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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

yeaG has a role in the adaptive response to sustained N starvation.(A) yeaG is upregulated in response to N starvation. Levels of yeaG expression in wild-type E. coli were calculated as relative mRNA amounts compared to the 16S control under N-replete (N+) and N-starved (N-) conditions. The insert is a schematic graph showing that E. coli growth arrest corresponds to N run out (NRO) in the media (orange line) and indicates the time points (N+ and N-) at which cells were sampled for total RNA extraction and analysis. (B) ‘Pre-starvation’ growth of ∆yeaG, ∆yeaH and ∆yeaGH mutant strains is similar to that of the wild-type strain. The growth curves were obtained by determining the OD600nm readings at half-hourly time points. (C) Transcription of yeaG occurs during sustained (24 h) N starvation. The level of yeaG expression in wild-type E. coli is expressed as fold change in mRNA levels between N+ and N-24 h starved conditions. In the presence of rifampicin, an inhibitor of RNA synthesis, no yeaG mRNA was detected in the N-24 h sample, which indicates that the yeaG mRNA expression observed in the absence of rifampicin is due to transcription of yeaG over the 24 h period of sustained N starvation. The insert is a schematic representation of the experiment. (D) ∆yeaG, ∆yeaH and ∆yeaGH mutant strains display shorter lag phase during ‘recovery’ growth following 24 h of N starvation compared to a wild-type strain. The ‘recovery’ growth curves were obtained by determining the OD600nm readings at half-hourly time points. The dotted black line indicates the ‘pre-starvation’ growth curve for the wild-type strain. The insert is a schematic representation of the experiment. (E) Measurement of ‘recovery’ growth of wild-type and ∆yeaG mutant strain by colony-forming unit (CFU) count at 0 h, 2 h, 4 h and 6 h. (F) The rate of ammonium consumption by the wild-type and ∆yeaG mutant strain is directly proportional to their respective ‘recovery’ growth characteristics. Error bars on all growth curves and graph represent sd (n = 3). Statistical analyses were performed by one-way ANOVA (**P < 0.01).
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f1: yeaG has a role in the adaptive response to sustained N starvation.(A) yeaG is upregulated in response to N starvation. Levels of yeaG expression in wild-type E. coli were calculated as relative mRNA amounts compared to the 16S control under N-replete (N+) and N-starved (N-) conditions. The insert is a schematic graph showing that E. coli growth arrest corresponds to N run out (NRO) in the media (orange line) and indicates the time points (N+ and N-) at which cells were sampled for total RNA extraction and analysis. (B) ‘Pre-starvation’ growth of ∆yeaG, ∆yeaH and ∆yeaGH mutant strains is similar to that of the wild-type strain. The growth curves were obtained by determining the OD600nm readings at half-hourly time points. (C) Transcription of yeaG occurs during sustained (24 h) N starvation. The level of yeaG expression in wild-type E. coli is expressed as fold change in mRNA levels between N+ and N-24 h starved conditions. In the presence of rifampicin, an inhibitor of RNA synthesis, no yeaG mRNA was detected in the N-24 h sample, which indicates that the yeaG mRNA expression observed in the absence of rifampicin is due to transcription of yeaG over the 24 h period of sustained N starvation. The insert is a schematic representation of the experiment. (D) ∆yeaG, ∆yeaH and ∆yeaGH mutant strains display shorter lag phase during ‘recovery’ growth following 24 h of N starvation compared to a wild-type strain. The ‘recovery’ growth curves were obtained by determining the OD600nm readings at half-hourly time points. The dotted black line indicates the ‘pre-starvation’ growth curve for the wild-type strain. The insert is a schematic representation of the experiment. (E) Measurement of ‘recovery’ growth of wild-type and ∆yeaG mutant strain by colony-forming unit (CFU) count at 0 h, 2 h, 4 h and 6 h. (F) The rate of ammonium consumption by the wild-type and ∆yeaG mutant strain is directly proportional to their respective ‘recovery’ growth characteristics. Error bars on all growth curves and graph represent sd (n = 3). Statistical analyses were performed by one-way ANOVA (**P < 0.01).

