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Microbiology 152 (2006), 1347-1359; DOI  10.1099/mic.0.28744-0
© 2006 Society for General Microbiology

Identification of the {sigma}E regulon of Salmonella enterica serovar Typhimurium

Henrieta Skovierova1, Gary Rowley2, Bronislava Rezuchova1, Dagmar Homerova1, Claire Lewis2, Mark Roberts2 and Jan Kormanec1

1 Institute of Molecular Biology, Centre of Excellence for Molecular Medicine, Slovak Academy of Science, Dubravska cesta 21, 845 51 Bratislava, Slovak Republic
2 Molecular Bacteriology Group, Institute of Comparative Medicine, Department of Veterinary Pathology, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH, UK

Correspondence
Jan Kormanec
jan.kormanec{at}savba.sk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The extracytoplasmic function sigma factor, {sigma}E, has been shown to play a critical role in virulence of Salmonella enterica serovar Typhimurium (S. Typhimurium). The previously optimized two-plasmid system has been used to identify S. Typhimurium promoters recognized by RNA polymerase containing {sigma}E. This method allowed identification of 34 {sigma}E-dependent promoters that direct expression of 62 genes in S. Typhimurium, 23 of which (including several specific for S. Typhimurium) have not been identified previously to be dependent upon {sigma}E in Escherichia coli. The promoters were confirmed in S. Typhimurium and transcriptional start points of the promoters were determined by S1-nuclease mapping. All the promoters contained sequences highly similar to the consensus sequence of {sigma}E-dependent promoters. The identified genes belonging to the S. Typhimurium {sigma}E-regulon encode proteins involved in primary metabolism, DNA repair systems and outer-membrane biogenesis, and regulatory proteins, periplasmic proteases and folding factors, proposed lipoproteins, and inner- and outer-membrane proteins with unknown functions. Several of these {sigma}E-dependent genes have been shown to play a role in virulence of S. Typhimurium.

Abbreviations: ECF, extracytoplasmic function; ESR, extracytoplasmic stress response; E{sigma}E, RNA polymerase holoenzyme containing {sigma}E; OPG, osmoregulated periplasmic glucan(s); OPP, oligopeptide permease; TSP, transcription start point


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Serovars of Salmonella enterica are intracellular pathogens of vertebrates that cause a wide spectrum of diseases. Salmonella enterica serovar Typhimurium (S. Typhimurium) causes a typhoid-like systemic infection in mice and enteritis in humans and other animals. Within its host and in the environment, Salmonella species, like other bacteria, are exposed to a wide variety of stresses. To survive these detrimental conditions, bacteria have evolved a number of stress response systems, including the so-called extracytoplasmic stress response (ESR). In the related species Escherichia coli, the ESR has been shown to be governed by at least three partially overlapping signal transduction pathways: the CpxRA and BaeSR two-component systems and the extracytoplasmic function (ECF) sigma factor RpoE ({sigma}E) (Ruiz & Silhavy, 2005Down). The rpoE gene is located in an operon that includes three downstream genes, rseA, rseB and rseC. The E. coli rpoE gene is essential for cell viability and its expression is autoregulated and induced under conditions leading to the misfolding of periplasmic and outer-membrane proteins, such as heat-shock, and ethanol and osmotic stress. The activity of {sigma}E is controlled by its specific membrane-bound anti-sigma factor, RseA, which, under non-stressed conditions, sequesters the majority of {sigma}E. In response to outer-membrane protein folding perturbations, RseA is cleaved by the successive action of two membrane proteases, DegS and YaeL (RseP), liberating the complex into the cytoplasm where RseA is degraded, freeing {sigma}E to complex with core RNA polymerase to govern expression of {sigma}E-dependent genes (reviewed by Alba & Gross, 2004Down). Two independent molecular genetic approaches have previously identified 58 members of the E. coli {sigma}E regulon, including periplasmic proteases and folding factors, several phospholipids and lipopolysaccharide (LPS) biosynthesis proteins, regulatory proteins, primary metabolism proteins and proteins with unknown function (Dartigalongue et al., 2001Down; Rezuchova et al., 2003Down). Recently, DNA microarray analysis after transient expression of rpoE in exponential- and early-stationary-phase E. coli has identified 156 genes that were significantly upregulated, including the previously reported 31 {sigma}E regulon genes (Kabir et al., 2005Down). This approach increased the number of genes in the {sigma}E regulon to 183, although many of these may be indirectly dependent upon {sigma}E (Kabir et al., 2005Down).

