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Large-scale filament formation inhibits the activity of CTP synthetase.

Barry RM, Bitbol AF, Lorestani A, Charles EJ, Habrian CH, Hansen JM, Li HJ, Baldwin EP, Wingreen NS, Kollman JM, Gitai Z - Elife (2014)

Bottom Line: Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation.This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels.We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, United States.

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Metabolomic analysis of wild-type and CtpSE277RE. coli after addition of 200 µg/ml C13-cytidine.Fold changes of metabolite levels of NCM3722 kanRE. coli and CtpSE277RE. coli were compared to wild type levels at 0 min. Hierarchical clustering of metabolites is shown.DOI:http://dx.doi.org/10.7554/eLife.03638.030
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fig8s1: Metabolomic analysis of wild-type and CtpSE277RE. coli after addition of 200 µg/ml C13-cytidine.Fold changes of metabolite levels of NCM3722 kanRE. coli and CtpSE277RE. coli were compared to wild type levels at 0 min. Hierarchical clustering of metabolites is shown.DOI:http://dx.doi.org/10.7554/eLife.03638.030

Mentions: To determine the impact of CtpSE277R on cell physiology, we replaced wild-type CtpS with CtpSE277R at its native locus in E. coli. This strain exhibited defective growth compared to wild type in rich (Figure 7C) and minimal media (Figure 7—figure supplement 3). Wild type doubling time was 51 min ± 1.5 min, while the CtpSE277R doubling time was 130 min ± 11 min in rich media. Immunoblotting confirmed that CtpSE277R was expressed at similar levels to wild-type CtpS (Figure 7—figure supplement 4). One possible explanation for the growth impairment is that CtpSE277R could not produce enough CTP to support robust growth. However, CTP levels, as measured by mass spectrometry, are not reduced in the CtpSE277R strain (Figure 8—figure supplement 1). In fact, CTP levels are modestly higher in the mutant than in wild type cells (1.6 ± 0.3-fold higher). Because average CTP levels are higher in these cells, CtpSE277R likely does not impair growth due to reduced CTP production. Rather, the elevated CTP levels and the observation that growth became particularly affected at mid-log phase support the hypothesis that the CtpSE277R mutant is defective in regulating CTP levels when adapting to changes in the cellular environment.


Large-scale filament formation inhibits the activity of CTP synthetase.

Barry RM, Bitbol AF, Lorestani A, Charles EJ, Habrian CH, Hansen JM, Li HJ, Baldwin EP, Wingreen NS, Kollman JM, Gitai Z - Elife (2014)

Metabolomic analysis of wild-type and CtpSE277RE. coli after addition of 200 µg/ml C13-cytidine.Fold changes of metabolite levels of NCM3722 kanRE. coli and CtpSE277RE. coli were compared to wild type levels at 0 min. Hierarchical clustering of metabolites is shown.DOI:http://dx.doi.org/10.7554/eLife.03638.030
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126345&req=5

fig8s1: Metabolomic analysis of wild-type and CtpSE277RE. coli after addition of 200 µg/ml C13-cytidine.Fold changes of metabolite levels of NCM3722 kanRE. coli and CtpSE277RE. coli were compared to wild type levels at 0 min. Hierarchical clustering of metabolites is shown.DOI:http://dx.doi.org/10.7554/eLife.03638.030
Mentions: To determine the impact of CtpSE277R on cell physiology, we replaced wild-type CtpS with CtpSE277R at its native locus in E. coli. This strain exhibited defective growth compared to wild type in rich (Figure 7C) and minimal media (Figure 7—figure supplement 3). Wild type doubling time was 51 min ± 1.5 min, while the CtpSE277R doubling time was 130 min ± 11 min in rich media. Immunoblotting confirmed that CtpSE277R was expressed at similar levels to wild-type CtpS (Figure 7—figure supplement 4). One possible explanation for the growth impairment is that CtpSE277R could not produce enough CTP to support robust growth. However, CTP levels, as measured by mass spectrometry, are not reduced in the CtpSE277R strain (Figure 8—figure supplement 1). In fact, CTP levels are modestly higher in the mutant than in wild type cells (1.6 ± 0.3-fold higher). Because average CTP levels are higher in these cells, CtpSE277R likely does not impair growth due to reduced CTP production. Rather, the elevated CTP levels and the observation that growth became particularly affected at mid-log phase support the hypothesis that the CtpSE277R mutant is defective in regulating CTP levels when adapting to changes in the cellular environment.

Bottom Line: Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation.This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels.We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, United States.

Show MeSH