ppGpp: magic beyond RNA polymerase

When faced with limiting nutrients, bacteria rapidly reallocate cellular resources by stopping the synthesis of DNA, stable RNAs, ribosomal proteins and membrane components and rapidly producing factors that are cru-cial for stress resistance, glycolysis and amino acid syn-thesis1. This so-called stringent response to nutrient stress is accomplished in part by a massive switch in the tran-scription profile, coordinated by the signalling nucleo-tides guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are also referred to as magic spot or the alarmone. The nucleotide ppGpp was discovered as a ‘magic spot’ on thin-layer chromato grams that were designed to analyse changes in cellular nucleotide pools in response to nutrient stress. Since then, extensive genetic and molecular analyses in Escherichia coli have established that ppGpp alters transcription globally by regulating RNA polymerase (RNAP) activity directly and regulating RNAP σ-factor activity indirectly1,2.

Two classes of enzyme control the cellular pool of ppGpp. Monofunctional synthetases, known as RelA proteins, use GTP and ATP to generate pppGpp, which is then converted to ppGpp (FIG. 1). Bifunctional synthetase–hydrolase enzymes, called SpoT, Rel or RSH (Rel–Spo homologue) proteins, can make ppGpp and pppGpp and can also hydrolyse the nucleotides to yield either GDP and pyrophosphate (PPi) or GTP and PPi, respectively; in this Review, ppGpp and pppGpp are collectively referred to as ppGpp, unless otherwise specified. The SpoT hydrolase activity is essential to bacterial cells, as high concentrations of ppGpp bring replication to a halt. Different species of bacteria encode a variety of synthetase and hydrolase

enzymes to control the concentrations of ppGpp1–3. To adapt rapidly to their environment, bacteria regulate the enzymes that synthesize and degrade ppGpp. For example, when E. coli encounters a shortage of amino acids, uncharged tRNAs stimulate RelA activity (FIG. 1). By contrast, bacteria require bifunctional SpoT enzymes to respond to a variety of nutrient stresses, such as phosphate, carbon, iron or fatty acid starvation.

ppGpp exerts many of its physiological effects by interacting directly with RNAP in cooperation with DnaK suppressor (DksA), a protein that binds in the second-ary channel of the enzyme and amplifies the impact of the alarmone (FIG. 1). Whether bound ppGpp and DksA act positively or negatively on transcription by RNAP is determined by properties that are intrinsic to the promoter in question4.

ppGpp and DksA can also regulate transcription by an indirect process known as σ-factor competition. In the logarithmic growth period, the vegetative σ-factor, σ70 (also known as RpoD), directs RNAP to initiate the transcription of operons that are fundamental to the synthesis of proteins, lipids and DNA. During a strin-gent response, high concentrations of ppGpp inhibit RNAP binding to strong σ70-dependent promoters, such as the promoters of ribosomal RNA and tRNA genes; consequently, more core RNAP is available to bind to the alternative σ-factors that accumulate in response to par-ticular stresses5. These alternative σ-factors then direct RNAP to transcribe genes that are devoted to coping with those conditions2. In addition to modulating pro-moter selection by RNAP, ppGpp can exert a broad influence on bacterial cells. For example, ppGpp coor-dinates the synthesis of ribosomes to suit the growth

1Department of Microbiology, University of Washington, Health Sciences Building K116, 1959 NE Pacific St., Box 357710, Seattle, Washington 98195-7710, USA.2Department of Microbiology & Immunology, University of Michigan Medical School, 6733 Medical Science Building II, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109–5620, USA.Correspondence to M.S.S. e-mail: [email protected]:10.1038/nrmicro2720

Stringent responseA stress response coordinated by guanosine tetraphosphate and guanosine pentaphosphate, in which cells rapidly inhibit synthesis of stable RNA, ribosomes and proteins, leading to growth arrest.

ppGpp: magic beyond RNA polymeraseZachary D. Dalebroux1 and Michele S. Swanson2

Abstract | During stress, bacteria undergo extensive physiological transformations, many of which are coordinated by ppGpp. Although ppGpp is best known for enhancing cellular resilience by redirecting the RNA polymerase (RNAP) to certain genes, it also acts as a signal in many other cellular processes in bacteria. After a brief overview of ppGpp biosynthesis and its impact on promoter selection by RNAP, we discuss how bacteria exploit ppGpp to modulate the synthesis, stability or activity of proteins or regulatory RNAs that are crucial in challenging environments, using mechanisms beyond the direct regulation of RNAP activity.

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Nature Reviews | Microbiology

ppGpp

(GTP or GDP) + ATP

(GTP or GDP) + PPi

Limiting phosphate, fatty acids, carbon or iron;osmotic shock

Expression of components for protein, lipid, RNA and DNA synthesis

Expression of components for stress resistance

Limiting amino acids

RNA polymerase

σ70 σ*

RelASpoT

DksA

DksA

AdaptorIn the context of post-translational regulation of protein stability, a protein that binds another protein and chaperones it to the proteasome for degradation; an example of such an adaptor is regulator of σS protein RssB.

rate in replicating E. coli cells6,7, and accumulated ppGpp inhibits DNA replication in Bacillus subtilis8. The alarmone also modulates the expression of virulence factors that equip bacterial pathogens to cope with envi-ronmental and metabolic stress3 (BOX 1). Furthermore, recent studies have revealed crosstalk between ppGpp regulation and other nucleotide-signalling pathways that are known to govern bacterial cell differentiation processes such as biofilm formation9,10. Recent reviews

have analysed how bacteria control their pools of ppGpp to direct RNAP to the appropriate genes and to control replication1; the mechanism of promoter control by ppGpp and DksA4; and the capacity of the stringent response machinery to enable a wide vari-ety of pathogens to summon virulence pathways that serve their metabolic needs3. In this Review, we focus on other modes of direct and indirect regulation by ppGpp, drawing examples from both model and patho-genic bacteria. In particular, we describe how ppGpp alters the stability, assembly and activity of certain target proteins and RNAs (TABLE 1), and how bacteria integrate production of the alarmone with production of other nucleotide signals to respond to metabolic or environmental cues.

