ppGpp inhibits the activity of Escherichia coli DnaG primase

Maciąg, Monika; Kochanowska, Maja; Łyżeń, Robert; Węgrzyn, Grzegorz; Szalewska-Pałasz, Agnieszka

doi:10.1016/j.plasmid.2009.11.002

Cited by 78 publications

(74 citation statements)

References 30 publications

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“…(p)ppGpp has been shown to directly impact many processes in other bacteria, including transcription of the translation machinery (26)(27)(28)(29), GTP and ATP levels (30)(31)(32)(33), DNA replication (34)(35)(36)(37), and metabolism (38)(39)(40). In general, the many effects of (p)ppGpp are complex and seem to vary greatly among different organisms.…”

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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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 Gram-negative bacteria, this transcriptional shift is mediated by (p)ppGpp interacting with the RNA polymerase (RNAP), which in combination with the transcription factor DksA, modulates gene expression on a transcriptional level (3,14). Aside from the RNAP, there are at least 15 other direct (p)ppGpp target proteins in Escherichia coli, such as the translation elongation factors EFG and EF-Tu, the DNA primase DnaG, and a number of amino acid decarboxylases that are involved in the acid stress response (15)(16)(17). These nucleotides also play major roles in controlling bacterial persistence in Gram-negative bacteria by activating toxin-antitoxin systems and triggering slow growth (9), leading to cells that persist in the host following antibiotic treatment.…”

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confidence: 99%

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

Corrigan

Bellows

Wood

et al. 2016

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

187

227

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The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Grampositive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Grampositive bacterium Staphylococcus aureus. We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases-RsgA, RbgA, Era, HflX, and ObgE-as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria.ribosome | stringent response | tolerance | ppGpp | Staphylococcus aureus

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“…During stress, the transcription of genes required for rapid growth, such as those encoding rRNAs, is limited by (p)ppGpp accumulation along with a concomitant activation of genes involved in amino acid biosyn-thesis, alternate nutrient transport, and general stress responses (3,5). In addition, (p)ppGpp leads to rapid physiological adjustments by inhibiting enzymes involved in protein synthesis, DNA replication, and nucleotide synthesis, especially GTP (6)(7)(8)(9)(10)(11).…”

mentioning

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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ppGpp inhibits the activity of Escherichia coli DnaG primase

Cited by 78 publications

References 30 publications

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

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

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

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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