Diversity in (p)ppGpp metabolism and effectors

Liu, Kuanqing; Bittner, Alycia N.; Wang, Jue D.

doi:10.1016/j.mib.2015.01.012

Cited by 175 publications

(177 citation statements)

References 74 publications

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“…The stringent response is a highly conserved mechanism that enables bacteria (1)(2)(3) and plant chloroplasts (4)(5)(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)(8)(9) as well as persister (10,11) and biofilm formation (12).…”

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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.

112

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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mentioning

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.

112

172

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“…Accumulation of (p)ppGpp induces a wide range of physiological alterations that affect many important cellular processes such as DNA replication, transcription, translation, and metabolism (27). In E. coli, (p)ppGpp and a coregulator, DksA, bind directly to RNA polymerase to inhibit or activate transcription (28,29).…”

Section: Discussionmentioning

confidence: 99%

“…(p)ppGpp has also been shown be important for the association of alternative sigma factors with the RNA polymerase core (30). In the Gram-positive bacterium B. subtilis, (p)ppGpp's effects on transcriptional regulation occur primarily through modulation of GTP levels (27). GTP is one of the substrates for (p)ppGpp syn- thesis, so as (p)ppGpp levels rise, GTP becomes depleted.…”

Section: Discussionmentioning

confidence: 99%

CodY Regulates SigD Levels and Activity by Binding to Three Sites in the fla / che Operon

Ababneh

Herman

2015

J Bacteriol

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Exponentially growing cultures of Bacillus subtilis (PY79) are composed primarily of nonmotile, chained cells. The alternative sigma factor, SigD, promotes the phenotypic switch from nonmotile, chained cells to unchained, motile cells. In the present work, we investigated the role of the GTP-sensing protein CodY in the regulation of SigD. Deletion of codY resulted in a significant increase in SigD accumulation and activity and shifted the proportion of unchained cells up from ϳ15% to ϳ75%, suggesting that CodY is an important regulator of SigD. CodY was previously shown to bind to the P D3 and P fla/che promoters located upstream of the first gene in the sigD-containing fla/che operon. Using electrophoretic mobility shift assays, we found that CodY also binds to two other previously uncharacterized sites within the fla/che operon. Mutations in any one of the three binding sites resulted in SigD levels similar to those seen with the ⌬codY mutant, suggesting that each site is sufficient to tip cells toward a maximal level of CodY-dependent SigD accumulation. However, mutations in all three sites were required to phenocopy the ⌬codY mutant's reduced level of cell chaining, consistent with the idea that CodY binding in the fla/che operon is also important for posttranslational control of SigD activity. IMPORTANCEOne way that bacteria adapt quickly and efficiently to changes in environmental quality is to employ global transcriptional regulators capable of responding allosterically to key cellular metabolites. In this study, we found that the conserved GTP-sensing protein CodY directly regulates cell motility and chaining in B. subtilis by controlling expression and activity of SigD. Our results suggest that B. subtilis becomes poised for cell dispersal as intracellular GTP levels are depleted. Genetically identical populations of bacteria often exhibit phenotypic heterogeneity, possessing cells that switch between distinct physiological and/or developmental states. Phenotypic switching is most widely understood in the context of highly regulated responses to changes in environmental cues, such as nutrient status and cell density. However, phenotypic switching can also occur spontaneously, and stochastic switching between two stable phenotypes at the subpopulation level is termed phenotypic bistability (1, 2). Bistability likely confers a selective advantage to a species under unpredictable environmental conditions by increasing the chance that at least one alternative cell state will survive under any given adverse condition (2-4). For example, in a given population of antibiotic-sensitive bacteria, small subpopulations of genetically identical and yet nongrowing and antibiotictolerant bacteria known as "persisters" are detectable (5, 6). Since the persister subpopulation is nongrowing, it is able to survive a sudden onslaught of antibiotic selection.Exponentially growing populations of Bacillus subtilis are comprised of both chained, nonmotile cells and unchained, motile cells, the latter of which are presu...

