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When faced with limiting nutrients, bacteria rapidly enzymes to control the concentrations of ppGpp1–3. To
Stringent response
A stress response coordinated reallocate cellular resources by stopping the synthesis of adapt rapidly to their environment, bacteria regulate
by guanosine tetraphosphate DNA, stable RNAs, ribosomal proteins and membrane the enzymes that synthesize and degrade ppGpp. For
and guanosine pentaphosphate, components and rapidly producing factors that are cru- example, when E. coli encounters a shortage of amino
in which cells rapidly inhibit cial for stress resistance, glycolysis and amino acid syn- acids, uncharged tRNAs stimulate RelA activity (FIG. 1).
synthesis of stable RNA,
ribosomes and proteins,
thesis1. This so-called stringent response to nutrient stress By contrast, bacteria require bifunctional SpoT enzymes
leading to growth arrest. is accomplished in part by a massive switch in the tran- to respond to a variety of nutrient stresses, such as
scription profile, coordinated by the signalling nucleo- phosphate, carbon, iron or fatty acid starvation.
tides guanosine tetraphosphate (ppGpp) and guanosine ppGpp exerts many of its physiological effects by
pentaphosphate (pppGpp), which are also referred interacting directly with RNAP in cooperation with DnaK
to as magic spot or the alarmone. The nucleotide suppressor (DksA), a protein that binds in the second-
ppGpp was discovered as a ‘magic spot’ on thin-layer ary channel of the enzyme and amplifies the impact of
chromatograms that were designed to analyse changes the alarmone (FIG. 1). Whether bound ppGpp and DksA
in cellular nucleotide pools in response to nutrient stress. act positively or negatively on transcription by RNAP
Since then, extensive genetic and molecular analyses is determined by properties that are intrinsic to the
in Escherichia coli have established that ppGpp alters promoter in question4.
transcription globally by regulating RNA polymerase ppGpp and DksA can also regulate transcription by
(RNAP) activity directly and regulating RNAP σ-factor an indirect process known as σ-factor competition. In
activity indirectly 1,2. the logarithmic growth period, the vegetative σ-factor,
1
Department of Microbiology, Two classes of enzyme control the cellular pool σ70 (also known as RpoD), directs RNAP to initiate
University of Washington, of ppGpp. Monofunctional synthetases, known as the transcription of operons that are fundamental to the
Health Sciences Building K116,
1959 NE Pacific St.,
RelA proteins, use GTP and ATP to generate pppGpp, synthesis of proteins, lipids and DNA. During a strin-
Box 357710, Seattle, which is then converted to ppGpp (FIG. 1). Bifunctional gent response, high concentrations of ppGpp inhibit
Washington 98195-7710, synthetase–hydrolase enzymes, called SpoT, Rel or RNAP binding to strong σ70-dependent promoters, such
USA. RSH (Rel–Spo homologue) proteins, can make ppGpp as the promoters of ribosomal RNA and tRNA genes;
2
Department of Microbiology
and pppGpp and can also hydrolyse the nucleotides consequently, more core RNAP is available to bind to the
& Immunology, University of
Michigan Medical School, to yield either GDP and pyrophosphate (PPi) or GTP alternative σ-factors that accumulate in response to par-
6733 Medical Science and PP i, respectively; in this Review, ppGpp and ticular stresses5. These alternative σ-factors then direct
Building II, 1150 West Medical pppGpp are collectively referred to as ppGpp, unless RNAP to transcribe genes that are devoted to coping
Center Drive, Ann Arbor, otherwise specified. The SpoT hydrolase activity is with those conditions2. In addition to modulating pro-
Michigan 48109–5620, USA.
