REVIEWS

ppGpp: magic beyond RNA

polymerase
Zachary 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.

Stringent response
A 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.
1
Department of Microbiology,
University of Washington,
Health Sciences Building K116,
1959 NE Pacific St.,
Box 357710, Seattle,
Washington 98195-7710,
USA.
2
Department 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: mswanson@umich.edu
doi:10.1038/nrmicro2720
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
chromato­grams 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 indirectly 1,2.
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 PP i, 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

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REVIEWS
Limiting
phosphate, (GTP or GDP) Limiting
fatty acids, amino acids
+ ATP
carbon or iron; SpoT RelA
osmotic shock
ppGpp
(GTP or GDP) + PPi
DksA
RNA polymerase
σ70 DksA σ*
Expression of components for protein, Expression of components
lipid, RNA and DNA synthesis for stress resistance
Figure 1 | ppGpp alters RNA polymerase promoter selection by σ-factors.
Nature Reviews | Microbiology
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. PPi, pyrophosphate.
rate in replicating E.  coli cells 6,7, and accumulated
ppGpp inhibits DNA replication in Bacillus subtilis 8. The
Adaptor
In the context of
alarmone also modulates the expression of virulence
post-translational regulation factors that equip bacterial pathogens to cope with envi-
of protein stability, a protein ronmental and metabolic stress3 (BOX 1). Furthermore,
that binds another protein recent studies have revealed crosstalk between ppGpp
and chaperones it to the
proteasome for degradation;
regulation and other nucleotide-signalling pathways
an example of such an adaptor that are known to govern bacterial cell differentiation
is regulator of σS protein RssB. processes such as biofilm formation9,10. Recent reviews
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 (LD50) 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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© 2012 Macmillan Publishers Limited. All rights reserved
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 stability
When 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-
ity 16. 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 activity 18,19. For example,
when phosphate is limiting, the ppGpp pool generated
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
swapping the AT-rich discriminator sequence of the iraP
promoter for a GC-rich sequence, implicating ppGpp
in activation of iraP transcription 18. 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
 REVIEWS

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 Bacillus subtilis, Staphylococcus aureus, 65–69
consumption Streptococcus pyogenes, Streptococcus
mutans and Listeria monocytogenes
Cyclic di-GMP Signalling nucleotide Cooperative induction of biofilm E. coli 77
matrix synthesis
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 Salmonella enterica subsp. enterica 45
binding serovar Typhimurium
PigR Transcription factor Increased binding to the complex Francisella tularensis 51
containing the transcription factors
MglA–SspA and RNA polymerase
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 54,55,57
Streptomyces coelicolor
LdcI Enhancer of acid tolerance Inhibition of activity E. coli 53
Exopolyphosphatase Polyphosphate degradation Inhibition of activity E. coli 65,66
enzyme
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.

Proteasome
A cytoplasmic complex that
unfolds and degrades proteins.
Anti-adaptor
In 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.
Discriminator
The 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 activity
A 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 stability
In 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 motility 28,29 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. pneumo­
phila, 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 out­compete

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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.

ppGpp iraP mRNA IraP RssB Regulators of virulence in pathogens

Inactive
In addition to regulating transcription by modulating
adapter the stability and activity of σ-factors, ppGpp controls the
activity of transcription factors that are dedicated to
RNAP Stress resistance and
iraP virulence factor production virulence genes in Salmonella enterica subsp. enterica
serovar Typhimurium and Francisella tularensis.

