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Regulation of Indole Signalling during the Transition of E. coli from Exponential to Stationary Phase.

Gaimster H, Summers D - PLoS ONE (2015)

Bottom Line: It has recently been shown that cell-associated indole transiently reaches a very high concentration (approx. 60 mM) during stationary phase entry, presumably because indole is being produced more rapidly than it can leave the cell.The magnitude and duration of the resulting indole pulse are dependent upon the availability of exogenous tryptophan.Indole production stops when all the available tryptophan is depleted and the cell-associated indole equilibrates with the supernatant.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, United Kingdom.

ABSTRACT
During the transition from exponential to stationary phase E. coli produces a substantial quantity of the small, aromatic signalling molecule indole. In LB medium the supernatant indole concentration reaches a maximum of 0.5-1 mM. At this concentration indole has been implicated in many processes inducing acid resistance and the modulation of virulence. It has recently been shown that cell-associated indole transiently reaches a very high concentration (approx. 60 mM) during stationary phase entry, presumably because indole is being produced more rapidly than it can leave the cell. It is proposed that this indole pulse inhibits growth and cell division, causing the culture to enter stationary phase before nutrients are completely exhausted, with benefits for survival in long-term stationary phase. This study asks how E. coli cells rapidly upregulate indole production during stationary phase entry and why the indole pulse has a duration of only 10-15 min. We find that at the start of the pulse tryptophanase synthesis is triggered by glucose depletion and that this is correlates with the up-regulation of indole synthesis. The magnitude and duration of the resulting indole pulse are dependent upon the availability of exogenous tryptophan. Indole production stops when all the available tryptophan is depleted and the cell-associated indole equilibrates with the supernatant.

No MeSH data available.


Related in: MedlinePlus

The addition of tryptophan to a stationary phase BW25113 culture leads to further indole synthesis.Stationary phase (24 hours) BW25113 cells were incubated with no additional tryptophan or 2 mM tryptophan added every subsequent 24 hours (arrows indicate times of addition). Samples were removed then centrifuged to remove cells. The supernatant was assayed for external indole using Kovacs assay. Data shown are the mean values ± standard deviation for three independent repeats.
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pone.0136691.g003: The addition of tryptophan to a stationary phase BW25113 culture leads to further indole synthesis.Stationary phase (24 hours) BW25113 cells were incubated with no additional tryptophan or 2 mM tryptophan added every subsequent 24 hours (arrows indicate times of addition). Samples were removed then centrifuged to remove cells. The supernatant was assayed for external indole using Kovacs assay. Data shown are the mean values ± standard deviation for three independent repeats.

Mentions: In order to test the hypothesis that indole synthesis is limited by the availability of free tryptophan, an overnight culture of BW25113 cells was diluted into fresh LB medium to OD600 = 0.05. The culture was grown at 37°C with shaking for 24 hours. The supernatant indole concentration in the stationary phase culture was assayed and it was divided into two. One sub-culture was incubated at 37°C with shaking for a further 144 hours (6 days). The other culture was also incubated with shaking at 37°C but 2 mM supplements of tryptophan were added at 24, 48 and 72 hours. Thus, in total, a further 6 mM tryptophan was added to the culture. Samples were taken from the cultures every 24 hours for 72 hours and one final sample at 168 hours. The supernatant indole in each sample was assayed (Fig 3). After 24 hours, when no additional tryptophan had been added to either culture, the concentrations of indole in the culture supernatants were the same (approx. 1 mM). In the unsupplemented culture, the external indole remained constant at approximately 1 mM for 6 days. In the supplemented cultures, where an additional 2 mM tryptophan was added at 24 hours, the supernatant indole had risen to approximately 3.5 mM by 48 hours. This represents complete conversion to indole of the 2 mM tryptophan supplement in addition to approx. 1 mM present originally in the LB medium. A further 2 mM tryptophan was added at 48 hours and the supernatant indole had risen to approximately 5 mM by 72 hours. Once again, this represents almost complete conversion of the tryptophan supplement to indole. Although final supplement of 2 mM tryptophan was added at 72 hours, the external indole increased only slightly and remained at approximately 5 mM even after 168 hours. This is consistent with the findings of Li and Young (2014) who reported stoichiometric conversion of tryptophan into indole, up to a maximum indole concentration of approx. 5 mM. The data of Fig 3 demonstrate that active tryptophanase remains present in stationary phase cells for at least two days. During this period tryptophan availability limits indole synthesis. It therefore seems likely that tryptophan depletion is responsible for termination of the indole pulse during the transition to stationary phase.