σ-factor stabilityWhen ppGpp alerts a bacterium to stress, σ-factors alter the transcription profile by directing RNAP to specific promoters. σS (also known as RpoS), a stationary-phase σ-factor that is crucial for stress resistance and the expression of virulence factors by a variety of patho-gens11,12, is regulated by ppGpp through an impressive array of direct and indirect mechanisms13–15.

σS is regulated at several levels: gene transcription, mRNA translation, protein stability and protein activ-ity16. Although the rpoS gene is transcribed and the mRNA translated during vegetative growth, the con-centration of σS in the cell remains low because regula-tor of σS protein RssB, an adaptor protein, directs σS to the ClpXP proteasome for degradation11,17 (FIG. 2a). In E. coli, ppGpp promotes σS protein stability by inducing expression of the anti-adaptor proteins IraP and IraD, which bind RssB and block its activity18,19. For example, when phosphate is limiting, the ppGpp pool generated by SpoT activates iraP transcription, thereby increas-ing σS stability18 (FIG. 2b). Induction of the σS-mediated stress response by phosphate limitation is abolished by swapping the AT-rich discriminator sequence of the iraP promoter for a GC-rich sequence, implicating ppGpp in activation of iraP transcription18. The ppGpp-induced activation of iraP is specific to phosphate stress and requires SpoT hydrolase activity but not SpoT synthetase activity, as in the presence of RelA and a synthetase-defective allele of SpoT, iraP induction in response to phosphate stress resembles that of wild-type bacteria. Likewise, neither overexpression of RelA nor increased ppGpp synthesis (triggered in response to amino acid stress caused by serine hydroxamate treat-ment) is sufficient to induce iraP expression18. Perhaps transcriptional induction of the iraP promoter during phosphate limitation requires a specific concentra-tion of the alarmone that can only be achieved by the bifunctional enzyme SpoT.

The alarmone also regulates the expression of IraD, a second anti-adaptor protein devoted to σS stability (FIG. 2c). Both iraD transcription and σS stability increase as E. coli enters stationary phase19,20. Although the mecha-nism involved remains to be defined, genetic experiments implicate the alarmone in this growth phase regulation. In particular, iraD expression is elevated in replicating

Figure 1 | ppGpp alters RNA polymerase promoter selection by σ-factors. In response to particular stresses, SpoT and RelA catalyse pyrophosphoryl transfer from ATP to GTP or GDP to synthesize the signalling nucleotides guanosine pentaphosphate and guanosine tetraphosphate, respectively; these nucleotides are collectively referred to as ppGpp. Together with DnaK suppressor (DksA), ppGpp directs transcription initiation at particular gene promoters through direct interaction with RNA polymerase. In part, ppGpp and DksA act by promoting the interaction of RNA polymerase with alternative σ-factors (σ*), such as σE. When metabolic precursors are plentiful, SpoT instead degrades ppGpp, and the vegetative σ-factor, σ70, directs RNA polymerase to genes that are crucial for bacterial replication. PP

i, pyrophosphate.

Box 1 | SpoT, not RelA, is required during infection

For several bacterial pathogens, the stringent response enzyme that is crucial for infection is SpoT, which has both hydrolase and synthetase activities for guanosine tetraphosphate and guanosine pentaphosphate (referred to here as ppGpp). Because the hydrolase activity of SpoT is essential to bacterial cells that generate ppGpp — high levels of ppGpp disrupt the cell cycle, so it must be degraded — the function of SpoT is deduced by comparing the phenotype of relA spoT double mutants (which, lacking both synthetases, do not produce ppGpp) to that of relA single mutants. Mice infected intragastrically with either wild-type or relA Salmonella enterica subsp. enterica serovar Typhimurium succumb to infection within 7–10 days, whereas mice infected with relA spoT bacteria show no signs of illness up to 30 days after inoculation at >100‑fold the lethal dose (LD

50) of wild-type bacteria90. For subcutaneous (bubonic) infection with

Yersinia pestis, relA spoT bacteria exhibit an ~100,000-fold increase in the LD50

relative to that for wild-type and relA bacteria91. During Francisella tularensis infection of macrophages, the number of viable relA spoT bacteria recovered is 100,000 fewer than the number of viable wild-type or relA bacteria recovered. Likewise, mice infected intradermally with wild-type and relA F. tularensis succumb within 5–7 days, whereas mice infected with relA spoT bacteria remain viable up to 20 days after infection54. Wild-type and relA Legionella pneumophila proliferate in macrophage cultures, but relA spoT mutants do not survive host-to-host transmission92,93. Additional experiments are needed to identify whether the hydrolase activity that is unique to SpoT accounts for its crucial role during infection. An alternative hypothesis that warrants testing is that the capacity of SpoT synthetase activity to be induced by the particular stresses encountered during infection explains why pathogens require SpoT but not RelA.

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ProteasomeA cytoplasmic complex that unfolds and degrades proteins.

Anti-adaptorIn the context of post-translational regulation of protein stability, a protein that binds an adaptor and blocks its chaperone activity; examples of anti-adaptors are IraP and IraD.

