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Recent functional insights into the role of (p)ppGpp in bacterial physiology

Key Points

  • The stringent response is a regulatory mechanism that is controlled by members of the RelA–SpoT homologue (RSH) protein family in response to stress in bacteria. It is mediated by two related alarmone nucleotides, guanosine tetraphosphate and guanosine pentaphosphate, which are collectively referred to as (p)ppGpp.

  • The RSH enzymes can be divided into two categories: long, multidomain proteins (RelA, Rel and SpoT) and short, single-domain enzymes (small alarmone synthetases (SASs) and small alarmone hydrolases (SAHs)). The enzymatic activity of the long enzymes is regulated by interactions with molecular effectors, such as 'starved' ribosomes in the case of RelA, and the activity of short RSHs is regulated at the transcriptional level.

  • The regulatory role of (p)ppGpp in general metabolism is exerted by direct and indirect mechanisms. The direct mechanisms rely on the alarmone binding to and regulating a molecular target, whereas indirect mechanisms rely on changes in the concentrations of GTP and ATP, elicited by (p)ppGpp production.

  • The primary target of (p)ppGpp-mediated regulation is transcription. In Escherichia coli, this regulation relies on direct binding of the alarmone to the β′- and ω-subunits of RNA polymerase (RNAP), whereas in Bacillus subtilis, regulation is indirect and relies on changes in the concentration of the initiator nucleotide.

  • In addition to transcription, (p)ppGpp production regulates several other cellular processes, such as protein biosynthesis, replication, the acid stress response, polyphosphate metabolism, biosynthesis and uptake of nucleotides, and (via a direct interaction with RelA) the stringent response itself.

  • The effects of (p)ppGpp span a continuum from acute survival responses elicited by stresses such as nutrient deprivation or heat shock, mediated by high (p)ppGpp levels, to the 'housekeeping' role of basal (p)ppGpp levels in normal metabolic proesses, such as the production of amino acids and nucleotides.

  • The stringent response has a key role in bacterial virulence and persistence (the formation of antibiotic-tolerant cells). This has prompted the recent development of specific inhibitors of this process, which serve as a starting point for the future development of novel antivirulence compounds.

Abstract

The alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively referred to as (p)ppGpp) are involved in regulating growth and several different stress responses in bacteria. In recent years, substantial progress has been made in our understanding of the molecular mechanisms of (p)ppGpp metabolism and (p)ppGpp-mediated regulation. In this Review, we summarize these recent insights, with a focus on the molecular mechanisms governing the activity of the RelA/SpoT homologue (RSH) proteins, which are key players that regulate the cellular levels of (p)ppGpp. We also discuss the structural basis of transcriptional regulation by (p)ppGpp and the role of (p)ppGpp in GTP metabolism and in the emergence of bacterial persisters.

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Figure 1: (p)ppGpp metabolism by RelA, SpoT and Rel.
Figure 2: Mechanism of action of RelA.
Figure 3: Direct regulation of RNAP activity by (p)ppGpp.
Figure 4: Role of (p)ppGpp in GTP homeostasis.

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Acknowledgements

The authors are grateful to V. Shingler for her comments on the manuscript and M. Valle for providing the cryo-EM images of RelA. This work was supported by the European Regional Development Fund through the Centre of Excellence in Chemical Biology (V.H. and T.T.); by the Estonian Science Foundation (grants ETF9012 and PUT37 (V.H.), and grant ETF9020 (G.C.A.)); by Umeå University, the Swedish Research Council, the Ragnar Söderberg Foundation and the Kempe Foundation (V.H.); by the US National Institutes of Health (grant GM087350 (K.S.M.)); and by a European Research Council Advanced Investigator Grant (294517; PERSIST) and a Novo Nordisk Foundation Laureate Research Grant (K.G.).

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Glossary

GTPases

Enzymes that bind and hydrolyse GTP to produce GDP.

Translocation

A step in the elongation cycle of the ribosome in which the mRNA–tRNA complex advances on the ribosome by a distance of one codon.

ThrRS, GTPase and SpoT domain

(TGS domain). A protein domain with a suggested ligand-binding function.

Aspartokinase, chorismate mutase and TyrA domain

(ACT domain). A protein domain that is involved in ligand binding and that is often present in metabolic enzymes.

A-site

The ribosome acceptor site, which accommodates tRNA and various protein factors during the translation cycle.

Deacylated tRNAs

tRNAs that are uncharged, as they lack an aminoacyl group at the 3′-CCA end.

Sarcin–ricin loop

(SRL). An essential structural element of the 23S ribosomal RNA; this loop interacts with various protein factors during translation.

A/T state

A structural conformation of an aminoacyl-tRNA, in which the charged tRNA becomes distorted as it occupies the ribosomal A-site while interacting with elongation factor Tu.

Elongation factor Tu

(EF-Tu). A GTPase that delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis.

Acyl carrier protein

(ACP). An essential component of the fatty acid synthesis pathway that stabilizes and transports the growing lipid chain. Specialized ACPs are also involved in other processes that require acyl transfer, such as the synthesis of polyketide antibiotics.

σW regulon

An operon that is regulated by the transcription factor σW and that is induced in response to various stresses.

Monophyletic

Pertaining to a clade in a phylogenetic tree: sharing a single common ancestor.

Paraphyletic

Pertaining to a clade in a phylogenetic tree: sharing a single common ancestor but excluding some descendent sequences.

Salvage pathway

A pathway in which nucleotides are synthesized from intermediates (nucleobases and ribonucleosides that are either the products of degradative processes within the cell or imported from the extracellular milieu), rather than de novo, from phosphoribosyl pyrophosphate as a starting compound.

ppGpp0 strain

A bacterial strain that is unable to produce (p)ppGpp, as it lacks functional large and small RelA–SpoT homologues.

Toxin–antitoxin module

(TA module). A bacterial system that involves a toxin and an antitoxin that neutralizes its cognate toxin (for type II TA systems). The toxins of these systems collectively inhibit several essential cellular functions, such as translation, DNA replication and cell wall synthesis.

Lon protease

An ATP-dependent protease that degrades aberrant and short-lived polypeptides.

Phenol-soluble modulins

(PSMs). A family of amphipathic α-helical peptides that have surfactant-like properties and multiple roles in staphylococcal virulence through their contribution to immune evasion and biofilm development.

A-factor

A signalling molecule (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) that triggers secondary metabolism and morphogenesis in Streptomyces spp.

Fruiting bodies

Multicellular aggregates of myxobacteria that form as a result of nutrient deprivation.

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Hauryliuk, V., Atkinson, G., Murakami, K. et al. Recent functional insights into the role of (p)ppGpp in bacterial physiology. Nat Rev Microbiol 13, 298–309 (2015). https://doi.org/10.1038/nrmicro3448

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