The starvation-stress response of Salmonella enterica serovar Typhimurium requires {sigma}E-, but not CpxR-regulated extracytoplasmic functions

William J. Kenyon1, D. Geary Sayers1, Sue Humphreys2, Mark Roberts2 and Michael P. Spector1

Department of Biomedical Sciences, University of South Alabama, Mobile, AL 36688, USA1
Department of Veterinary Pathology, Glasgow University Veterinary School, Glasgow G61 1QH, UK2

Author for correspondence: Michael P. Spector. Tel: +1 251 380 2688. Fax: +1 251 380 2711. e-mail: mspector{at}usouthal.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Starvation of Salmonella enterica serovar Typhimurium (S. Typhimurium) for an exogenous source of carbon and energy (C-starvation) induces the starvation-stress response (SSR). The SSR functions to (i) maintain viability during long-term C-starvation and (ii) generate cross-resistance to other environmental stresses. The SSR is, at least partially, under the control of the alternative sigma factor, {sigma}S. It is hypothesized that C-starvation causes cell envelope stresses that could induce the {sigma}E and/or Cpx regulons, both of which control extracytoplasmic functions and, thus, may play a role in the regulation of the SSR. In support of this hypothesis, Western blot analysis showed that the relative levels of {sigma}E increased during C-starvation, peaking after approximately 72 h of C-starvation; in contrast, CpxR levels remained relatively constant from exponential phase up to 72 h of C-starvation. To determine if {sigma}E, and thus the regulon it controls, is an essential component of the SSR, several mutant strains were compared for their abilities to survive long-term C-starvation and to develop C-starvation-induced (CSI) cross-resistances. An rpoE mutant strain was significantly impaired in both long-term C-starvation survival (LT-CSS) and in CSI cross-resistance to challenges with 20 mM H2O2 for 40 min, 55 °C for 16 min, pH 3·1 for 60 min and 870·2 USP U polymyxin B ml-1 (PmB) for 60 min, to varying degrees. These results suggest that C-starvation can generate signals that induce the rpoE regulon and that one or more members of the {sigma}E regulon are required for maximal SSR function. Furthermore, evidence suggests that the {sigma}E and {sigma}S regulons function through separate mechanisms in the SSR. In contrast, C-starvation does not appear to generate signals required for Cpx regulon induction which support the findings that it is not required for LT-CSS or cross-resistance to H2O2, pH 3·1 or PmB challenges. However, it was required to achieve maximal cross-resistance to 55 °C. Therefore, {sigma}E is a key regulatory component of the SSR and represents an additional {sigma} factor required for the SSR of Salmonella.

Keywords: rpoE, extracytoplasmic stress, sigma factors

Abbreviations: CRP, cAMP receptor protein; CSI, C-starvation-inducible/-induced; ECF, extracytoplasmic function; LT-CSS, long-term C-starvation survival; MS hiPCN, MOPS-buffered salts medium non-limiting in glucose, phosphate and nitrogen; MS loC, MOPS-buffered salts medium limiting for glucose (C-source); PmB, polymyxin B; S. Typhimurium, Salmonella enterica serovar Typhimurium; SSR, starvation-stress response


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Serovars of Salmonella enterica encounter various microenvironments in the process of cycling through their numerous host and non-host niches. Many of these environs are less than optimal for growth and can even threaten viability. Thus Salmonella, and related bacteria, must be capable of sensing and responding to such stresses to survive. Starvation for a carbon and energy source (C-starvation) is one of the most common stresses confronted by bacterial cells in environments outside of the laboratory (Foster & Spector, 1995 ; Spector, 1998 ). Salmonella enterica serovar Typhimurium (S. Typhimurium) and many other non-spore-forming bacteria respond to C-starvation with major alterations in gene expression that result in morphologically and physiologically different and generally stress-resistant cells, when compared to growing cells. This is referred to as the starvation-stress response (SSR) (Spector & Cubitt, 1992 ; Foster & Spector, 1995 ; Spector, 1998 ). Unlike the stationary-phase response, the SSR is elicited (a) in response to a single specific parameter, carbon-energy source starvation, and (b) at cell densities 1 to 2 logs lower than those measured under typical stationary-phase conditions.

The SSR has two functions: (a) to combat the long-term effects of C-starvation and (b) to provide a general resistance to other environmental stresses such as extremes in temperature and pH, and exposure to H2O2 and antimicrobial peptides. A subset of SSR loci essential to long-term C-starvation survival (LT-CSS) and general stress resistance is under the control of the alternative {sigma} factor encoded by the rpoS gene, {sigma}S (O’Neal et al., 1994 ; Hengge-Aronis, 1996 ; Muffler et al., 1997 ; Loewen et al., 1998 ; Spector, 1998 ; Spector et al., 1999 ). In addition, {sigma}S is essential for full oral virulence of Salmonella in the mouse virulence model system (Fang et al., 1992 ; Loewen et al., 1998 ; Ibanez-Ruiz et al., 2000 ).

