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Pushing the envelope: extracytoplasmic stress responses in bacterial pathogens Gary Rowley*¶ ||, Michael Spector ‡||, Jan Kormanec§ and Mark Roberts*

Abstract | Despite being nutrient rich, the tissues and fluids of vertebrates are hostile to microorganisms, and most bacteria that attempt to take advantage of this environment are rapidly eliminated by host defences. Pathogens have evolved various means to promote their survival in host tissues, including stress responses that enable bacteria to sense and adapt to adverse conditions. Many different stress responses have been described, some of which are responsive to one or a small number of cues, whereas others are activated by a broad range of insults. The surface layers of pathogenic bacteria directly interface with the host and can bear the brunt of the attack by the host armoury. Several stress systems that respond to perturbations in the microbial cell outside of the cytoplasm have been described and are known collectively as extracytoplasmic or envelope stress responses (ESRs). Here, we review the role of the ESRs in the pathogenesis of Gram-negative bacterial pathogens.

*Molecular Bacteriology Group, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK. ‡ Department of Biomedical Sciences, University of South Alabama, Mobile, Alabama 36688-0002, USA. § Institute of Molecular Biology, Centre of Excellence for Molecular Medicine, Slovak Academy of Science, Dubravska cesta 21, 845 51 Bratislava, Slovak Republik. ¶ Present address: Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK. || These authors contributed equally to this work. Correspondence to M.R. e-mail: [email protected] doi:10.1038/nrmicro1394 Published online 10 April 2006

The ability to perceive changes in the internal and external environment and modify the activity of genes or metabolism is important for most life forms. Bacterial pathogens need to sense their proximity and location within the host. This allows them to modulate the appropriate production of virulence factors. Bacterial pathogens also need to defend themselves against stress that originates from within the infected host as well as from the external environment between hosts. The surface of bacterial pathogens is the first point of contact with the host and is a major target for the antibacterial armament of the host. Many different stress-response systems have now been described, and several have been studied in detail. However, most of these studies have been performed on laboratory strains, and the importance of the stress responses to bacteria has largely been studied in vitro. This article reviews a group of stress responses known collectively as extracytoplasmic (or envelope) stress responses (ESRs) and discusses their importance to Gram-negative bacterial pathogens. The role of ESRs in the biology of Gram-negative bacteria has received the most attention, but analogous responses have also been identified in Gram-positive and acid-fast bacteria. In Gram-negative bacteria, these responses target stresses that affect components of the cell envelope, including periplasmic and outer-membrane proteins1. The best characterized of these responses are those mediated

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by the alternative sigma factor σE (RpoE)2–5 and the twocomponent regulator CpxAR6–9. However, Gram-negative bacteria have two additional ESRs: the BaeSR-regulated response10 and the phage shock response11–14.

Extracytoplasmic-function sigma factors The extracytoplasmic function (ECF) family of alternative sigma factors controls the expression of genes, the products of which function outside of the cytoplasm15,16 (TABLES 1,2). The best-studied ECF sigma factor is the Escherichia coli rpoE gene product, σE. In laboratory strains of E. coli, σE is an essential sigma factor2 but this is not the case for most bacteria17–20. Membership of the E. coli σE regulon has grown significantly over the past few years21–23 (TABLE 1). In addition to itself (rpoE) and the downstream genes in the same operon (rseABC, see below), many other genes have been shown to be regulated by σE. These include genes that encode proteins involved in the folding or degradation of polypeptides in the periplasm, such as proteases, peptidyl-prolylisomerases (PPIases), chaperones and thiol:disulfide oxido-reductases (for example, protein disulfide isomerase II, which is involved in disulfide-bond formation), as well as genes involved in lipopolysaccharide (LPS) biogenesis and/or modification, and several genes of unknown function22–26 (TABLE 1, Supplementary information S1 (table)). Interestingly, σE also positively regulates VOLUME 4 | MAY 2006 | 383

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REVIEWS Table 1 | Selected members of the σE regulon in Escherichia coli Gene

Gene function

σ / σ regulation and other σ factors E

E

rpoD

Housekeeping sigma factor D (σD or σ70)

rpoE

ECF sigma factor E

rpoH

Heat-shock sigma factor H (σH or σ32); cytoplasmic branch of heat-shock response

rpoN

Nitrogen metabolism sigma factor D (σ54)

rseA

Anti-sigma factor that binds and sequesters σE (IMP)

rseB

Binds to RseA; minor negative regulator of σE activity (PP)

rseC

Minor positive regulator of σE activity (IMP); has a role in thiamine biosynthesis; negative control of SoxR-dependent response

rseP (yaeL/ecfE)

IMP; metalloprotease/carboxypeptidase that cleaves RseA at cytoplasmic site

Primary metabolism, sensory-regulatory functions cca

tRNA nucleotidyl transferase

clpX

ATP-dependent specificity component of ClpP protease

cutC

CP involved in copper tolerance and sensing

dnaE

α-subunit of DNA polymerase III

ftsZ

Cell-division protein

fusA

Elongation factor G

greA

Transcription elongation factor

lhr

ATP-dependent helicase

malQ

4-α-glucanotransferase

mdoGH

Osmoregulated periplasmic glucan synthesis

pioA

Putative general secretion pathway protein A

prfB

Peptide chain release factor RF-2

recJ

Exonuclease; DNA repair

rnhB

Ribonuclease HII

PPs — proteases/chaperones/folding enzymes asnB

Periplasmic l-asparaginase

dsbC

Thiol:disulfide oxido-reductase/protein disulfide isomerase II

fkpA

FKBP-family PPIase

htrA (degP)

Serine protease and chaperone

skp

Chaperone involved in OMP assembly and folding; in operon with lpxDA (skp lpxDA fabZ)

surA

Parvulin-family PPIase and chaperone

Phospholipid and LPS biogenesis/modification ahpF

Alkyl hydroperoxide reductase; lipid detoxification

fadD

Acyl-coenzyme A synthase

lpxA

UDP-N-acetylglucosamine acetyltransferase involved in lipid A biosynthesis; in operon with lpxD (lpxDA fabZ)

lpxD

UDP-3-O-(3-hydroxymyristoyl)-glucosamine N-acyltransferase involved in lipid A biosynthesis; in operon with lpxD (lpxDA fabZ)

lpxP (ddg)

Cold-shock-induced palmitoleoyl transferase; adds palmitoleate to nascent lipid A of LPS

plsB

Glycerol-3-phosphate acyltransferase

psd

Phosphatidylserine decarboxylase; converts phosphatidylserine to phophatidylethanolamine

rfaD (htrM)

ADP-l-glycerol-d-mannoheptose-6-epimerase; first gene of rfaDFCL operon involved in LPS biosynthesis

IMPs — transport, unknown function bacA

Undecaprenyl pyrophosphate phosphatase; involved in bacitracin A resistance

ptsN

Phosphotransferase system enzyme IIA

sbmA

Putative transporter protein possibly involved in microcin B17 and J25 uptake; homologues in Sinorhizobium meliloti and Brucella abortus are involved in intracellular survival; in operon with yaiW (sbmA yaiW)

For the listed genes, the σE -dependent promoter has been identified, details are given in REFS 21–23. CP, cytoplasmic protein; ECF, extracytoplasmic function; IMP, inner-membrane protein; LPS, lipopolyscaccharide; OMP, outer-membrane protein; PP, periplasmic protein; PPIase, peptidyl-prolyl-isomerase.

