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Adaptation to sustained nitrogen starvation by Escherichia coli requires the eukaryote-like serine/threonine kinase YeaG.

Figueira R, Brown DR, Ferreira D, Eldridge MJ, Burchell L, Pan Z, Helaine S, Wigneshweraraj S - Sci Rep (2015)

Bottom Line: The mechanism by which yeaG acts, involves the transcriptional repression of two toxin/antitoxin modules, mqsR/mqsA and dinJ/yafQ.This, consequently, has a positive effect on the expression of rpoS, the master regulator of the general bacterial stress response.Overall, results indicate that yeaG is required to fully execute the rpoS-dependent gene expression program to allow E. coli to adapt to sustained N starvation and unravels a novel facet to the regulatory basis that underpins adaptive response to N stress.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Molecular Microbiology and Infection, Imperial College London, UK.

ABSTRACT
The Escherichia coli eukaryote-like serine/threonine kinase, encoded by yeaG, is expressed in response to diverse stresses, including nitrogen (N) starvation. A role for yeaG in bacterial stress response is unknown. Here we reveal for the first time that wild-type E. coli displays metabolic heterogeneity following sustained periods of N starvation, with the metabolically active population displaying compromised viability. In contrast, such heterogeneity in metabolic activity is not observed in an E. coli ∆yeaG mutant, which continues to exist as a single and metabolically active population and thus displays an overall compromised ability to survive sustained periods of N starvation. The mechanism by which yeaG acts, involves the transcriptional repression of two toxin/antitoxin modules, mqsR/mqsA and dinJ/yafQ. This, consequently, has a positive effect on the expression of rpoS, the master regulator of the general bacterial stress response. Overall, results indicate that yeaG is required to fully execute the rpoS-dependent gene expression program to allow E. coli to adapt to sustained N starvation and unravels a novel facet to the regulatory basis that underpins adaptive response to N stress.

No MeSH data available.