Unlike in E. coli, the rpoE gene in S. Typhimurium is not essential for cell viability, even at high temperature. However, S. typhimurium {sigma}E has been shown to be required for oxidative stress resistance, stationary-phase survival and pathogenicity. S. Typhimurium rpoE mutants are defective in survival and proliferation in macrophage and epithelial cell lines, and are highly attenuated for virulence in a mouse model (Humphreys et al., 1999Down; Kenyon et al., 2002Down; Testerman et al., 2002Down). The reduced virulence of the mutant is partially due to the increased sensitivity to reactive oxygen species produced by host macrophages (Humphreys et al., 1999Down; Testerman et al., 2002Down). S. Typhimurium {sigma}E also plays an important role in resistance to non-oxidative mammalian host defence mechanisms such as antimicrobial peptides (Humphreys et al., 1999Down; Crouch et al., 2005Down). Moreover, the S. Typhimurium rpoE gene has been shown to be up-regulated in S. Typhimurium within macrophages in vitro and murine tissues in vivo (Eriksson et al., 2003Down; Rollenhagen et al., 2004Down). These results indicate that the genes of the {sigma}E regulon should play roles in all these processes. Thus, identification and characterization of the S. Typhimurium {sigma}E regulon may reveal new genes involved in the virulence and survival of S. Typhimurium in the host.

Although organization of the S. Typhimurium rpoE operon resembles its counterpart in E. coli, its regulation is slightly different. Expression of S. Typhimurium rpoE is governed by three promoters, including one, rpoEp3, directly recognized by RNA polymerase holoenzyme containing {sigma}E (E{sigma}E). Like its E. coli counterpart, the rpoEp3 promoter is partially induced by heat shock and osmotic stress, but it is most strongly induced by cold shock and entry into stationary phase (Miticka et al., 2003Down). By using the {sigma}E-dependent rpoEp3 promoter, we optimized the previously established E. coli two-plasmid system for the identification of promoters recognized by S. Typhimurium {sigma}E. The S. Typhimurium rpoE gene was cloned in an expression plasmid under the control of an inducible promoter and the rpoEp3 promoter was cloned upstream of a reporter gene in a compatible promoter-probe plasmid. The promoter was active in the E. coli two-plasmid system only after induced expression of S. Typhimurium rpoE, with a transcription start point (TSP) identical to that in S. Typhimurium (Miticka et al., 2003Down). In the present paper, we have used this optimized E. coli two-plasmid system to identify and locate S. Typhimurium {sigma}E-dependent promoters directing expression of genes which belong to the S. Typhimurium {sigma}E regulon. Moreover, the deduced functions of the identified genes are discussed in relation to the virulence of S. Typhimurium.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Bacterial strains, plasmids and culture conditions.
S. Typhimurium SL1344 (Hoiseth & Stocker, 1981Down) was used for chromosomal DNA preparation. E. coli XL-1 Blue (Stratagene) was used as a host for cloning experiments. The E. coli plasmid pSB40 (Park et al., 1989Down) was kindly provided by Dr M. K. Winson, University of Nottingham. The expression plasmid pAC7 has been described by Rezuchova & Kormanec (2001)Down. Plasmid pAC-rpoEST4 containing the S. Typhimurium rpoE gene under the control of the arabinose-inducible PBAD promoter has been described by Miticka et al. (2003)Down. For RNA isolation, E. coli with the corresponding plasmids was inoculated in LB medium (Ausubel et al., 1995Down) supplemented with ampicillin (50 µg ml–1) and chloramphenicol (40 µg ml–1) and grown at 37 °C to exponential phase (OD600=0·3). Expression of S. Typhimurium rpoE was induced for 3 h with 0·0002 % (w/v) arabinose. To grow S. Typhimurium with rpoE artificially expressed for RNA isolation, S. Typhimurium SL1344 containing pAC-rpoEST4 or pAC7 (as negative control) were grown in LB with 40 µg chloramphenicol ml–1 to exponential phase (OD600=0·24) and expression of rpoE was induced for 3 h with 0·2 % (w/v) arabinose. Conditions for E. coli growth and transformation were as described by Ausubel et al. (1995)Down.