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REVIEWS Table 2 | Examples of known or putative extracytoplasmic function (ECF) sigma factors in bacteria Bacteria

ECF sigma Reported associated functions/mutant phenotypes* factor

Gram-negative bacteria Actinobacillus pleuropneumoniae

σE

Possible role in porcine pleuropneumonia

Azotobacter vinelandii

AlgT (AlgU) Reduced alginate biosynthesis

Escherichia coli

σE

High- and low-temperature sensitivity; ethanol sensitivity; abnormalities in lipopolysaccharide; defective biofilm formation

Haemophilus influenzae

σE

In non-typeable strains, expressed intracellularly and required for survival within murine macrophages

Myxococcus xanthus

CarQ

Defective light-induced carotenoid biosynthesis

Pseudomonas spp.

AlgU (AlgT, σ22)

Reduced alginate biosynthesis; reduced resistance to high temperature, reactive oxygen species; defective biofilm formation; important for respiratory but not systemic infection

Pseudomonas syringae

HrpL

Attenuated virulence in plant hosts; defective biofilm formation

Salmonella enterica

σE

Reduced long-term C-starvation-survival/C-starvation-inducible crossresistance to H2O2, acid pH, high temperature, polymyxin B; defects in diauxic shifts from glucose to certain alternative C sources; reduced survival in macrophages; attenuated mouse virulence

Vibrio cholerae

σE

Attenuated infant mouse virulence; sensitivity to 3% ethanol

Vibrio angustum

σ28

Reduced survival during starvation and exposure to high temperature, reactive oxygen species, detergents

Yersinia enterocolitica

σE

Activated in vivo; defective osmotic stress response to high levels of nonmetabolizable sugars

σX

High-temperature sensitivity; impaired resistance to reactive oxygen species

σW

Defective alkaline-pH-shock response

σ

Decreased resistance to acid pH, ethanol, cell-wall active antimicrobials, superoxide ions

Deinococcus radiodurans

Sig1

Reduced survival during heat stress and ethanol stress

Streptomyces coelicolor

σ

Altered peptidoglycan; hypersensitivity to muramidases; overproduction of actinorhodin; requirement of high levels of Mg2+ for growth and sporulation

σR

Sensitivity to H2O2 and diamide (thiol-specific oxidant)

σC

Homologue of M. tuberculosis ECF sigma factor σC; no reported phenotypes

σE

Homologue of M. tuberculosis ECF sigma factor σE; no reported phenotypes

σ

Increased sensitivity to H2O2 and diamide; attenuated mouse virulence

σE

Hypersensitivity to detergent surface stress, high temperature and reactive oxygen species; attenuated macrophage growth and survival; attenuated virulence in mouse model systems

σH

Increased sensitivity to H2O2 and diamide; attenuated lung immune/ inflammatory responses in mouse model systems

Gram-positive bacteria Bacillus subtilis

M

Outer-membrane proteins Polypeptides in Gram-negative bacteria that are located in the outer membrane.

Acid-fast bacteria Mycobacterium leprae

Sigma factor A subunit of RNA polymerase that determines the DNAbinding specificity depending on the sigma factor bound.

E

Mycobacterium tuberculosis

C

Two-component regulator Consists of a histidine kinase (HK) and a response regulator (RR). The HK senses the change in environment and relays the message to the RR through a phosphorylation event. The phosphorylated RR binds to specific DNA-binding sites that regulate transcription of a subset of genes.

Chaperones Molecules that participate in the folding/assembly of other proteins but do not form part of the final structure themselves.

*See text for further explanation and references.

the expression of the genes that encode the housekeeping sigma factor RpoD and the heat-shock sigma factor RpoH (TABLE 1). The activity of σE is negatively controlled by its cognate membrane-bound anti-sigma factor RseA, which binds σE, rendering it inactive. The activation and regulation of σE in E. coli has recently been reviewed24 and is illustrated in FIG. 1. The release of σE from RseA allows it to bind to core RNA polymerase and redirect the RNA polymerase


holoenzyme to σE-dependent promoters (consensus sequence: 5′-ggAACtt-N16-TCnaA-3′, REFS 22,23,27), enabling σE-dependent gene expression (TABLE 1; FIG. 1, Supplementary information S1 (table)). One such promoter lies upstream of the rpoE rseABC operon4,28. Increased transcription from this promoter not only leads to increased levels of σE but also of the regulator RseA, which will function homeostatically to switch off the σE response once the activating stimulus is removed. VOLUME 4 | MAY 2006 | 385


REVIEWS σE and its homologues, to varying degrees, control bacterial functions that are required both for their pathogenesis (as discussed below) and for their development of maximal resistance to various environmental stresses. For example, in S. typhimurium, σE is required for maximal protection against reactive oxygen species (ROS) and antimicrobial peptides, for survival in stationary phase, for long-term carbon-starvation survival and for carbon-starvation-inducible crossresistance to H2O2, acidic pH, high temperature and polymyxin B18,33,34. The role of σE in bacterial pathogenesis is complex, varies from pathogen to pathogen and could also be important for infection at one host site but not another. These complexities are discussed below.

