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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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Activity of selected promoters in response to salt, ethanol or in stationary phase.A. Sequences of selected promoters. The transcriptional start sites mapped previously are in bold and underlined [22, 29]. The putative -35 and -10 boxes are highlighted in grey. B. (left panel) Fold-change values from microarray experiments. (middle panel) Luciferase activity of luxCDABE transcriptional fusions to selected promoters in the wild type (WT), ecfG1 mutant (ΔecfG1), ecfG2 mutant (ΔecfG2), double ecfG1 ecfG2 mutant (Δ2), sextuple mutant (Δ6) or phyR mutant (ΔphyR). Cultures were treated with 1% ethanol (ethanol), 20 mM salt (salt) or H2O (control) in exponential phase 60 minutes prior to measurement, or were grown to stationary phase. The right panel shows the same stationary phase values with different axis ranges in order to better see the differences between the strains. AU, arbitrary units.
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pone.0152519.g003: Activity of selected promoters in response to salt, ethanol or in stationary phase.A. Sequences of selected promoters. The transcriptional start sites mapped previously are in bold and underlined [22, 29]. The putative -35 and -10 boxes are highlighted in grey. B. (left panel) Fold-change values from microarray experiments. (middle panel) Luciferase activity of luxCDABE transcriptional fusions to selected promoters in the wild type (WT), ecfG1 mutant (ΔecfG1), ecfG2 mutant (ΔecfG2), double ecfG1 ecfG2 mutant (Δ2), sextuple mutant (Δ6) or phyR mutant (ΔphyR). Cultures were treated with 1% ethanol (ethanol), 20 mM salt (salt) or H2O (control) in exponential phase 60 minutes prior to measurement, or were grown to stationary phase. The right panel shows the same stationary phase values with different axis ranges in order to better see the differences between the strains. AU, arbitrary units.

Mentions: To confirm the microarray results, we analyzed five target promoters (Fig 3A) using luxCDABE transcriptional fusions in the different backgrounds in response to ethanol and salt treatment, in order to test whether the same induction patterns were observed for another stress. Based on microarray results, the promoters chosen seemed to depend only on σEcfG1, or σEcfG1 and σEcfG2, with or without contribution of the remaining σEcfG proteins (Fig 3B, left panels). All five promoters tested were inducible in exponential phase by salt or ethanol in the wild-type strain (Fig 3B, middle panel), and induction was abolished in the ΔecfG1, Δ2, Δ6 or ΔphyR backgrounds, but not in the ΔecfG2 background, although induction in the latter background appeared reduced compared to the wild type (Fig 3B, middle panel). In exponential phase without stress, the basal luciferase activity of the fusions was not changed in the different backgrounds compared to the wild type for 2126p and 3874p, whereas 1696p, 4255p, 5204p showed lower activity in ΔecfG1, Δ2, Δ6 or ΔphyR backgrounds (Fig 3B, middle panel). Thus, in exponential phase, contributions of σEcfG1 and σEcfG2 but not of the other σEcfG proteins to the expression of the different genes were observed. Since the contribution of the minor σEcfG sigma factors was not evident in response to acute stress for the promoters tested, we sought to analyze the response in stationary phase. In this condition, 1696p and 4255p were induced, whereas activity of the other promoters was the same as in exponential phase or even reduced (Fig 3B, middle panel). Importantly, for all promoters tested, the expression levels were lower in the Δ6 strain compared to the Δ2 strain (Fig 3B, right panel). Altogether, these data suggest that each target is controlled by a combination of several σEcfG sigma factors, with σEcfG1 playing a major role in activation of gene expression in response to acute stress. The finding that σEcfG contribution apparently depends on the conditions raises the possibility that different σEcfG sigma factors are regulated in response to different signals. We next explored how the σEcfG proteins could be regulated.


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)

Activity of selected promoters in response to salt, ethanol or in stationary phase.A. Sequences of selected promoters. The transcriptional start sites mapped previously are in bold and underlined [22, 29]. The putative -35 and -10 boxes are highlighted in grey. B. (left panel) Fold-change values from microarray experiments. (middle panel) Luciferase activity of luxCDABE transcriptional fusions to selected promoters in the wild type (WT), ecfG1 mutant (ΔecfG1), ecfG2 mutant (ΔecfG2), double ecfG1 ecfG2 mutant (Δ2), sextuple mutant (Δ6) or phyR mutant (ΔphyR). Cultures were treated with 1% ethanol (ethanol), 20 mM salt (salt) or H2O (control) in exponential phase 60 minutes prior to measurement, or were grown to stationary phase. The right panel shows the same stationary phase values with different axis ranges in order to better see the differences between the strains. AU, arbitrary units.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152519.g003: Activity of selected promoters in response to salt, ethanol or in stationary phase.A. Sequences of selected promoters. The transcriptional start sites mapped previously are in bold and underlined [22, 29]. The putative -35 and -10 boxes are highlighted in grey. B. (left panel) Fold-change values from microarray experiments. (middle panel) Luciferase activity of luxCDABE transcriptional fusions to selected promoters in the wild type (WT), ecfG1 mutant (ΔecfG1), ecfG2 mutant (ΔecfG2), double ecfG1 ecfG2 mutant (Δ2), sextuple mutant (Δ6) or phyR mutant (ΔphyR). Cultures were treated with 1% ethanol (ethanol), 20 mM salt (salt) or H2O (control) in exponential phase 60 minutes prior to measurement, or were grown to stationary phase. The right panel shows the same stationary phase values with different axis ranges in order to better see the differences between the strains. AU, arbitrary units.
Mentions: To confirm the microarray results, we analyzed five target promoters (Fig 3A) using luxCDABE transcriptional fusions in the different backgrounds in response to ethanol and salt treatment, in order to test whether the same induction patterns were observed for another stress. Based on microarray results, the promoters chosen seemed to depend only on σEcfG1, or σEcfG1 and σEcfG2, with or without contribution of the remaining σEcfG proteins (Fig 3B, left panels). All five promoters tested were inducible in exponential phase by salt or ethanol in the wild-type strain (Fig 3B, middle panel), and induction was abolished in the ΔecfG1, Δ2, Δ6 or ΔphyR backgrounds, but not in the ΔecfG2 background, although induction in the latter background appeared reduced compared to the wild type (Fig 3B, middle panel). In exponential phase without stress, the basal luciferase activity of the fusions was not changed in the different backgrounds compared to the wild type for 2126p and 3874p, whereas 1696p, 4255p, 5204p showed lower activity in ΔecfG1, Δ2, Δ6 or ΔphyR backgrounds (Fig 3B, middle panel). Thus, in exponential phase, contributions of σEcfG1 and σEcfG2 but not of the other σEcfG proteins to the expression of the different genes were observed. Since the contribution of the minor σEcfG sigma factors was not evident in response to acute stress for the promoters tested, we sought to analyze the response in stationary phase. In this condition, 1696p and 4255p were induced, whereas activity of the other promoters was the same as in exponential phase or even reduced (Fig 3B, middle panel). Importantly, for all promoters tested, the expression levels were lower in the Δ6 strain compared to the Δ2 strain (Fig 3B, right panel). Altogether, these data suggest that each target is controlled by a combination of several σEcfG sigma factors, with σEcfG1 playing a major role in activation of gene expression in response to acute stress. The finding that σEcfG contribution apparently depends on the conditions raises the possibility that different σEcfG sigma factors are regulated in response to different signals. We next explored how the σEcfG proteins could be regulated.

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