Out of our interest in studying the SSR of Salmonella, we became interested in further understanding the effects of C-starvation on extracytoplasmic functions (ECFs). Two pathways have been characterized in growing Escherichia coli cells that respond to extracytoplasmic stress and control ECF. One pathway is under the control of the alternative sigma factor, {sigma}E, encoded by the rpoE gene, and the other by the two-component sensor–regulatory system CpxRA. In E. coli, as well as Salmonella, {sigma}E is activated by stresses to the cell envelope and then directs the transcription of genes with ECFs (Erickson & Gross, 1989 ; Connolly et al., 1997 ; Missiakas & Raina, 1998 ; Raivio & Silhavy, 1999 ). Unlike in E. coli, Salmonella strains lacking {sigma}E are not temperature-sensitive and {sigma}E is not an essential sigma factor during exponential-phase growth (Mecsas et al., 1993 ; Hiratsu et al., 1995 ; De Las Penas et al., 1997a ; Humphreys et al., 1999 ). They are, however, sensitive to oxidizing agents and polymyxin B (PmB) when analysed using disk susceptibility tests on agar media (Humphreys et al., 1999 ). Mutants of rpoE and at least one of the genes it is known to regulate in E. coli, htrA (also known as degP), exhibit significant attenuation in the murine salmonellosis model (Humphreys et al., 1999 ). In E. coli, the rpoE regulon is believed to sense and respond to misfolded proteins in the outer membrane and/or periplasm, which can be caused by high temperature, ethanol and perhaps other stresses (Mecsas et al., 1993 ; Hiratsu et al., 1995 ; Raina et al., 1995 ; Rouviere et al., 1995 ; Missiakas et al., 1997 ). In addition to the HtrA protease, members of the E. coli {sigma}E regulon include functions needed for envelope protein refolding (FkpA and SurA), rpoE/{sigma}E regulation, virulence and other unidentified functions (De Las Penas et al., 1997b ; Danese & Silhavy, 1997 ; Missiakas et al., 1997 ; Missiakas & Raina, 1997a , b ; Pallen & Wren, 1997 ; Betton et al., 1998 ; Humphreys et al., 1999 ; Spiess et al., 1999 ; Nitta et al., 2000 ).

In E. coli, the two-component regulator–sensor CpxRA system constitutes another extracytoplasmic stress response system (Pogliano et al., 1997 ; Raivio & Silhavy, 1999 ). Some members of the CpxR regulon such as CpxP, HtrA and DsbC have been assigned anti-stress functions (Dartigalongue et al., 2001 ; Danese & Silhavy, 1998 ; Skorko-Glonek et al., 1999 ). Homologues of these genes have been identified in Salmonella. A major role attributed to the Cpx regulon is to assist in biosynthesis of P pili by dealing with misfolding or aggregation of subunits within the periplasm (Raivio & Silhavy, 1999 ).

Because C-starvation could result in cell envelope stresses and/or cause misfolding of cell envelope proteins that can activate the {sigma}E and/or Cpx regulatory pathways, we investigated the role of the alternative sigma factor {sigma}E and the two-component regulator–sensor CpxRA system in the SSR of S. Typhimurium. To determine if either or both of these regulons are required for the SSR, knock-out mutants of rpoE and cpxR were tested for their roles in the two known functions of the SSR, (a) LT-CSS and (b) generation of C-starvation-inducible (CSI) cross-resistance to other environmental stresses. The results reported here indicate that C-starvation generates signals capable of inducing the {sigma}E regulon, but not the CpxRA regulon, indicating that in addition to {sigma}S and cAMP-CRP (cAMP receptor protein), {sigma}E is a key regulator of the SSR of S. Typhimurium.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and transductions.
The bacterial strains used in this study were all derivatives of the mouse-virulent strain of S. Typhimurium, SL1344, and are listed in Table 1. Desired mutations were transferred to appropriate strains via transduction with the high transducing derivative of P22 bacteriophage, P22 HT int-105 (Chan et al., 1972 ). Transductants were determined to be non-lysogens by demonstrating sensitivity to the H5 derivative of P22 bacteriophage on Green Agar (Maloy, 1990 ).