Outer membrane

Periplasm

Misfolded protein COOH

DegS PDZ

COOH

a

RseB

b

RseA Inner membrane

c

RseP σE

σE RseA peptides

Transcription of σE -dependent genes: rpoE rseABC, htrA, surA, fkpA, skp, dsbC, lpxP

g

Cytoplasm

σE

d

SspB

ClpP

f

ClpX

e σE

σE

σE

σE

Figure 1 | Model for regulation of σE activity by regulated intramembrane proteolysis (RIP) in Escherichia coli. The major mechanism for σE activation in response to extracytoplasmic stress is the degradation of RseA by the successive action of two proteases, DegS and RseP. In the absence of misfolded proteins, the PDZ domain of DegS prevents degradation of RseA at periplasmic cleavage sites. In the presence of misfolded outer-membrane proteins (OMPs), the PDZ domain of DegS interacts with a motif in the carboxyl end of the OMPs (a). This activates DegS protease activity, which leads to RseA cleavage at a periplasmic site (b). In turn, the RseP protease can then cleave RseA at a cytoplasmic cleavage site (c), resulting in release of a σE–RseA inhibitory complex into the cytoplasm, where it binds to SspB (d), which directs this complex to ClpXP (e). ClpXP specifically degrades the RseA portion, leading to release of σE (f). This allows σE to bind to core RNA polymerase with transcription of σE-dependent genes (g)24,117.

PDZ domain Protein domain found in signalling proteins of bacteria and eukaryotes. The PDZ domain consists of 80–90 amino acids typically arranged as 6 β-strands and 2 α-helices. The PDZ domain recognizes peptide sequences typically in the C terminus of the target protein. Most proteins with PDZ domains are located in the plasma membrane.

Regulated intra-membrane proteolysis (RIP). A conserved mechanism for controlling several important signalling pathways in both prokaryotes and eukaryotes. It normally involves the release of a cytoplasmic membrane-bound transcription factor, which in this context is a sigma factor.

Based on bioinformatics analysis, it is likely that this model of σE regulation is common to many bacteria, particularly members of the γ-proteobacteria, as putative orthologues of σE, RseA, RseB, DegS and RseP (YaeL) are present in their genomes24. The mechanism of proteolytic cleavage of RseA to activate σE through the successive actions of two proteases, DegS and RseP, is characteristic of a novel mechanism to activate signalling pathways used by both prokaryotic and eukaryotic life forms (including humans) termed regulated intra-membrane proteolysis (RIP)29. The stress conditions that activate the σE pathway in E. coli and/or Salmonella enterica serovar Typhimurium (S. typhimurium) and other bacteria are listed in TABLE 3. It has been proposed, but in most cases has not been formally demonstrated, that all of these conditions lead to the periplasmic accumulation of one or more outermembrane proteins that are the signal for σE activation through DegS. It is possible that there are additional signals that lead to σE activation, as there is evidence in both S. typhimurium and E. coli of DegS-independent pathways of σE activation30–32.

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Role of σ E in bacterial pathogenesis Pseudomonas aeruginosa. Pseudomonas aeruginosa is a Gram-negative bacterium that is ubiquitous in the environment and is an important opportunistic pathogen of humans, animals and plants. The importance of σE to bacterial pathogenesis first became apparent from the study of its orthologue, AlgU (AlgT), in P. aeruginosa35. σE of E. coli and AlgU are functionally equivalent, and the organization of the algU operon is identical to that found in enteric bacteria — algU mucA mucB mucC are equivalent to rpoE rseA rseB rseC36,37. P. aeruginosa is a major cause of respiratory infection in patients with cystic fibrosis (CF) 35 . P. aeruginosa isolated from CF patients is usually mucoid owing to constitutive overproduction of the extracellular polysaccharide, alginate35. AlgU directly regulates the production of alginate by directing transcription of the alginate-biosynthesis operon (first gene algD) from an AlgU-dependent promoter, and also indirectly, by upregulating the expression of genes that encode transcriptional activators of the algD operon, including algR and algZ 35,38,39. Conversion of non-mucoid strains to mucoid strains (constitutive alginate production) occurs predominantly owing to mutations in mucA (rseA), resulting in constitutive activation of AlgU40. Several roles for alginate in the pathogenesis of respiratory P. aeruginosa infection in CF patients have been proposed, including promotion of adhesion and biofilm formation, inhibition of leukocyte phagocytosis and the respiratory burst, and salvaging of ROS35. Environmental strains of P. aeruginosa (believed to be the source of infection for CF patients) and isolates from other sites of infection are not normally mucoid. This indicates that the environment of the CF respiratory tract selects for mutants in which AlgU regulon expression, and in particular alginate production, is constitutive35. Surprisingly, algU mutants of P. aeruginosa are not attenuated in murine models of systemic P. aeruginosa infection41. Also, earlier studies showed that mucoid strains (that presumably have constitutive AlgU activation) have reduced systemic virulence42. One possible explanation for these findings is the negative effect of AlgU on expression of the type III secretion system (TTSS), which is an important virulence determinant43. However, in contrast to the systemic-infection model, mucoid www.nature.com/reviews/micro


REVIEWS Type III secretion system (TTSS). Used by many pathogenic bacteria to inject virulence proteins directly into host cells through needle-like structures. Also used by the bacteria to export flagellumprotein subunits.

strains of P. aeruginosa were cleared less efficiently from the respiratory tract of aerosol-infected mice 40. Therefore, AlgU and AlgU-regulated genes seem to be important for respiratory but not systemic P. aeruginosa infection. Does AlgU contribute to the pathology of P. aeruginosa infection in CF patients as well as promoting the respiratory survival of P. aeruginosa? Long-term persistence of P. aeruginosa in the respiratory tract of CF patients is characterized by chronic inflammation with high levels of neutrophils and inflammatory

cytokines/chemokines44,45. Alginate itself stimulates the production of IL-1 and TNF-α35. Also, lipopeptides corresponding to the N terminus of two AlgU-regulated lipoproteins (LptA and LptB) stimulate production of the proinflammatory chemokine IL-8 by human macrophages46. The lptA gene is essential for P. aeruginosa pulmonary infection in rats47. Finally, production of hydrogen cyanide (HCN) is a virulence factor for P. aeruginosa in the Caenorhabditis elegans nematode infection model48, and (through AlgR) AlgU positively regulates the HCN-synthesis genes (hcnAB)49.