c Steady state The SlyA regulator of S. Typhimurium. Following

(GTP or GDP) + ATP phagocytosis, S. Typhimurium activates the two-
ClpXP component virulence regulatory system PhoPQ. Once
RelA SpoT σS activated, the histidine kinase PhoQ phosphorylates the
iraD mRNA cytosolic response regulator PhoP, which then binds
IraD RssB
DNA and stimulates the transcription of genes involved
ppGpp Inactive in remodelling host cell vacuoles and resistance to
adapter
cationic antimicrobial peptides (CAMPs) produced by
RNAP ? the host42 (FIG. 3b). One gene induced by PhoPQ is slyA43,
iraD
which encodes a multiple antibiotic resistance protein R
(MarR) family transcriptional regulator that controls the
Figure 2 | ppGpp activates transcription of anti-adaptor proteins to stabilize σS in transcription of genes which are essential for virulence in
Escherichia coli. a | During logarithmic growth, regulatorNature Reviews
of σS protein | Microbiology
RssB, an adaptor S. Typhimurium44–46. SlyA participates in a feed-forward
protein, directs the stationary phase σ-factor, σ , to the ClpXP proteosome for degradation.
S
loop with PhoP47 in which each component responds
b | During phosphate stress, SpoT hydrolase activity is inhibited, altering the levels of
guanosine pentaphosphate and guanosine tetraphosphate (collectively, ppGpp) and to a distinct signal. Whereas PhoPQ senses external
activating IraP, an anti-adaptor that sequesters RssB from σS and prevents delivery of the cues for activation, SlyA relies on cytosolic ppGpp48.
σ-factor to ClpXP. c | Genetic studies indicate that ppGpp also promotes transcription of The alarmone facilitates SlyA dimerization and binding
iraD, which encodes another Escherichia coli anti-adaptor protein. PPi, pyrophosphate; to target promoters, as evidenced by both in vitro and
RNAP, RNA polymerase. 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
target mRNAs for CsrA binding 35 (FIG. 3a). Thus, control S. Typhimurium encounters low pH, a low concentration
over the expression of regulatory RNAs is crucial for of Mg 2+ or a high concentration of CAMP49, and the cel-
bacteria to modulate CsrA activity. lular ppGpp pools are sufficient, SlyA activates transcrip-
Serine hydroxamate In Pseudomonas aeruginosa, E. coli and L. pneumo­ tion in one direction while PhoP stimulates transcription
A structural analogue of phila, the concentrations of the regulatory RNAs that in the other direction. Consequently, the bacterium coor-
l‑serine that induces the
counteract CsrA increase as bacteria enter stationary dinates expression of several factors, including those that
stringent response by inhibiting
charging of seryl-tRNA
phase31,32,36–39. In L. pneumophila, the increase in tran- confer resistance to serum or antimicrobial peptides. This
synthetase. scription of the regulatory RNA RsmZ during station- is a striking example of how a bacterial pathogen inte-
ary phase requires the ppGpp synthetase, RelA, and the grates signalling by ppGpp with other inputs to control
rpoB bifunctional synthetase–hydrolase, SpoT25. In addi- the complex regulatory cascades that govern virulence
The gene encoding the
tion, the expression of both RsmY (another regulatory gene expression.
β-subunit of RNA polymerase;
this subunit is involved in RNA) and RsmZ is rapidly stimulated in an engineered
transcription initiation through strain of L. pneumophila that synthesizes ppGpp dur- The PigR regulator of F. tularensis. To control the
its interaction with the σ-factor. ing log phase25,40. However, the precise mechanism of expression of genes that are essential for growth in
The commonly used antibiotic transcriptional activation is complex, as it requires both macro­phages, F. tularensis relies on the macrophage
rifampicin targets this β-subunit
to inhibit transcription, and
σS and the Legionella transmission activator–Legionella growth locus–stringent starvation protein A (MglA–SspA)
mutations in rpoB commonly transmission sensor (LetA–LetS) two-component complex 50 (FIG. 3c). Both MglA and SspA are members
confer rifampicin resistance. phosphorelay system that is crucial for the expression of the SspA family 51,52 and associate with RNAP in

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 REVIEWS

a Nutrient starvation Nutrient abundance

LetS

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

Transmission b Acidic pH, high CAMP or low Mg2+

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
controlled by a particular
regulatory protein.
Stringent starvation protein A
A 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 other 53. 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. tula­
rensis 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.