Mentions: Previously we reported that NtrC and RNA polymerase bind to the promoter region of the yeaGH operon in N-starved E. coli4. To establish whether these binding events lead to transcription of the yeaGH operon we measured levels of yeaG mRNA by quantitative real-time PCR. Bacteria were grown in batch cultures in minimal media that was supplemented with a limiting amount (3 mM) of ammonium as the sole source of N. Under these conditions, we previously showed that bacterial growth arrest directly coincides with the acquisition of a bona fide N starved state (indicated as N- in the schematic in Fig. 1A where OD600 is ~0.85 and [NH4Cl] is <0.000625 mM) that occurs 20 min after ammonium has run out in the media (indicated as NRO in the schematic in Fig. 1A)4. As expected, yeaG mRNA levels in N-starved E. coli were 36-fold (±2.3 SD) higher than in bacteria from nitrogen-replete conditions (indicated as N+ in the schematic in Fig. 1A where OD600 is ~0.3 and [NH4Cl] is ~2.5 mM) (Fig. 1A). Having established that yeaG is expressed in N-starved E. coli shortly after sensing N starvation, we next determined the growth characteristics of a ∆yeaG mutant E. coli strain in minimal media containing limiting amount (3 mM) of ammonium as the sole N source. Under these conditions (henceforth referred to as ‘pre-starvation’ growth) no growth difference was detected between the ∆yeaG mutant and wild-type E. coli and both strains ceased growth when ammonium ran out in the media (Fig. 1B). Even though the expression of yeaG occurs shortly after the cells sense N starvation (Fig. 1A), it appears to be continuously expressed for up to 24 h into N starvation (Fig. 1C), we considered whether yeaG could have a role in adaptation to sustained (here defined as 24 h, unless otherwise indicated) N starvation. To investigate this, wild-type and ∆yeaG mutant bacteria were subjected to 24 h of N starvation before being sub-cultured into fresh media and their growth monitored (henceforth referred to as ‘recovery’ growth). As shown in Fig. 1D, the wild-type strain displayed a considerably increased lag phase (by 2.3 h) during ‘recovery’ growth compared to ‘pre-starvation’ growth, which is characteristic of bacterial adaptation to the new growth environment. In marked contrast to the wild-type strain, the ∆yeaG mutant strain displayed a much shorter lag phase (by 32 min or 13% less than the length of wild-type lag phase) during ‘recovery’ growth (Fig. 1D and Table S1). However, once in the exponential phase of ‘recovery’ growth, the doubling times of both strains were comparable (Fig. 1D and Table S1). Similar results were obtained with E. coli strains lacking yeaH or both yeaG and yeaH genes, indicating that yeaG and yeaH are functionally linked (Fig. 1D and Table S1). Control experiments in which we either measured the number of viable cells at different times during ‘recovery’ growth by plate count of colony forming units (CFU) (Fig. 1E) or monitored the rate of ammonium consumption during ‘recovery’ growth (Fig. 1F) independently validated the shorter lag phase of the ∆yeaG mutant compared to the wild-type strain. Importantly, the difference in the length of the lag phase between the wild-type and ∆yeaG strain during ‘recovery’ growth was directly proportional to the length of time spent under N starvation conditions (Fig. 2), which further emphasizes the role for yeaG in sustained N starvation, even though the expression of yeaG occurs shortly after the cells experience N starvation.


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)

