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Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography
Published on: June 4, 2019
Basal levels of (p)ppGpp in Enterococcus faecalis: the magic beyond the stringent response.
Anthony O Gaca1, Jessica K Kajfasz, James H Miller
1Center for Oral Biology and Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA.
Basal levels of guanosine tetraphosphate (p)ppGpp are crucial for bacterial metabolism and antibiotic survival, even below stringent response thresholds. Understanding these basal pools offers new antimicrobial therapy targets.
Area of Science:
- Bacterial Physiology
- Molecular Microbiology
- Biochemistry
Background:
- The stringent response (SR), regulated by (p)ppGpp, is vital for bacterial survival under stress.
- In Enterococcus faecalis, RSH and RelQ proteins control (p)ppGpp production, impacting stress survival and virulence.
- The role of basal (p)ppGpp levels during normal bacterial growth is not well understood.
Purpose of the Study:
- To investigate the significance of basal (p)ppGpp levels in bacterial metabolism and antibiotic survival.
- To explore how modest alterations in (p)ppGpp impact gene expression and cellular processes.
- To identify potential antimicrobial targets by understanding (p)ppGpp regulation.
Main Methods:
- Microarray analysis of Enterococcus faecalis strains with varying (p)ppGpp levels.
- Analysis of fermentation profiles and hydrogen peroxide production.
- Enzyme activity assays for GTP biosynthesis and GTP homeostasis studies.
Main Results:
- Low basal (p)ppGpp levels lead to a "transcriptionally relaxed" state, inducing energy generation genes.
- Altered fermentation and increased H2O2 production observed in strains lacking basal (p)ppGpp.
- (p)ppGpp directly inhibits GTP biosynthesis enzymes, and its absence dysregulates GTP homeostasis.
Conclusions:
- Basal (p)ppGpp pools play a critical role in maintaining metabolic balance during balanced growth.
- Antibiotic survival is linked to basal (p)ppGpp levels, not solely the stringent response.
- Targeting basal (p)ppGpp levels may offer novel strategies against drug-resistant bacteria.
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