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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

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© 2012 Macmillan Publishers Limited. All rights reserved


(GTP or GDP) + ATP
Limiting
amino acids RelA RNAP
ppGpp,
Ribosome
pppGpp
Ribosomal-protein genes
Ribosomal proteins
Free amino acids
Lon
P P P P P P PP P P P P P P P PP P Exopolyphosphatase
P P P P P P PP P Pi
P P P P P P PP P ATP
Polyphosphate Polyphosphate kinase
Figure 5 | Involvement of ppGpp and pppGpp in the degradation of ribosomal
proteins. In response to amino acid depletion, Escherichia coli synthesizes
Nature Reviews | guanosine
Microbiology
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.
Pi, inorganic phosphate; RNAP, RNA polymerase.
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 Lon-dependent
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
Second messenger ppGpp, pppGpp and polyphosphate enable bacteria to
A molecule that relays signals tolerate nutrient stress and to induce virulence traits
from a receiver or receptor
by exerting an effect on a
when conditions no longer favour replication, knowl-
downstream cellular factor edge of the corresponding regulatory circuitries will
or process. probably inform new therapeutic strategies.
NATURE REVIEWS | MICROBIOLOGY VOLUME 10 | MARCH 2012 | 209
© 2012 Macmillan Publishers Limited. All rights reserved
REVIEWS
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
patho­genic firmicutes, such as Staphylococcus aureus 72,
Streptococcus pyogenes 73, Streptococcus mutans 74 and
Listeria monocytogenes 75.
Crosstalk between ppGpp and cyclic dinucleotides
The 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 host 78–80. 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
REVIEWS

a GTP depletion b Deactivation of ribosomal-gene transcription

Nutrient starvation: Nutrient starvation:
GTP[low]
(GTP or GDP) + ATP RelQ (GTP or GDP) + ATP
RelP
Initiating
RelA RNAP
nucleotide
AGUG rrnB
ppGpp

c Deactivation of the CodY repressor

Nutrient abundance:
GTP GTP
Purine pathway GTP
GTP Transcriptional repression of
RNAP
CodY-dependent genes
CodY
AMP IMP XMP GMP

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-
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 matrix 9. 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.
Conclusion
The 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

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© 2012 Macmillan Publishers Limited. All rights reserved

 REVIEWS

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 σ S (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 spatio­temporal
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 melano­
gaster 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.