yeaG has a role in the adaptive response to sustained N starvation.(A) yeaG is upregulated in response to N starvation. Levels of yeaG expression in wild-type E. coli were calculated as relative mRNA amounts compared to the 16S control under N-replete (N+) and N-starved (N-) conditions. The insert is a schematic graph showing that E. coli growth arrest corresponds to N run out (NRO) in the media (orange line) and indicates the time points (N+ and N-) at which cells were sampled for total RNA extraction and analysis. (B) ‘Pre-starvation’ growth of ∆yeaG, ∆yeaH and ∆yeaGH mutant strains is similar to that of the wild-type strain. The growth curves were obtained by determining the OD600nm readings at half-hourly time points. (C) Transcription of yeaG occurs during sustained (24 h) N starvation. The level of yeaG expression in wild-type E. coli is expressed as fold change in mRNA levels between N+ and N-24 h starved conditions. In the presence of rifampicin, an inhibitor of RNA synthesis, no yeaG mRNA was detected in the N-24 h sample, which indicates that the yeaG mRNA expression observed in the absence of rifampicin is due to transcription of yeaG over the 24 h period of sustained N starvation. The insert is a schematic representation of the experiment. (D) ∆yeaG, ∆yeaH and ∆yeaGH mutant strains display shorter lag phase during ‘recovery’ growth following 24 h of N starvation compared to a wild-type strain. The ‘recovery’ growth curves were obtained by determining the OD600nm readings at half-hourly time points. The dotted black line indicates the ‘pre-starvation’ growth curve for the wild-type strain. The insert is a schematic representation of the experiment. (E) Measurement of ‘recovery’ growth of wild-type and ∆yeaG mutant strain by colony-forming unit (CFU) count at 0 h, 2 h, 4 h and 6 h. (F) The rate of ammonium consumption by the wild-type and ∆yeaG mutant strain is directly proportional to their respective ‘recovery’ growth characteristics. Error bars on all growth curves and graph represent sd (n = 3). Statistical analyses were performed by one-way ANOVA (**P < 0.01).
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f1: yeaG has a role in the adaptive response to sustained N starvation.(A) yeaG is upregulated in response to N starvation. Levels of yeaG expression in wild-type E. coli were calculated as relative mRNA amounts compared to the 16S control under N-replete (N+) and N-starved (N-) conditions. The insert is a schematic graph showing that E. coli growth arrest corresponds to N run out (NRO) in the media (orange line) and indicates the time points (N+ and N-) at which cells were sampled for total RNA extraction and analysis. (B) ‘Pre-starvation’ growth of ∆yeaG, ∆yeaH and ∆yeaGH mutant strains is similar to that of the wild-type strain. The growth curves were obtained by determining the OD600nm readings at half-hourly time points. (C) Transcription of yeaG occurs during sustained (24 h) N starvation. The level of yeaG expression in wild-type E. coli is expressed as fold change in mRNA levels between N+ and N-24 h starved conditions. In the presence of rifampicin, an inhibitor of RNA synthesis, no yeaG mRNA was detected in the N-24 h sample, which indicates that the yeaG mRNA expression observed in the absence of rifampicin is due to transcription of yeaG over the 24 h period of sustained N starvation. The insert is a schematic representation of the experiment. (D) ∆yeaG, ∆yeaH and ∆yeaGH mutant strains display shorter lag phase during ‘recovery’ growth following 24 h of N starvation compared to a wild-type strain. The ‘recovery’ growth curves were obtained by determining the OD600nm readings at half-hourly time points. The dotted black line indicates the ‘pre-starvation’ growth curve for the wild-type strain. The insert is a schematic representation of the experiment. (E) Measurement of ‘recovery’ growth of wild-type and ∆yeaG mutant strain by colony-forming unit (CFU) count at 0 h, 2 h, 4 h and 6 h. (F) The rate of ammonium consumption by the wild-type and ∆yeaG mutant strain is directly proportional to their respective ‘recovery’ growth characteristics. Error bars on all growth curves and graph represent sd (n = 3). Statistical analyses were performed by one-way ANOVA (**P < 0.01).
Mentions: Previously we reported that NtrC and RNA polymerase bind to the promoter region of the yeaGH operon in N-starved E. coli4. To establish whether these binding events lead to transcription of the yeaGH operon we measured levels of yeaG mRNA by quantitative real-time PCR. Bacteria were grown in batch cultures in minimal media that was supplemented with a limiting amount (3 mM) of ammonium as the sole source of N. Under these conditions, we previously showed that bacterial growth arrest directly coincides with the acquisition of a bona fide N starved state (indicated as N- in the schematic in Fig. 1A where OD600 is ~0.85 and [NH4Cl] is <0.000625 mM) that occurs 20 min after ammonium has run out in the media (indicated as NRO in the schematic in Fig. 1A)4. As expected, yeaG mRNA levels in N-starved E. coli were 36-fold (±2.3 SD) higher than in bacteria from nitrogen-replete conditions (indicated as N+ in the schematic in Fig. 1A where OD600 is ~0.3 and [NH4Cl] is ~2.5 mM) (Fig. 1A). Having established that yeaG is expressed in N-starved E. coli shortly after sensing N starvation, we next determined the growth characteristics of a ∆yeaG mutant E. coli strain in minimal media containing limiting amount (3 mM) of ammonium as the sole N source. Under these conditions (henceforth referred to as ‘pre-starvation’ growth) no growth difference was detected between the ∆yeaG mutant and wild-type E. coli and both strains ceased growth when ammonium ran out in the media (Fig. 1B). Even though the expression of yeaG occurs shortly after the cells sense N starvation (Fig. 1A), it appears to be continuously expressed for up to 24 h into N starvation (Fig. 1C), we considered whether yeaG could have a role in adaptation to sustained (here defined as 24 h, unless otherwise indicated) N starvation. To investigate this, wild-type and ∆yeaG mutant bacteria were subjected to 24 h of N starvation before being sub-cultured into fresh media and their growth monitored (henceforth referred to as ‘recovery’ growth). As shown in Fig. 1D, the wild-type strain displayed a considerably increased lag phase (by 2.3 h) during ‘recovery’ growth compared to ‘pre-starvation’ growth, which is characteristic of bacterial adaptation to the new growth environment. In marked contrast to the wild-type strain, the ∆yeaG mutant strain displayed a much shorter lag phase (by 32 min or 13% less than the length of wild-type lag phase) during ‘recovery’ growth (Fig. 1D and Table S1). However, once in the exponential phase of ‘recovery’ growth, the doubling times of both strains were comparable (Fig. 1D and Table S1). Similar results were obtained with E. coli strains lacking yeaH or both yeaG and yeaH genes, indicating that yeaG and yeaH are functionally linked (Fig. 1D and Table S1). Control experiments in which we either measured the number of viable cells at different times during ‘recovery’ growth by plate count of colony forming units (CFU) (Fig. 1E) or monitored the rate of ammonium consumption during ‘recovery’ growth (Fig. 1F) independently validated the shorter lag phase of the ∆yeaG mutant compared to the wild-type strain. Importantly, the difference in the length of the lag phase between the wild-type and ∆yeaG strain during ‘recovery’ growth was directly proportional to the length of time spent under N starvation conditions (Fig. 2), which further emphasizes the role for yeaG in sustained N starvation, even though the expression of yeaG occurs shortly after the cells experience N starvation.

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