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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 catalytic activities of the AAA+ and eSTK domains of YeaG are required for its role in adaptation to sustained N starvation in E. coli.(A,B) Growth curves show complementation of the phenotype of the ∆yeaG mutant strain with plasmid-borne yeaG (pBAD18-yeaG), but not with mutant variants of yeaG containing catalytically deleterious mutations in the AAA+ domain (in A. pBAD18-yeaG K116A; pBAD18-yeaG R232A) or eSTK domain (in B. pBAD18-yeaG K426A; pBAD18-yeaG Y382Stop). Error bars on all growth curves represent sd (n = 3). Statistical analyses were performed by one-way ANOVA (**P < 0.01). (C) Protein levels of wild-type and mutant variants of YeaG assessed by anti-His immunoblotting.
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f3: The catalytic activities of the AAA+ and eSTK domains of YeaG are required for its role in adaptation to sustained N starvation in E. coli.(A,B) Growth curves show complementation of the phenotype of the ∆yeaG mutant strain with plasmid-borne yeaG (pBAD18-yeaG), but not with mutant variants of yeaG containing catalytically deleterious mutations in the AAA+ domain (in A. pBAD18-yeaG K116A; pBAD18-yeaG R232A) or eSTK domain (in B. pBAD18-yeaG K426A; pBAD18-yeaG Y382Stop). Error bars on all growth curves represent sd (n = 3). Statistical analyses were performed by one-way ANOVA (**P < 0.01). (C) Protein levels of wild-type and mutant variants of YeaG assessed by anti-His immunoblotting.

Mentions: We were able to revert the growth characteristics of the ∆yeaG mutant strain to that of the wild-type strain by complementing with inducible plasmid-borne yeaG (∆yeaG pBAD-yeaG), thereby demonstrating that the altered growth of the mutant strain is not due to any undesired effects caused by the deletion of the yeaG gene (Fig. 3A,B). To determine if the catalytic activity of the AAA+ and/or STK domains of YeaG are required for function, we constructed mutant variants of YeaG based on amino acid sequence alignments with AAA+ and STK domains of well-characterized proteins (Figure S1B and Figure S1C). These mutants contained catalytically deleterious single amino acid substitutions either in the AAA+ (pBAD18-yeaG K116A and pBAD18-yeaG R232A) or STK (pBAD18-yeaG K426A) domain. We also generated a truncated version of YeaG by introducing a stop codon at amino acid position Y382 (pBAD18-yeaG Y382stop), which results in only the AAA+ domain being expressed. As shown in Fig. 3A–C, even though the mutant variants were produced at 64% (yeaG K116A), 48% (yeaG R232A), 100% (yeaG K426A) and 96% (yeaG Y382stop) of wild-type levels, none of them were able to complement the phenotype of the ∆yeaG mutant strain under our experimental conditions. Overall, the results in Figs 1, 2, 3 clearly demonstrate that yeaG (and yeaH) has a role in the adaptation of E. coli to sustained N starvation and that the catalytic activities of both, the AAA+ and STK domains of YeaG, are required in this process.


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 catalytic activities of the AAA+ and eSTK domains of YeaG are required for its role in adaptation to sustained N starvation in E. coli.(A,B) Growth curves show complementation of the phenotype of the ∆yeaG mutant strain with plasmid-borne yeaG (pBAD18-yeaG), but not with mutant variants of yeaG containing catalytically deleterious mutations in the AAA+ domain (in A. pBAD18-yeaG K116A; pBAD18-yeaG R232A) or eSTK domain (in B. pBAD18-yeaG K426A; pBAD18-yeaG Y382Stop). Error bars on all growth curves represent sd (n = 3). Statistical analyses were performed by one-way ANOVA (**P < 0.01). (C) Protein levels of wild-type and mutant variants of YeaG assessed by anti-His immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664914&req=5

f3: The catalytic activities of the AAA+ and eSTK domains of YeaG are required for its role in adaptation to sustained N starvation in E. coli.(A,B) Growth curves show complementation of the phenotype of the ∆yeaG mutant strain with plasmid-borne yeaG (pBAD18-yeaG), but not with mutant variants of yeaG containing catalytically deleterious mutations in the AAA+ domain (in A. pBAD18-yeaG K116A; pBAD18-yeaG R232A) or eSTK domain (in B. pBAD18-yeaG K426A; pBAD18-yeaG Y382Stop). Error bars on all growth curves represent sd (n = 3). Statistical analyses were performed by one-way ANOVA (**P < 0.01). (C) Protein levels of wild-type and mutant variants of YeaG assessed by anti-His immunoblotting.
Mentions: We were able to revert the growth characteristics of the ∆yeaG mutant strain to that of the wild-type strain by complementing with inducible plasmid-borne yeaG (∆yeaG pBAD-yeaG), thereby demonstrating that the altered growth of the mutant strain is not due to any undesired effects caused by the deletion of the yeaG gene (Fig. 3A,B). To determine if the catalytic activity of the AAA+ and/or STK domains of YeaG are required for function, we constructed mutant variants of YeaG based on amino acid sequence alignments with AAA+ and STK domains of well-characterized proteins (Figure S1B and Figure S1C). These mutants contained catalytically deleterious single amino acid substitutions either in the AAA+ (pBAD18-yeaG K116A and pBAD18-yeaG R232A) or STK (pBAD18-yeaG K426A) domain. We also generated a truncated version of YeaG by introducing a stop codon at amino acid position Y382 (pBAD18-yeaG Y382stop), which results in only the AAA+ domain being expressed. As shown in Fig. 3A–C, even though the mutant variants were produced at 64% (yeaG K116A), 48% (yeaG R232A), 100% (yeaG K426A) and 96% (yeaG Y382stop) of wild-type levels, none of them were able to complement the phenotype of the ∆yeaG mutant strain under our experimental conditions. Overall, the results in Figs 1, 2, 3 clearly demonstrate that yeaG (and yeaH) has a role in the adaptation of E. coli to sustained N starvation and that the catalytic activities of both, the AAA+ and STK domains of YeaG, are required in this process.

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