1. Potrykus, K. & Cashel, M. (p)ppGpp: still magical?
Annu. Rev. Microbiol. 62, 35–51 (2008).
2. Jishage, M., Kvint, K., Shingler, V. & Nystrom, T.
Regulation of σ factor competition by the alarmone
ppGpp. Genes Dev. 16, 1260–1270 (2002).
Genetic and biochemical studies that establish the
concept of σ-factor competition.
3. Dalebroux, Z. D., Svensson, S. L., Gaynor, E. C. &
Swanson, M. S. ppGpp conjures bacterial virulence.
Microbiol. Mol. Biol. Rev. 74, 171–199 (2010).
4. Haugen, S. P., Ross, W. & Gourse, R. L. Advances in
bacterial promoter recognition and its control by
factors that do not bind DNA. Nature Rev. Microbiol.
6, 507–519 (2008).
5. Osterberg, S., Del Peso-Santos, T. & Shingler, V.
Regulation of alternative sigma factor use. Annu. Rev.
Microbiol. 65, 37–55 (2011).
6. Murray, H. D., Schneider, D. A. & Gourse, R. L. Control
of rRNA expression by small molecules is dynamic and
nonredundant. Mol. Cell 12, 125–134 (2003).
7. Potrykus, K., Murphy, H., Philippe, N. & Cashel, M.
ppGpp is the major source of growth rate control in
E. coli. Environ. Microbiol. 13, 563–575 (2011).
8. Wang, J. D., Sanders, G. M. & Grossman, A. D.
Nutritional control of elongation of DNA replication
by (p)ppGpp. Cell 128, 865–875 (2007).
An analysis of DNA replication in E. coli cultures
and with purified components identifies direct
inhibition of DNA primase activity by ppGpp.
9. Boehm, A. et al. Second messenger signalling governs
Escherichia coli biofilm induction upon ribosomal
stress. Mol. Microbiol. 72, 1500–1516 (2009).
A genetic study demonstrating synergy between
the ppGpp and c‑di‑GMP signalling pathways.
10. Rao, F. et al. YybT is a signaling protein that contains a
cyclic dinucleotide phosphodiesterase domain and a
GGDEF domain with ATPase activity. J. Biol. Chem.
285, 473– 482 (2010).
A biochemical analysis showing that ppGpp inhibits
the degradation of c‑di‑AMP by YybT.
11. Hengge-Aronis, R. Signal transduction and regulatory
mechanisms involved in control of the σS (RpoS)
subunit of RNA polymerase. Microbiol. Mol. Biol. Rev.
66, 373–395 (2002).
12. Dong, T. & Schellhorn, H. E. Role of RpoS in virulence
of pathogens. Infect. Immun. 78, 887–897 (2010).
13. Shingler, V. Signal sensory systems that impact
σ54-dependent transcription. FEMS Microbiol. Rev.
35, 425–440 (2010).
14. Gummesson, B. et al. Increased RNA polymerase
availability directs resources towards growth at the
expense of maintenance. EMBO J. 28, 2209–2219
(2009).
15. Battesti, A., Majdalani, N. & Gottesman, S. The RpoS-
mediated general stress response in Escherichia coli.
Annu. Rev. Microbiol. 65, 189–213 (2011).
16. Klauck, E., Typas, A. & Hengge, R. The σS subunit of
RNA polymerase as a signal integrator and network
master regulator in the general stress response in
Escherichia coli. Sci. Prog. 90, 103–127 (2007).
17. Bougdour, A., Wickner, S. & Gottesman, S. Modulating
RssB activity: IraP, a novel regulator of σS stability in
Escherichia coli. Genes Dev. 20, 884–897 (2006).
A molecular genetics investigation which finds that
stabilization of σS during phosphate starvation is
mediated by SpoT-induced activation of iraP
transcription.
18. Bougdour, A. & Gottesman, S. ppGpp regulation of