DiscriminatorThe DNA sequence between the −10-box hexamer of a promoter and the transcriptional start site at nucleotide +1. Promoters that are activated by guanosine tetraphosphate and guanosine pentaphosphate acting with DnaK suppressor (DksA) are typically AT-rich in this region, whereas repressed targets are typically GC-rich.

rpoB mutants (which have an RNAP subunit-β with an altered function) that exhibit a stringent response profile in the absence of ppGpp.

σ-factor activityA second σ-factor that is tightly regulated in E. coli is σE (also known as RpoE), the σ-factor that coordinates the cellular response to the presence of misfolded proteins in the periplasm or outer membrane. However, rather than stabilize the σE protein, ppGpp enhances its activ-ity. σE activity is inhibited by σE factor regulatory protein (RseA), an inner-membrane protein that sequesters the σ-factor. In response to cell envelope stress, such as the presence of misfolded outer-membrane porins, RseA is degraded by a proteolyic cascade, thereby releasing σE (REF. 21) to direct RNAP to genes that are important for outer-membrane biogenesis.

Activation of the σE regulon in E. coli also occurs in response to metabolic stress that is signalled by ppGpp. The amount of rpoE mRNA increases promptly when a stringent response is induced pharmacologically by serine hydroxamate22. Nevertheless, σE protein levels remain constant when cells accumulate ppGpp, and the rpoE promoter is not required to observe increased σE activity in stationary phase23,24. Instead, σE-mediated transcription initiation of certain genes in the σE regu-lon is stimulated directly by ppGpp and DksA, as meas-ured by in vitro transcription reactions using purified components24. As bacteria transition to stationary phase

and the alarmone accumulates, transcription mediated by σE is probably further enhanced when the RNAP that is released from ribosomal RNA operons becomes avail-able to alternative σ-factors24. Thus, by exerting its influ-ence on both σ-factor activity and the pool of free RNAP, ppGpp drives the expression of genes in the σE regulon in E. coli as a rapid response to outer-membrane damage or metabolic stress.

Transcript stabilityIn Legionella pneumophila and E. coli, ppGpp induces the expression of non-coding regulatory RNAs25,26 to regulate the stability of certain mRNAs indirectly via the carbon storage regulator (CsrA) regulatory pathway.

CsrA is an RNA-binding protein that controls a vari-ety of bacterial processes, ranging from biofilm forma-tion27 and motility28,29 to protein secretion by the type III (REF. 30), type IV (REF. 31) and type VI32 secretion systems. For example, in the intracellular pathogen L. pneumophila, CsrA represses the expression of crucial virulence factors that promote bacterial transmission, such as fac-tors that are responsible for lysosome evasion. CsrA targets include components of the Dot–Icm type IV secretion system and the flagellar secretion system31,33. Through its physical interaction with leader sequences and ribosome-binding sites on target mRNAs, CsrA pre-vents the translation of these transcripts and facilitates their degradation34. CsrA activity is inhibited by spe-cialized non-coding regulatory RNAs that out compete

Table 1 | ppGpp targets other than RNA polymerase

Target Cellular function Effect of ppGpp on target Species Refs

Indirect mechanisms

σS Transcription factor Increased stability Escherichia coli 15,16

CsrA Translation regulator Inhibition of activity E. coli and Legionella pneumophila 22,23,37

CodY Transcription repressor Inhibition of activity owing to GTP consumption

Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus mutans and Listeria monocytogenes

65–69

Cyclic di-GMP Signalling nucleotide Cooperative induction of biofilm matrix synthesis

E. coli 77

Cyclic di-AMP Signalling nucleotide Increased concentration B. subtilis 80

σE Transcription factor Increased activity E. coli 20

Direct mechanisms

SlyA Transcription factor Increased dimerization and DNA binding

Salmonella enterica subsp. enterica serovar Typhimurium

45

PigR Transcription factor Increased binding to the complex containing the transcription factors MglA–SspA and RNA polymerase

Francisella tularensis 51

DNA primase Initiator of DNA replication Inhibition of activity B. subtilis 7

EF-Tu, EF-G and IF2 Translation factors Inhibition of activity E. coli and Geobacillus stearothermophilus 52,88

PNPase Promoter of RNA turnover Inhibition of activity Nonomuraea sp. ATCC 39727 and Streptomyces coelicolor

54,55,57

LdcI Enhancer of acid tolerance Inhibition of activity E. coli 53

Exopolyphosphatase Polyphosphate degradation enzyme

Inhibition of activity E. coli 65,66

CsrA, carbon storage regulator; EF, elongation factor; IF2, translation initiation factor 2; LdcI, lysine decarboxylase, inducible; MglA, macrophage growth locus; PNPase, polynucleotide phosphorylase; ppGpp, guanosine tetraphosphate and guanosine pentaphosphate; SspA, stringent starvation protein A.

R E V I E W S




(GTP or GDP) + ATP (GTP or GDP) + PPi

Phosphate limitation

Logarithmic growth and nutrient abundancea

b

c

Activeadapter

RssBIraP

iraP

iraP mRNA

RssB

Stress resistance and virulence factor production

RNAP

ppGpp

Phosphate stress

P

RssB

P

SpoT

(GTP or GDP) + ATP

Steady state

RssBIraD

iraD

iraD mRNA

?RNAP

ppGpp

SpoTRelA

Inactiveadapter

Inactiveadapter

σS

σS

σS

σS

ClpXP

ClpXP

ClpXPσS

Serine hydroxamateA structural analogue of l-serine that induces the stringent response by inhibiting charging of seryl-tRNA synthetase.

rpoBThe gene encoding the β-subunit of RNA polymerase; this subunit is involved in transcription initiation through its interaction with the σ-factor. The commonly used antibiotic rifampicin targets this β-subunit to inhibit transcription, and mutations in rpoB commonly confer rifampicin resistance.

target mRNAs for CsrA binding35 (FIG. 3a). Thus, control over the expression of regulatory RNAs is crucial for bacteria to modulate CsrA activity.