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Table 1. Bacterial strains and plasmids used or constructed in this study

 
Growth media and conditions used.
The minimal media used in this study were modified MOPS-buffered salts (MS) media (Neidhardt et al., 1974 ) containing either 0·4% (w/v) glucose (MS hiPCN) or 0·03% (w/v) glucose (MS loC) and are described in detail elsewhere (Spector & Cubitt, 1992 ; O’Neal et al., 1994 ). Histidine was added to a final concentration of 0·2 mM. Ampicillin (Ap) and kanamycin (Km) were used at final concentrations of 30 and 50 µg ml-1, respectively. Luria–Bertani (LB) agar (Difco) was used as a rich medium for viability plating.

LT-CSS assay.
As described previously (Spector et al., 1999 ), desired strains were grown overnight in MS hiPCN at 37 °C with aeration. Overnight cultures were diluted 1:100 into fresh MS hiPCN and grown to an OD600 of 0·3–0·4 (exponential phase). One millilitre of this culture was then added to 9 ml MS medium lacking glucose (MS noC) in a 50 ml screw-cap flask. These cultures were then incubated at 37 °C with aeration for 21 d. At desired time points, aliquots of each culture were serially diluted in MS buffer and plated in triplicate onto LB agar containing the necessary antibiotic(s) at 37 °C to determine viable counts. Survival was measured as the percentage of maximum viability achieved for the culture, typically ca. 5x108 c.f.u. ml-1. Results are presented as mean values of at least three separate trials±SEM.

Western blot analysis.
Pellets of exponential-phase or C-starved cells were lysed with 0·1% SDS and total protein was determined (Bio-Rad; Bradford, 1976 ). Proteins were separated by SDS-PAGE (Tris/HCl 4–15% Ready Gels; Bio-Rad) and transferred to a PVDF membrane (Bio-Rad). The membrane was incubated overnight at 4 °C with anti-{sigma}E antibody (polyclonal rabbit antibody) or anti-CpxR antibody (polyclonal rabbit antibody). The membrane was washed three times and then incubated at room temperature for 1 h in anti-rabbit HRP-conjugated antibody (Amersham Pharmacia Biotech). Following five washes, ECL reagents 1 and 2 (Amersham Pharmacia Biotech) were mixed 1:1 (v/v) and poured onto the surface of the membrane. After 1·5 min, the membrane was exposed to X-ray film (Kodak Biomax MR) and/or type 667 film (Polaroid) for various time intervals. The film was developed and the molecular mass of proteins was determined by reference to prestained standards (Gibco-BRL; very low range).

Assays for CSI cross-resistance to environmental stresses.
Strains were grown overnight in MS hiPCN medium at 37 °C with aeration. Overnight cultures were then diluted 1:100 into either MS hiPCN to generate exponential-phase cells or into MS loC to generate C-starved cells. C-starvation was indicated by cessation of growth due to exhaustion of glucose from the MS loC cultures (final OD600 of 0·3–0·4). Exponential-phase, 4–5 h C-starved and 24 h C-starved cells were challenged by diluting the culture 1:100 into one of the following challenge media: (a) MS buffer containing 20 mM H2O2, (b) NCE buffer (Neidhardt et al., 1974 ) at pH 3·1, (c) MS buffer at 55 °C or (d) MS buffer containing 870·2 USP U PmB ml-1. Viable counts were determined at various time intervals during each challenge as appropriate. Percentage survival was calculated by dividing the c.f.u. ml-1 at the desired time point by the c.f.u. ml-1 at time zero (100% viability; typically ca. 3–5x106 c.f.u. ml-1) and multiplying by 100. Results are presented as mean values of at least three separate trials±SEM.

ß-Galactosidase assay.
Exponential-phase cells and 5, 24, 48 and 72 h C-starved cells carrying either pTFP1 or pTFP2 were assayed for ß-galactosidase activity according to the method of Miller (1972) .


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
{sigma}E, but not CpxR, is required for LT-CSS
One of the functions of the SSR is to allow S. Typhimurium to survive long periods of carbon-energy source starvation (Foster & Spector, 1995 ; Spector, 1998 ). It should be noted that we are starving cells at cell densities (approx. 108 c.f.u. ml-1 or less) where cross-feeding from dead-lysed cells would be minimal (Spector & Cubitt, 1992 ). We hypothesized that long-termC-starvation could generate cell envelope or extracytoplasmic damage that must be dealt with to maintain cell viability. To test this hypothesis, we determined whether strains carrying knockout mutations in rpoE (encoding the alternative sigma factor, {sigma}E) or cpxR (encoding the regulator CpxR) exhibit LT-CSS comparable to the wild-type parent. Both {sigma}E and CpxR have been shown in E. coli to be major regulators of ECFs in response to extracytoplasmic stress (Erickson & Gross, 1989 ; Connolly et al., 1997 ; Missiakas & Raina, 1998 ; Raivio & Silhavy, 1999 ).

