Journal
JOURNAL OF BACTERIOLOGY
Volume 197, Issue 18, Pages 2908-2919Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/JB.00324-15
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Funding
- NIDCR training program in oral sciences [T90 DE021985]
- NIH [R01GM084003]
- Kennedy Shriver National Institute of Child Health and Human Development intramural program of the National Institutes of Health
- Polish National Science Center [DEC-2013/10/E/NZ1/00657]
- Czech Science Foundation [15-11711S]
- European Regional Development Fund through the Centre of Excellence in Chemical Biology, Estonian Science Foundation [ETF9012, PUT37]
- Ragnar Soderberg Foundation
- Swedish Research Council
- Kempe Foundation
- Umea University
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The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p) ppGpp. In Enterococcus faecalis, (p) ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (Rel(Ef)) and the small alarmone synthetase (SAS) RelQ(Ef). Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQ(Ef) synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p) ppGpp synthesis from GDP and GTP, RelQ(Ef) also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p) ppGpp. We found that pGpp, like (p) ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQ(Ef) was activated only by ppGpp. Furthermore, enzymatic activity of RelQ(Ef) is insensitive to relacin, a (p) ppGpp analog developed as an inhibitor of long RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p) ppGpp. IMPORTANCE Accumulation of the nucleotide second messengers (p) ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p) ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p) ppGpp synthetase RelQ of Enterococcus faecalis (RelQ(Ef)), we found that, in addition to (p) ppGpp, RelQ(Ef) is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p) ppGpp. These findings provide a new regulatory feature of RelQ(Ef) and suggest that pGpp may represent a new member of the (pp) pGpp family of alarmones.
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