DNA manipulations.
DNA manipulations in E. coli were performed as described by Ausubel et al. (1995)Down. Nucleotide sequencing was performed by the chemical method (Maxam & Gilbert, 1980Down) and by the dideoxy chain-termination method (Sanger et al., 1977Down), using a TaqTrack kit (Promega). An S. Typhimurium SL1344 genomic library was prepared by cloning 0·5–1·2 kb partial Sau3AI chromosomal DNA fragments into the BamHI site of pSB40. About 120 000 original clones obtained from the transformation of E. coli XL-1 Blue were used for total plasmid isolation by using a Qiagen plasmid purification kit. The clones were statistically checked for the presence of insert and all the picked clones contained fragments in the range 0·5–1·2 kb.

Detection of E. coli clones containing the rpoE-dependent promoter fragment.
The S. Typhimurium SL1344 genomic library was transformed into E. coliXL-1 Blue containing the compatible plasmid pAC-rpoEST4. The clones were selected on LBACX plates (LB medium with 5 g lactose l–1, 100 µg ampicillin ml–1, 40 µg chloramphenicol ml–1, 20 µg X-Gal ml–1) with 2 µg arabinose ml–1 as described by Miticka et al. (2003)Down. The colonies were screened after 24 h growth at 37 °C. Blue clones were inoculated in parallel onto two LBACX plates containing either 2 µg arabinose ml–1 (LBACX-ARA) or 2 mg glucose ml–1 (LBACX-GLU), respectively. Clones that were blue on LBACX-ARA and white on LBACX-GLU were inoculated into 1 ml LB with 100 µg ampicillin ml–1 and grown overnight at 37 °C. Cells were pelleted, resuspended in 200 µl STE buffer (0·1 M NaCl, 10 mM Tris/HCl, pH 8, 1 mM EDTA) with 0·5 mg lysozyme ml–1, incubated for 5 min at room temperature, boiled for 1·5 min and centrifuged for 10 min at 16 000 g. One microlitre of supernatant was transformed in parallel into E. coli XL-1 Blue strains harbouring either pAC-rpoEST4 or pAC7 and plated onto LBACX-ARA.

Isolation of RNA and S1-nuclease mapping.
After finishing growth, cell suspensions of E. coli or S. Typhimurium were immediately poured into 50 ml Falcon tubes containing about 15 ml crushed ice prechilled to –80 °C, then the cells were centrifuged, washed with DEPC-treated ice-cold 0·15 M NaCl and total RNA was prepared as described by Kormanec (2001)Down. High-resolution S1-nuclease mapping was performed according to Kormanec (2001)Down. Samples (40 µg) of RNA were hybridized to approximately 0·02 pmol of a suitable DNA probe labelled at one 5' end with [{gamma}-32P]ATP [approx. 3x106 c.p.m. (pmol probe)–1] and treated with 120 U S1-nuclease. The probes for S1-nuclease mapping of the proposed S. Typhimurium {sigma}E-dependent promoters in the E. coli two-plasmid system were prepared by PCR amplification from the corresponding pEST plasmid (pEST1-pEST124) isolated from the positive clone using the 5' end-labelled universal oligonucleotide primer –47 from the lacZ{alpha}-coding region of the pEST plasmid, and primer mut80 from the 5' region flanking the BamHI cloning site of pSB40 (Table 1Down). The probes for S1-nuclease mapping for in vivo verification in S. Typhimurium were prepared by PCR amplification from the corresponding pEST plasmid using the 5' end-labelled internal reverse primer from the corresponding coding region (Table 1Down), and the direct primer mut80. Oligonucleotides were labelled at their 5' ends with [{gamma}-32P] (4500 Ci mmol–1; ICN) and T4 polynucleotide kinase. The labelled DNA fragments were isolated from polyacrylamide gels as described by Kormanec (2001)Down. The RNA-protected DNA fragments were analysed on DNA sequencing gels together with G+A and T+C sequencing ladders derived from the end-labelled fragments (Maxam & Gilbert, 1980Down).