Correspondence to M.S.S.
essential to bacterial cells, as high concentrations moter selection by RNAP, ppGpp can exert a broad
e‑mail: mswanson@umich.edu of ppGpp bring replication to a halt. Different species of influence on bacterial cells. For example, ppGpp coor-
doi:10.1038/nrmicro2720 bacteria encode a variety of synthetase and hydrolase dinates the synthesis of ribosomes to suit the growth
Figure 1 | ppGpp alters RNA polymerase promoter selection by σ-factors. σ-factor stability
Nature Reviews | Microbiology
In response to particular stresses, SpoT and RelA catalyse pyrophosphoryl transfer from When ppGpp alerts a bacterium to stress, σ-factors alter
ATP to GTP or GDP to synthesize the signalling nucleotides guanosine pentaphosphate the transcription profile by directing RNAP to specific
and guanosine tetraphosphate, respectively; these nucleotides are collectively referred
promoters. σS (also known as RpoS), a stationary-phase
to as ppGpp. Together with DnaK suppressor (DksA), ppGpp directs transcription
initiation at particular gene promoters through direct interaction with RNA polymerase. σ-factor that is crucial for stress resistance and the
In part, ppGpp and DksA act by promoting the interaction of RNA polymerase with expression of virulence factors by a variety of patho-
alternative σ-factors (σ*), such as σE. When metabolic precursors are plentiful, SpoT gens11,12, is regulated by ppGpp through an impressive
instead degrades ppGpp, and the vegetative σ-factor, σ70, directs RNA polymerase to array of direct and indirect mechanisms13–15.
genes that are crucial for bacterial replication. PPi, pyrophosphate. σS is regulated at several levels: gene transcription,
mRNA translation, protein stability and protein activ-
ity 16. Although the rpoS gene is transcribed and the
rate in replicating E. coli cells 6,7, and accumulated mRNA translated during vegetative growth, the con-
ppGpp inhibits DNA replication in Bacillus subtilis 8. The centration of σS in the cell remains low because regula-
Adaptor
In the context of
alarmone also modulates the expression of virulence tor of σS protein RssB, an adaptor protein, directs σS to
post-translational regulation factors that equip bacterial pathogens to cope with envi- the ClpXP proteasome for degradation11,17 (FIG. 2a). In
of protein stability, a protein ronmental and metabolic stress3 (BOX 1). Furthermore, E. coli, ppGpp promotes σS protein stability by inducing
that binds another protein recent studies have revealed crosstalk between ppGpp expression of the anti-adaptor proteins IraP and IraD,
and chaperones it to the
proteasome for degradation;
regulation and other nucleotide-signalling pathways which bind RssB and block its activity 18,19. For example,
an example of such an adaptor that are known to govern bacterial cell differentiation when phosphate is limiting, the ppGpp pool generated
is regulator of σS protein RssB. processes such as biofilm formation9,10. Recent reviews by SpoT activates iraP transcription, thereby increas-
ing σS stability 18 (FIG. 2b). Induction of the σS-mediated
stress response by phosphate limitation is abolished by
Box 1 | SpoT, not RelA, is required during infection swapping the AT-rich discriminator sequence of the iraP
promoter for a GC-rich sequence, implicating ppGpp
For several bacterial pathogens, the stringent response enzyme that is crucial for
infection is SpoT, which has both hydrolase and synthetase activities for guanosine
in activation of iraP transcription 18. The ppGpp-
tetraphosphate and guanosine pentaphosphate (referred to here as ppGpp). Because induced activation of iraP is specific to phosphate
the hydrolase activity of SpoT is essential to bacterial cells that generate ppGpp — high stress and requires SpoT hydrolase activity but not
levels of ppGpp disrupt the cell cycle, so it must be degraded — the function of SpoT SpoT synthetase activity, as in the presence of RelA and
is deduced by comparing the phenotype of relA spoT double mutants (which, lacking a synthetase-defective allele of SpoT, iraP induction in
both synthetases, do not produce ppGpp) to that of relA single mutants. Mice infected response to phosphate stress resembles that of wild-type
intragastrically with either wild-type or relA Salmonella enterica subsp. enterica serovar bacteria. Likewise, neither overexpression of RelA nor
Typhimurium succumb to infection within 7–10 days, whereas mice infected with relA increased ppGpp synthesis (triggered in response to
spoT bacteria show no signs of illness up to 30 days after inoculation at >100‑fold the amino acid stress caused by serine hydroxamate treat-
lethal dose (LD50) of wild-type bacteria90. For subcutaneous (bubonic) infection with
ment) is sufficient to induce iraP expression18. Perhaps
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
transcriptional induction of the iraP promoter during
macrophages, the number of viable relA spoT bacteria recovered is 100,000 fewer than phosphate limitation requires a specific concentra-
the number of viable wild-type or relA bacteria recovered. Likewise, mice infected tion of the alarmone that can only be achieved by the
intradermally with wild-type and relA F. tularensis succumb within 5–7 days, whereas bifunctional enzyme SpoT.