Related in: MedlinePlus

The absence of yeaG reduces bacterial cell viability during sustained N starvation.(A) The viability of wild-type, ∆yeaG mutant and ∆yeaG mutant complemented with plasmid-borne YeaG (∆yeaG pBAD18-yeaG) during sustained N starvation was determined by colony-forming unit (CFU) count over 5 days. Error bars represent s.e.m. (n = 3). Statistical analyses were performed by one-way ANOVA (*P < 0.05). (B) The viability of wild-type, ∆yeaG, ∆yeaG complemented with plasmid-borne YeaG (∆yeaG pBAD18-yeaG) or ∆yeaG mutant strain expressing variants of YeaG with deleterious mutations in the eSTK catalytic domain (pBAD18-yeaG K426A; pBAD18-yeaG Y382Stop) was determined by colony-forming unit (CFU) count after 20 min, 72 h & 120 h in N starvation. Error bars represent sd (n = 3). Statistical analyses were performed by one-way ANOVA (*P < 0.05). (C) Competitive Index (CI) analysis of Salmonella Typhimurium ∆yeaGH. C57 BL/6 mice (n = 5) were inoculated intragastrically with equal numbers of wild-type and mutant strain. Bacteria were recovered from infected spleens after 5 days post-inoculation and strains distinguished based on antibiotic resistance by replica plating. A CI value of 0.64 (p = 0.037), indicating moderate attenuation of the ∆yeaGH mutant strain in vivo, was obtained by calculating the ratio of wild-type to ∆yeaGH bacteria recovered (output), divided by the ratio of wild-type to ∆yeaGH mutant bacteria present in the inoculum (input). The scatter plot displays values obtained for individual mice and the mean is indicated. (D) Metabolically-inactive bacteria are more likely to withstand long-term stress. Representative histogram of GFP fluorescence shows wild-type population distribution according to metabolic activity at selected time points. The insert diagram shows the levels of GFP fluorescence measured at 3.5 h following induction of GFP expression during ‘recovery’ growth. Bacteria were subsequently transferred to media without inducer or N and GFP fluorescence was assessed again after 24 h in starvation conditions (see text for details).
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f6: The absence of yeaG reduces bacterial cell viability during sustained N starvation.(A) The viability of wild-type, ∆yeaG mutant and ∆yeaG mutant complemented with plasmid-borne YeaG (∆yeaG pBAD18-yeaG) during sustained N starvation was determined by colony-forming unit (CFU) count over 5 days. Error bars represent s.e.m. (n = 3). Statistical analyses were performed by one-way ANOVA (*P < 0.05). (B) The viability of wild-type, ∆yeaG, ∆yeaG complemented with plasmid-borne YeaG (∆yeaG pBAD18-yeaG) or ∆yeaG mutant strain expressing variants of YeaG with deleterious mutations in the eSTK catalytic domain (pBAD18-yeaG K426A; pBAD18-yeaG Y382Stop) was determined by colony-forming unit (CFU) count after 20 min, 72 h & 120 h in N starvation. Error bars represent sd (n = 3). Statistical analyses were performed by one-way ANOVA (*P < 0.05). (C) Competitive Index (CI) analysis of Salmonella Typhimurium ∆yeaGH. C57 BL/6 mice (n = 5) were inoculated intragastrically with equal numbers of wild-type and mutant strain. Bacteria were recovered from infected spleens after 5 days post-inoculation and strains distinguished based on antibiotic resistance by replica plating. A CI value of 0.64 (p = 0.037), indicating moderate attenuation of the ∆yeaGH mutant strain in vivo, was obtained by calculating the ratio of wild-type to ∆yeaGH bacteria recovered (output), divided by the ratio of wild-type to ∆yeaGH mutant bacteria present in the inoculum (input). The scatter plot displays values obtained for individual mice and the mean is indicated. (D) Metabolically-inactive bacteria are more likely to withstand long-term stress. Representative histogram of GFP fluorescence shows wild-type population distribution according to metabolic activity at selected time points. The insert diagram shows the levels of GFP fluorescence measured at 3.5 h following induction of GFP expression during ‘recovery’ growth. Bacteria were subsequently transferred to media without inducer or N and GFP fluorescence was assessed again after 24 h in starvation conditions (see text for details).

Mentions: Since σ38-dependent gene expression is central to full adaptation to diverse stress conditions, including N starvation, and thus ultimately to the survival of the bacterial cell, we next investigated how the deletion of yeaG impacts bacterial survival following sustained N starvation. Wild-type and ∆yeaG mutant bacterial numbers were assessed by plate count of CFU during N starvation over 5 days. As shown in Fig. 6A, following an initial period of little change, bacterial numbers began to drop after 72 h of N starvation for both strains. For the wild-type strain there is a slow but steady decrease in bacterial numbers. However, in comparison, the viability of the ∆yeaG mutant strain declines significantly faster. This faster rate of death was not observed in the ∆yeaG mutant strain that was complemented with a plasmid-borne wild-type copy of yeaG, whose bacterial numbers were comparable to the wild-type strain throughout sustained N starvation (Fig. 6A). However, as expected, a similar decrease in the number of viable bacterial cells was observed after 72 h of N starvation when the ∆yeaG mutant strain was complemented with catalytic mutant variants of yeaG (Fig. 6B). This observation further corroborates previous results (Fig. 3), which suggests that the catalytic activity of the AAA+ and STK domain of YeaG is important for its function. We thus conclude that absence of yeaG impairs the ability of E. coli to survive sustained periods of N starvation, most likely because the σ38-dependent adaptive response cannot be fully executed. Consistent with a role for YeaG in allowing bacteria to adapt to adverse growth environments, a comparative analysis of virulence carried out with a S. Typhimurium ∆yeaGH and wild-type strains indicated that mutant bacteria displayed lower proliferation in the spleen (Fig. 6C), thus implying that yeaG could be a determinant the overall fitness of S. Typhimurium.