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  		Table 1. Primers used in this study

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Identification of the S. Typhimurium promoters recognized by E{sigma}E using the E. coli two-plasmid system
To identify S. Typhimurium {sigma}E-dependent promoters, we used the optimized E. coli two-plasmid screening system that was successfully used for the identification of the E. coli {sigma}E regulon (Rezuchova et al., 2003Down). This method assumes that the E. coli RNA polymerase core enzyme will interact with a particular heterologous sigma factor expressed from one plasmid, and that the resulting holoenzyme can recognize a promoter present in a library of chromosomal fragments cloned in the second compatible plasmid, upstream of a reporter gene. This E. coli two-plasmid system was optimized using the S. Typhimurium {sigma}E-dependent rpoEp3 promoter. The S. Typhimurium rpoE gene was cloned into expression plasmid pAC7 under the control of an arabinose-inducible PBAD promoter, resulting in plasmid pAC-rpoEST4. Following arabinose-induced expression of S. Typhimurium rpoE, E. coli RNA polymerase holoenzyme containing S. Typhimurium {sigma}E (E{sigma}E) was able to recognize the rpoEp3 promoter cloned upstream of the lacZ{alpha} reporter gene in the second compatible plasmid. Moreover, the transcription of the rpoEp3 promoter was initiated from the identical TSP as in S. Typhimurium (Miticka et al., 2003Down). These results indicated that this optimized E. coli two-plasmid system could be used for identification of S. Typhimurium {sigma}E-dependent promoters. For this purpose, an S. Typhimurium genomic library cloned into pSB40 was used to transform E. coli XL-1 Blue containing pAC-rpoEST4. After screening of about 120 000 colonies on LBACX-ARA plates, 5040 blue clones that represented promoters active in E. coli (including {sigma}E-dependent promoters) were picked up. After further selection of the identified blue clones on LBACX-ARA and LBACX-GLU plates, the plasmids were isolated from 1020 clones and transformed in parallel into E. coli XL-1 Blue with pAC7 and pAC-rpoEST4, respectively. Colonies were screened on LBACX-ARA plates. Clones containing plasmids with {sigma}E-dependent promoters were blue in E. coli XL-1 Blue with pAC7-rpoEST4 and white in E. coli XL-1 Blue containing pAC7. Clones with {sigma}E-independent promoters were blue in both strains. With this last screen we identified 124 positive clones containing putative {sigma}E-dependent promoters (plasmids pEST1–pEST124). Sequencing of the DNA fragments revealed 34 representatives. Although the quality of the library was high (about 120 000 original clones correspond to a calculated probability greater than 0·99999), we cannot rule out that the S. Typhimurium library used covered the complete genome. Several representatives of the {sigma}E-dependent promoters were found more than 10 times and some were found only once. Therefore, the number of identified {sigma}E-dependent promoters may not be complete.