mice infected with relA spoT bacteria remain viable up to 20 days after infection54. The alarmone also regulates the expression of IraD,
Wild-type and relA Legionella pneumophila proliferate in macrophage cultures, but relA a second anti-adaptor protein devoted to σS stability
spoT mutants do not survive host-to-host transmission92,93. Additional experiments are (FIG. 2c). Both iraD transcription and σS stability increase
needed to identify whether the hydrolase activity that is unique to SpoT accounts for as E. coli enters stationary phase19,20. Although the mecha-
its crucial role during infection. An alternative hypothesis that warrants testing is that nism involved remains to be defined, genetic experiments
the capacity of SpoT synthetase activity to be induced by the particular stresses
implicate the alarmone in this growth phase regulation.
encountered during infection explains why pathogens require SpoT but not RelA.
In particular, iraD expression is elevated in replicating
rpoB mutants (which have an RNAP subunit-β with an and the alarmone accumulates, transcription mediated
altered function) that exhibit a stringent response profile by σE is probably further enhanced when the RNAP that
in the absence of ppGpp. is released from ribosomal RNA operons becomes avail-
able to alternative σ-factors24. Thus, by exerting its influ-
σ-factor activity ence on both σ-factor activity and the pool of free RNAP,
A second σ-factor that is tightly regulated in E. coli is σE ppGpp drives the expression of genes in the σE regulon
(also known as RpoE), the σ-factor that coordinates the in E. coli as a rapid response to outer-membrane damage
cellular response to the presence of misfolded proteins or metabolic stress.
in the periplasm or outer membrane. However, rather
Proteasome than stabilize the σE protein, ppGpp enhances its activ- Transcript stability
A cytoplasmic complex that ity. σE activity is inhibited by σE factor regulatory protein In Legionella pneumophila and E. coli, ppGpp induces
unfolds and degrades proteins. (RseA), an inner-membrane protein that sequesters the the expression of non-coding regulatory RNAs25,26 to
σ-factor. In response to cell envelope stress, such as regulate the stability of certain mRNAs indirectly via
Anti-adaptor
In the context of the presence of misfolded outer-membrane porins, RseA the carbon storage regulator (CsrA) regulatory pathway.
post-translational regulation of is degraded by a proteolyic cascade, thereby releasing σE CsrA is an RNA-binding protein that controls a vari-
protein stability, a protein that (REF. 21) to direct RNAP to genes that are important for ety of bacterial processes, ranging from biofilm forma-
binds an adaptor and blocks its outer-membrane biogenesis. tion27 and motility 28,29 to protein secretion by the type III
chaperone activity; examples of
anti-adaptors are IraP and IraD.
Activation of the σE regulon in E. coli also occurs in (REF. 30), type IV (REF. 31) and type VI32 secretion systems.
response to metabolic stress that is signalled by ppGpp. For example, in the intracellular pathogen L. pneumo
Discriminator The amount of rpoE mRNA increases promptly when phila, CsrA represses the expression of crucial virulence
The DNA sequence between a stringent response is induced pharmacologically by factors that promote bacterial transmission, such as fac-
the −10‑box hexamer of a
serine hydroxamate22. Nevertheless, σE protein levels tors that are responsible for lysosome evasion. CsrA
promoter and the transcriptional
start site at nucleotide +1. remain constant when cells accumulate ppGpp, and the targets include components of the Dot–Icm type IV
Promoters that are activated rpoE promoter is not required to observe increased σE secretion system and the flagellar secretion system31,33.
