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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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Phenotypic analysis of ecfG mutants.A. Sensitivity to methylglyoxal of the wild type (WT), phyR mutant (ΔphyR), ecfG single mutants (ΔecfG1, ΔecfG2, ΔecfG3, ΔecfG4, ΔecfG5, and ΔecfG6) and ecfG multiple mutants (Δ4, Δ5, Δ2, Δ5(ecfG1), Δ5(ecfG2), Δ6n). B. Growth of the same strains on MM supplemented with NaCl (middle panel) or ethanol (right panel) compared to growth on MM (left panel). Ten-fold serial dilutions are shown from left to right, starting from undiluted samples. C and D. Sensitivity to methylglyoxal of the sextuple mutant expressing each ecfG from its own promoter on pCM62 (C) or from the strong mxaF promoter on pCM80 (D).
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pone.0152519.g001: Phenotypic analysis of ecfG mutants.A. Sensitivity to methylglyoxal of the wild type (WT), phyR mutant (ΔphyR), ecfG single mutants (ΔecfG1, ΔecfG2, ΔecfG3, ΔecfG4, ΔecfG5, and ΔecfG6) and ecfG multiple mutants (Δ4, Δ5, Δ2, Δ5(ecfG1), Δ5(ecfG2), Δ6n). B. Growth of the same strains on MM supplemented with NaCl (middle panel) or ethanol (right panel) compared to growth on MM (left panel). Ten-fold serial dilutions are shown from left to right, starting from undiluted samples. C and D. Sensitivity to methylglyoxal of the sextuple mutant expressing each ecfG from its own promoter on pCM62 (C) or from the strong mxaF promoter on pCM80 (D).

Mentions: We previously demonstrated the involvement of ecfG1 in the GSR of M. extorquens AM1: σEcfG1 was shown to regulate a subset of the PhyR regulon and to interact with NepR [4]. Despite these observations, an ecfG1 mutant does not exhibit stress sensitivity, in contrast to a phyR mutant, suggesting that other σEcfG proteins are involved in the GSR. To test this hypothesis, we constructed single and multiple ecfG mutants and analyzed their sensitivity to different stresses. As shown in Fig 1A and 1B, no increased sensitivity to methylglyoxal, salt or ethanol was observed for any single mutant or for the quadruple mutant (Δ4, corresponding to ΔecfG1 ΔecfG3 ΔecfG4 ΔecfG5) compared to the wild-type strain. In contrast, the quintuple mutant (Δ5, corresponding to ΔecfG1 ΔecfG3 ΔecfG4 ΔecfG5 ΔecfG2) was more sensitive than the wild type to methylglyoxal, salt and ethanol, to the same extent as a sextuple mutant (Fig 1A and 1B). Since the difference between the Δ4 and Δ5 strains is the deletion of ecfG2, we tested whether an ecfG1 ecfG2 double mutant (Δ2) was stress sensitive. Indeed, this mutant showed increased sensitivity to all stresses tested compared to the wild type, albeit the strain was not as stress sensitive as the quintuple and sextuple mutants (Fig 1A and 1B). To rule out that the combination of deletions of one or several of the four remaining ecfG genes together with the deletion of either ecfG1 or ecfG2 is sufficient to confer stress sensitivity, a ΔecfG3 ΔecfG4 ΔecfG5 ΔecfG6 deletion mutant (Δ4n) was first constructed, followed by deletion of ecfG1 or ecfG2. None of these quintuple mutants [Δ5(ecfG1) and Δ5(ecfG2)] showed increased stress sensitivity. Only deletion of both ecfG1 and ecfG2 in the Δ4n strain (Δ6n) resulted in a stress sensitive strain (Fig 1A), indicating that deletion of either ecfG1 or ecfG2 in combination with the remaining ecfG genes is not sufficient to confer stress sensitivity.


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)

Phenotypic analysis of ecfG mutants.A. Sensitivity to methylglyoxal of the wild type (WT), phyR mutant (ΔphyR), ecfG single mutants (ΔecfG1, ΔecfG2, ΔecfG3, ΔecfG4, ΔecfG5, and ΔecfG6) and ecfG multiple mutants (Δ4, Δ5, Δ2, Δ5(ecfG1), Δ5(ecfG2), Δ6n). B. Growth of the same strains on MM supplemented with NaCl (middle panel) or ethanol (right panel) compared to growth on MM (left panel). Ten-fold serial dilutions are shown from left to right, starting from undiluted samples. C and D. Sensitivity to methylglyoxal of the sextuple mutant expressing each ecfG from its own promoter on pCM62 (C) or from the strong mxaF promoter on pCM80 (D).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4814048&req=5

pone.0152519.g001: Phenotypic analysis of ecfG mutants.A. Sensitivity to methylglyoxal of the wild type (WT), phyR mutant (ΔphyR), ecfG single mutants (ΔecfG1, ΔecfG2, ΔecfG3, ΔecfG4, ΔecfG5, and ΔecfG6) and ecfG multiple mutants (Δ4, Δ5, Δ2, Δ5(ecfG1), Δ5(ecfG2), Δ6n). B. Growth of the same strains on MM supplemented with NaCl (middle panel) or ethanol (right panel) compared to growth on MM (left panel). Ten-fold serial dilutions are shown from left to right, starting from undiluted samples. C and D. Sensitivity to methylglyoxal of the sextuple mutant expressing each ecfG from its own promoter on pCM62 (C) or from the strong mxaF promoter on pCM80 (D).
Mentions: We previously demonstrated the involvement of ecfG1 in the GSR of M. extorquens AM1: σEcfG1 was shown to regulate a subset of the PhyR regulon and to interact with NepR [4]. Despite these observations, an ecfG1 mutant does not exhibit stress sensitivity, in contrast to a phyR mutant, suggesting that other σEcfG proteins are involved in the GSR. To test this hypothesis, we constructed single and multiple ecfG mutants and analyzed their sensitivity to different stresses. As shown in Fig 1A and 1B, no increased sensitivity to methylglyoxal, salt or ethanol was observed for any single mutant or for the quadruple mutant (Δ4, corresponding to ΔecfG1 ΔecfG3 ΔecfG4 ΔecfG5) compared to the wild-type strain. In contrast, the quintuple mutant (Δ5, corresponding to ΔecfG1 ΔecfG3 ΔecfG4 ΔecfG5 ΔecfG2) was more sensitive than the wild type to methylglyoxal, salt and ethanol, to the same extent as a sextuple mutant (Fig 1A and 1B). Since the difference between the Δ4 and Δ5 strains is the deletion of ecfG2, we tested whether an ecfG1 ecfG2 double mutant (Δ2) was stress sensitive. Indeed, this mutant showed increased sensitivity to all stresses tested compared to the wild type, albeit the strain was not as stress sensitive as the quintuple and sextuple mutants (Fig 1A and 1B). To rule out that the combination of deletions of one or several of the four remaining ecfG genes together with the deletion of either ecfG1 or ecfG2 is sufficient to confer stress sensitivity, a ΔecfG3 ΔecfG4 ΔecfG5 ΔecfG6 deletion mutant (Δ4n) was first constructed, followed by deletion of ecfG1 or ecfG2. None of these quintuple mutants [Δ5(ecfG1) and Δ5(ecfG2)] showed increased stress sensitivity. Only deletion of both ecfG1 and ecfG2 in the Δ4n strain (Δ6n) resulted in a stress sensitive strain (Fig 1A), indicating that deletion of either ecfG1 or ecfG2 in combination with the remaining ecfG genes is not sufficient to confer stress sensitivity.

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