Characterization of S. Typhimurium promoters recognized by E{sigma}E
To locate the TSP of the identified S. Typhimurium {sigma}E-dependent promoters, high-resolution S1-nuclease mapping was performed using RNA isolated from E. coli XL-1 Blue, containing a corresponding pEST plasmid bearing a particular {sigma}E-dependent promoter and pAC-rpoEST4, grown to exponential phase and induced by arabinose. The 5'-labelled DNA probes were prepared from the corresponding pEST plasmid with external primers, enabling the location of the putative {sigma}E-dependent promoter only in the pEST plasmid-bearing DNA fragment. In all cases, RNA-protected fragments were identified only using RNA from E. coli XL-1 Blue with the corresponding pEST plasmid and pAC-rpoEST4 grown under conditions inducing S. Typhimurium rpoE. No RNA-protected fragment was identified with a control RNA from E. coli containing a particular pEST plasmid and pAC7 grown under similar conditions. To investigate the activities of these putative {sigma}E-dependent promoters in their chromosomal location in S. Typhimurium, and to confirm their dependence upon {sigma}E, high-resolution S1-nuclease mapping was performed using the 5'-labelled probes prepared from the corresponding pEST plasmids using internal reverse primers from the coding regions of the corresponding {sigma}E-dependent S. Typhimurium genes and RNA isolated from S. Typhimurium SL1344 containing pAC-rpoEST4 or pAC7, respectively, grown to exponential phase and induced for 3 h with arabinose. RNA-protected fragments were identified using RNA isolated from S. Typhimurium SL1344 containing pAC-rpoEST4, and grown to exponential phase with rpoE expression artificially induced with arabinose (Fig. 1Down, lanes 1). No RNA-protected fragment was identified with control RNA from S. Typhimurium SL1344 containing pAC7, grown to exponential phase and also induced with arabinose (Fig. 1Down, lanes 2). The TSPs of the identified promoters were in the identical positions as for the {sigma}E-dependent promoter in the E. coli two-plasmid system located on the corresponding pEST plasmid. Thus, these results indicated that the chromosomally located promoters are dependent in vivo on {sigma}E in S. Typhimurium. By using this strategy, 34 {sigma}E-dependent promoters were localized and verified in vivo in S. Typhimurium. Comparison of the nucleotide sequences upstream of the identified TSPs (Fig. 2Down) revealed a consensus promoter sequence that is similar to that of {sigma}E of E. coli (Rezuchova et al., 2003Down). Interestingly, based on the generated sequence logo (Fig. 2bDown), another residue, a G preceding the –10 region, appeared to be conserved in the {sigma}E-dependent promoters, thus suggesting a new {sigma}E-consensus sequence, GGAACTT-N15-GTCTAA. The generated logo and the conservation of nucleotides within the –35 and –10 regions (Fig. 2Down) correlates well with our experimental analysis of the importance of specific bases within the S. Typhimurium {sigma}E-dependent rpoEp3 promoter for binding with E{sigma}E. This mutagenesis analysis identified the bases shown in upper case letters as the most important in the –35 (ggAActt) and –10 (TctaA) regions (Miticka et al., 2004Down). Interestingly, as in E. coli (Rezuchova et al., 2003Down), in almost all cases (except surAp), strictly conserved spacing between the –10 and –35 recognition sites was found in S. Typhimurium {sigma}E-dependent promoters (Fig. 2Down). In several cases, additional {sigma}E-independent promoters were identified, in addition to the corresponding {sigma}E-dependent promoter, that direct expression of the corresponding gene of the S. Typhimurium {sigma}E regulon (Fig. 1Down).


Figure 1
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  		Fig. 1. Examples of TSP determination for S. Typhimurium {sigma}E-dependent promoters by high-resolution S1-nuclease mapping. The particular 5'-labelled DNA fragment was hybridized with 40 µg RNA isolated from exponentially grown S. Typhimurium SL1344 containing pAC-rpoEST4 (lanes 1) or pAC7 (lanes 2) and induced for 3 h with 0·2 % arabinose. The RNA-protected DNA fragments were analysed on DNA sequencing gels together with G+A (lane A) and T+C (lane T) sequencing ladders derived from end-labelled fragments (Maxam & Gilbert, 1980Down). Thin horizontal arrows indicate the positions of RNA-protected fragments and thick angled arrows indicate the nucleotide corresponding to TSP. Before assigning the TSP, 1·5 nt was subtracted from the length of the protected fragment to account for the difference in the 3' ends resulting from S1-nuclease digestion and the chemical sequencing reactions. In some cases, the thick angled arrows indicate {sigma}E-independent promoters. All S1-nuclease mapping experiments were performed twice with independent sets of RNA with similar results.

 

Figure 2
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  		Fig. 2. (a) Nucleotide sequence alignment of the S. Typhimurium {sigma}E-dependent promoters. The corresponding –10 and –35 regions are depicted in bold. The TSP is in bold and underlined. The S. Typhimurium {sigma}E consensus sequence is shown below the alignment. (b) Determination of the S. Typhimurium {sigma}E consensus sequence. The aligned promoter sequences were analysed using the WebLogo program (http://weblogo.berkeley.edu/). The sequences were trimmed at the 3' end to make them all the same length as required by the program. Also, a ‘C’ residue in the spacer region of the surAp promoter was removed to make it the same length as the rest of the promoters. The height of a stack indicates sequence conservation (2=100 % conservation) and the height of each individual nucleotide within the stack indicates its relative frequency at that position.