RpoS degradation via anti-adaptor protein IraP. Proc.
Natl Acad. Sci. USA 104, 12896–12901 (2007).
19. Merrikh, H., Ferrazzoli, A. E. & Lovett, S. T. Growth
phase and (p)ppGpp control of IraD, a regulator of
RpoS stability, in Escherichia coli. J. Bacteriol. 191,
7436–7446 (2009).
20. Merrikh, H., Ferrazzoli, A. E., Bougdour, A., Olivier-
Mason, A. & Lovett, S. T. A DNA damage response in
Escherichia coli involving the alternative sigma factor,
RpoS. Proc. Natl Acad. Sci. USA 106, 611–616 (2009).
21. Ades, S. E. Regulation by destruction: design of the σE
envelope stress response. Curr. Opin. Microbiol. 11,
535–540 (2008).
22. Durfee, T., Hansen, A. M., Zhi, H., Blattner, F. R. & Jin,
D. J. Transcription profiling of the stringent response in
Escherichia coli. J. Bacteriol. 190, 1084–1096 (2008).
23. Costanzo, A. & Ades, S. E. Growth phase-dependent
regulation of the extracytoplasmic stress factor, σE, by
guanosine 3′,5′-bispyrophosphate (ppGpp).
J. Bacteriol. 188, 4627–4634 (2006).
24. Costanzo, A. et al. ppGpp and DksA likely regulate the
activity of the extracytoplasmic stress factor σE in
Escherichia coli by both direct and indirect
mechanisms. Mol. Microbiol. 67, 619–632 (2008).
A biochemical experiment determining that ppGpp
directly stimulates σEactivity, together with an
in vivo analysis that indicates enhanced
competition for RNAP also occurs.
25. Dalebroux, Z. D. “Magic Spot” Conjures Transmission
of Legionella pneumophila. Thesis, Univ. Michigan
(2010).
26. Edwards, A. N. et al. Circuitry linking the Csr and
stringent response global regulatory systems. Mol.
Microbiol. 80, 1561–1580 (2011).
27. Jackson, D. W. et al. Biofilm formation and dispersal
under the influence of the global regulator CsrA of
Escherichia coli. J. Bacteriol. 184, 290–301 (2002).
28. Wei, B. L. et al. Positive regulation of motility and
flhDC expression by the RNA-binding protein CsrA
of Escherichia coli. Mol. Microbiol. 40, 245–256
(2001).
29. Molofsky, A. B. & Swanson, M. S. Legionella
pneumophila CsrA is a pivotal repressor of
transmission traits and activator of replication.
Mol. Microbiol. 50, 445–461 (2003).
30. Bhatt, S. et al. The RNA binding protein CsrA is a
pleiotropic regulator of the locus of enterocyte
effacement pathogenicity island of enteropathogenic
Escherichia coli. Infect. Immun. 77, 3552–3568
(2009).
31. Rasis, M. & Segal, G. The LetA‑RsmYZ‑CsrA regulatory
cascade, together with RpoS and PmrA, post-
transcriptionally regulates stationary phase activation
of Legionella pneumophila Icm/Dot effectors. Mol.
Microbiol. 72, 995–1010 (2009).
32. Brencic, A. & Lory, S. Determination of the regulon
and identification of novel mRNA targets of
Pseudomonas aeruginosa RsmA. Mol. Microbiol. 72,
612–632 (2009).
33. Molofsky, A. B. & Swanson, M. S. Differentiate to
thrive: lessons from the Legionella pneumophila life
cycle. Mol. Microbiol. 53, 29–40 (2004).
34. Lapouge, K., Schubert, M., Allain, F. H. & Haas, D.
Gac/Rsm signal transduction pathway of
γ-proteobacteria: from RNA recognition to regulation
of social behaviour. Mol. Microbiol. 67, 241–253
(2008).
35. Babitzke, P. & Romeo, T. CsrB sRNA family:
sequestration of RNA-binding regulatory proteins.
Curr. Opin. Microbiol. 10, 156–163 (2007).

