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A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues
Published on: February 19, 2019
Global transcriptional analysis of the stringent response in Enterococcus faecalis
Anthony O Gaca1, Jacqueline Abranches1, Jessica K Kajfasz1
1Center for Oral Biology and Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.
Microbiology (Reading, England)
|June 2, 2012
Summary
Enterococcus faecalis uses RelA and RelQ enzymes to produce guanosine tetraphosphate/guanosine pentaphosphate [(p)ppGpp], crucial for the stringent response. RelA is primary under stress, but RelQ is vital for a complete and timely response, impacting virulence and metabolism.
Area of Science:
- Microbiology
- Bacterial Physiology
- Molecular Biology
Background:
- Guanylate cyclase activity, producing guanosine tetraphosphate/guanosine pentaphosphate [(p)ppGpp], is central to the stringent response in Enterococcus faecalis.
- This response is mediated by RelA (bifunctional synthetase/hydrolase) and RelQ (monofunctional synthetase), with RelA previously identified as the primary enzyme under stress.
Purpose of the Study:
- To elucidate the specific roles of RelA and RelQ in (p)ppGpp synthesis and the stringent response in E. faecalis.
- To investigate the impact of (p)ppGpp production on bacterial virulence and homeostasis.
- To perform global transcriptome analysis in a (p)ppGpp(0) Gram-positive bacterium to understand transcriptional regulation.
Main Methods:
- Comparative analysis of (p)ppGpp profiles in wild-type, ΔrelA, ΔrelQ, and ΔrelAΔrelQ E. faecalis strains.
- Assessment of bacterial virulence using macrophage survival assays (J774A.1) and Galleria mellonella killing assays.
- Global transcriptome analysis (RNA sequencing) under starvation and mupirocin-induced stress conditions.
Main Results:
- The ΔrelAΔrelQ strain exhibited attenuated virulence, unlike ΔrelA or ΔrelQ single mutants.
- Transcriptome profiling revealed RelA as the major driver of (p)ppGpp-mediated repression of macromolecular biosynthesis genes.
- RelQ was found to be essential for a full and timely stringent response, despite rapid RelA-dependent (p)ppGpp accumulation.
- The (p)ppGpp(0) strain showed altered energy metabolism, suggesting impaired fitness due to metabolic imbalance.
Conclusions:
- RelA is the primary enzyme for (p)ppGpp synthesis and stringent response initiation in E. faecalis.
- RelQ plays a critical, albeit previously uncharacterized, role in ensuring the completeness and timeliness of the stringent response.
- The absence of both RelA and RelQ leads to impaired virulence and potentially unbalanced metabolism, highlighting the complex interplay of these enzymes in bacterial survival and pathogenesis.
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