As presented in Fig. 1, an rpoE knockout mutant, GVB311 (Humphreys et al., 1999 ), showed a relatively rapid decline in culture viability and exhibited about 10-fold lower survival after 21 d of C-starvation, compared to its wild-type parent. In contrast, the cpxR knockout mutation did not appear to significantly impair LT-CSS, compared to the wild-type parent strain. This indicates that one or more members of the {sigma}E regulon, but not the CpxR regulon, plays an important role in LT-CSS.



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Fig. 1. LT-CSS of S. Typhimurium wild-type, rpoE and cpxR mutant strains. Strains SL1344 (wild-type; {bullet}; n=8), GVB311 (rpoE mutant; {blacksquare}; n=8) and GVB368 (cpxR mutant; {blacktriangleup}; n=7) were starved for a carbon and energy source for a total of 21 d as described in Methods. The data shown are mean values±SEM from the indicated number (n) of separate experiments.

 
Western blot analysis shows that {sigma}E accumulates during C-starvation
The finding that an rpoE mutant was defective in LT-CSS prompted us to examine the relative levels of {sigma}E during long-term C-starvation. Western blot analysis of exponential-phase (LP) and C-starved wild-type cells of S. Typhimurium (SL1344) showed that the amount of {sigma}E protein increased relative to total cellular protein during C-starvation (Fig. 2a). In fact, {sigma}E levels continued to increase, peaking at about 48 h of C-starvation (Fig. 2a) and staying at that relative level for at least 144 h of C-starvation (data not shown). This relative accumulation of {sigma}E correlated well with the observed kinetics of the loss of viability of the rpoE mutant (Fig. 1), supporting a proposed role for one or more {sigma}E-regulated loci in coping with C-starvation.



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Fig. 2. Western blot analysis of {sigma}E levels and transcriptional activity of {sigma}E during C-starvation. Wild-type S. Typhimurium SL1344 was grown and treated as described in the Methods. (a) Ten micrograms of whole-cell protein was loaded per lane, electrophoresed, transferred to PVDF membrane and probed with antibody to {sigma}E. The blot shown is representative of at least three separate trials. (b) Wild-type S. Typhimurium possessing either pTFP1 ({bullet}) or pTFP2 ({blacksquare}) was grown to exponential phase (LP) and C-starved (C-st) for the time periods indicated. ß-Galactosidase activity was measured by the procedure of Miller (1972) . The data shown are mean values±SEM from at least three separate experiments.

 
{sigma}E-dependent transcription increases during C-starvation, indicating that all or part of the accumulated {sigma}E is active
Although Western blot analysis showed that {sigma}E protein accumulated during C-starvation, we wanted to determine if all or part of this protein was active (not sequestered by the anti-sigma factor RseA) in the starved cell. For this, strains carrying the pTFP1 or pTFP2 plasmids were grown and C-starved for up to 144 h. pTFP1 contains the {sigma}E-independent P1 promoter of the rpoE rseABC operon fused to a promoterless lacZ gene, while pTFP2 contains the {sigma}E-dependent P2 promoter of the rpoE rseABC operon fused to a promoterless lacZ gene (T. Testerman & F. Fang, unpublished). In this way, we were able to monitor and compare the transcription from a {sigma}E-dependent and -independent promoter over the same time period that we observed {sigma}E accumulation.

Results, presented in Fig. 2(b), demonstrate that transcription from the {sigma}E-dependent promoter increased 4·5-fold during the first 5 h of C-starvation compared to exponential phase; this was followed by a further approximately 1·5-fold increase over the next 67 h of C-starvation. This, for the most part, mirrored the increase in {sigma}E observed during Western blot analysis. In contrast, transcription from the {sigma}E-independent promoter remained relatively constant from exponential phase through 72 h of C-starvation. This indicates that a significant portion of the accumulated {sigma}E protein is active in C-starved cells.

These data also show that the rpoE rseABC operon is transcriptionally induced during C-starvation in a {sigma}E-dependent manner.

{sigma}E is needed for maximal CSI cross-resistance to H2O2, 55 °C, acid pH and the antimicrobial peptide PmB
Another consequence of induction of the SSR is a remarkable increase in the resistance of C-starved cells to a variety of other stresses (Hengge-Aronis, 1996 ; McLeod & Spector, 1996 ; Seymour et al., 1996 ; Spector, 1998 ; Spector et al., 1999 ). Therefore, we wanted to determine if the {sigma}E regulon, in addition to its role in LT-CSS, is important for the development of CSI cross-resistance to other environmental stresses.