Table 3 | Examples of stresses and conditions proposed or known to activate envelope stress responses (ESRs) in bacteria ESR

Stress/condition

Bacteria

ECF σ-mediated

Heat shock

Escherichia coli, Salmonella enterica serovar Typhimurium (S. typhimurium), Pseudomonas spp., Vibrio angustum, Bacillus subtilis, Deinococcus radiodurans, Mycobacterium tuberculosis

Cold shock

E. coli, S. typhimurium

Oxidative stress

E. coli, S. typhimurium, Pseudomonas spp., V. angustum, B. subtilis

Ethanol exposure

Streptomyces coelicolor, D. radiodurans, M. tuberculosis, E. coli, Vibrio cholerae, B. subtilis

Exposure to detergents

V. angustum, M. tuberculosis

Hyperosmolarity, osmotic stress

E. coli, Yersinia enterocolitica

Carbon-energy-source starvation

S. typhimurium, V. angustum

Glucose to maltose, citrate or succinate shifts

S. typhimurium

Stationary phase (LB medium)


Biofilm formation

E. coli, Pseudomonas spp.

Overexpression of OMPs


Overexpression of OmpC-like terminal peptides

E. coli

Growth in vivo (pathogenesis)

S. typhimurium, P. aeruginosa, V. cholerae. Haemophilus influenzae (non-typeable), Actinobacillus pleuropneumoniae

Light

Myxococcus xanthus

CpxRA-mediated Presence of abiotic surfaces, adhesion

E. coli

Alkaline pH

E. coli

Overproduction of OM lipoproteins (for example, NlpE)

E. coli

Increased expression of certain pili (for example, P pili)

E. coli (UPEC, EPEC)

Pathogenesis

E. coli (UPEC, EPEC), S. typhimurium, Shigella sonnei, Legionella pneumophila

BaeRS-mediated

Unknown

E. coli

Phage shock response

Infection with filamentous phage (for example, f1)

E. coli

Dissipation of proton-motive force

E. coli, Y. enterocolitica, S. typhimurium

High temperature

E. coli, Y. enterocolitica, S. typhimurium

Osmotic stress

E. coli, Y. enterocolitica

Ethanol/organic-solvent exposure

E. coli, Y. enterocolitica

Stationary phase (LB medium) at alkaline pH

E. coli

Pathogenesis

Y. enterocolitica, S. typhimurium, Shigella flexneri

ECF, extracytoplasmic function; EPEC, enteropathogenic E. coli; OMP, outer-membrane protein, UPEC, uropathogenic E. coli.


VOLUME 4 | MAY 2006 | 387 © 2006 Nature Publishing Group

REVIEWS Salmonella: a model intracellular pathogen. Salmonella spp. are important pathogens of humans as well as domestic and wild animals. They can cause infections ranging from localized enteritis (Salmonella food poisoning) to systemic infections such as typhoid fever in humans. In a few individuals, Salmonella spp. can persist for long periods in host tissues. Salmonellae are invasive pathogens and can promote their own uptake into various eukaryotic cell types and then proliferate and/or survive within a modified membrane-bound vacuole. The rpoE gene is upregulated and is crucial for survival of S. typhimurium in macrophages in vitro and in host tissue in vivo18,34,50–52. For S. typhimurium, there is a good correlation between intramacrophage survival and virulence in mice, and this holds true for S. typhimurium rpoE mutants18,53. Inactivation of rpoE significantly reduces the ability of S. typhimurium to cause disease by either oral or parenteral (intravenous or intraperitoneal) routes of infection18,34,52. Lack of RpoE renders S. typhimurium more sensitive to killing by several antimicrobial agents that the bacteria are likely to encounter in vivo, including ROS and antimicrobial peptides18,34. The capacity of an S. typhimurium rpoE mutant to cause lethal infection in mice through the intraperitoneal route is greatly increased if the phagocytes of the infected mice are unable to generate ROS (owing to mutations in genes encoding the phagocyte NADPH oxidase)34. When given orally, however, the S. typhimurium rpoE mutant remains attenuated, which has been attributed to the inability of this mutant to defend itself against host antimicrobial peptides52. The first σE-regulated gene found to be important for Salmonella pathogenesis was htrA53,54. The HtrA protein is active in the bacterial periplasm and can function as either a serine protease or a chaperone55. S. typhimurium htrA mutants exhibit increased sensitivity to killing by ROS and reduced survival and/or replication within macrophages18,53,54. These mutants are also highly attenuated in the murine model of systemic Salmonella infection18,56. Surprisingly, when given orally to calves, an S. typhimurium htrA mutant was not attenuated (although it was attenuated when administered parenterally)57. In cattle, S. typhimurium infection presents predominantly as enteritis57. These findings indicate that HtrA is dispensable for the enteric phase of Salmonella infection but is necessary for growth at systemic sites. In support of this hypothesis, htrA mutants of S. typhimurium can efficiently colonize intestinal lymphoid tissue of mice but are unable to survive and proliferate in the liver and spleen18,56. Since these seminal studies in Salmonella, HtrA has been found to be important for the virulence of a wide range of Gram-negative and Gram-positive bacterial pathogens18,54,58–65. SurA and FkpA are σE-regulated proteins that are involved in the process of protein folding in the periplasm of S. typhimurium6,21,66. Both proteins are PPIases that catalyse the normally slow cis-trans isomerization around proline residues in polypeptides67. Like HtrA, they can also function as chaperones independent of their enzymatic activity68,69. surA mutants of S. typhimurium 388 | MAY 2006 | VOLUME 4

are defective in both invasion and intracellular survival within eukaryotic cells in vitro, are attenuated in a mouse model of infection and make efficacious live vaccines66,70. Null mutations in fkpA were also expected to attenuate Salmonella, as an fkpA mutant of a particular biotype of S. typhimurium (Copenhagen) was reported to be defective for intracellular replication, and FkpA homologues are important for infection of host cells by other intracellular pathogens, including Legionella pneumophila and Chlamydia trachomatis 67,71,72. However, contrary to these expectations, fkpA mutants of two different S. typhimurium strains were not attenuated in mice66. It is probable that there are other σE-regulated S. typhimurium genes that are important for Salmonella pathogenesis, and identification of these genes is under active investigation. S. typhimurium rpoE, surA and htrA mutants are completely attenuated in mice infected through the oral route of infection. Interestingly, whereas S. typhimurium htrA and surA mutants function as excellent live oral anti-Salmonella vaccines, S. typhimurium rpoE mutants do not18,56,69. This is probably because S. typhimurium rpoE mutants are too attenuated to illicit a significant host immune response; they survive significantly less well in murine tissues than S. typhimurium htrA or surA mutants. Other organisms. The role of σE in the pathogenesis of several other organisms has been investigated. Nontypeable Haemophilus influenzae (NTHi) causes various infections in humans, including otitis media, sinusitis, bronchitis and pneumonia. In NTHi strains, rpoE is highly expressed in murine macrophages and is required for intracellular survival in these cells17. Actinobacillus pleuropneumoniae is related to H. influenzae and causes pleuropneumonia in pigs, a severe and often fatal infection. Interestingly, in this organism, inactivation of rseA (mlcA) but not rpoE was attenuating in porcine lungs73. The absence of σE compromised the ability of both classical and El Tor strains of Vibrio cholerae to colonize the intestinal tract of infant mice19. A degS mutant of uropathogenic E. coli (UPEC) was attenuated in murine systemic and urinary-tract infection models74. It was assumed that this phenotype was due to the degS mutation preventing activation of the σE regulon.

