From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

Gaca, Anthony O.; Kudrin, Pavel; Colomer-Winter, Cristina; Beljantseva, Jelena; Liu, Kuanqing; Anderson, Brent; Wang, Jue D.; Rejman, Dominik; Potrykus, Katarzyna; Cashel, Michael; Hauryliuk, Vasili; Lemos, José A.

doi:10.1128/jb.00324-15

Cited by 90 publications

(154 citation statements)

References 68 publications

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“…As we have shown previously (19), the enzymatic activity of E. faecalis RelQ is insensitive to the addition of E. coli 70S ribosomes (Fig. 1A).…”

Section: Resultsmentioning

confidence: 57%

“…Enzymatic assays with E. faecalis RelQ were performed following the method of Gaca et al (19). DRaCALA assays were performed as described by Roelofs et al (25).…”

Section: Methodsmentioning

confidence: 99%

“…Crystallographic analysis of the Bacillus subtilis RelQ (SAS1) revealed that it forms a tetramer that binds two pppGpp molecules at the interface between subunits, leading to an allosteric activation of the enzyme's catalytic activity (18). Activation by both ppGpp and pppGpp has been reported for RelQ from Enterococcus faecalis (19).…”

mentioning

confidence: 99%

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Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA

Beljantseva

Kudrin

Andresen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, pathogenicity, and antibiotic tolerance. We show that the tetrameric small alarmone synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis is a sequence-specific RNA-binding protein. RelQ's enzymatic and RNA binding activities are subject to intricate allosteric regulation. (p)ppGpp synthesis is potently inhibited by the binding of single-stranded RNA. Conversely, RelQ's enzymatic activity destabilizes the RelQ:RNA complex. pppGpp, an allosteric activator of the enzyme, counteracts the effect of RNA. Tetramerization of RelQ is essential for this regulatory mechanism, because both RNA binding and enzymatic activity are abolished by deletion of the SAS-specific C-terminal helix 5α. The interplay of pppGpp binding, (p)ppGpp synthesis, and RNA binding unites two archetypal regulatory paradigms within a single protein. The mechanism is likely a prevalent but previously unappreciated regulatory switch used by the widely distributed bacterial SAS enzymes. stringent response | (p)ppGpp | RNA-protein interaction | allosteric regulation | nucleotide signaling

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“…As we have shown previously (19), the enzymatic activity of E. faecalis RelQ is insensitive to the addition of E. coli 70S ribosomes (Fig. 1A).…”

Section: Resultsmentioning

confidence: 57%

“…Enzymatic assays with E. faecalis RelQ were performed following the method of Gaca et al (19). DRaCALA assays were performed as described by Roelofs et al (25).…”

Section: Methodsmentioning

confidence: 99%

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Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA

Beljantseva

Kudrin

Andresen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Even though transcription of rel does not change during Mn limitation (see Fig. S3 in the supplemental material), Rel is known to use Mn as the required cofactor during (p)ppGpp hydrolysis (39,40). Therefore, it is tempting to speculate that (p)ppGpp synthetized by RelQ or Rel itself would steadily accumulate due to the presence of a poorly active or inactive Rel hydrolase.…”

Section: Discussionmentioning

confidence: 99%

Association of Metal Homeostasis and (p)ppGpp Regulation in the Pathophysiology of Enterococcus faecalis

