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The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p) ppGpp.

ABSTRACT: The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp, regulate bacterial responses to nutritional and other stresses. There is evidence for potential existence of a third alarmone, guanosine-5'-monophosphate-3'-diphosphate (pGpp), with less-clear functions. Here, we demonstrate the presence of pGpp in bacterial cells, and perform a comprehensive screening to identify proteins that interact respectively with pGpp, ppGpp and pppGpp in Bacillus species. Both ppGpp and pppGpp interact with proteins involved in inhibition of purine nucleotide biosynthesis and with GTPases that control ribosome assembly or activity. By contrast, pGpp interacts with purine biosynthesis proteins but not with the GTPases. In addition, we show that hydrolase NahA (also known as YvcI) efficiently produces pGpp by hydrolyzing (p)ppGpp, thus modulating alarmone composition and function. Deletion of nahA leads to reduction of pGpp levels, increased (p)ppGpp levels, slower growth recovery from nutrient downshift, and loss of competitive fitness. Our results support the existence and physiological relevance of pGpp as a third alarmone, with functions that can be distinct from those of (p)ppGpp.

SUBMITTER: Yang J

PROVIDER: S-EPMC7584652 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p) ppGpp.

Yang Jin J Anderson Brent W BW Turdiev Asan A Turdiev Husan H Stevenson David M DM Amador-Noguez Daniel D Lee Vincent T VT Wang Jue D JD

Nature communications 20201023 1

The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp, regulate bacterial responses to nutritional and other stresses. There is evidence for potential existence of a third alarmone, guanosine-5'-monophosphate-3'-diphosphate (pGpp), with less-clear functions. Here, we demonstrate the presence of pGpp in bacterial cells, and perform a comprehensive screening to identify proteins that interact respectively with pGpp, ppGpp and pppGpp in Bacillus spec ...[more]

PMID: 33097692

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Project description:UnlabelledThe 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 (RelEf) and the small alarmone synthetase (SAS) RelQEf. 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 RelQEf synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEf 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 RelQEf was activated only by ppGpp. Furthermore, enzymatic activity of RelQEf 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.ImportanceAccumulation 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 (RelQEf), we found that, in addition to (p)ppGpp, RelQEf 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 RelQEf and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.

Project description:Gene expression and proper downstream cellular functions upon facing environmental shifts depend on the combined and cooperative regulation of genetic networks. Here, we identified cAMP receptor protein (CRP) as a master regulator of (p)ppGpp (guanosine tetra- and penta-phosphate) homeostasis. Via CRP-mediated direct transcriptional regulation of the (p)ppGpp synthetase/hydrolase RelA and SpoT, cAMP-CRP stimulates pervasive accumulation of (p)ppGpp under glucose-limiting conditions. Notably, CRP exerts a nonclassical property as a translational regulator through YfiQ-dependent acetylation of ribosome protein S1 at K247, which further enhances the translation of RelA, SpoT, and CRP itself. From a synthetic biology perspective, this self-activating feedback loop for (p)ppGpp synthesis highlights the function of CRP-mediated dual enhancement (CMDE) in controlling bacterial gene expression, which enables stable activation of genetic circuits. CMDE applied in synthetic circuits leads to a stable increase in p-coumaric acid, cinnamic acid, and pinosylvin production. Our findings showed that CRP-mediated dual circuits for (p)ppGpp regulation enable robust activation that could address bioproduction and other biotechnological needs.IMPORTANCETranscriptional-translational coordination is fundamental for rapid and efficient gene expression in most bacteria. Here, we uncovered the roles of cAMP-CRP in this process. We found that CRP distinctly increases RelA and SpoT transcription and translation, and that acetylation of S1 at K247 accelerates the self-activation of the leading CRP under glucose-limiting conditions. We further found that elevated (p)ppGpp significantly impedes the formation of the cAMP-CRP complex, an active form responsible for transcriptional activation. A model was created in which cAMP-CRP and (p)ppGpp cooperate to dynamically modulate the efficiency of transcriptional-translational coordination responses to stress. More broadly, productive activation in synthetic circuits was achieved through the application of CRP-mediated dual enhancement (CMDE), promising to inspire new approaches for the development of cell-based biotechnologies.

Dataset Information

The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p) ppGpp.

Publications

The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p) ppGpp.

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