by guanosine tetraphosphate activity in stationary phase23,24. Instead, σE-mediated Through its physical interaction with leader sequences
and guanosine pentaphosphate transcription initiation of certain genes in the σE regu- and ribosome-binding sites on target mRNAs, CsrA pre-
acting with DnaK suppressor
(DksA) are typically AT‑rich in
lon is stimulated directly by ppGpp and DksA, as meas- vents the translation of these transcripts and facilitates
this region, whereas repressed ured by in vitro transcription reactions using purified their degradation34. CsrA activity is inhibited by spe-
targets are typically GC‑rich. components24. As bacteria transition to stationary phase cialized non-coding regulatory RNAs that outcompete
a Logarithmic growth and nutrient abundance of numerous factors in stationary phase25,31,38,39 (FIG. 3a).
P Similarly, in E. coli both ppGpp and the cofactor pro-
RssB P tein DksA activate transcription of the analogous non-
RssB coding RNAs, CsrB and CsrC26. However, a previous
ClpXP genetic study indicated that transcription of CsrB is
σS σS
induced independently of ppGpp in stationary phase
Active E. coli cells41. Although the basis for this discrepancy is
adapter not clear, the previous E. coli study 41 relied on analysis of
relA spoT-mutant strains, which frequently acquire sup-
pressor mutations that can mimic the effects of ppGpp.
σS Whether P. aeruginosa and other pathogens also use
b Phosphate limitation
ppGpp to relieve CsrA-mediated repression of stationary
(GTP or GDP) + ATP (GTP or GDP) + PPi phase and virulence genes by inducing the expression of
ClpXP
SpoT Phosphate stress σS
regulatory RNAs is an open question.
P ppGpp [high]
ppGpp [low]
RsmZ RNA CsrA
P LetA
σS
CsrA CsrA
CsrA
P LetA RNAP
AUG AUG
5ʹ 5ʹ CsrA
LetA box rsmZ mRNA mRNA
Transmission traits activated Transmission traits repressed
PhoQ
P
L. pneumophila
Phagosome escape P PhoP
CAMP resistance
(+)
slyA
F. tularensis S. Typhimurium
SlyA
SlyA
Sif Actin
c ppGpp
ppGpp
Phagosome escape and
intracellular replication (+) SlyA SlyA P PhoP (+)
MglA pagD SlyA box PhoP box pagC
PigR
SspA RNAP
CAMP resistance and intracellular replication
iglA
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
Nature Reviews | Microbiology
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.
Regulon
All of the genes and operons
for which expression is complex with each other 53. PigR (a homologue of the lacking, the alarmone has a crucial role in controlling
controlled by a particular Francisella novicida protein FevR) is a DNA-binding PigR–MglA–SspA-mediated expression of virulence
regulatory protein.
transcription factor that is essential for the growth of genes in F. tularensis. Thus, both PigR from F. tula
Stringent starvation protein A F. tularensis in macrophages and for pathogenesis in rensis and SlyA from S. Typhimurium illustrate that
A protein that is synthesized mice54, and seems to activate the MglA–SspA–RNAP ppGpp can act by mechanisms beyond a direct inter-
in response to amino acid complex by a direct interaction (FIG. 3c). PigR binding is action with RNAP. Considering these precedents, it is
starvation and associates with promoted by ppGpp, as more PigR can be crosslinked tempting to propose that other pathogens which are
RNA polymerase to control
transcription. It promotes
to MglA–SspA–RNAP in wild-type F. tularensis than known to exploit the alarmone to control virulence3
survival during acid stress and in relA spoT mutants54. Although biochemical evi- also employ regulatory proteins for which activity is
nutrient limitation in bacteria. dence for a direct PigR–ppGpp interaction is still governed by ppGpp.