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“…In addition, RNAP dissociation can allow alternative sigma factors to bind RNAP, leading to the regulation of distinct promoter sets important for adjustment to new environmental conditions (3, 6). In some bacteria, ppGpp executes the stringent response in a different way, not involving RNAP binding (7,8).The overall processes of the stringent response are similar in a wide variety of bacteria, but structural and functional variations occur. Some bacterial genomes carry a single spoT/relA gene that encodes a bifunctional protein comprising a ppGpp synthetase and a hydrolase domain (9-11).…”

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confidence: 99%

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confidence: 99%

Contributions of Sinorhizobium meliloti Transcriptional Regulator DksA to Bacterial Growth and Efficient Symbiosis with Medicago sativa

Wippel

Long

2016

J Bacteriol

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The stringent response, mediated by the (p)ppGpp synthetase RelA and the RNA polymerase-binding protein DksA, is triggered by limiting nutrient conditions. For some bacteria, it is involved in regulation of virulence. We investigated the role of two DksA-like proteins from the Gram-negative nitrogen-fixing symbiont Sinorhizobium meliloti in free-living culture and in interaction with its host plant Medicago sativa. The two paralogs, encoded by the genes SMc00469 and SMc00049, differ in the constitution of two major domains required for function in canonical DksA: the DXXDXA motif at the tip of a coiled-coil domain and a zinc finger domain. Using mutant analyses of single, double, and triple deletions for SMc00469 (designated dksA), SMc00049, and relA, we found that the ⌬dksA mutant but not the ⌬SMc00049 mutant showed impaired growth on minimal medium, reduced nodulation on the host plant, and lower nitrogen fixation activity in early nodules, while its nod gene expression was normal. The ⌬relA mutant showed severe pleiotropic phenotypes under all conditions tested. Only S. meliloti dksA complemented the metabolic defects of an Escherichia coli dksA mutant. Modifications of the DXXDXA motif in SMc00049 failed to establish DksA function. Our results imply a role for transcriptional regulator DksA in the S. meliloti-M. sativa symbiosis. IMPORTANCEThe stringent response is a bacterial transcription regulation process triggered upon nutritional stress. Sinorhizobium meliloti, a soil bacterium establishing agriculturally important root nodule symbioses with legume plants, undergoes constant molecular adjustment during host interaction. Analyzing the components of the stringent response in this alphaproteobacterium helps understand molecular control regarding the development of plant interaction. Using mutant analyses, we describe how the lack of DksA influences symbiosis with Medicago sativa and show that a second paralogous S. meliloti protein cannot substitute for this missing function. This work contributes to the field by showing the similarities and differences of S. meliloti DksA-like proteins to orthologs from other species, adding information to the diversity of the stringent response regulatory system. Bacteria employ the stringent response as a mechanism to adjust global gene expression to adverse nutrient conditions. For example, when Escherichia coli encounters amino acid starvation, the ribosome-bound protein RelA (1, 2) recognizes uncharged tRNA molecules and synthesizes guanosine tetraphosphate and guanosine pentaphosphate (referred to here as ppGpp) from GTP and ATP (3). The alarmone ppGpp and the regulatory protein DksA subsequently bind RNA polymerase (RNAP) and alter the kinetic properties of promoter/RNAP complexes (4). In particular, ppGpp and DksA reduce the lifetime of promoter/RNAP complexes by inhibiting the transition from closed to intermediate complex formation on promoters depending on the primary sigma factor RpoD ( 70 ) (4, 5). In addition, RNAP dissociation can allow alternative...

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Diversity in (p)ppGpp metabolism and effectors

Cited by 175 publications

References 74 publications

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

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

CodY Regulates SigD Levels and Activity by Binding to Three Sites in the fla / che Operon

Contributions of Sinorhizobium meliloti Transcriptional Regulator DksA to Bacterial Growth and Efficient Symbiosis with Medicago sativa

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