MS_RHII-RSD, a Dual-Function RNase HII-(p)ppGpp Synthetase from Mycobacterium smegmatis

Murdeshwar, Maya S; Chatterji, Dipankar

doi:10.1128/jb.00258-12

Cited by 54 publications

(89 citation statements)

References 55 publications

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“…This is consistent with recent studies by Bag and colleagues, who demonstrated that Rv1366 is unable to synthesize ppGpp in vitro (50). In contrast, the Mycobacterium smegmatis SAS, MSM_5849, exhibited weak (p)ppGpp synthetase activity as well as Mn(II)-dependent RNase HII activity (51). Of note, this SAS-RNase HII fusion appears to occur with some frequency among species of mycobacteria.…”

Section: Changing Paradigms: the Discovery Of "Short" Rela/spot Homologssupporting

confidence: 80%

Many Means to a Common End: the Intricacies of (p)ppGpp Metabolism and Its Control of Bacterial Homeostasis

2015

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In nearly all bacterial species examined so far, amino acid starvation triggers the rapid accumulation of the nucleotide second messenger (p)ppGpp, the effector of the stringent response. While for years the enzymes involved in (p)ppGpp metabolism and the significance of (p)ppGpp accumulation to stress survival were considered well defined, a recent surge of interest in the field has uncovered an unanticipated level of diversity in how bacteria metabolize and utilize (p)ppGpp to rapidly synchronize a variety of biological processes important for growth and stress survival. In addition to the classic activation of the stringent response, it has become evident that (p)ppGpp exerts differential effects on cell physiology in an incremental manner rather than simply acting as a biphasic switch that controls growth or stasis. Of particular interest is the intimate relationship of (p)ppGpp with persister cell formation and virulence, which has spurred the pursuit of (p)ppGpp inhibitors as a means to control recalcitrant infections. Here, we present an overview of the enzymes responsible for (p)ppGpp metabolism, elaborate on the intricacies that link basal production of (p)ppGpp to bacterial homeostasis, and discuss the implications of targeting (p)ppGpp synthesis as a means to disrupt long-term bacterial survival strategies. While analyzing nucleotide extracts of Escherichia coli, Cashel and Gallant visualized two spots by thin-layer chromatography that could be implicated in the inhibition of stable RNA accumulation provoked by amino acid starvation, which they dubbed "magic spots" (1). These magic spots were later identified as the hyperphosphorylated guanosine derivatives ppGpp (GDP, 3=-diphosphate) and pppGpp (GTP, 3=-diphosphate), collectively referred to as (p)ppGpp, or alarmones (2, 3). Subsequent studies revealed that (p)ppGpp is responsible for activation of the stringent response (SR), a highly conserved stress response to nutrient starvation (4, 5). Generally speaking, accumulation of (p)ppGpp induces large-scale transcriptional alterations leading to general repression of genes required for rapid growth, such as rRNA genes, and concomitant activation of genes involved in amino acid biosynthesis, nutrient acquisition, and stress survival. In addition to transcriptional control, (p)ppGpp has been shown to directly inhibit the activity of several enzymes, including DNA primase, translation factors, and enzymes involved in GTP biosynthesis (6) (Fig. 1). Ultimately, the SR reallocates cellular resources toward adaptation to a semidormant state, facilitating survival under unfavorable conditions (5, 7). Although initially defined as a response to amino acid and carbon starvation, the term SR has since been expanded to include any regulatory effect exerted by cellular (p)ppGpp accumulation irrespective of the triggering mechanism (4).The broad physiological alterations induced by (p)ppGpp accumulation rely heavily upon transcriptional alterations. In Gammaproteobacteria, such as the Gram-negative paradigmatic orga...