NATURE REVIEWS | MICROBIOLOGY VOLUME 10 | MARCH 2012 | 211

© 2012 Macmillan Publishers Limited. All rights reserved

REVIEWS
36. Gudapaty, S., Suzuki, K., Wang, X., Babitzke, P. &
Romeo, T. Regulatory interactions of Csr components:
the RNA binding protein CsrA activates csrB
transcription in Escherichia coli. J. Bacteriol. 183,
6017–6027 (2001).
37. Weilbacher, T. et al. A novel sRNA component of the
carbon storage regulatory system of Escherichia coli.
Mol. Microbiol. 48, 657–670 (2003).
38. Hovel-Miner., G. et al. σS controls multiple pathways
associated with intracellular multiplication of Legionella
pneumophila. J. Bacteriol. 191, 2461–2473 (2009).
39. Sahr, T. et al. Two small ncRNAs jointly govern
virulence and transmission in Legionella pneumophila.
Mol. Microbiol. 72, 741–762 (2009).
40. Dalebroux, Z. D., Yagi, B. F., Sahr, T., Buchrieser, C. &
Swanson, M. S. Distinct roles of ppGpp and DksA in
Legionella pneumophila differentiation. Mol.
Microbiol. 76, 200–219 (2010).
41. Jonas, K., Tomenius, H., Romling, U., Georgellis, D. &
Melefors, O. Identification of YhdA as a regulator of
the Escherichia coli carbon storage regulation system.
FEMS Microbiol. Lett. 264, 232–237 (2006).
42. Kato, A. & Groisman, E. A. The PhoQ/PhoP regulatory
network of Salmonella enterica. Adv. Exp. Med. Biol.
631, 7–21 (2008).
43. Norte, V. A., Stapleton, M. R. & Green, J. PhoP-
responsive expression of the Salmonella enterica
serovar typhimurium slyA gene. J. Bacteriol. 185,
3508–3514 (2003).
44. Miller, S. I., Kukral, A. M. & Mekalanos, J. J. A two-
component regulatory system (phoP phoQ) controls
Salmonella typhimurium virulence. Proc. Natl Acad.
Sci. USA 86, 5054–5058 (1989).
45. Libby, S. J. et al. A cytolysin encoded by Salmonella is
required for survival within macrophages. Proc. Natl
Acad. Sci. USA 91, 489–493 (1994).
46. Ellison, D. W. & Miller, V. L. Regulation of virulence by
members of the MarR/SlyA family. Curr. Opin.
Microbiol. 9, 153–159 (2006).
47. Shi, Y., Latifi, T., Cromie, M. J. & Groisman, E. A.
Transcriptional control of the antimicrobial peptide
resistance ugtL gene by the Salmonella PhoP and SlyA
regulatory proteins. J. Biol. Chem. 279, 38618–38625
(2004).
48. Zhao, G., Weatherspoon, N., Kong, W., Curtiss, R., &
Shi, Y. A dual-signal regulatory circuit activates
transcription of a set of divergent operons in
Salmonella typhimurium. Proc. Natl Acad. Sci. USA
105, 20924–20929 (2008).
Genetic and biochemical experiments which
demonstrate that ppGpp binds the transcription
factor SlyA and promotes its dimerization, DNA
binding and activity as a transcriptional activator.
49. Prost, L. R., Sanowar, S. & Miller, S. I. Salmonella
sensing of anti-microbial mechanisms to promote
survival within macrophages. Immunol. Rev. 219,
55–65 (2007).
50. Lauriano, C. M. et al. MglA regulates transcription of
virulence factors necessary for Francisella tularensis
intraamoebae and intramacrophage survival. Proc.
Natl Acad. Sci. USA 101, 4246–4249 (2004).
51. Ishihama, A. & Saitoh, T. Subunits of RNA polymerase
in function and structure. IX. Regulation of RNA
polymerase activity by stringent starvation protein
(SSP). J. Mol. Biol. 129, 517–530 (1979).
52. Williams, M. D., Ouyang, T. X. & Flickinger, M. C.
Starvation-induced expression of SspA and SspB: the
effects of a null mutation in sspA on Escherichia coli
protein synthesis and survival during growth and
prolonged starvation. Mol. Microbiol. 11, 1029–1043
(1994).
53. Guina, T. et al. MglA regulates Francisella tularensis
subsp. novicida (Francisella novicida) response to
starvation and oxidative stress. J. Bacteriol. 189,
6580–6586 (2007).
54. Charity, J. C., Blalock, L. T., Costante-Hamm, M. M.,
Kasper, D. L. & Dove, S. L. Small molecule control of
virulence gene expression in Francisella tularensis.
PLoS Pathog 5, e1000641 (2009).
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.
55. Cashel, M. & Rudd, K.E. in Escherichia coli and
Salmonella typhimurium: Cellular and Molecular
Biology (eds Neidhardt, F. C. et al.) 1458–1496
(American Society for Microbiology, Washington DC,