Adaptation to sustained nitrogen starvation by Escherichia coli requires the eukaryote-like serine/threonine kinase YeaG.

Figueira R, Brown DR, Ferreira D, Eldridge MJ, Burchell L, Pan Z, Helaine S, Wigneshweraraj S - Sci Rep (2015)

The absence of yeaG reduces bacterial cell viability during sustained N starvation.(A) The viability of wild-type, ∆yeaG mutant and ∆yeaG mutant complemented with plasmid-borne YeaG (∆yeaG pBAD18-yeaG) during sustained N starvation was determined by colony-forming unit (CFU) count over 5 days. Error bars represent s.e.m. (n = 3). Statistical analyses were performed by one-way ANOVA (*P < 0.05). (B) The viability of wild-type, ∆yeaG, ∆yeaG complemented with plasmid-borne YeaG (∆yeaG pBAD18-yeaG) or ∆yeaG mutant strain expressing variants of YeaG with deleterious mutations in the eSTK catalytic domain (pBAD18-yeaG K426A; pBAD18-yeaG Y382Stop) was determined by colony-forming unit (CFU) count after 20 min, 72 h & 120 h in N starvation. Error bars represent sd (n = 3). Statistical analyses were performed by one-way ANOVA (*P < 0.05). (C) Competitive Index (CI) analysis of Salmonella Typhimurium ∆yeaGH. C57 BL/6 mice (n = 5) were inoculated intragastrically with equal numbers of wild-type and mutant strain. Bacteria were recovered from infected spleens after 5 days post-inoculation and strains distinguished based on antibiotic resistance by replica plating. A CI value of 0.64 (p = 0.037), indicating moderate attenuation of the ∆yeaGH mutant strain in vivo, was obtained by calculating the ratio of wild-type to ∆yeaGH bacteria recovered (output), divided by the ratio of wild-type to ∆yeaGH mutant bacteria present in the inoculum (input). The scatter plot displays values obtained for individual mice and the mean is indicated. (D) Metabolically-inactive bacteria are more likely to withstand long-term stress. Representative histogram of GFP fluorescence shows wild-type population distribution according to metabolic activity at selected time points. The insert diagram shows the levels of GFP fluorescence measured at 3.5 h following induction of GFP expression during ‘recovery’ growth. Bacteria were subsequently transferred to media without inducer or N and GFP fluorescence was assessed again after 24 h in starvation conditions (see text for details).
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f6: The absence of yeaG reduces bacterial cell viability during sustained N starvation.(A) The viability of wild-type, ∆yeaG mutant and ∆yeaG mutant complemented with plasmid-borne YeaG (∆yeaG pBAD18-yeaG) during sustained N starvation was determined by colony-forming unit (CFU) count over 5 days. Error bars represent s.e.m. (n = 3). Statistical analyses were performed by one-way ANOVA (*P < 0.05). (B) The viability of wild-type, ∆yeaG, ∆yeaG complemented with plasmid-borne YeaG (∆yeaG pBAD18-yeaG) or ∆yeaG mutant strain expressing variants of YeaG with deleterious mutations in the eSTK catalytic domain (pBAD18-yeaG K426A; pBAD18-yeaG Y382Stop) was determined by colony-forming unit (CFU) count after 20 min, 72 h & 120 h in N starvation. Error bars represent sd (n = 3). Statistical analyses were performed by one-way ANOVA (*P < 0.05). (C) Competitive Index (CI) analysis of Salmonella Typhimurium ∆yeaGH. C57 BL/6 mice (n = 5) were inoculated intragastrically with equal numbers of wild-type and mutant strain. Bacteria were recovered from infected spleens after 5 days post-inoculation and strains distinguished based on antibiotic resistance by replica plating. A CI value of 0.64 (p = 0.037), indicating moderate attenuation of the ∆yeaGH mutant strain in vivo, was obtained by calculating the ratio of wild-type to ∆yeaGH bacteria recovered (output), divided by the ratio of wild-type to ∆yeaGH mutant bacteria present in the inoculum (input). The scatter plot displays values obtained for individual mice and the mean is indicated. (D) Metabolically-inactive bacteria are more likely to withstand long-term stress. Representative histogram of GFP fluorescence shows wild-type population distribution according to metabolic activity at selected time points. The insert diagram shows the levels of GFP fluorescence measured at 3.5 h following induction of GFP expression during ‘recovery’ growth. Bacteria were subsequently transferred to media without inducer or N and GFP fluorescence was assessed again after 24 h in starvation conditions (see text for details).
Mentions: Since σ38-dependent gene expression is central to full adaptation to diverse stress conditions, including N starvation, and thus ultimately to the survival of the bacterial cell, we next investigated how the deletion of yeaG impacts bacterial survival following sustained N starvation. Wild-type and ∆yeaG mutant bacterial numbers were assessed by plate count of CFU during N starvation over 5 days. As shown in Fig. 6A, following an initial period of little change, bacterial numbers began to drop after 72 h of N starvation for both strains. For the wild-type strain there is a slow but steady decrease in bacterial numbers. However, in comparison, the viability of the ∆yeaG mutant strain declines significantly faster. This faster rate of death was not observed in the ∆yeaG mutant strain that was complemented with a plasmid-borne wild-type copy of yeaG, whose bacterial numbers were comparable to the wild-type strain throughout sustained N starvation (Fig. 6A). However, as expected, a similar decrease in the number of viable bacterial cells was observed after 72 h of N starvation when the ∆yeaG mutant strain was complemented with catalytic mutant variants of yeaG (Fig. 6B). This observation further corroborates previous results (Fig. 3), which suggests that the catalytic activity of the AAA+ and STK domain of YeaG is important for its function. We thus conclude that absence of yeaG impairs the ability of E. coli to survive sustained periods of N starvation, most likely because the σ38-dependent adaptive response cannot be fully executed. Consistent with a role for YeaG in allowing bacteria to adapt to adverse growth environments, a comparative analysis of virulence carried out with a S. Typhimurium ∆yeaGH and wild-type strains indicated that mutant bacteria displayed lower proliferation in the spleen (Fig. 6C), thus implying that yeaG could be a determinant the overall fitness of S. Typhimurium.