2017

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In Enterococcus faecalis, the regulatory nucleotides pppGpp and ppGpp, collectively, (p)ppGpp, are required for growth in blood, survival within macrophages, and virulence. However, a clear understanding of how (p)ppGpp promotes virulence in E. faecalis and other bacterial pathogens is still lacking. In the host, the essential transition metals iron (Fe) and manganese (Mn) are not readily available to invading pathogens because of a host-driven process called nutritional immunity. Considering its central role in adaptation to nutritional stresses, we hypothesized that (p)ppGpp mediates E. faecalis virulence through regulation of metal homeostasis. Indeed, supplementation of serum with either Fe or Mn restored growth and survival of the Δrel ΔrelQ [(p)ppGpp 0 ] strain to wild-type levels. Using a chemically defined medium, we found that (p)ppGpp accumulates in response to either Fe depletion or Mn depletion and that the (p)ppGpp 0 strain has a strong growth requirement for Mn that is alleviated by Fe supplementation. Although inactivation of the nutrient-sensing regulator codY restored some phenotypes of the (p)ppGpp 0 strain, transcriptional analysis showed that the (p)ppGpp/CodY network does not promote transcription of known metal transporters. Interestingly, physiologic and enzymatic investigations suggest that the (p)ppGpp 0 strain requires higher levels of Mn in order to cope with high levels of endogenously produced reactive oxygen species (ROS). Because (p)ppGpp mediates antibiotic persistence and virulence in several bacteria, our findings have broad implications and provide new leads for the development of novel therapeutic and preventive strategies against E. faecalis and beyond. KEYWORDS (p)ppGpp, Enterococcus, manganese, metal homeostasis, nutritional immunity, oxidative stress E nterococcus faecalis is a common member of the human gut microbiota but also a leading cause of a number of hospital-acquired infections such as endocarditis and surgical wound and urinary tract infections (1). The association of this opportunistic pathogen with disease relies on its exceptional resilience, which allows it to prevail in the unfavorable hospital setting while providing a competitive advantage over other bacteria during both infection and treatment.Among the most prominent regulators involved in bacterial adaptation to stress are the nucleotide second messengers guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively, (p)ppGpp, best known as the effectors of the stringent response (SR) (2). While initially discovered to accumulate in response to amino acid starvation, (p)ppGpp has also been shown to accumulate in response to restrictions in carbon, fatty acids, and iron, as well as in response to other nonnutritional stresses (3). During the SR, (p)ppGpp accumulation triggers transcriptional alterations that lead to general repression of rapid growth and simultaneous activation of stress survival, nutrient uptake, and biosynthesis pathways. In addition to its involvement in...

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“…S3D) (25,28). To understand this phenomenon in greater detail, we performed an in-depth kinetic analysis of SAS1 (Fig.…”

Section: Sas1 Binds Its Substrates In An Ordered Sequence During Alarmentioning

confidence: 99%

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone

Steinchen

Schuhmacher

Altegoer

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

110

172

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Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (S N 2) reaction. We show that pppGpp-but not ppGpp-positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles. stringent response | (p)ppGpp | hydrogen-deuterium exchange mass spectrometry | alarmone | crystallography T he ability of living organisms to adapt their metabolism to nutrient limitation or environmental changes is of utmost importance to survival. The stringent response is a highly conserved mechanism that enables bacteria (1-3) and plant chloroplasts (4-6) to respond to nutrient (i.e., amino acid) limitations. However, recent work has indicated that guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] also impact other, nonstringent response processes such as virulence (7-9) as well as persister (10, 11) and biofilm formation (12). Realization of the importance of (p)ppGpp has also opened new avenues for the design of antimicrobial agents (13,14).Central to these processes is the synthesis of two alarmones, pppGpp and ppGpp, which globally reprogram the transcription and translation associated with a variety of cellular processes (summarized in refs. 9 and 15) and which also control the elongation of DNA replication (16). Until now, both alarmones have been collectively named "(p)ppGpp," because knowledge of their individual roles remained mysterious. Only recently has a study indicated that either alarmone might execute disparate biological functions (17).Alarmone synthesis is carried out by synthetases of RelA/SpoT homology (RSH) (18) that catalyze the transfer of pyrophosphate (β-, γ-phosphates) from ATP to the ribose 3′-OH of GDP or GTP to synthesize ppGpp or pppGpp, respectively. An in-depth analysis of the catalytic mechanism for this reaction is currently not available. Only one structure describes the GDP-bound state of an RSH synthetase domain, bearing remarkable simil...

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From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

Cited by 90 publications

References 68 publications

Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA

Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA

Association of Metal Homeostasis and (p)ppGpp Regulation in the Pathophysiology of Enterococcus faecalis

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone

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