Regulation of Indole Signalling during the Transition of E. coli from Exponential to Stationary Phase.

Gaimster H, Summers D - PLoS ONE (2015)

The addition of tryptophan to a stationary phase BW25113 culture leads to further indole synthesis.Stationary phase (24 hours) BW25113 cells were incubated with no additional tryptophan or 2 mM tryptophan added every subsequent 24 hours (arrows indicate times of addition). Samples were removed then centrifuged to remove cells. The supernatant was assayed for external indole using Kovacs assay. Data shown are the mean values ± standard deviation for three independent repeats.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4557909&req=5

pone.0136691.g003: The addition of tryptophan to a stationary phase BW25113 culture leads to further indole synthesis.Stationary phase (24 hours) BW25113 cells were incubated with no additional tryptophan or 2 mM tryptophan added every subsequent 24 hours (arrows indicate times of addition). Samples were removed then centrifuged to remove cells. The supernatant was assayed for external indole using Kovacs assay. Data shown are the mean values ± standard deviation for three independent repeats.
Mentions: In order to test the hypothesis that indole synthesis is limited by the availability of free tryptophan, an overnight culture of BW25113 cells was diluted into fresh LB medium to OD600 = 0.05. The culture was grown at 37°C with shaking for 24 hours. The supernatant indole concentration in the stationary phase culture was assayed and it was divided into two. One sub-culture was incubated at 37°C with shaking for a further 144 hours (6 days). The other culture was also incubated with shaking at 37°C but 2 mM supplements of tryptophan were added at 24, 48 and 72 hours. Thus, in total, a further 6 mM tryptophan was added to the culture. Samples were taken from the cultures every 24 hours for 72 hours and one final sample at 168 hours. The supernatant indole in each sample was assayed (Fig 3). After 24 hours, when no additional tryptophan had been added to either culture, the concentrations of indole in the culture supernatants were the same (approx. 1 mM). In the unsupplemented culture, the external indole remained constant at approximately 1 mM for 6 days. In the supplemented cultures, where an additional 2 mM tryptophan was added at 24 hours, the supernatant indole had risen to approximately 3.5 mM by 48 hours. This represents complete conversion to indole of the 2 mM tryptophan supplement in addition to approx. 1 mM present originally in the LB medium. A further 2 mM tryptophan was added at 48 hours and the supernatant indole had risen to approximately 5 mM by 72 hours. Once again, this represents almost complete conversion of the tryptophan supplement to indole. Although final supplement of 2 mM tryptophan was added at 72 hours, the external indole increased only slightly and remained at approximately 5 mM even after 168 hours. This is consistent with the findings of Li and Young (2014) who reported stoichiometric conversion of tryptophan into indole, up to a maximum indole concentration of approx. 5 mM. The data of Fig 3 demonstrate that active tryptophanase remains present in stationary phase cells for at least two days. During this period tryptophan availability limits indole synthesis. It therefore seems likely that tryptophan depletion is responsible for termination of the indole pulse during the transition to stationary phase.

Bottom Line: It has recently been shown that cell-associated indole transiently reaches a very high concentration (approx. 60 mM) during stationary phase entry, presumably because indole is being produced more rapidly than it can leave the cell.The magnitude and duration of the resulting indole pulse are dependent upon the availability of exogenous tryptophan.Indole production stops when all the available tryptophan is depleted and the cell-associated indole equilibrates with the supernatant.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, United Kingdom.

ABSTRACT
During the transition from exponential to stationary phase E. coli produces a substantial quantity of the small, aromatic signalling molecule indole. In LB medium the supernatant indole concentration reaches a maximum of 0.5-1 mM. At this concentration indole has been implicated in many processes inducing acid resistance and the modulation of virulence. It has recently been shown that cell-associated indole transiently reaches a very high concentration (approx. 60 mM) during stationary phase entry, presumably because indole is being produced more rapidly than it can leave the cell. It is proposed that this indole pulse inhibits growth and cell division, causing the culture to enter stationary phase before nutrients are completely exhausted, with benefits for survival in long-term stationary phase. This study asks how E. coli cells rapidly upregulate indole production during stationary phase entry and why the indole pulse has a duration of only 10-15 min. We find that at the start of the pulse tryptophanase synthesis is triggered by glucose depletion and that this is correlates with the up-regulation of indole synthesis. The magnitude and duration of the resulting indole pulse are dependent upon the availability of exogenous tryptophan. Indole production stops when all the available tryptophan is depleted and the cell-associated indole equilibrates with the supernatant.

No MeSH data available.


Related in: MedlinePlus