 
Identification of S. Typhimurium {sigma}E-dependent genes
Comparison of the nucleotide sequence downstream of identified promoters with the complete sequence of S. Typhimurium LT2 (http://genomeold.wustl.edu/projects/bacterial/styphimurium/) and almost completed sequence of S. Typhimurium SL1344 (www.sanger.ac.uk/Projects/Salmonella) revealed the genes directed by the identified S. Typhimurium {sigma}E-dependent promoters (Table 2Down). The 34 identified {sigma}E-dependent promoters control expression of 62 genes found in both Salmonella genomes, including 18 single genes and 13 proposed operons. Possible operon structures of the {sigma}E-dependent genes were predicted on the basis of close gene arrangements and transcription direction in the genomic sequence of S. Typhimurium (in many cases ORFs in operons were translationally coupled). One operon (stm1250, stm1251) was controlled by two tandem {sigma}E-dependent promoters. Interestingly, 13 {sigma}E-dependent promoters were located in the coding region of the upstream convergent genes. Based on these data, these 62 {sigma}E-dependent genes probably constitute the {sigma}E regulon in S. Typhimurium.


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  		Table 2. Function and genetic organization of genes directed by {sigma}E in S. Typhimurium SL1344

IM, Inner membrane; OM, outer membrane; HP, hypothetical protein; asterisks indicate the presence of an internal {sigma}E-dependent promoter; in all cases putative or known promoters lie to the left of the leftmost genes.

 
Members of the {sigma}E regulon common in S. Typhimurium and E. coli
Three independent approaches have already identified 183 {sigma}E-dependent genes in E. coli (Dartigalongue et al., 2001Down; Kabir et al., 2005Down; Rezuchova et al., 2003Down). As in E. coli, the genes regulated by S. Typhimurium {sigma}E fall into similar functional categories, including periplasmic proteases and folding factors, proteins involved in cell membrane integrity and in phospholipid and LPS biosynthesis, regulatory proteins, primary metabolism proteins and membrane or periplasmic proteins of unknown function (Table 2Up). Of the 62 identified S. Typhimurium {sigma}E-dependent genes, 39 orthologues (rpoE, rseA, rseB, rseC, rpoH, rpoD, fusA, tufA, htrA, recB, surA, pdxA, ksgA, apaG, apaH, fkpA, plsB, psd, yjeP, lpxP, yaeT, hlpA, lpxD, yfiO, tolA, tolB, pal, ybgF, yabI, ycbK, ycbL, yeaY, yfeY, yiiD, sbmA, yaiW, yraP, ygiM, yggN) have been shown previously to be {sigma}E-dependent in E. coli. Recently, two of the identified members of the {sigma}E regulon, YaeT and YfiO, have been shown to form a multicomponent complex together with YfgL and NlpB proteins which is essential for the assembly of proteins in the outer membrane of E. coli (Wu et al., 2005Down), thus assigning a role of these two previously uncharacterized proteins in outer-membrane biogenesis.

Three of these {sigma}E-dependent genes have hitherto been shown to be involved in Salmonella virulence. The surA gene, encoding a periplasmic peptidylprolyl-cis-trans-isomerase (PPIase) involved in protein folding, has a role in adherence and invasion of host eukaryotic cells. Furthermore, the S. Typhimurium surA mutant was attenuated when administrated orally or intravenously to BALB/c mice and the S. Typhimurium surA mutant demonstrated potential as a vaccine candidate (Sydenham et al., 2000Down). In contrast, the other {sigma}E-dependent PPIase-encoding gene, fkpA, has only a minor effect on the ability of S. Typhimurium to invade and survive within epithelial cells and macrophages and cause infection in mice. However, the effect of the fkpA mutation on S. Typhimurium virulence was more profound if the mutation was combined with a mutation in surA, or in another member of the {sigma}E regulon, htrA (Humphreys et al., 2003Down). The htrA (degP) gene encodes a periplasmic protease essential for degradation of damaged proteins. In E. coli, HtrA is required for survival at high temperatures (Strauch et al., 1989Down); in contrast, S. typhimurium htrA mutant strains are not temperature-sensitive, but are more sensitive to oxidizing agents and are required for survival within macrophages and for virulence in mice (Johnson et al., 1991Down; Humphreys et al., 1999Down). However, the difference in S. Typhimurium rpoE and htrA mutants in terms of the degree of their attenuation in mice and their sensitivity to noxious agents indicated that additional genes in the {sigma}E regulon should play a critical role in virulence and in combating a variety of stresses (Humphreys et al., 1999Down).