Bottom Line: The mechanism by which yeaG acts, involves the transcriptional repression of two toxin/antitoxin modules, mqsR/mqsA and dinJ/yafQ.This, consequently, has a positive effect on the expression of rpoS, the master regulator of the general bacterial stress response.Overall, results indicate that yeaG is required to fully execute the rpoS-dependent gene expression program to allow E. coli to adapt to sustained N starvation and unravels a novel facet to the regulatory basis that underpins adaptive response to N stress.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Molecular Microbiology and Infection, Imperial College London, UK.

ABSTRACT
The Escherichia coli eukaryote-like serine/threonine kinase, encoded by yeaG, is expressed in response to diverse stresses, including nitrogen (N) starvation. A role for yeaG in bacterial stress response is unknown. Here we reveal for the first time that wild-type E. coli displays metabolic heterogeneity following sustained periods of N starvation, with the metabolically active population displaying compromised viability. In contrast, such heterogeneity in metabolic activity is not observed in an E. coli ∆yeaG mutant, which continues to exist as a single and metabolically active population and thus displays an overall compromised ability to survive sustained periods of N starvation. The mechanism by which yeaG acts, involves the transcriptional repression of two toxin/antitoxin modules, mqsR/mqsA and dinJ/yafQ. This, consequently, has a positive effect on the expression of rpoS, the master regulator of the general bacterial stress response. Overall, results indicate that yeaG is required to fully execute the rpoS-dependent gene expression program to allow E. coli to adapt to sustained N starvation and unravels a novel facet to the regulatory basis that underpins adaptive response to N stress.

No MeSH data available.


Related in: MedlinePlus