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Nitrogen stress response and stringent response are coupled in Escherichia coli.

Brown DR, Barton G, Pan Z, Buck M, Wigneshweraraj S - Nat Commun (2014)

Bottom Line: In addition, nitrogen-starved E. coli cells synthesize a signal molecule, guanosine tetraphosphate (ppGpp), which serves as an effector molecule of many processes including transcription to initiate global physiological changes, collectively termed the stringent response.The regulatory mechanisms leading to elevated ppGpp levels during nutritional stresses remain elusive.Here, we show that transcription of relA, a key gene responsible for the synthesis of ppGpp, is activated by NtrC during nitrogen starvation.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
Assimilation of nitrogen is an essential process in bacteria. The nitrogen regulation stress response is an adaptive mechanism used by nitrogen-starved Escherichia coli to scavenge for alternative nitrogen sources and requires the global transcriptional regulator NtrC. In addition, nitrogen-starved E. coli cells synthesize a signal molecule, guanosine tetraphosphate (ppGpp), which serves as an effector molecule of many processes including transcription to initiate global physiological changes, collectively termed the stringent response. The regulatory mechanisms leading to elevated ppGpp levels during nutritional stresses remain elusive. Here, we show that transcription of relA, a key gene responsible for the synthesis of ppGpp, is activated by NtrC during nitrogen starvation. The results reveal that NtrC couples these two major bacterial stress responses to manage conditions of nitrogen limitation, and provide novel mechanistic insights into how a specific nutritional stress leads to elevating ppGpp levels in bacteria.

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Related in: MedlinePlus

Establishing N-starved growth conditions in E. coli.(a) The growth arrest of wild-type E. coli NCM3722 cells coincides with ammonium run out (at t=NRO) in the minimal Gutnick medium. The time points at which the E. coli cells were sampled for downstream analysis are indicated (t=N+ and t=N− represents growth under nitrogen replete and starved conditions, respectively). (b) The growth curves of wild-type NCM3722, NCM3722:glnG-FLAG (NtrC-3xFLAG), NCM3722:ΔglnG and NCM3722:ΔglnG::glnGind (−/+ IPTG). The quantitation of the doubling times is also given. (c) Graph showing the relative levels of glnK mRNA expression as fold-change in cells sampled at t=N+ and t=N−. Error bars on all growth curves represent s.d. (where n=3). Statistical significant relationships from One-way ANOVA analysis are denoted (****P<0.0001).
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f1: Establishing N-starved growth conditions in E. coli.(a) The growth arrest of wild-type E. coli NCM3722 cells coincides with ammonium run out (at t=NRO) in the minimal Gutnick medium. The time points at which the E. coli cells were sampled for downstream analysis are indicated (t=N+ and t=N− represents growth under nitrogen replete and starved conditions, respectively). (b) The growth curves of wild-type NCM3722, NCM3722:glnG-FLAG (NtrC-3xFLAG), NCM3722:ΔglnG and NCM3722:ΔglnG::glnGind (−/+ IPTG). The quantitation of the doubling times is also given. (c) Graph showing the relative levels of glnK mRNA expression as fold-change in cells sampled at t=N+ and t=N−. Error bars on all growth curves represent s.d. (where n=3). Statistical significant relationships from One-way ANOVA analysis are denoted (****P<0.0001).

Mentions: To identify genome regions preferentially associated with NtrC, we introduced an in-frame fusion encoding three repeats of the FLAG epitope at the 3′ end of glnG in E. coli strain NCM3722, a prototrophic E. coli K-12 strain. To establish N-starved conditions, we grew batch cultures of the wild-type NCM3722 strain in Gutnick minimal medium supplemented with a limiting amount (3 mM) of NH4Cl as the sole N source and monitored bacterial growth as a function of ammonium consumption. In Fig. 1a, the time when wild-type NCM3722 stops growing (t=NRO) coincides with the ammonium run out ([ammonium]<0.000625, mM) in the growth medium. To establish that the FLAG epitope had not compromised the activity of NtrC, we measured the doubling times of wild-type NCM3722, NCM3722:glnG-FLAG and NCM3722:ΔglnG strains under our growth conditions. The doubling time of the NCM3722:glnG-FLAG strain is close to that of the wild-type NCM3722 strain; however, as expected, the doubling time of the NCM3722:ΔglnG strain is longer than that of the wild-type NCM3722 strain (Fig. 1b). Further, the expression levels of glnK mRNA, an Ntr stress response gene directly activated by NtrC, at t=N− (that is, the time-point 20 min after N runs out) are similar in the wild-type NCM3722 and NCM3722:glnG-FLAG strains, and as expected is not readily detected in the NCM3722:ΔglnG strain (Fig. 1c). Complementation of the NCM3722:ΔglnG strain with inducible plasmid-borne glnG restored both the doubling time and glnK mRNA expression (Fig. 1b,c). In summary, we conclude that (i) FLAG-tag has not adversely affected biological activity of NtrC and (ii) under our experimental conditions t=N− is representative of the time when the E. coli cells are starved for N.