Two-component regulatory systems and the ESR Two-component signal-transduction systems are commonly used by bacteria to sense and respond to extracytoplasmic conditions. These systems are composed of a (transmembrane) sensor kinase and a (cytoplasmic) response regulator. The prototypic two-component regulator involved in combating envelope stress is the CpxAR (Cpx) system8,75–77. In E. coli, the Cpx system is believed to control the expression of more than 100 proteins in response to certain envelope stress conditions, some of which overlap with the σE regulon77,78. Members of the Cpx regulon (TABLE 4) include HtrA, the periplasmic thiol:disulphide oxido-reductases or protein disulphide isomerase DsbA, the periplasmic PPIases PpiA and PpiD, as well as the CpxR, CpxA and CpxP proteins that www.nature.com/reviews/micro


REVIEWS Table 4 | Known or putative members of the Cpx regulon in Escherichia coli Type of regulation

Gene

Function of gene product*

cpxP

Negative regulator of CpxA kinase activity (PP)

cpxRA

Response regulator (CP)/sensor kinase (IMP) components of Cpx two-component system

Positive regulation Cpx proteins and regulation

PPs — proteases/ dsbA chaperones/folding enzymes htrA (degP)

Thiol:disulfide oxido-reductase/protein disulphide isomerase II Serine protease and chaperone

ppiA

Cyclophilin-family PPIase

ppiD

Parvulin-family PPIase and chaperone

skp

Chaperone involved in OMP assembly and folding; in operon with lpxDA (skp lpxDA fabZ)

spy

PP induced by spheroplasting and oxidative stress; also controlled by BaeSR system

Protein secretion

secA

Preprotein translocase; ATPase secretion component

Phospholipid and LPS biogenesis/modification

psd

Phosphatidylserine decarboxylase; converts phosphatidylserine to phophatidylethanolamine

Virulence loci or adherence factors

bfp

Type IV bundle-forming pili of EPEC

mviM

Proposed virulence factor (exact function unclear)

pap

P pili subunit components, biogenesis (see below)

OMPs

ompC

Major outer-membrane porin 1 protein

Other loci of known and unknown function

aroK

Shikimate kinase I; aromatic-amino-acid biosynthesis

ung

Uracil-DNA glycosylase

rpoD

Housekeeping sigma factor D (σD or σ70)

rpoE

ECF sigma factor E

rpoH

Heat-shock sigma factor H (σH or σ32), cytoplasmic branch of heat-shock response

rseA

IMP; anti-sigma factor that binds and sequesters σE

rseB

PP; binds to RseA; minor negative regulator of σE activity

rseC

IMP with roles as a minor positive regulator of σE activity; some role in thiamine biosynthesis; negative control of SoxR-dependent response

Virulence loci or adherence factors

csgBAC

Curli fimbriae subunit and production proteins

pap

Phase variation; inhibits transition from OFF to ON phase

Chemotaxis and motility

aer

Aerotaxis sensor receptor; senses cellular redox state or proton-motive force

motABcheAW

Proton conductor component of motor/flagellar rotation; chemotactic sensory His kinase; positive regulator of CheA

tsr

Serine chemoreceptor

minCDE

Cell-division inhibition; control of FtsZ ring formation

Negative regulation Sigma factors and regulation

Cell division

*See text for further explanation and references78. CP, cytoplasmic protein; ECF, extracytoplasmic function; EPEC, enteropathogenic E. coli; IMP, inner-membrane protein; LPS, lipopolysaccharide; OMP, outer-membrane protein; PP, periplasmic protein; PPIase, peptidyl-prolyl-isomerase.

function, respectively, as a response regulator, a sensor kinase and a negative-feedback regulator of the system (FIG. 2). Additional known or putative members of the Cpx regulon are listed in TABLE 4. A model for Cpx -regulon regulation in E. coli is given in FIG. 2 (REFS 7,75,79,80). The Cpx ESR is activated by envelope stresses (TABLE 3) that cause CpxP to disassociate from CpxA, activating CpxA kinase activity and, in turn, phosphorylation of CpxR7,8,75,79. Phosphorylated NATURE REVIEWS | MICROBIOLOGY

CpxR regulates gene expression by binding to a specific recognition site (consensus sequence: 5′-GTAAA-N5GTAAA-3′; REF. 78) that is located upstream of genes encoding the members of the Cpx regulon (TABLE 4). Conditions that activate the Cpx system are listed in TABLE 3 (REFS 7,9,75,81–84). There is some overlap in both the genes belonging to the σE and Cpx regulons and in the activating stimuli, indicating that the two pathways might function cooperatively; however, the Cpx system can VOLUME 4 | MAY 2006 | 389


REVIEWS pH stress

Surface adhesion

Outer membrane

Misfolded proteins

Accumulated/ aggregated pilin proteins + CpxP Periplasm

+

+

NIpE Overproduced NIpE (?)

CpxA

ATP

Inner membrane

ADP CpxR

P CpxR

P +

rpoE rseABC Cytoplasm

degP cpxRA dsbA cpxP ppiA ppiD

Figure 2 | Model for regulation of the CpxRA pathway. Signals generated by some form of envelope stress (pH stress; surface adhesion; overproduced, aggregated or misfolded proteins) are somehow sensed and transduced, presumably through the periplasmic domain of CpxA. This activates CpxA kinase, which triggers autophosphorylation of a conserved histidine residue in its cytoplasmic domain. Phosphorylated CpxA then transfers the phosphate group to an aspartate residue in the N-terminal domain of the response regulator CpxR. CpxR-P then binds to specific sites on DNA. CpxP-R binding activates (and in some cases represses) the transcription of genes in the Cpx regulon77. Similar to the σE regulon, the cpxRA genes are positively autoregulated by CpxR-P, enabling amplification of the response. However, CpxR-P also upregulates cpxP, which encodes a small periplasmic protein, CpxP, that binds to CpxA, inhibiting its kinase activity and providing feedback inhibition. This inhibition would then be relieved by envelope stresses that cause CpxP to dissociate from CpxA. Other negative regulators of CpxA activity, represented by a question mark, also seem to be involved75.