Previous studies showed that cells must C-starve for 4–5 h to exhibit maximal CSI cross-resistance to most other environmental stresses (Jenkins et al., 1988 ; McLeod & Spector, 1996 ; Seymour et al., 1996 ; Spector et al., 1999 ). Therefore, desired strains were assayed for CSI cross-resistance to a (i) 40 min challenge with 20 mM H2O2, (ii) 16 min challenge at 55 °C, (iii) 60 min challenge at pH 3·1 and (iv) 60 min challenge with 870·2 USP U PmB ml-1. Cells were challenged during exponential-phase growth and after 3–5 and 24 h of C-starvation.

As reported previously (Seymour et al., 1996 ), survival of wild-type exponential-phase cells was undetectable, while 5 and 24 h C-starved wild-type cells exhibited 50–70% survival after 40 min of H2O2 challenge (Table 2), a classic CSI cross-resistance phenotype. In comparison, survival of the rpoE mutant was impaired 10-fold in 5 h C-starved cells but only about 5- to 6-fold in 24 h C-starved cells. Complementation of the rpoE mutation through expression of {sigma}E from pSH117 in strain GVB372 (Humphreys et al., 1999 ) restored CSI cross-resistance to wild-type levels (Table 2), indicating that the rpoE mutation is causing a real and reproducible effect. This indicates that one or more {sigma}E regulon members is essential for full CSI cross-resistance to H2O2, although this appears to become less essential the longer the cells are C-starved (Table 2, 24 h C-starved rpoE mutant cells).


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Table 2. Effect of an rpoE, cpxR or rpoS mutation on CSI cross-resistance to various environmental stresses

 
Thermotolerance has been demonstrated previously in C-starved cells (Jenkins et al., 1988 ; Hengge-Aronis, 1996 ; Spector, 1998 ; Spector et al., 1999 ). This, coupled with our findings that rpoE mutants are deficient in LT-CSS and CSI cross-resistance to H2O2, raised the question as to whether the {sigma}E regulon is required for CSI thermotolerance. Interestingly, previous studies found that rpoE mutants are temperature-sensitive in E. coli, but not in Salmonella (Mecsas et al., 1993 ; Hiratsu et al., 1995 ; De Las Penas et al., 1997a ; Humphreys et al., 1999 ), and of the four known loci regulated by {sigma}E, three, rpoH, htrA and fkpA, are involved in thermotolerance in E. coli (Jenkins et al., 1991 ; Mecsas et al., 1993 ; Hiratsu et al., 1995 ; Raina et al., 1995 ; Rouviere et al., 1995 ; Yura et al., 1996 ; Missiakas & Raina, 1998 ; Raivio & Silhavy, 1999 ). Therefore, we investigated the role of {sigma}E in CSI cross-resistance to a 16 min challenge at 55 °C. As illustrated in Table 2, survival of C-starved rpoE mutants was reduced approximately 11- to 36-fold in both 5 and 24 h C-starved cells, respectively, when compared to the wild-type strain. Plasmid-borne {sigma}E again restored survival to wild-type levels in strain GVB372, indicating once more that these effects are real and reproducible (Table 2). Again, this demonstrates that one or more members of the {sigma}E regulon are needed for the generation of maximal protection against the damaging effects of thermal stress in C-starved cells.

C-starvation has also been shown to generate cross-resistance to acid pH (Foster & Spector, 1995 ; Spector, 1998 ; Spector et al., 1999 ). Since we have already demonstrated a defective phenotype for an rpoE mutant in terms of various aspects of SSR function, we wanted to determine whether the {sigma}E regulon is required for CSI cross-resistance to a 40 min challenge at pH 3·1. In the case of wild-type cells, 5 and 24 h of C-starvation provided approximately 50-fold increased resistance to an external pH of 3·1, compared to exponential-phase cells (Table 2). Introduction of an rpoE knockout mutation reduced acid tolerance in 5 and 24 h C-starved cells by some 22- to 30-fold, respectively, compared to 5 and 24 h C-starved wild-type cells (Table 2). The finding that expression of {sigma}E from the rpoE+ containing plasmid pSH117 (GVB372) again restored cross-resistance to wild-type levels (Table 2) provides support for a role for the {sigma}E regulon in CSI cross-resistance to pH 3·1.

