C-terminal triple FLAG-tagged versions of NepR or NepR paralogues. The NepR and MexAM1_META2p0735 triple FLAG-tagged versions were functional as judged by methylglyoxal sensitivity assays (S4B Fig and see below for comparison with the untagged versions). As shown in Fig 6A, PhyR could be co-immunoprecipitated with NepR, MexAM1_META1p1275 and MexAM1_META2p0735; no co-immunoprecipitation of PhyR was observed for MexAM1_META1p2700 or for a control strain bearing the empty pCM80 plasmid. Altogether, these data indicate that in addition to NepR, PhyR interacts with MexAM1_META1p1275 and MexAM1_META2p0735. However, our data suggest that these proteins do not act as anti-sigma factor since they were not found to interact with any σEcfG protein, although it is possible that interactions were missed in our analysis. In case absence of interaction was true, NepR homologues might then rather act as anti-anti-anti-sigma factors.


Multiple σEcfG and NepR Proteins Are Involved in the General Stress Response in Methylobacterium extorquens.

Francez-Charlot A, Frunzke J, Zingg J, Kaczmarczyk A, Vorholt JA - PLoS ONE (2016)

Characterization of NepR paralogues.A. Interactions between NepR paralogues and PhyR. Co-immunoprecipitation of C-terminal triple FLAG-tagged NepR paralogues. The control strain (WT) bears the empty plasmid pCM80. The input (I), flow-through (FT), last washing step (W) and elution (E) fractions were analyzed by Western blot using anti-PhyR (upper panel) or anti-FLAG (middle panel) antibodies, or stained with Coomassie Blue (lower panel). B. Stress sensitivity of strains overexpressing nepR paralogues. Methylglyoxal sensitivity of the wild-type strains overexpressing nepR (WT/nepR) or one of the putative nepR homologues (WT/1275, WT/2700, WT/735) from the mxaF promoter, or bearing the empty plasmid (WT/80). Data are displayed as means +/- SD of three independent biological replicates. C. Putative σEcfG-dependent promoter of META2_0735. The -35 and -10 boxes are highlighted in grey. D. Activity of the 735p::luxCDABE transcriptional fusion in response to ethanol or salt in the wild-type, Δ6 or ΔphyR strain. Values are given as means +/- SD of two independent biological replicates.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4814048&req=5

pone.0152519.g006: Characterization of NepR paralogues.A. Interactions between NepR paralogues and PhyR. Co-immunoprecipitation of C-terminal triple FLAG-tagged NepR paralogues. The control strain (WT) bears the empty plasmid pCM80. The input (I), flow-through (FT), last washing step (W) and elution (E) fractions were analyzed by Western blot using anti-PhyR (upper panel) or anti-FLAG (middle panel) antibodies, or stained with Coomassie Blue (lower panel). B. Stress sensitivity of strains overexpressing nepR paralogues. Methylglyoxal sensitivity of the wild-type strains overexpressing nepR (WT/nepR) or one of the putative nepR homologues (WT/1275, WT/2700, WT/735) from the mxaF promoter, or bearing the empty plasmid (WT/80). Data are displayed as means +/- SD of three independent biological replicates. C. Putative σEcfG-dependent promoter of META2_0735. The -35 and -10 boxes are highlighted in grey. D. Activity of the 735p::luxCDABE transcriptional fusion in response to ethanol or salt in the wild-type, Δ6 or ΔphyR strain. Values are given as means +/- SD of two independent biological replicates.
Mentions: Because no interactions between the putative NepR homologues and the σEcfG proteins could be observed, we wondered whether they were involved in the PhyR cascade by other means. Most NepR residues known to interact with PhyR based on C. crescentus and S. melonis PhyR-NepR complexes are conserved in these proteins (S2C Fig), suggesting they can bind PhyR, and for two of them (MexAM1_META1_2700 and MexAM1_META2_0735), putative σEcfG-type promoters are found in the upstream region of their encoding genes, linking them to the GSR. Interactions between PhyR and NepR homologues were analyzed using co-immunoprecipitation experiments using C-terminal triple FLAG-tagged versions of NepR or NepR paralogues. The NepR and MexAM1_META2p0735 triple FLAG-tagged versions were functional as judged by methylglyoxal sensitivity assays (S4B Fig and see below for comparison with the untagged versions). As shown in Fig 6A, PhyR could be co-immunoprecipitated with NepR, MexAM1_META1p1275 and MexAM1_META2p0735; no co-immunoprecipitation of PhyR was observed for MexAM1_META1p2700 or for a control strain bearing the empty pCM80 plasmid. Altogether, these data indicate that in addition to NepR, PhyR interacts with MexAM1_META1p1275 and MexAM1_META2p0735. However, our data suggest that these proteins do not act as anti-sigma factor since they were not found to interact with any σEcfG protein, although it is possible that interactions were missed in our analysis. In case absence of interaction was true, NepR homologues might then rather act as anti-anti-anti-sigma factors.

Bottom Line: We identify distinct levels of regulation for the different sigma factors, as well as two NepR paralogues that interact with PhyR.Our results suggest that in M. extorquens AM1, ecfG and nepR paralogues have diverged in order to assume new roles that might allow integration of positive and negative feedback loops in the regulatory system.Comparison of the core elements of the GSR regulatory network in Methylobacterium species provides evidence for high plasticity and rapid evolution of the GSR core network in this genus.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, ETH Zurich, Zurich, Switzerland.

ABSTRACT
In Alphaproteobacteria, the general stress response (GSR) is controlled by a conserved partner switch composed of the sigma factor σ(EcfG), its anti-sigma factor NepR and the anti-sigma factor antagonist PhyR. Many species possess paralogues of one or several components of the system, but their roles remain largely elusive. Among Alphaproteobacteria that have been genome-sequenced so far, the genus Methylobacterium possesses the largest number of σ(EcfG) proteins. Here, we analyzed the six σ(EcfG) paralogues of Methylobacterium extorquens AM1. We show that these sigma factors are not truly redundant, but instead exhibit major and minor contributions to stress resistance and GSR target gene expression. We identify distinct levels of regulation for the different sigma factors, as well as two NepR paralogues that interact with PhyR. Our results suggest that in M. extorquens AM1, ecfG and nepR paralogues have diverged in order to assume new roles that might allow integration of positive and negative feedback loops in the regulatory system. Comparison of the core elements of the GSR regulatory network in Methylobacterium species provides evidence for high plasticity and rapid evolution of the GSR core network in this genus.

Show MeSH
Related in: MedlinePlus