In Pseudomonas aeruginosa, E. coli and L. pneumophila, the concentrations of the regulatory RNAs that counteract CsrA increase as bacteria enter stationary phase31,32,36–39. In L. pneumophila, the increase in tran-scription of the regulatory RNA RsmZ during station-ary phase requires the ppGpp synthetase, RelA, and the bifunctional synthetase–hydrolase, SpoT25. In addi-tion, the expression of both RsmY (another regulatory RNA) and RsmZ is rapidly stimulated in an engineered strain of L. pneumophila that synthesizes ppGpp dur-ing log phase25,40. However, the precise mechanism of transcriptional activation is complex, as it requires both σS and the Legionella transmission activator–Legionella transmission sensor (LetA–LetS) two-component phosphorelay system that is crucial for the expression

of numerous factors in stationary phase25,31,38,39 (FIG. 3a). Similarly, in E. coli both ppGpp and the cofactor pro-tein DksA activate transcription of the analogous non-coding RNAs, CsrB and CsrC26. However, a previous genetic study indicated that transcription of CsrB is induced independently of ppGpp in stationary phase E. coli cells41. Although the basis for this discrepancy is not clear, the previous E. coli study41 relied on analysis of relA spoTmutant strains, which frequently acquire sup-pressor mutations that can mimic the effects of ppGpp. Whether P. aeruginosa and other pathogens also use ppGpp to relieve CsrA-mediated repression of stationary phase and virulence genes by inducing the expression of regulatory RNAs is an open question.

Regulators of virulence in pathogensIn addition to regulating transcription by modulating the stability and activity of σ-factors, ppGpp controls the activity of transcription factors that are dedicated to virulence genes in Salmonella enterica subsp. enterica serovar Typhimurium and Francisella tularensis.

The SlyA regulator of S. Typhimurium. Following phagocytosis, S. Typhimurium activates the two- component virulence regulatory system PhoPQ. Once activated, the histidine kinase PhoQ phosphorylates the cytosolic response regulator PhoP, which then binds DNA and stimulates the transcription of genes involved in remodelling host cell vacuoles and resistance to cationic antimicrobial peptides (CAMPs) produced by the host42 (FIG. 3b). One gene induced by PhoPQ is slyA43, which encodes a multiple antibiotic resistance protein R (MarR) family transcriptional regulator that controls the transcription of genes which are essential for virulence in S. Typhimurium44–46. SlyA participates in a feed-forward loop with PhoP47 in which each component responds to a distinct signal. Whereas PhoPQ senses external cues for activation, SlyA relies on cytosolic ppGpp48. The alarmone facilitates SlyA dimerization and binding to target promoters, as evidenced by both in vitro and in vivo assays48 (FIG. 3b). The S. Typhimurium chromo-some contains at least seven divergent operons for which transcription is controlled by both SlyA and PhoP. When S. Typhimurium encounters low pH, a low concentration of Mg2+ or a high concentration of CAMP49, and the cel-lular ppGpp pools are sufficient, SlyA activates transcrip-tion in one direction while PhoP stimulates transcription in the other direction. Consequently, the bacterium coor-dinates expression of several factors, including those that confer resistance to serum or antimicrobial peptides. This is a striking example of how a bacterial pathogen inte-grates signalling by ppGpp with other inputs to control the complex regulatory cascades that govern virulence gene expression.

The PigR regulator of F. tularensis. To control the expression of genes that are essential for growth in macro phages, F. tularensis relies on the macrophage growth locus–stringent starvation protein A (MglA–SspA) complex50 (FIG. 3c). Both MglA and SspA are members of the SspA family51,52 and associate with RNAP in

Figure 2 | ppGpp activates transcription of anti-adaptor proteins to stabilize σS in Escherichia coli. a | During logarithmic growth, regulator of σS protein RssB, an adaptor protein, directs the stationary phase σ-factor, σS, to the ClpXP proteosome for degradation. b | During phosphate stress, SpoT hydrolase activity is inhibited, altering the levels of guanosine pentaphosphate and guanosine tetraphosphate (collectively, ppGpp) and activating IraP, an anti-adaptor that sequesters RssB from σS and prevents delivery of the σ-factor to ClpXP. c | Genetic studies indicate that ppGpp also promotes transcription of iraD, which encodes another Escherichia coli anti-adaptor protein. PP

i, pyrophosphate;

RNAP, RNA polymerase.

R E V I E W S




LetS

ppGppppGpp

ppGpp

[low][high]

PhoQ

SlyASlyA

SlyA SlyA PhoP

PhoP box pagCpagD

(+)(+)

SlyA box

slyA

RsmZ RNA

a

b

c

rsmZ

LetA

LetA

LetA box

Transmission

Phagosome escape CAMP resistance

F. tularensis S. Typhimurium

Actin

L. pneumophila

P

P

P

Sif

P

RNAP

CsrA

CsrA

CsrA

AUGmRNA

CsrA

5ʹAUG

mRNATransmission traits repressedTransmission traits activated

CAMP resistance and intracellular replication

Acidic pH, high CAMP or low Mg2+

(+)

5ʹ

iglA

PigRSspA

MglA

RNAP

Phagosome escape and intracellular replication

ppGpp

CsrA

P

P

σS

Nutrient starvation Nutrient abundance

PhoP

RegulonAll of the genes and operons for which expression is controlled by a particular regulatory protein.