The cationic antimicrobial polypeptide PmB causes disruption of the outer membrane of Gram-negative bacteria (Daugelavicius et al., 2000 ) and may introduce or cause extracytoplasmic damage. We have previously reported that C-starvation can generate cross-resistance to PmB (McLeod & Spector, 1996 ). Interestingly, unlike CSI cross-resistance to H2O2, 55 °C and pH 3·1, this form of cross-resistance is independent of the {sigma}S regulon and was found to develop after only 30 min of C-starvation, compared to 4–5 h for cross-resistance to other stresses, adding to the uniqueness of this form of CSI cross-resistance (McLeod & Spector, 1996 ). Therefore, we wanted to determine if {sigma}E functions in CSI cross-resistance to a 60 min challenge with 870·2 USP U PmB ml-1. As reported previously (McLeod & Spector, 1996 ), wild-type cells showed a substantial increase in resistance to a 60 min challenge with PmB after 3 and 24 h of C-starvation, respectively, when compared to exponential-phase cells. The rpoE mutant strain (GVB311) displayed a significant reduction in PmB resistance, particularly in 24 h C-starved cells, which was restored to wild-type levels by plasmid-borne expression of {sigma}E in strain GVB372 (Table 2). The rpoE mutant exhibited a reproducible 6-fold reduction in PmB resistance after 3 h of C-starvation and a more than 30-fold reduction in 24 h C-starved cells. Thus, one or more {sigma}E-regulated loci appear to be essential for maximal CSI cross-resistance to PmB, especially in 24 h C-starved cells. A role for the {sigma}E regulon in CSI cross-resistance to PmB suggests that at least part of the {sigma}E regulon functions through separate pathways from the {sigma}S regulon in the SSR, since the {sigma}S regulon plays no apparent role in CSI cross-resistance to PmB (McLeod & Spector, 1996 ).

Interestingly, exponential-phase rpoE mutant cells are hypersensitive to 20 mM H2O2, pH 3·1 and PmB challenges. This is similar to the effect that an rpoS mutation has on exponential-phase cells upon challenges with other environmental stresses (Table 2; Lange & Hengge-Aronis, 1991 ; McCann et al., 1991 ; Seymour et al., 1996 ; Foster & Spector, 1995 ; Spector et al., 1999 ). Thus, like an rpoS mutant, an rpoE mutant is defective in all aspects of the SSR and hypersensitive to certain environmental stresses during exponential-phase growth.

CpxR is needed for CSI cross-resistance to 55 °C but not to H2O2, pH 3·1 or PmB
Although the cpxR mutation did not affect LT-CSS (Fig. 1), and according to Western blot analysis CpxR levels appear to decline slightly from exponential-phase levels and remain relatively constant during C-starvation (Fig. 3), we nonetheless wanted to examine the potential role for the Cpx regulon in CSI cross-resistance. Results presented in Table 2 indicate that the cpxR mutant was not significantly different from the wild-type parent in terms of CSI cross-resistance to a 40 min challenge with 20 mM H2O2, a 60 min challenge at pH 3·1 or a 60 min challenge with 870·2 USP U PmB ml-1.



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Fig. 3. Western blot analysis of CpxR levels during C-starvation. Wild-type S. Typhimurium SL1344 was grown and treated as described in Methods. Three micrograms of whole-cell protein was loaded per lane, electrophoresed, transferred to PVDF membrane and probed with antibody to CpxR. The presented blot is representative of at least two separate trials.

 
Interestingly, the cpxR mutant was defective in CSI cross-resistance to a 16 min challenge at 55 °C. As illustrated in Table 2, the cpxR mutant exhibited similar reductions in percentage survival levels as the rpoE mutant, indicating that the Cpx regulon may play a role in thermotolerance generated in C-starved cells.

{sigma}E-regulated functions appear to act through different ‘pathways’ than {sigma}S-regulated functions in various aspects of the SSR in Salmonella
As previously shown in E. coli and Salmonella (reviewed by Foster & Spector, 1995 ; Hengge-Aronis, 1996 ; Spector, 1998 ), an rpoS knockout mutant exhibits deficiencies in both functions of the SSR, i.e. LT-CSS and CSI cross-resistance to other stresses, as well as showing hypersensitivity to environmental stresses in exponential phase. To get an idea of the interrelationship of the {sigma}S and {sigma}E regulons in the SSR, we performed similar LT-CSS assays on an rpoS mutant (SMS438) and rpoE rpoS double knockout mutant (SMS592). As shown in Fig. 4, the rpoS mutant exhibited its typical 75- to 100-fold reduced level of survival after 21 d of C-starvation. The rpoE rpoS double mutant exhibited an even more rapid decrease in culture viability during C-starvation than the rpoS or rpoE mutant, ultimately displaying about 600-fold reduced survival after 21 d of C-starvation, compared to its wild-type parent. This suggests that the {sigma}S and {sigma}E regulons act through different ‘pathways’ or mechanisms to maintain cell viability during long-term C-starvation. However, it is possible that they may overlap in some components.