Inhibition of core pathway enzymes turnover of stable RNAs relative to turnover in the wild-
In addition to shaping the transcriptional profile, ppGpp type strain57. Likewise, a relA-mutant strain of S. coeli
directly regulates several other core bacterial processes. color exhibits decreased mRNA half-life in stationary
For example, DNA replication and protein synthesis phase, whereas induction of relA expression increases
are downregulated by ppGpp through direct inhibition mRNA half-life58. Therefore, when actinomycetes are
of DNA primase and the activity of translation elonga- stressed, ppGpp increases the stability of bulk mRNA
tion factors, and potentially also the activity of trans- by directly inhibiting PNPase, which would preserve the
lation initiation factors55,56. The alarmone also directly mRNAs for crucial cellular processes.
inhibits the activity of enzymes that are crucial for In E. coli, PNPase activity is not inhibited by ppGpp57,58.
RNA degradation by actinomycetes, for acid tolerance Instead, to regulate mRNA half-life, E. coli uses a com-
of gammaproteobacteria and for the accumulation of mon mechanism of stringent regulation: ppGpp directly
polyphosphate by E. coli 57–60. inhibits transcription of pcnB, a gene that encodes
poly(A) polymerase and so is crucial for polyadenylation
ppGpp-mediated regulation of mRNA half-life. of mRNAs (which promotes their degradation). Thus,
Actinomycetes use ppGpp to preserve their mRNA. In ppGpp also increases the stability of mRNA in E. coli,
Nonomuraea sp. ATCC 39727 and Streptomyces coelicolor, albeit by an alternative mechanism.
ppGpp inhibits polynucleotide phosphorylase (PNPase)
activity 57,58. PNPase is crucial for RNA turnover in ppGpp regulation of the acid stress response.
these bacteria, which lack RNase II and RNase R61. The Cytoplasmic LdcI (lysine decarboxylase, inducible; also
enzyme catalyses both phosphorolysis (through 3′–5′ known as CadA) of E. coli is induced in response to acid
DNA primase exonuclease activity) and polymerization (through stress and is crucial for survival in low-pH environ-
An enzyme that generates 3′-polyribonucleotide polymerase activity) of mRNAs to ments62. Cytoplasmic pH is increased when LdcI con-
short RNA primers that are facilitate transcript degradation. In vitro, ppGpp inhibits sumes a proton as it catalyses decarboxylation of l‑lysine
elongated by DNA polymerase both the phosphorolysis and polymerization activities of to cadaverine and carbon dioxide62 (FIG. 4a). Cadaverine,
during chromosome
replication. DNA primase is
PNPase by an allosteric mechanism57. After prolonged a basic polyamine, is then excreted in exchange for lysine
essential for the replication of starvation in broth, Nonomuraea sp. mutants that via the inner-membrane protein lysine–cadaverine anti-
chromosomes and plasmids. have reduced ppGpp concentrations exhibit increased porter (CadB). Enteropathogens such as Vibrio cholerae
and S. Typhimurium use their homologous CadA–
CadB systems to survive conditions that are typical of
a Extreme acid stress b Mild acid stress or neutral the intestine, an anaerobic environment rich in volatile
CO2 Cadaverine fatty acids63,64.
Cadaverine CO2 CO2 Recently, the X‑ray crystallographic structure of E. coli
Cadaverine L-lysine Cadaverine L-lysine LdcI was determined, revealing the ppGpp-binding
Extracellular space pH 2–3 pH 4–5
sites59. The protein forms a ringed decamer of five dimers.