Stringent starvation protein AA protein that is synthesized in response to amino acid starvation and associates with RNA polymerase to control transcription. It promotes survival during acid stress and nutrient limitation in bacteria.

complex with each other53. PigR (a homologue of the Francisella novicida protein FevR) is a DNA-binding transcription factor that is essential for the growth of F. tularensis in macrophages and for pathogenesis in mice54, and seems to activate the MglA–SspA–RNAP complex by a direct interaction (FIG. 3c). PigR binding is promoted by ppGpp, as more PigR can be crosslinked to MglA–SspA–RNAP in wild-type F. tularensis than in relA spoT mutants54. Although biochemical evi-dence for a direct PigR–ppGpp interaction is still

lacking, the alarmone has a crucial role in controlling PigR–MglA–SspA-mediated expression of virulence genes in F. tularensis. Thus, both PigR from F. tularensis and SlyA from S. Typhimurium illustrate that ppGpp can act by mechanisms beyond a direct inter-action with RNAP. Considering these precedents, it is tempting to propose that other pathogens which are known to exploit the alarmone to control virulence3

also employ regulatory proteins for which activity is governed by ppGpp.

Figure 3 | Intracellular bacterial pathogens require ppGpp to control the activity of key virulence regulators. To survive within phagocytes, pathogens temporally regulate specialized virulence systems through guanosine tetraphosphate and guanosine pentaphosphate (collectively, ppGpp). a | In Legionella pneumophila, ppGpp controls the activation of two non-coding regulatory RNAs, RsmY (not shown) and RsmZ, that antagonize the global repressor and mRNA-binding protein carbon storage regulator (CsrA). By undefined mechanisms, ppGpp activates the stationary phase σ-factor, σS, and the Legionella transmission activator–Legionella transmission sensor (LetA–LetS) two-component system to stimulate transcription of rsmY and rsmZ. Under stress conditions, RsmY and RsmZ bind and sequester CsrA protein to relieve the CsrA-mediated translational repression of mRNAs that are important for bacterial spread. b | In Salmonella enterica subsp. enterica serovar Typhimurium, ppGpp promotes dimerization and DNA binding of the transcription factor SlyA, which participates in a feed-forward loop with the response regulator PhoP. PhoP is activated by the membrane sensor kinase PhoQ in response to acidic pH, elevated levels of cationic antimicrobial peptides (CAMPs) or low levels of Mg2+. In the presence of ppGpp, SlyA and PhoP activate transcription from several divergent operons to promote CAMP resistance and intracellular replication of the pathogen. (+) indicates positive regulation. c | In Francisella tularensis, ppGpp promotes the interaction between the DNA-binding transcription factor PigR and the RNA polymerase (RNAP)-associated macrophage growth locus–stringent starvation protein A (MglA–SspA) complex to activate the transcription of genes encoding factors that mediate phagosome escape and intracellular replication of the bacterium, such as intracellular growth locus (iglA). Sif, Salmonella-induced filament.

R E V I E W S




Cytosol

Extracellular space pH 2–3 pH 4–5

Peptidoglycan

Cadaverine Cadaverine

Cadaverine

Amino acid starvationAmino acid starvation

pH ↑

a Extreme acid stress b Mild acid stress or neutral

Amino acids consumed Amino acids preserved

Cadaverine L-lysine

L-lysineLdcI LdcIL-lysine L-lysine

L-lysine

L-lysine

L-lysineL-lysine

pH 4–5 pH 6–7

CO2CO

2

CO2

CO2

CO2

CO2

Cadaverine

Cadaverine

Cadaverine

ppGppppGpp

CadB CadB

DNA primaseAn enzyme that generates short RNA primers that are elongated by DNA polymerase during chromosome replication. DNA primase is essential for the replication of chromosomes and plasmids.

Inhibition of core pathway enzymesIn addition to shaping the transcriptional profile, ppGpp directly regulates several other core bacterial processes. For example, DNA replication and protein synthesis are downregulated by ppGpp through direct inhibition of DNA primase and the activity of translation elonga-tion factors, and potentially also the activity of trans-lation initiation factors55,56. The alarmone also directly inhibits the activity of enzymes that are crucial for RNA degradation by actinomycetes, for acid tolerance of gamma proteobacteria and for the accumulation of polyphosphate by E. coli57–60.

ppGpp-mediated regulation of mRNA half-life. Actinomycetes use ppGpp to preserve their mRNA. In Nonomuraea sp. ATCC 39727 and Streptomyces coelicolor, ppGpp inhibits polynucleotide phosphorylase (PNPase) activity57,58. PNPase is crucial for RNA turn over in these bacteria, which lack RNase II and RNase R61. The enzyme catalyses both phosphorolysis (through 3′–5′ exonuclease activity) and polymerization (through 3′-polyribonucleotide polymerase activity) of mRNAs to facilitate transcript degradation. In vitro, ppGpp inhibits both the phosphorolysis and polymerization activities of PNPase by an allosteric mechanism57. After prolonged starvation in broth, Nonomuraea sp. mutants that have reduced ppGpp concentrations exhibit increased

turn over of stable RNAs relative to turnover in the wild-type strain57. Likewise, a relA-mutant strain of S. coelicolor exhibits decreased mRNA half-life in stationary phase, whereas induction of relA expression increases mRNA half-life58. Therefore, when actinomycetes are stressed, ppGpp increases the stability of bulk mRNA by directly inhibiting PNPase, which would preserve the mRNAs for crucial cellular processes.