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Fig. 4. LT-CSS of S. Typhimurium wild-type, rpoE, rpoS and rpoE rpoS (double) mutant strains. Strains SL1344 (wild-type; {bullet}; n=8), GVB311 (rpoE mutant; {blacksquare}; n=8), SMS438 (rpoS mutant; {blacktriangleup}; n=1) and SMS592 (rpoE rpoS double mutant, {diamondsuit}; n=3) were starved for a carbon and energy source for a total of 21 d as described in Methods. The data shown are mean values±SEM from the indicated number (n) of separate experiments (except for the rpoS strain).

 
To characterize the interrelationship of the {sigma}E and {sigma}S regulons with respect to CSI cross-resistance, we subjected the rpoS mutant and rpoE rpoS double mutant to the various stress challenges after exponential-phase growth and 5 and 24 h of C-starvation. As reported previously (Jenkins et al., 1988 ; Seymour et al., 1996 ; Spector et al., 1999 ) an rpoS mutant is severely deficient in CSI cross-resistance to H2O2 and heat challenge, exhibiting undetectable levels of viability after 40 min of H2O2 challenge or 16 min at 55 °C (Table 2). As a result of the drastic effect of the rpoS mutation alone, no additional effect could be discerned in the rpoE rpoS double mutant (Figs 3 and 4).

Interestingly, the rpoE rpoS mutant exhibited a comparable decrease in acid tolerance after 5 h of C-starvation, as did 5 h C-starved rpoE mutant cells. This is consistent with the finding that the rpoS mutant is not defective in CSI cross-resistance to pH 3·1 after 5 h of C-starvation (Table 2). However, in 24 h C-starved cells the affect of knocking out both {sigma}S and {sigma}E, in general, has a more dramatic effect than knocking-out each separately (Table 2), supporting the idea that the {sigma}S and {sigma}E regulons act through separate pathways or mechanisms in the SSR and the CSI cross-resistance to acid pH challenge.

It was reported previously (McLeod & Spector, 1996 ) that CSI cross-resistance to PmB challenge was {sigma}S-independent. So, we did not test an rpoS mutant or rpoE rpoS double mutant for CSI cross-resistance to PmB since such experiments would not yield additional useful information.

Additional findings that support the hypothesis that the {sigma}E and {sigma}S regulons appear to act through separate pathways has come from Western blot analysis of {sigma}E and {sigma}S accumulation in rpoS and rpoE mutants, respectively. {sigma}E accumulation during C-starvation was not significantly different in the rpoS mutant compared to the wild-type cell; similarly {sigma}S accumulation during C-starvation was not significantly different in the rpoE mutant compared to the wild-type strain (data not shown).


   DISCUSSION
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The results presented here indicate that C-starvation appears to generate signals capable of inducing the {sigma}E regulon and that one or more members of the {sigma}E regulon are required for the two known functions of the SSR, LT-CSS and CSI cross-resistance to other environmental stresses in S. Typhimurium. A previous report (Humphreys et al., 1999 ) suggested that rpoE mutants might be defective in the utilization of certain C-sources. This was inferred from the findings that an rpoE mutant, compared to wild-type strains, exhibited a longer lag phase and failed to reach as high an optical density after overnight growth in LB or M9 succinate medium in contrast to LB glucose or M9 glucose medium, respectively. This suggests that rpoE mutants might be deficient in the use of oxidatively metabolizable C-sources. Whether this deficiency is relevant to the reason why rpoE mutants survive long-term C-starvation poorly is unclear at this time.

The role of the {sigma}E regulon has been examined in stationary-phase cells where it has been proposed that {sigma}E controls the lysis of dead cells (Nitta et al., 2000 ). However, these studies were performed on cultures at much higher cell densities and cells being subjected to multiple stress conditions simultaneously, e.g. pH stress, oxygen stress or excreted toxic metabolites, depending on the medium used. Nutrient starvation may or may not be the most important stress or the major limiting stress under these conditions. The studies described here were performed on cultures grown to much lower cell densities, up to 2 to 3 logs lower cell densities in some cases, and cells specifically subjected to starvation for an available exogenous carbon-energy source. For this reason it is unlikely that we are examining conditions similar to those described in Nitta et al. (2000) . In that study the authors suggest that {sigma}E controls cell lysis and the amount of nutrients released from dead cells to maintain the viability of a subpopulation of viable cells. This could be hypothesized at the very high cell densities examined in that report where sufficient numbers of cells are dying and lysing to provide nutrients to support growth of other cells in the culture. However, at the cell densities examined here, such cross-feeding is unlikely to occur to any great extent (Spector & Cubitt, 1992 ).