Pili/fimbriae Filamentous surface structures that are more rigid, but thinner and shorter, than flagella. They mediate adhesion to both biotic and abiotic surfaces.

Curli A type of fimbriae that mediate binding to components of the extracellular matrix, often implicated in biofilm formation.

negatively regulate the σE pathway21,22,28,78,85. Interestingly, periplasmic accumulation of one P pili subunit (PapG) activates both the σE and Cpx pathways, whereas another (PapE) activates only the Cpx pathway 86. A major role of the Cpx system seems to be regulating the production of macromolecular surface structures (see below). The Cpx pathway and pathogenesis. The Cpx regulon has been implicated in the virulence of several bacterial pathogens. In particular, the Cpx system regulates the expression of surface structures that are associated with bacterial virulence, including pili/fimbriae and TTSSs. However, for most organisms, the role of the Cpx system during infection has not been investigated.

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Escherichia coli. There are several studies showing that Cpx affects the expression of different types of pili/fimbriae in E. coli — Curli (negative regulation)87, P/PAP pili of UPEC (positive or negative regulation)84,88 and type IV bundle-forming pili (BFP) of enteropathogenic E. coli (EPEC; positive regulation)89. As well as directly regulating pili/fimbrial genes, Cpx-regulated genes such as dsbA and ppiA might assist in the assembly of pili/fimbriae. There is only one report that disruption of Cpx regulation of pili expression affects the ability of E. coli to adhere to eukaryotic cells; an EPEC cpxR mutant produced less BFP and was less able to adhere to eukaryotic cells in vitro89. Salmonella serovars. The Cpx system affects the invasiveness of Salmonella in a pH-dependent fashion. Invasion of eukaryotic cells is mediated by effector proteins that are delivered by the TTSS encoded by the Salmonella pathogenicity island 1 (SPI-1). HilA is a positive regulator of the SPI-1 TTSS genes, and CpxA (but not CpxR) is required by S. typhimurium for expression of hilA at pH 6.0 but not pH 8.0 (REF. 90). At pH 6.0, the invasiveness of an S. typhimurium cpxA mutant is reduced. Conversely, at alkaline pH (pH 8.0), S. typhimurium cpxA and cpxR null mutants are slightly (but significantly) more invasive than wild-type strains for reasons that are unknown85,90. Also, a transposon (TnphoA) insertion in cpxA of S. typhi reduced the ability of the strain to adhere to, and invade, epithelial cells in vitro91. In-frame deletions in the region of cpxA that encodes the periplasmic sensor domain render CpxA signal blind and constitutively activate the Cpx pathway, a so-called cpxA* mutation8. A cpxA* mutation greatly reduced the ability of S. typhimurium to adhere to — and as a consequence invade — eukaryotic cells in vitro85. Survival and replication of S. typhimurium within macrophages in vitro does not require the Cpx system85. In mice, S. typhimurium cpxA and cpxA* mutants are attenuated when administered by both the oral and parenteral routes of infection but, interestingly, a cpxR mutant is not85. This indicates that CpxA and CpxR can function independently75,90. Constitutive activation of the Cpx pathway had a much greater effect on S. typhimurium virulence than the loss of cpxA85. Salmonella serovars, including S. typhimurium, have the potential to express a diverse number of pili/fimbriae, most of which have only been shown to be expressed in vivo92. Preliminary evidence suggests that S. typhimurium CpxAR negatively regulates expression of Curli and possibly other pili (M.R., unpublished observations), and this could contribute to the reduced adhesiveness and/or virulence of the S. typhimurium cpxA* strain. However, as Cpx can repress the expression of rpoE, it is possible that the attenuation of the S. typhimurium cpxA* strain results (at least partially) from interference with rpoE expression28,78,85. Shigella spp. Shigella spp. are the causative agents of bacillary dysentery. Shigella spp. are invasive pathogens and their entry into eukaryotic cells is mediated by a virulence-plasmid-encoded TTSS. Similar to Salmonella spp., the Cpx system is involved in pH-dependent www.nature.com/reviews/micro


REVIEWS Type IV secretion system (TFSS). Used to deliver proteins or DNA across the bacterial cell envelope.

regulation of Shigella TTSS invasion genes. Expression of the TTSS genes is tightly controlled by the virulenceplasmid-encoded virF and invE (virB) genes93. VirF activates transcription of invE and, in turn, InvE switches on transcription of the TTSS genes93. In response to external pH, Cpx activates expression of virF and invE and, consequently, expression of the TTSS genes94–96. Furthermore, the Cpx system also enhances post-transcriptional production of InvE94. Legionella pneumophila. L. pneumophila is the cause of Legionnaire’s disease in humans. The organisms can live within phagocytic cells such as macrophages in humans and amoebae in the environment. The L. pneumophila icm-dot genes are necessary for infection of amoebae, macrophages and animals 97,98. They encode a type IV secretion system (TFSS) that translocates effector proteins into host cells to modulate the intracellular environment and facilitate intracellular survival97,98. CpxR positively affects transcription of several of the icm-dot genes99. However, neither cpxR nor rpoE are required for L. pneumophila infection, survival or growth within macrophages or amoebae99. It is not known if L. pneumophila htrA is regulated by σE or Cpx; however, htrA is required for infection of mice and replication in macrophages and epithelial

Secretion defects

High temperature

Ethanol/ hydrophobic organic solvents

Proton ionophores

Filamentous phage infection or f1 pIV protein

Stationaryphase at high pH

Outer membrane

Periplasm + +

+ PspE

+

+ +

LZM

PMF PspD PspB

PspA

(?) PspA

PspF

(–)

(+)

(+)

RNA-P σ54

Inner membrane

PspC

pspA B

C

D

E

Figure 3 | Model for phage shock protein (PSP) response regulation. Signals generated by protein-secretion defects, hydrophobic organic solvents, proton ionophores, filamentous phage infection/f1 protein pIV, stationary-phase at alkaline pH and high temperature are somehow sensed and transduced intracellularly to increase the expression of the pspABCD operon. In this model based on Escherichia coli, PspB and PspC are positive regulators of PspA. PspA has a role in maintenance of the proton-motive force (PMF) and also negatively controls PspF activity as an enhancer-binding protein activator, whichenhances σ54-dependent transcription of the pspABCD operon and pspG. The roles/ functions of PspE (which is not present in all organisms), PspD and/or PspG in this system have yet to be defined. LZM, leucine zipper motif; RNA-P, RNA polymerase.


cells but, interestingly, the gene is not necessary for infection of amoebae63. In summary, Cpx is involved in regulating the expression of surface virulence factors such as pili/fimbriae, TTSSs and TFSSs. Control of expression of these structures is complex, involving several transcriptional regulators. However, so far, no virulence factor has been shown to absolutely require the Cpx system for expression. The Cpx pathway been shown to have an in vivo role in only one organism, S. typhimurium. BaeSR. A second two-component system that consists of BaeS, the sensor kinase, and BaeR, the response regulator, was recently reported to represent a third envelope-stress signal-transduction pathway10. Clues to the physiological function(s) of the Bae system in E. coli have come from studies showing that overexpression of BaeR increases resistance to β-lactam antibiotics, novobiocin and bile salts (for example, deoxycholate)100–102. So far, there are no reports showing the involvement of the BaeSR system in bacterial pathogenesis, and this system will not be considered further here.