In E. coli, PNPase activity is not inhibited by ppGpp57,58. Instead, to regulate mRNA half-life, E. coli uses a com-mon mechanism of stringent regulation: ppGpp directly inhibits transcription of pcnB, a gene that encodes poly(A) polymerase and so is crucial for polyadenylation of mRNAs (which promotes their degradation). Thus, ppGpp also increases the stability of mRNA in E. coli, albeit by an alternative mechanism.

ppGpp regulation of the acid stress response. Cytoplasmic LdcI (lysine decarboxylase, inducible; also known as CadA) of E. coli is induced in response to acid stress and is crucial for survival in low-pH environ-ments62. Cytoplasmic pH is increased when LdcI con-sumes a proton as it catalyses decarboxylation of l-lysine to cadaverine and carbon dioxide62 (FIG. 4a). Cadaverine, a basic polyamine, is then excreted in exchange for lysine via the inner-membrane protein lysine–cadaverine anti-porter (CadB). Enteropathogens such as Vibrio cholerae and S. Typhimurium use their homologous CadA–CadB systems to survive conditions that are typical of the intestine, an anaerobic environment rich in volatile fatty acids63,64.

Recently, the X-ray crystallographic structure of E. coli LdcI was determined, revealing the ppGpp-binding sites59. The protein forms a ringed decamer of five dimers. Within each ring, an electron density corres ponding to ppGpp was identified at the interface between neigh-bouring monomers, giving ten ppGpp-binding sites per decamer. The LdcI–ppGpp interaction is important for enzyme activity, as in mildly acidic conditions (that is, an extracellular pH of 4–5, or a cytoplasmic pH of 6–7), ppGpp acts as an allosteric inhibitor of LdcI59 (FIG. 4b). By contrast, in extremely acidic conditions (that is, an extra-cellular pH of 2–3, or a cytoplasmic pH of 4–5), LdcI continues to convert lysine to cadaverine despite binding ppGpp (FIG. 4a). Targeted point mutations in LdcI that were designed to abrogate the LdcI–ppGpp interaction render the enzyme insensitive to inhibition by ppGpp at pH 6.5. In addition, ppGpp appears to inhibit lysine decarboxylation in vivo, as on starvation and mild pH stress, E. coli cells expressing ppGpp-insensitive variants of LdcI neutralize bacteriological medium more rap-idly and robustly than bacteria expressing the wild-type allele59.

The interaction between ppGpp and LdcI indicates that E. coli cells coordinate their response to acid and nutrient stress by various mechanisms. By inhibiting enzyme activity during mild acid stress or at a neutral pH, starved bacteria preserve their cytoplasmic pool of lysine. Under extreme acid stress, additional regulatory factors may be involved, as ppGpp interacts with LdcI but does not inhibit its activity. It will be interesting to

Figure 4 | ppGpp acts as an allosteric inhibitor of lysine decarboxylase in Escherichia coli. a | When amino acids are limiting, guanosine tetraphosphate and guanosine pentaphosphate (collectively, ppGpp) are synthesized. Under conditions of amino acid limitation coupled with extreme acid stress, the interaction of LdcI (lysine decarboxylase, inducible) with ppGpp is not sufficient to inhibit the activity of the enzyme, so LdcI increases cytoplasmic pH by consuming a proton as it converts cytosolic lysine to cadaverine, a polyamine. Cadaverine is exported from the cell by the inner-membrane protein lysine–cadaverine antiporter (CadB). b | When amino acids are limiting but the pH is mildly acidic or neutral, ppGpp binds to LdcI and inhibits its activity, preserving the pool of cytoplasmic lysine.

R E V I E W S




ppGpp, pppGpp

(GTP or GDP) + ATPLimiting amino acids

Ribosome

Free amino acids

Polyphosphate Polyphosphate kinase

ATPP

i

Exopolyphosphatase

Ribosomal-protein genes

Ribosomal proteins

P PP P PPPPP

LonP PP P PPPPP

P PP P PPPPPP PP P PPPPP

RNAPRelA

Second messengerA molecule that relays signals from a receiver or receptor by exerting an effect on a downstream cellular factor or process.

determine whether ppGpp also controls other acid-induced decarboxylases such as glutamic acid, arginine or ornithine decarboxylases.

ppGpp-mediated regulation of exopolyphosphatase. All cells synthesize polyphosphate, an anionic polymer of orthophosphate that bacteria use as an energy source and a signalling molecule60. Polyphosphate is generated from ATP by polyphosphate kinase and degraded to inorganic phosphate by exopolyphosphatase60. Although E. coli expresses both enzymes constitutively, the activ-ity of exopolyphosphatase is inhibited by pppGpp and, less so, by ppGpp65. Thus, in response to nutrient stress, bacteria accumulate not only the alarmone but also polyphosphate.

By coupling accumulation of the alarmone and of polyphosphate, E. coli can respond to amino acid stress by inhibiting expression of ribosomal genes while recy-cling free ribosomal proteins (FIG. 5). Polyphosphate binds particular ribosomal proteins and activates the Lon protease in vitro, and coordinates Londependent degradation of ribosomal proteins in vivo66. Therefore, by directly inhibiting exopolyphosphatase activity, ppGpp and pppGpp equip bacteria to salvage amino acids from components of the protein synthesis machinery.

Polyphosphate also contributes to the virulence of a wide variety of bacterial pathogens. Although the mech-anisms involved remain to be determined, Helicobacter pylori, Mycobacterim tuberculosis, Neisseria meningitidis, P. aeruginosa, S. Typhimurium and S. flexneri require polyphosphate kinase to express various traits, such as motility, stress resistance and colonization60. Because ppGpp, pppGpp and polyphosphate enable bacteria to tolerate nutrient stress and to induce virulence traits when conditions no longer favour replication, knowl-edge of the corresponding regulatory circuitries will probably inform new therapeutic strategies.