Nitrogen stress response and stringent response are coupled in Escherichia coli.

Brown DR, Barton G, Pan Z, Buck M, Wigneshweraraj S - Nat Commun (2014)

Establishing N-starved growth conditions in E. coli.(a) The growth arrest of wild-type E. coli NCM3722 cells coincides with ammonium run out (at t=NRO) in the minimal Gutnick medium. The time points at which the E. coli cells were sampled for downstream analysis are indicated (t=N+ and t=N− represents growth under nitrogen replete and starved conditions, respectively). (b) The growth curves of wild-type NCM3722, NCM3722:glnG-FLAG (NtrC-3xFLAG), NCM3722:ΔglnG and NCM3722:ΔglnG::glnGind (−/+ IPTG). The quantitation of the doubling times is also given. (c) Graph showing the relative levels of glnK mRNA expression as fold-change in cells sampled at t=N+ and t=N−. Error bars on all growth curves represent s.d. (where n=3). Statistical significant relationships from One-way ANOVA analysis are denoted (****P<0.0001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4066584&req=5

f1: Establishing N-starved growth conditions in E. coli.(a) The growth arrest of wild-type E. coli NCM3722 cells coincides with ammonium run out (at t=NRO) in the minimal Gutnick medium. The time points at which the E. coli cells were sampled for downstream analysis are indicated (t=N+ and t=N− represents growth under nitrogen replete and starved conditions, respectively). (b) The growth curves of wild-type NCM3722, NCM3722:glnG-FLAG (NtrC-3xFLAG), NCM3722:ΔglnG and NCM3722:ΔglnG::glnGind (−/+ IPTG). The quantitation of the doubling times is also given. (c) Graph showing the relative levels of glnK mRNA expression as fold-change in cells sampled at t=N+ and t=N−. Error bars on all growth curves represent s.d. (where n=3). Statistical significant relationships from One-way ANOVA analysis are denoted (****P<0.0001).
Mentions: To identify genome regions preferentially associated with NtrC, we introduced an in-frame fusion encoding three repeats of the FLAG epitope at the 3′ end of glnG in E. coli strain NCM3722, a prototrophic E. coli K-12 strain. To establish N-starved conditions, we grew batch cultures of the wild-type NCM3722 strain in Gutnick minimal medium supplemented with a limiting amount (3 mM) of NH4Cl as the sole N source and monitored bacterial growth as a function of ammonium consumption. In Fig. 1a, the time when wild-type NCM3722 stops growing (t=NRO) coincides with the ammonium run out ([ammonium]<0.000625, mM) in the growth medium. To establish that the FLAG epitope had not compromised the activity of NtrC, we measured the doubling times of wild-type NCM3722, NCM3722:glnG-FLAG and NCM3722:ΔglnG strains under our growth conditions. The doubling time of the NCM3722:glnG-FLAG strain is close to that of the wild-type NCM3722 strain; however, as expected, the doubling time of the NCM3722:ΔglnG strain is longer than that of the wild-type NCM3722 strain (Fig. 1b). Further, the expression levels of glnK mRNA, an Ntr stress response gene directly activated by NtrC, at t=N− (that is, the time-point 20 min after N runs out) are similar in the wild-type NCM3722 and NCM3722:glnG-FLAG strains, and as expected is not readily detected in the NCM3722:ΔglnG strain (Fig. 1c). Complementation of the NCM3722:ΔglnG strain with inducible plasmid-borne glnG restored both the doubling time and glnK mRNA expression (Fig. 1b,c). In summary, we conclude that (i) FLAG-tag has not adversely affected biological activity of NtrC and (ii) under our experimental conditions t=N− is representative of the time when the E. coli cells are starved for N.

Bottom Line: In addition, nitrogen-starved E. coli cells synthesize a signal molecule, guanosine tetraphosphate (ppGpp), which serves as an effector molecule of many processes including transcription to initiate global physiological changes, collectively termed the stringent response.The regulatory mechanisms leading to elevated ppGpp levels during nutritional stresses remain elusive.Here, we show that transcription of relA, a key gene responsible for the synthesis of ppGpp, is activated by NtrC during nitrogen starvation.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
Assimilation of nitrogen is an essential process in bacteria. The nitrogen regulation stress response is an adaptive mechanism used by nitrogen-starved Escherichia coli to scavenge for alternative nitrogen sources and requires the global transcriptional regulator NtrC. In addition, nitrogen-starved E. coli cells synthesize a signal molecule, guanosine tetraphosphate (ppGpp), which serves as an effector molecule of many processes including transcription to initiate global physiological changes, collectively termed the stringent response. The regulatory mechanisms leading to elevated ppGpp levels during nutritional stresses remain elusive. Here, we show that transcription of relA, a key gene responsible for the synthesis of ppGpp, is activated by NtrC during nitrogen starvation. The results reveal that NtrC couples these two major bacterial stress responses to manage conditions of nitrogen limitation, and provide novel mechanistic insights into how a specific nutritional stress leads to elevating ppGpp levels in bacteria.

Show MeSH
Related in: MedlinePlus