Phage-shock-protein response and ESR A proposed fourth ESR system is the phage shock protein (PSP) response originally described for its induction in bacteria following infection with filamentous phage103. In E. coli, the PSP response is mediated by the pspABCDE operon. The pspA gene (the first gene in the operon) is also induced by several environmental stress conditions11,14,103–107 (TABLE 3). In fact, the psp operon is required for long-term stationary-phase survival at pH 9 (REF. 14). A model for the PSP response system is illustrated in FIG. 3. The physiological function of the psp operon is only beginning to be elucidated. Based on the various stresses and conditions that lead to pspA induction as well the observed phenotypes of pspA mutants in E. coli, S. typhimurium and Yersinia enterocolitica, the PspA protein (or PSP response system in general) is proposed to function in the maintenance of the proton-motive force (PMF) across the inner membrane13,105,108. Recently, it was found that σE, as well as PSP, is necessary for maintenance of PMF, and that PSP compensates for the absence of σE in an S. typhimurium rpoE mutant during stationary phase. Again, this is an excellent example of how the individual ESR pathways interact with each other109. The PSP regulon and virulence. Little is known about the role of the PSP regulon in bacterial virulence. The only studies so far have focused on Y. enterocolitica. Y. enterocolitica psp mutants are attenuated in mice110–112. The psp genes of Y. enterocolitica are induced and necessary for normal growth when the plasmid-encoded Ysc TTSS is expressed in vitro. Y. enterocolitica psp mutants are thought to be attenuated because of growth inhibition when the Ysc TTSS is expressed in vivo110–112. There is generally a good correlation between the effect of overexpression of the TTSS secretin gene yscC on the growth of different Y. enterocolitica psp mutants in vitro and their level of attenuation111,112. VOLUME 4 | MAY 2006 | 391


REVIEWS Conclusions Pathogens need to respond to various insults to survive in the environment of the host and for infection to be successful. Integration of the signals generated into coherent messages, which the organism can counter with both transcriptional and post-translational responses, is paramount. These responses involve several global regulatory systems, including ESRs. This also applies to survival of pathogens in the environment. Interestingly, the clinically relevant environment of a biofilm requires the ESR to integrate signals. A transcriptome analysis of E. coli revealed that many ESR genes belonging to the σE, CpxAR and PSP regulons are upregulated during biofilm formation113. A great deal has been learned about the ESRs but, so far, most studies have focused on organisms that are incapable of surviving outside of the lab. As such, it is crucially important to determine if the findings obtained in the laboratory also pertain to the ‘wild’. Although the involvement of ESRs in bacterial pathogenesis or environmental survival has only been investigated in a small number of pathogens, it is already apparent that a particular regulator or regulon member can be important for the biology of one pathogen but not another, and can have a role in one stage of an infection cycle but not another. It is therefore also important to use appropriate model systems to study the role of ESRs (or other stress responses) in pathogenesis.

Ruiz, N., Kahne, D. & Silhavy, T. J. Advances in understanding bacterial outer-membrane biogenesis. Nature Rev. Microbiol. 4, 57–66 (2006). A thorough and interesting review of the biogenesis of the bacterial outer membrane, combined with an overview of the techniques involved in achieving this understanding. 2. De Las, P., Connolly, L. & Gross, C. A. σE is an essential sigma factor in Escherichia coli. J Bacteriol. 179, 6862–6864 (1997). 3. Erickson, J. W. & Gross, C. A. Identification of the σE subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression. Genes Dev. 3, 1462–1471 (1989). A seminal paper for those interested in the ESR of enterics. The first study to identify the σE subunit through its role in E. coli high-temperature regulation. This same group then began to map the E. coli rpoE operon and dissect its regulation. 4. Rouviere, P. E. et al. rpoE, the gene encoding the second heat-shock sigma factor, σE, in Escherichia coli. EMBO J. 14, 1032–1042 (1995). 5. Raivio, T. L. Envelope stress responses and Gramnegative bacterial pathogenesis. Mol. Microbiol. 56, 1119–1128 (2005). 6. Danese, P. N. & Silhavy, T. J. The σE and the Cpx signal transduction systems control the synthesis of periplasmic protein-folding enzymes in Escherichia coli. Genes Dev. 11, 1183–1193 (1997). 7. Raivio, T. L., Popkin, D. L. & Silhavy, T. J. The Cpx envelope stress response is controlled by amplification and feedback inhibition. J. Bacteriol. 181, 5263–5272 (1999). 8. Raivio, T. L. & Silhavy, T. J. Transduction of envelope stress in Escherichia coli by the Cpx two-component system. J. Bacteriol. 179, 7724–7733 (1997). 9. Snyder, W. B., Davis, L. J., Danese, P. N., Cosma, C. L. & Silhavy, T. J. Overproduction of NlpE, a new outer membrane lipoprotein, suppresses the toxicity of periplasmic LacZ by activation of the Cpx signal transduction pathway. J. Bacteriol. 177, 4216–4223 (1995). 10. Raffa, R. G. & Raivio, T. L. A third envelope stress signal transduction pathway in Escherichia coli. Mol. Microbiol. 45, 1599–1611 (2002). This group identified the BaeSR two-component system as a third member of the ESR which shares some overlap with the CpxRA pathway in regulating certain genes. 1.

11.

12.

13.

14.

15. 16.

17.

18.

19.