Within each ring, an electron density corresponding to
ppGpp was identified at the interface between neigh-
bouring monomers, giving ten ppGpp-binding sites per
Peptidoglycan
decamer. The LdcI–ppGpp interaction is important for
CadB CadB enzyme activity, as in mildly acidic conditions (that is,
Cytosol pH 4–5 pH 6–7 an extracellular pH of 4–5, or a cytoplasmic pH of 6–7),
ppGpp acts as an allosteric inhibitor of LdcI59 (FIG. 4b). By
CO2
CO2 contrast, in extremely acidic conditions (that is, an extra-
Cadaverine L-lysine Cadaverine L-lysine
cellular pH of 2–3, or a cytoplasmic pH of 4–5), LdcI
Cadaverine CO2 continues to convert lysine to cadaverine despite binding
pH ↑
ppGpp (FIG. 4a). Targeted point mutations in LdcI that
Amino acid starvation
Amino acid starvation were designed to abrogate the LdcI–ppGpp interaction
render the enzyme insensitive to inhibition by ppGpp
ppGpp
ppGpp at pH 6.5. In addition, ppGpp appears to inhibit lysine
LdcI LdcI decarboxylation in vivo, as on starvation and mild pH
L-lysine L-lysine
L-lysine stress, E. coli cells expressing ppGpp-insensitive variants
Amino acids consumed L-lysine Amino acids preserved of LdcI neutralize bacteriological medium more rap-
idly and robustly than bacteria expressing the wild-type
Figure 4 | ppGpp acts as an allosteric inhibitor of lysine decarboxylase in allele59.
Nature
Escherichia coli. a | When amino acids are limiting, guanosine Reviews | Microbiology
tetraphosphate and The interaction between ppGpp and LdcI indicates
guanosine pentaphosphate (collectively, ppGpp) are synthesized. Under conditions of that E. coli cells coordinate their response to acid and
amino acid limitation coupled with extreme acid stress, the interaction of LdcI (lysine
nutrient stress by various mechanisms. By inhibiting
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 enzyme activity during mild acid stress or at a neutral
cytosolic lysine to cadaverine, a polyamine. Cadaverine is exported from the cell by the pH, starved bacteria preserve their cytoplasmic pool of
inner-membrane protein lysine–cadaverine antiporter (CadB). b | When amino acids are lysine. Under extreme acid stress, additional regulatory
limiting but the pH is mildly acidic or neutral, ppGpp binds to LdcI and inhibits its activity, factors may be involved, as ppGpp interacts with LdcI
preserving the pool of cytoplasmic lysine. but does not inhibit its activity. It will be interesting to
Nutrient starvation:
ADP GDP
GTP[low]
CodY Derepression of CodY
ATP GTP Activation of stress response
RNAP
Figure 6 | ppGpp synthesis affects GTP-dependent gene expression in firmicutes. a | In both firmicutes and
gammaproteobacteria, synthesis of guanosine tetraphosphate and guanosine pentaphosphate Nature Reviews | ppGpp)
(collectively, Microbiology
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.
urinary tract or in the lungs of patients with cystic fibro- Interactions between ppGpp and c‑di-AMP. The mes-
sis81. Biofilms pose a clinical challenge, as some patho- senger c‑di-AMP was recently discovered as a nucleotide
gens induce their formation in response to antibiotics that signals DNA damage in B. subtilis83. The molecule is
at subinhibitory concentrations82. Thus, understanding synthesized by a diadenylyl cyclase (DAC) domain that
the signalling pathways that govern biofilm communities is widespread among bacteria84. As is the case for ppGpp
will inform antimicrobial therapies. and c‑di-GMP, bacteria possess enzymes for both synthe-
Low doses of translation inhibitors, such as chloram- sis and hydrolysis of c‑di-AMP. The cytoplasmic portion
phenicol, erythromycin or tetracycline, induce the for- of YybT, a transmembrane protein of B. subtilis, exhibits
mation of E. coli biofilms by a mechanism that requires phosphodiesterase activity towards c‑di-AMP10. c-di-
SpoT and the c‑di-GMP-synthesizing diguanylyl cyclase AMP signalling is likely to be influenced by the strin-
YdeH9. By compromising the translation machinery, gent response, as ppGpp is a potent inhibitor of the YybT
these antibiotics prompt SpoT to degrade ppGpp, lead- phosphodiesterase activity in vitro10. Accordingly, ppGpp
ing to derepression of pgaA, which encodes a protein is predicted to preserve the cellular pool of c‑di-AMP.