Differentially regulated genes of the {sigma}E regulon in S. Typhimurium and E. coli
Intriguingly, the {sigma}E-dependent promoters of several S. Typhimurium genes were different to their counterparts previously identified in E. coli. The expression of the rpoE, rseA, rseB, rseC operon is governed by a {sigma}E-dependent promoter located in an almost identical position to a highly similar sequence (identical –35 and –10 conserved regions) in both S. Typhimurium and E. coli (Miticka et al., 2003Down). Likewise, S. Typhimurium {sigma}E-dependent promoters rpoHp, htrAp, sbmAp, fkpAp, fusAp, psdp, lpxP, yeaYp and yggNp were highly similar (with almost identical –35 and –10 conserved regions) and located in almost identical positions to their counterparts in E. coli. However, the sequences and locations of both S. Typhimurium {sigma}E-dependent rpoDp promoters were different from their counterpart (rpoDp3) in E. coli (Dartigalongue et al., 2001Down). We found that the reported E. coli {sigma}E-dependent rpoDp3 promoter is located in a similar position to the previously located {sigma}H-dependent promoter in the E. coli rpoD gene (Taylor et al., 1984Down). Moreover, we have identified this {sigma}H-dependent promoter in an identical position in S. Typhimurium (Fig. 1Up). A similar discrepancy has been found for the S. Typhimurium {sigma}E-dependent promoters yfiOp, yraPp and ygiMp. The {sigma}E-dependent promoters of all their E. coli counterparts (ecfDp, ecfHp and ecfGp) have been located further downstream (Dartigalongue et al., 2001Down) and display only very weak similarity to the {sigma}E consensus sequence (Rezuchova et al., 2003Down; Miticka et al., 2004Down). The signals located by Dartigalongue et al. (2001)Down may thus correspond to the premature termination of the reverse transcriptase, as they used primer extension analysis for the location of the {sigma}E-dependent promoters. In our case, we verified {sigma}E-dependent promoters using the more reliable S1-nuclease mapping technique (Kormanec, 2001Down). Moreover, we found sequences highly similar to S. Typhimurium {sigma}E-dependent promoters (with identical –35 and –10 regions) in similar positions upstream of E. coli genes, suggesting they may correspond to the {sigma}E-dependent promoters in this species. However, we cannot rule out the possibility that these {sigma}E-dependent genes are differentially expressed in the two species. This also may be the case for surA and the yaeL (ecfE), yaeT (ecfK), hlpA (skp), lpxD operon. In E. coli, expression of surA is proposed to be governed by a {sigma}E-dependent promoter (which does not fit the {sigma}E consensus sequence) 176 bp upstream of the imp (ostA) gene that is located upstream of surA (Dartigalongue et al., 2001Down). However in S. Typhimurium, the {sigma}E-dependent surAp promoter has been located at the 3' end of the imp (ostA) coding region.