Indirect control of CodY in firmicutes The ability of ppGpp to affect the activity and stability of important regulators such as σS, σE and CsrA illustrates the extensive integration of ppGpp signalling with more specialized regulatory networks. The activity of CodY, a master regulator in Gram-positive firmicutes, is also influenced by the alarmone, but by a mechanism that is different from that used to control σS, σE and CsrA.

Although nearly all firmicutes encode ortho-logues of RelA and SpoT, ppGpp signalling differs in these bacteria67. In both gammaproteobacteria and firmicutes, ppGpp inhibits an enzyme that is crucial for GTP synthesis68,69. In addition, ppGpp synthesis consumes GTP1 (FIG. 6a). Consequently, researchers must address whether the effects ascribed to increased ppGpp production result from a concomitant reduction in the cellular GTP pool, rather than direct action of ppGpp. As some firmicutes rely on GTP as the initiating nucleotide for transcription of rRNA operons, low GTP concentrations result in cessation of rRNA synthesis70 (FIG. 6b). Furthermore, for a variety of firmicutes, GTP availability influences protein and antibiotic synthesis, genetic competence, flagellar production, sporulation and virulence, as each of these pathways is controlled by CodY, a DNA-binding transcriptional repressor that is activated by GTP71. During a stringent response, increased ppGpp production reduces the pool of GTP-bound CodY, effectively releasing the repressor from its target promoters and permitting their transcriptional activation71 (FIG. 6c). Although the interplay between CodY and ppGpp has been best studied in B. subtilis, this interaction also regulates gene expression in patho genic firmicutes, such as Staphylococcus aureus72, Streptococcus pyogenes73, Streptococcus mutans74 and Listeria monocytogenes75.

Crosstalk between ppGpp and cyclic dinucleotidesThe alarmone ppGpp modulates signalling by other nucleotide messengers through mechanisms other than its effect on the GTP pool. In recent years, the nucleo-tide messenger cyclic di-GMP (c-di-GMP) has gained recognition for its control of the switch between sessile and sedentary bacterial lifestyles76,77. Although c-di-GMP is ubiquitous among bacteria, this molecule has not been identified in archaea or eukaryotes to date. Indeed, c-di-GMP and the structurally related mol-ecule cyclic di-AMP (c-di-AMP) act as microorganism- associated molecular patterns (MAMPs), which are broadly conserved microbial molecules that trigger innate immune signalling in the host78–80. Biochemical and genetic studies in E. coli and B. subtilis suggest that a dialogue between ppGpp and cyclic dinucleotides enables bacteria to fine-tune second messenger levels in response to stress.

Interactions between ppGpp and c-di-GMP. Persistent pathogens such as P. aeruginosa and uropathogenic E. coli (UPEC) establish biofilm-like communities dur-ing infection. Within these multicellular structures, resident bacteria are protected from immune defences and therapeutics and can seed recurrent infections in the

Figure 5 | Involvement of ppGpp and pppGpp in the degradation of ribosomal proteins. In response to amino acid depletion, Escherichia coli synthesizes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp). ppGpp inhibits transcription of the genes encoding ribosomal proteins, and both ppGpp and pppGpp inhibit the activity of exopolyphosphatase. Polyphosphate therefore accumulates and binds directly to free ribosomal proteins and to the Lon protease. Consequently, E. coli generates free amino acids that can be incorporated into biosynthetic enzymes. P

i, inorganic phosphate; RNAP, RNA polymerase.

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ppGpp

(GTP or GDP) + ATP

Purine pathway

IMPAMP XMP GMP

GDP

GTP

ADP

ATP

(GTP or GDP) + ATPGTP

[low]

Nutrient starvation: Nutrient starvation:

Nutrient abundance:

Transcriptional repression of CodY-dependent genes

Derepression of CodY Activation of stress response

Initiatingnucleotide

GTP depletiona Deactivation of ribosomal-gene transcriptionb

Deactivation of the CodY repressorc

RelA

CodY

RelP

RelQ

RNAP

rrnBAGUG

GTP[low]

RNAP

GTP

GTP

GTPGTP

Nutrient starvation:

CodYRNAP

urinary tract or in the lungs of patients with cystic fibro-sis81. Biofilms pose a clinical challenge, as some patho-gens induce their formation in response to antibiotics at subinhibitory concentrations82. Thus, understanding the signalling pathways that govern biofilm communities will inform antimicrobial therapies.

Low doses of translation inhibitors, such as chloram-phenicol, erythromycin or tetracycline, induce the for-mation of E. coli biofilms by a mechanism that requires SpoT and the c-di-GMP-synthesizing diguanylyl cyclase YdeH9. By compromising the translation machinery, these antibiotics prompt SpoT to degrade ppGpp, lead-ing to derepression of pgaA, which encodes a protein required for the synthesis of poly-β-1,6-N-acetyl glu-cosamine (poly-GlcNAc), an essential component of the E. coli biofilm matrix9. Maximal poly-GlcNAc pro-duction also requires c-di-GMP formation by YdeH. Both ppGpp and c-di-GMP increase expression of independent components of the poly-GlcNAc synthe-sis pathway by post-transcriptional mechanisms that remain to be defined in detail9. The demand for low ppGpp and high c-di-GMP for biofilm formation in response to antibiotic treatment suggests that bacteria integrate second messenger cues to erect a barrier to antimicrobial agents.