required for the synthesis of poly‑β-1,6‑N-acetyl glu- The discovery of the direct and indirect interactions
cosamine (poly-GlcNAc), an essential component of between the signalling pathways mediated by ppGpp and
the E. coli biofilm matrix 9. Maximal poly-GlcNAc pro- by other nucleotides underscores the broad influence of
duction also requires c‑di-GMP formation by YdeH. the alarmone, not only on individual bacterial cells, but
Both ppGpp and c‑di-GMP increase expression of also within microbial communities.
independent components of the poly-GlcNAc synthe-
sis pathway by post-transcriptional mechanisms that Conclusion
remain to be defined in detail9. The demand for low The alarmone ppGpp coordinates bacterial physiology
ppGpp and high c‑di-GMP for biofilm formation in through an impressive array of mechanisms. By modu-
response to antibiotic treatment suggests that bacteria lating the expression, stability or activity of transcription
integrate second messenger cues to erect a barrier to factors and enzymes, ppGpp enables bacteria to alter their
antimicrobial agents. physiology in order to accommodate fluctuating nutrient
supplies and environmental stresses. Indeed, even within different cohort of target enzymes. How ppGpp, after
the classical mechanism by which ppGpp alters RNAP binding to its target, alters the biophysical properties
promoter selection, variations have recently come to light. of transcription-regulatory proteins such as SlyA and
By capturing a series of complete transcriptional profiles PigR or enzymes such as YybT and LdcI requires more
as E. coli cells respond to nutrient limitation, research- detailed structure–function studies.
ers have identified genetic hierarchies within the strin- It is not yet known whether the consumption of GTP
gent response regulon. For example, low concentrations during ppGpp biosynthesis indirectly impedes c-di-
of ppGpp are sufficient to induce the leucine-responsive GMP generation and subsequent signalling, as observed
regulatory protein (Lrp) pathway, whereas higher doses for CodY-mediated repression in firmicutes. Another
are needed to stimulate the σS regulon85. wide open question is, how widespread is ppGpp syn-
The strategy of tuning regulatory circuits accord- ergism, or antagonism, with cyclic nucleotide messen-
ing to ppGpp concentration might explain the strict gers? The discovery that c-di-GMP and c-di-AMP are
requirement for SpoT not only during induction of the not only crucial second messengers but also MAMPs
anti-adaptor protein IraP, which stabilizes σ S (FIG. 2b), that are recognized by host innate immune systems
but also during infection by intracellular patho- has opened another avenue of research into nucleotide
gens (BOX 1). The use of mutant spoT alleles encod- signalling.
ing enzymes that are defective in either synthesis or Recently, proteins that function as SpoT-like hydro-
hydrolysis of ppGpp will aid our understanding of how lases were discovered in human and Drosophila melano
bifunctional stringent response enzymes equip bacteria gaster cells, and flies that lack the corresponding gene are
with the ability to fine-tune their transcriptional profiles more vulnerable to amino acid starvation89. Therefore,
by modulating ppGpp pools in response to particular like bacteria and plants, metazoans might also exploit
environments. The development of a fluorescent che- ppGpp to vary the expression, stability or activity of pro-
mosensor for ppGpp86,87, like the biosensor for c-di- teins. The discovery of a variety of regulatory proteins
GMP88, is an important step towards spatiotemporal and enzymes for which activity is modulated directly
analysis of ppGpp in live cells. It is conceivable that by the alarmone will spur researchers to extend their
RelA and SpoT generate ppGpp in distinct subcellu- sights beyond the promoter to discover the full scope
lar domains, thereby each modulating the activity of a of ppGpp magic.
1. Potrykus, K. & Cashel, M. (p)ppGpp: still magical? 13. Shingler, V. Signal sensory systems that impact A biochemical experiment determining that ppGpp
Annu. Rev. Microbiol. 62, 35–51 (2008). σ54-dependent transcription. FEMS Microbiol. Rev. directly stimulates σEactivity, together with an
2. Jishage, M., Kvint, K., Shingler, V. & Nystrom, T. 35, 425–440 (2010). in vivo analysis that indicates enhanced
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55. Cashel, M. & Rudd, K.E. in Escherichia coli and (p)ppGpp and CodY in Streptococcus mutans. The authors declare no competing financial interests.
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