1996).
56. Srivatsan, A. & Wang, J. D. Control of bacterial
transcription, translation and replication by (p)ppGpp.
Curr. Opin. Microbiol. 11, 100–105 (2008).
212 | MARCH 2012 | VOLUME 10 www.nature.com/reviews/micro
57. Siculella, L. et al. Guanosine 5′-diphosphate
3′-diphosphate (ppGpp) as a negative modulator of
polynucleotide phosphorylase activity in a ‘rare’
actinomycete. Mol. Microbiol. 77, 716–729 (2010).
In vitro and in vivo experiments which establish that
ppGpp binds and inhibits the activity of an enzyme
that destabilizes mRNAs.
58. Gatewood, M. L. & Jones, G. H. (p)ppGpp inhibits
polynucleotide phosphorylase from streptomyces but
not from Escherichia coli and increases the stability of
bulk mRNA in Streptomyces coelicolor. J. Bacteriol.
192, 4275–4280 (2010).
A biochemical study demonstrating that direct
binding by ppGpp inhibits enzymes that destabilize
mRNA.
59. Kanjee, U. et al. Linkage between the bacterial acid
stress and stringent responses: the structure of the
inducible lysine decarboxylase. EMBO J. 30, 931–944
(2011).
The crystalography and enzymatic assays that
establish ppGpp as a direct regulator of enyzme
activity.
60. Rao, N. N., Gomez-Garcia, M. R. & Kornberg, A.
Inorganic polyphosphate: essential for growth and
survival. Annu. Rev. Biochem. 78, 605–647 (2009).
61. Alifano, P., Bruni, C. B. & Carlomagno, M. S. Control of
mRNA processing and decay in prokaryotes. Genetica
94, 157–172 (1994).
62. Zhao, B. & Houry, W. A. Acid stress response in
enteropathogenic gammaproteobacteria: an aptitude
for survival. Biochem. Cell Biol. 88, 301–314 (2010).
63. Merrell, D. S. & Camilli, A. Regulation of Vibrio
cholerae genes required for acid tolerance by a
member of the “ToxR-like” family of transcriptional
regulators. J. Bacteriol. 182, 5342–5350 (2000).
64. Park, Y. K., Bearson, B., Bang, S. H., Bang, I. S. &
Foster, J. W. Internal pH crisis, lysine decarboxylase
and the acid tolerance response of Salmonella
typhimurium. Mol. Microbiol. 20, 605–611 (1996).
65. Kuroda, A., Murphy, H., Cashel, M. & Kornberg, A.
Guanosine tetra- and pentaphosphate promote
accumulation of inorganic polyphosphate in
Escherichia coli. J. Biol. Chem. 272, 21240–21243
(1997).
66. Kuroda, A. et al. Role of inorganic polyphosphate in
promoting ribosomal protein degradation by the Lon
protease in E. coli. Science 293, 705–708 (2001).
67. Wolz, C., Geiger, T. & Goerke, C. The synthesis and
function of the alarmone (p)ppGpp in firmicutes. Int.
J. Med. Microbiol. 300, 142–147 (2010).
68. Gallant, J., Irr, J. & Cashel, M. The mechanism of
amino acid control of guanylate and adenylate
biosynthesis. J. Biol. Chem. 246, 5812–5816 (1971).
A seminal study demonstrating that ppGpp is a
second messenger which accumulates during amino
acid starvation and inhibits the activity of enzymes
that initiate ATP and GTP biosynthesis.
69. Lopez, J. M., Dromerick, A. & Freese, E. Response of
guanosine 5′-triphosphate concentration to nutritional
changes and its significance for Bacillus subtilis
sporulation. J. Bacteriol. 146, 605–613 (1981).
70. Krasny, L. & Gourse, R. L. An alternative strategy for
bacterial ribosome synthesis: Bacillus subtilis rRNA
transcription regulation. EMBO J. 23, 4473–4483
(2004).
The molecular genetic and biochemical experiments
which establish that the consumption of GTP during
ppGpp synthesis indirectly decreases rRNA
synthesis in B. subtiliis.
71. Sonenshein, A. L. CodY, a global regulator of
stationary phase and virulence in Gram-positive
bacteria. Curr. Opin. Microbiol. 8, 203–207 (2005).
72. Geiger, T. et al. Role of the (p)ppGpp synthase RSH,
a RelA/SpoT homolog, in stringent response and
virulence of Staphylococcus aureus. Infect. Immun.
78, 1873–1883 (2010).
73. Malke, H., Steiner, K., McShan, W. M. & Ferretti, J. J.
Linking the nutritional status of Streptococcus
pyogenes to alteration of transcriptional gene
expression: the action of CodY and RelA. Int. J. Med.
Microbiol. 296, 259–275 (2006).
74. Lemos, J. A., Nascimento, M. M., Lin, V. K.,
Abranches, J. & Burne, R. A. Global regulation by