Interactions between ppGpp and c-di-AMP. The mes-senger c-di-AMP was recently discovered as a nucleotide that signals DNA damage in B. subtilis83. The molecule is synthesized by a diadenylyl cyclase (DAC) domain that is widespread among bacteria84. As is the case for ppGpp and c-di-GMP, bacteria possess enzymes for both synthe-sis and hydrolysis of c-di-AMP. The cytoplasmic portion of YybT, a transmembrane protein of B. subtilis, exhibits phosphodiesterase activity towards c-di-AMP10. c-di-AMP signalling is likely to be influenced by the strin-gent response, as ppGpp is a potent inhibitor of the YybT phosphodiesterase activity in vitro10. Accordingly, ppGpp is predicted to preserve the cellular pool of c-di-AMP. The discovery of the direct and indirect interactions between the signalling pathways mediated by ppGpp and by other nucleotides underscores the broad influence of the alarmone, not only on individual bacterial cells, but also within microbial communities.

ConclusionThe alarmone ppGpp coordinates bacterial physiology through an impressive array of mechanisms. By modu-lating the expression, stability or activity of transcription factors and enzymes, ppGpp enables bacteria to alter their physiology in order to accommodate fluctuating nutrient

Figure 6 | ppGpp synthesis affects GTP-dependent gene expression in firmicutes. a | In both firmicutes and gamma proteobacteria, synthesis of guanosine tetraphosphate and guanosine pentaphosphate (collectively, ppGpp) causes a reduction in the cellular GTP pool through enzymatic consumption, while the alarmone itself (ppGpp) inhibits an enzyme (or enzymes) that is crucial for GTP synthesis (the bold pathway in the box). RelP and RelQ are two recently identified RelA family proteins called small alarmone synthetases, found in Streptococcus mutans and other firmicutes. b | As a result of GTP reduction in Bacillus subtilis, transcription of ribosomal RNA (illustrated by rrnB) is deactivated, because the initiating nucleotide for these operons is GTP. c | When GTP is abundant, the GTP-binding transcriptional repressor CodY, encoded by many firmicutes, negatively regulates genes that are dedicated to antibiotic synthesis, genetic competence, flagella production, sporulation and virulence. When GTP pools are depleted by ppGpp synthesis, CodY releases from its DNA targets, and the regulon is derepressed. IMP, inosine monophosphate; RNAP, RNA polymerase; XMP, xanthosine monophosphate.

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supplies and environmental stresses. Indeed, even within the classical mechanism by which ppGpp alters RNAP promoter selection, variations have recently come to light. By capturing a series of complete transcriptional profiles as E. coli cells respond to nutrient limitation, research-ers have identified genetic hierarchies within the strin-gent response regulon. For example, low concentrations of ppGpp are sufficient to induce the leucine-responsive regulatory protein (Lrp) pathway, whereas higher doses are needed to stimulate the σS regulon85.

The strategy of tuning regulatory circuits accord-ing to ppGpp concentration might explain the strict requirement for SpoT not only during induction of the anti-adaptor protein IraP, which stabilizes σS (FIG. 2b), but also during infection by intracellular patho-gens (BOX 1). The use of mutant spoT alleles encod-ing enzymes that are defective in either synthesis or hydrolysis of ppGpp will aid our understanding of how bifunctional stringent response enzymes equip bacteria with the ability to fine-tune their transcriptional profiles by modulating ppGpp pools in response to particular environments. The development of a fluorescent che-mosensor for ppGpp86,87, like the biosensor for c-di-GMP88, is an important step towards spatio temporal analysis of ppGpp in live cells. It is conceivable that RelA and SpoT generate ppGpp in distinct subcellu-lar domains, thereby each modulating the activity of a

different cohort of target enzymes. How ppGpp, after binding to its target, alters the biophysical properties of transcription-regulatory proteins such as SlyA and PigR or enzymes such as YybT and LdcI requires more detailed structure–function studies.

It is not yet known whether the consumption of GTP during ppGpp biosynthesis indirectly impedes c-di-GMP generation and subsequent signalling, as observed for CodY-mediated repression in firmicutes. Another wide open question is, how widespread is ppGpp syn-ergism, or antagonism, with cyclic nucleotide messen-gers? The discovery that c-di-GMP and c-di-AMP are not only crucial second messengers but also MAMPs that are recognized by host innate immune systems has opened another avenue of research into nucleotide signalling.

Recently, proteins that function as SpoT-like hydro-lases were discovered in human and Drosophila melanogaster cells, and flies that lack the corresponding gene are more vulnerable to amino acid starvation89. Therefore, like bacteria and plants, metazoans might also exploit ppGpp to vary the expression, stability or activity of pro-teins. The discovery of a variety of regulatory proteins and enzymes for which activity is modulated directly by the alarmone will spur researchers to extend their sights beyond the promoter to discover the full scope of ppGpp magic.

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Molecular genetic, biochemical and cell biological analyses determining that ppGpp binding promotes assembly and activity of a transcriptional complex which is crucial for intracellular growth.

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In vitro and in vivo experiments which establish that ppGpp binds and inhibits the activity of an enzyme that destabilizes mRNAs.

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A biochemical study demonstrating that direct binding by ppGpp inhibits enzymes that destabilize mRNA.

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AcknowledgementsThe authors’ research on virulence regulation in L. pneumophila has been supported by the US National Institutes of Health (grant 2 R01 AI44212 to M.S.S.) and the University of Michigan, Ann Arbor, USA (a Rackham Predoctoral Fellowship to Z.D.D.).

Competing interests statementThe authors declare no competing financial interests.

FURTHER INFORMATIONMichele S. Swanson’s homepage: www.med.umich.edu/microbio/bio/swanson_m.htm

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ppGpp: magic beyond RNA polymerase