Despite the huge advances in our knowledge of the ESR over the past 10 years, there is still much to learn, including completing the role call of each ESR regulon and determining the functional role of each member. In the case of pathogens, determining which ESR genes have a role in pathogenesis, and the nature of that role, is an important aim. This goal is of practical as well as fundamental interest. ESR genes and their products could be exploited to develop new vaccines and chemotherapeutic agents. For example, S. enterica htrA mutants (alone or in combination with other mutations) are safe, effective live oral vaccines, and immunization with purified HtrA provides partial protection against otitis media in several animal models of NTHi infection56,114,115. As many of the known members of ESR regulons are enzymes, they are also potential targets for novel antibacterial strategies. Furthermore, the ESR might also have a significant role in the development of antibiotic resistance, as studies have shown that inhibition of particular ESR pathways can lead to increased sensitivity to specific antibiotics, and overexpression of σE in E. coli greatly increases the minimum inhibitory concentration (MIC) of several novel antibiotic agents116. The clinical relevance of these findings, and whether activation of the ESR could help pathogens resist the action of antibiotics, has not been investigated but is clearly an area worthy of study.

Jones, S. E., Lloyd, L. J., Tan, K. K. & Buck, M. Secretion defects that activate the phage shock response of Escherichia coli. J. Bacteriol. 185, 6707–6711 (2003). Darwin, A. J. The phage-shock-protein response. Mol. Microbiol. 57, 621–628 (2005). An excellent overview of the PSP response in enteric bacteria, discussing signal induction and transduction combined with possible physiological roles. Maxson, M. E. & Darwin, A. J. Identification of inducers of the Yersinia enterocolitica phage shock protein system and comparison to the regulation of the RpoE and Cpx extracytoplasmic stress responses. J. Bacteriol. 186, 4199–4208 (2004). Weiner, L. & Model, P. Role of an Escherichia coli stress-response operon in stationary-phase survival. Proc. Natl Acad. Sci. USA 91, 2191–2195 (1994). Helmann, J. D. The extracytoplasmic function (ECF) sigma factors. Adv. Microb. Physiol. 46, 47–110 (2002). Lonetto, M. A., Brown, K. L., Rudd, K. E. & Buttner, M. J. Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions. Proc. Natl Acad. Sci. USA 91, 7573–7577 (1994). Craig, J. E., Nobbs, A. & High, N. J. The extracytoplasmic sigma factor, final σE, is required for intracellular survival of nontypeable Haemophilus influenzae in J774 macrophages. Infect. Immun. 70, 708–715 (2002). Humphreys, S., Stevenson, A., Bacon, A., Weinhardt, A. B. & Roberts, M. The alternative sigma factor, σE, is critically important for the virulence of Salmonella typhimurium. Infect. Immun. 67, 1560–1568 (1999). A seminal paper discussing the σE regulon in a pathogen showing that an rpoE mutant is viable in S. typhimurium, but is highly attenuated in a mouse model. This study led the way for later work in S. typhimurium and other organisms. Kovacikova, G. & Skorupski, K. The alternative sigma factor σE plays an important role in intestinal survival and virulence in Vibrio cholerae. Infect. Immun. 70, 5355–5362 (2002).

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20. Martin, D. W., Holloway, B. W. & Deretic, V. Characterization of a locus determining the mucoid status of Pseudomonas aeruginosa: AlgU shows sequence similarities with a Bacillus sigma factor. J. Bacteriol. 175, 1153–1164 (1993). 21. Dartigalongue, C., Missiakas, D. & Raina, S. Characterization of the Escherichia coli σE regulon. J. Biol. Chem. 276, 20866–20875 (2001). Until this publication, relatively few genes were known to be under the control of RpoE. Using promoter-fusion libraries, 20 RpoE-regulated promoters in E. coli were identified from various functional categories. 22. Rezuchova, B., Miticka, H., Homerova, D., Roberts, M. & Kormanec, J. New members of the Escherichia coli σE regulon identified by a two-plasmid system. FEMS Microbiol. Lett. 225, 1–7 (2003). 23. Rhodius, V. A., Suh, W. C., Nonaka, G., West, J. & Gross, C. A. Conserved and variable functions of the σE stress response in related genomes. PLoS Biol. 4, e2 (2005). 24. Alba, B. M. & Gross, C. A. Regulation of the Escherichia coli sigma-dependent envelope stress response. Mol. Microbiol. 52, 613–619 (2004). 25. Dartigalongue, C., Loferer, H. & Raina, S. EcfE, a new essential inner membrane protease: its role in the regulation of heat shock response in Escherichia coli. EMBO J. 20, 5908–5918 (2001). 26. Duguay, A. R. & Silhavy, T. J. Quality control in the bacterial periplasm. Biochim. Biophys. Acta 1694, 121–134 (2004). 27. Skovierova, H. et al. Identification of the σE regulon of Salmonella enterica serovar 1 Typhimurium. Microbiology (in the press). 28. Miticka, H. et al. Transcriptional analysis of the rpoE gene encoding extracytoplasmic stress response sigma factor σE in Salmonella enterica serovar Typhimurium. FEMS Microbiol. Lett. 226, 307–314 (2003). 29. Ehrmann, M. & Clausen, T. Proteolysis as a regulatory mechanism. Annu. Rev. Genet. 38, 709–724 (2004). 30. Grigorova, I. L. et al. Fine-tuning of the Escherichia coli σE envelope stress response relies on multiple mechanisms to inhibit signal-independent proteolysis of the transmembrane anti-sigma factor, RseA. Genes Dev. 18, 2686–2697 (2004).


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Competing interests statement The authors declare no competing financial interests.

DATABASES The following terms in this article are linked online to: Entrez Genome Project: http://www.ncbi.nlm.nih.gov/ entrez/query.fcgi?db=genomeprj Actinobacillus pleuropneumoniae | Caenorhabditis elegans | Chlamydia trachomatis | Escherichia coli | Haemophilus influenzae | Legionella pneumophila | Pseudomonas aeruginosa | Salmonella enterica serovar Typhimurium | Vibrio cholerae | Yersinia enterocolitica UniProtKB: http://ca.expasy.org/sprot AlgR | AlgU | BaeR | BaeS | CpxA | CpxP | CpxR | DegS | DsbA | FkpA | HilA | HtrA | IL-8 | InvE | PapE | PapG | PpiA | PpiD | PspA | RpoE | RseA | RseB | SurA | TNF-α | VirF | YaeL

FURTHER INFORMATION Mark Roberts’ homepage: http://www.gla.ac.uk/vet/ resesarch/iandi/bacteriology/salmonella.html

SUPPLEMENTARY INFORMATION See online article: S1 (table) Access to this links box is available online.