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Section: Changing Paradigms: the Discovery Of "Short" Rela/spot Homologssupporting

confidence: 80%

Many Means to a Common End: the Intricacies of (p)ppGpp Metabolism and Its Control of Bacterial Homeostasis

2015

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“…Therefore, the question regarding the role of Rv1366 in M. tuberculosis remains unanswered. Murdeshwar et al recently reported a study of the M. smegmatis SAS homolog, which they term MS_RHII-RSD because, unlike the M. tuberculosis homolog, MS_RHII-RSD encodes an amino-terminal RNase HII domain (41). The authors show that a small amount of (p)ppGpp is produced in an M. smegmatis ⌬rel Msm mutant that is dependent on MS_RHII-RSD; however, the importance of this activity for survival has not been explored.…”

Section: Discussionmentioning

confidence: 99%

Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

Weiss

Stallings

2013

J Bacteriol

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In Mycobacterium tuberculosis, the stringent response to amino acid starvation is mediated by the M. tuberculosis Rel (Rel Mtb ) enzyme, which transfers a pyrophosphate from ATP to GDP or GTP to synthesize ppGpp and pppGpp, respectively. (p)ppGpp then influences numerous metabolic processes. Rel Mtb also encodes a second, distinct catalytic domain that hydrolyzes (p)ppGpp into pyrophosphate and GDP or GTP. Rel Mtb is required for chronic M. tuberculosis infection in mice; however, it is unknown which catalytic activity of Rel Mtb mediates pathogenesis and whether (p)ppGpp itself is necessary. In order to individually investigate the roles of (p)ppGpp synthesis and hydrolysis during M. tuberculosis pathogenesis, we generated Rel Mtb point mutants that were either synthetase dead (Rel Mtb H344Y ) or hydrolase dead (Rel Mtb H80A ). M. tuberculosis strains expressing the synthetasedead Rel Mtb H344Y mutant did not persist in mice, demonstrating that the Rel Mtb (p)ppGpp synthetase activity is required for maintaining bacterial titers during chronic infection. Deletion of a second predicted (p)ppGpp synthetase had no effect on pathogenesis, demonstrating that Rel Mtb was the major contributor to (p)ppGpp production during infection. Interestingly, expression of an allele encoding the hydrolase-dead Rel Mtb mutant, Rel Mtb H80A , that is incapable of hydrolyzing (p)ppGpp but still able to synthesize (p)ppGpp decreased the growth rate of M. tuberculosis and changed the colony morphology of the bacteria. In addition, Rel Mtb H80A expression during acute or chronic M. tuberculosis infection in mice was lethal to the infecting bacteria. These findings highlight a distinct role for Rel Mtb -mediated (p)ppGpp hydrolysis that is essential for M. tuberculosis pathogenesis.

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“…However, there are other explanations for removal of the 5= ␥-phosphate from pppGpp, generating ppGpp, such as activities of nonspecific phosphohydrolases, including polyphosphate phosphatase, Nudix family hydrolases, or GTP-requiring enzymes for protein synthesis such as EF-G or EF-Tu (37)(38)(39)(40). A preference for GDP over GTP as an in vitro substrate has been observed for SASs from Mycobacterium smegmatis and S. aureus (24,41). Of note, the ability of RelQ Ef to synthesize (p)ppGpp in vitro was previously documented, but the relative efficiency of substrate utilization has not been (40).Therefore, we determined the efficiency of GTP versus GDP as a substrate for RelQ Ef synthesis of pppGpp and ppGpp ( Table 1).…”

Section: Relq Ef Prefers Gdp Over Gtp As a Substrate And Is Insensitimentioning

confidence: 99%

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

et al. 2015

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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. 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 (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. In order to survive under adverse environmental conditions, such as nutrient starvation, bacteria have evolved complex, interconnected regulatory networks that sense and integrate internal and external metabolic cues to activate cellular responses that enhance bacterial survival. One critical and highly conserved mechanism employed by bacteria to cope with nutritional as well as a variety of environmental and chemical stresses is the stringent response (SR). The SR is mediated by the accumulation of two guanine analog...

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MS_RHII-RSD, a Dual-Function RNase HII-(p)ppGpp Synthetase from Mycobacterium smegmatis

Cited by 54 publications

References 55 publications

Many Means to a Common End: the Intricacies of (p)ppGpp Metabolism and Its Control of Bacterial Homeostasis

Many Means to a Common End: the Intricacies of (p)ppGpp Metabolism and Its Control of Bacterial Homeostasis

Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

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

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