(p)ppGpp and CodY in Streptococcus mutans.
J. Bacteriol. 190, 5291–5299 (2008).
75. Bennett, H. J. et al. Characterization of relA and codY
mutants of Listeria monocytogenes: identification of
the CodY regulon and its role in virulence. Mol.
Microbiol. 63, 1453–1467 (2007).
76. Romling, U. & Simm, R. Prevailing concepts of c‑di‑GMP
signaling. Contrib. Microbiol. 16, 161–181 (2009).
© 2012 Macmillan Publishers Limited. All rights reserved
77. Hengge, R. Principles of c‑di‑GMP signalling in
bacteria. Nature Rev. Microbiol. 7, 263–273 (2009).
78. McWhirter, S. M. et al. A host type I interferon
response is induced by cytosolic sensing of the
bacterial second messenger cyclic-di‑GMP. J. Exp.
Med. 206, 1899–1911 (2009).
The first work to demonstrate that treatment with
cyclic nucleotides stimulates innate immune
mechanisms of mammalian cells.
79. Woodward, J. J., Iavarone, A. T. & Portnoy, D. A.
c‑di‑AMP secreted by intracellular Listeria
monocytogenes activates a host type I interferon
response. Science 328, 1703–1705 (2010).
An investigation which uses bacterial genetics to
establish that the production of cyclic nucleotides
by an intracellular pathogen stimulates host innate
immune defence pathways.
80. Vance, R. E., Isberg, R. R. & Portnoy, D. A. Patterns
of pathogenesis: discrimination of pathogenic and
nonpathogenic microbes by the innate immune
system. Cell Host Microbe 6, 10–21 (2009).
81. Hall-Stoodley, L. & Stoodley, P. Evolving concepts in
biofilm infections. Cell. Microbiol. 11, 1034–1043
(2009).
82. Hoffman, L. R. et al. Aminoglycoside antibiotics induce
bacterial biofilm formation. Nature 436, 1171–1175
(2005).
83. Witte, G., Hartung, S., Buttner, K. & Hopfner, K. P.
Structural biochemistry of a bacterial checkpoint
protein reveals diadenylate cyclase activity regulated
by DNA recombination intermediates. Mol. Cell 30,
167–178 (2008).
84. Romling, U. Great times for small molecules: c‑di‑AMP,
a second messenger candidate in Bacteria and
Archaea. Sci. Signal 1, pe39 (2008).
85. Traxler, M. F. et al. Discretely calibrated regulatory
loops controlled by ppGpp partition gene induction
across the ‘feast to famine’ gradient in Escherichia
coli. Mol. Microbiol. 79, 830–845 (2011).
A transcriptional profile analysis identifying genes
that require different amounts of ppGpp for their
induction, thus illustrating a dynamic range for this
regulatory pathway.
86. Rhee, H. W. et al. Selective fluorescent chemosensor
for the bacterial alarmone (p)ppGpp. J. Am. Chem.
Soc. 130, 784–785 (2008).
87. Gao, W. et al. Two novel point mutations in clinical
Staphylococcus aureus reduce linezolid susceptibility
and switch on the stringent response to promote
persistent infection. PLoS Pathog. 6, e1000944 (2010).
88. Christen, M. et al. Asymmetrical distribution of the
second messenger c‑di‑GMP upon bacterial cell
division. Science 328, 1295–1297 (2010).
89. Sun, D. et al. A metazoan ortholog of SpoT hydrolyzes
ppGpp and functions in starvation responses. Nature
Struct. Mol. Biol. 17, 1188–1194 (2010).
The first molecular and functional studies to
identify stringent response components in
D. melanogaster and human cells.
90. Pizarro-Cerda, J. & Tedin, K. The bacterial signal
molecule, ppGpp, regulates Salmonella virulence
gene expression. Mol. Microbiol. 52, 1827–1844
(2004).
91. Sun, W., Roland, K. L., Branger, C. G., Kuang, X. &
Curtiss, R. The role of relA and spoT in Yersinia pestis
KIM5 pathogenicity. PLoS ONE 4, e6720 (2009).
92. Zusman, T., Gal-Mor, O. & Segal, G. Characterization
of a Legionella pneumophila relA insertion mutant and
toles of RelA and RpoS in virulence gene expression.
J. Bacteriol. 184, 67–75 (2002).
93. Dalebroux, Z. D., Edwards, R. L. & Swanson, M. S.
SpoT governs Legionella pneumophila differentiation
in host macrophages. Mol. Microbiol. 71, 640–658
(2009).
Acknowledgements
The 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 statement
The authors declare no competing financial interests.
FURTHER INFORMATION
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