The current model for the activation of the {sigma}E regulatory pathway in E. coli involves several steps involving up to three membrane and periplasmic proteins, encoded by rseABC, themselves part of the {sigma}E regulon (Connolly et al., 1997 ; De Las Penas et al., 1997b ; Missiakas et al., 1997 ; Missiakas & Raina, 1997a , 1998 ; Raivio & Silhavy, 1999 ). Basically in this model, the cytoplasmic domain of the membrane-bound anti-sigma factor RseA sequesters {sigma}E, making it unavailable to bind to RNA polymerase core enzyme. Extracytoplasmic stress leads to the release of {sigma}E from RseA by one or a combination of two possible mechanisms. In the first, RseB binds to the periplasmic domain of RseA and then dissociates from RseA when it ‘senses’ and binds misfolded proteins in the cell envelope. This causes a conformational change in RseA that results in {sigma}E release into the cytoplasm (Missiakas et al., 1997 ; De Las Penas et al., 1997b ). In the second mechanism, a periplasmic protease, DegS, in response to misfolded proteins in the cell envelope, degrades RseA, causing the release of {sigma}E into the cytoplasm (Ades et al., 1999 ). In both cases, {sigma}E is made available for core RNA polymerase re-programming to increase transcription from {sigma}E-dependent promoters. This results in increased expression of {sigma}E itself as well as RseA and RseB. In addition to the rpoE rseABC operon, other reported members of the {sigma}E regulon in E. coli include rpoH, htrA (or degP), fkpA and other unidentified loci (Danese et al., 1995 ; Connolly et al., 1997 ; Danese & Silhavy, 1997 ; Missiakas & Raina, 1997a , 1998 ; Raivio & Silhavy, 1999 ).

Data presented in this paper indicate that (a) {sigma}E, but not CpxR, accumulates during C-starvation and (b) the {sigma}E regulon, but not the CpxR regulon, is required for LT-CSS (Fig. 1) and for CSI cross-resistances to other stresses in S. Typhimurium (Table 2). Therefore, we propose that C-starvation generates extracytoplasmic stress signals capable of activating the {sigma}E regulon, but not the CpxR regulon. If the mechanisms of activation described in E. coli are similar for Salmonella, then C-starvation generates extracytoplasmic signals that cause {sigma}E release from RseA, allowing for increased expression of {sigma}E and its regulon members (Fig. 2).

The finding that the {sigma}E regulon plays a key role in all aspects of the SSR tested to date suggests that C-starvation may lead to misfolding of or damage to proteins in the cell envelope, which would act as signals to induce the {sigma}E regulon. Many of the functions of known {sigma}E regulon members in E. coli involve repair of misfolded proteins such as the peptidyl prolyl isomerase (PPI) activities of FkpA and SurA (Dartigalongue et al., 2001 ). Whether this is true for Salmonella is currently under investigation.

This study represents the first report indicating that the {sigma}E regulon, but not the Cpx regulon, is required for the maximal function of the SSR in Salmonella. The data presented here clearly show that {sigma}E plays a key role in regulating elements of the SSR of S. Typhimurium. As such, {sigma}E (along with {sigma}S and cAMP-CRP) functions as one of the master regulators of the SSR of S. Typhimurium. These data gives us some new insight into the types of stresses encountered by the bacterial cell at the molecular level during C-starvation. Furthermore, like {sigma}S and cAMP-CRP, {sigma}E has previously been found to be required for the full virulence of S. Typhimurium (Humphreys et al., 1999 ). This further supports the hypothesis for a link between the SSR and virulence in this bacterium.


   ACKNOWLEDGEMENTS
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The authors would like to thank John Foster and Bruce White (University of South Alabama) for technical help and useful discussions. The authors are particularly grateful to Carol Gross and Susan Ades (University of California, San Francisco) for their kind gift of antibody to {sigma}E and Thomas Silhavy (Princeton University) for his kind gift of antibody to CpxR. The authors also wish to thank Traci Testerman and Ferric Fang (University of Colorado Health Science Center) for providing the pTFP1 and pTFP2 plasmids prior to publication. Portions of this work were supported by NIH grant no. AI/OD-41170-01 and NSF grant no. MCB-9985981 (to M.P.S.)and BBSRC grants BFP11355 and PRS12222 (to M.R.)


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Received 19 July 2001; revised 24 September 2001; accepted 5 October 2001.