In the case of the {sigma}E-dependent yaeL (ecfE), yaeT (ecfK), hlpA (skp), lpxD operon in E. coli, three proposed {sigma}E-dependent promoters were identified, none of which fit the {sigma}E consensus sequence (Dartigalongue et al., 2001Down). The first proposed {sigma}E-dependent promoter, ecfEp, was located upstream of yaeL (ecfE), the second, skpp, was located upstream of the hlpA (skp) gene, and the third, lpxDp2, was located at the end of the hlpA (skp) coding region, upstream of the lpxD gene (Dartigalongue et al., 2001Down). However, in S. Typhimurium, we have identified only one {sigma}E-dependent promoter in this region, yaeTp, which is located in the yaeL coding region upstream of the yaeT gene. We have analysed the whole of the S. Typhimurium yaeL, yaeT, hlpA, lpxD operon region for other potential {sigma}E-dependent promoters but we were unable to identify any {sigma}E-dependent promoters in the regions corresponding to the proposed E. coli {sigma}E-dependent promoters, although there were several {sigma}E-independent promoters (data not shown). As with the previous cases, we have identified sequences highly similar to the {sigma}E-dependent promoter yaeTp (with almost identical conserved –35 and –10 regions) in a similar position in E. coli, indicating that this is likely to be the {sigma}E-dependent promoter directing expression of yaeT and downstream genes in E. coli. However, as for the previous promoters, we cannot rule out that there may be differences in the regulation of {sigma}E-dependent genes between S. Typhimurium and E. coli.

Members of the {sigma}E regulon specific for S. Typhimurium
We identified 23 S. Typhimurium {sigma}E-dependent genes (ptr, recD, tolR, oppA, oppB, oppC, oppD, oppF, stm1741, eno, yggT, yggU, yggV, yggW, yjfO, yjfN, yiaD, dedD, ydcG, yfeK, yfeL, stm1250, stm1251) that have not been previously identified to be dependent upon {sigma}E in E. coli. The inferred functions of some of these new members of {sigma}E regulon fall broadly into the same categories as previously described for the {sigma}E regulon (Dartigalongue et al., 2001Down; Kabir et al., 2005Down; Rezuchova et al., 2003Down). Interestingly, in addition to the well characterized periplasmic serine protease HtrA (DegP), another periplasmic protease, Protease III, belongs to the {sigma}E regulon in S. Typhimurium. Protease III (Pitrilysis), the product of the ptr gene, is a periplasmic metalloprotease with specificity towards insulin and other low-molecular-mass substrates. The physiological role of Protease III is not known (Dykstra & Kushner, 1985Down; Swamy & Goldberg, 1982Down). It is thought that Protease III is involved in the turnover of proteins in the periplasmic space (Baneyx & Georgiou, 1991Down; Betton et al., 1998Down; Cornista et al., 2004Down). Thus, increased levels of Protease III may be needed after envelope stress. Expression of the ptr gene has been partially characterized in E. coli. A single promoter, 127 bp upstream from the start codon of ptr, has been identified in the upstream region (Claverie-Martin et al., 1987Down). Interestingly, no signal corresponding to this promoter region was identified in S. Typhimurium, although another {sigma}E-independent promoter was identified downstream of the {sigma}E-dependent ptrp promoter in the recC coding region 582 bp upstream from the start codon of ptr (data not shown). This indicates that ptr is differentially expressed in E. coli and S. Typhimurium and that ptr may not belong to the {sigma}E regulon in E. coli. As in E. coli, the S. Typhimurium ptr gene is intriguingly located between the recC and recBD genes which encode subunits of exonuclease V involved in DNA repair and genetic recombination. As the stop and start codons of ptr, recB and recD overlap, it is suggested that these genes may be part of an operon. The {sigma}E-dependent ptrp promoter may therefore also regulate expression of the downstream recBD genes, indicating a new role for the {sigma}E regulon in DNA repair and recombination. Actually, the recB gene has been recently found to be dependent upon {sigma}E in E. coli (Kabir et al., 2005Down). Interestingly, mutants of S. Typhimurium lacking the recBC function are avirulent in mice and unable to grow inside macrophages, and it has been suggested that S. Typhimurium uses this RecBCD recombination pathway to repair DNA double-strand breaks produced during growth inside macrophages (Buchmeier et al., 1993Down). Thus, recBC may be additional genes in the {sigma}E regulon that have a critical role in virulence.

One of the identified S. Typhimurium {sigma}E-dependent promoters has been located in the coding region of the tolQ gene in the ybgC, tolQ, tolR, tolA, tolB, pal, ybgF gene cluster. In E. coli, the genes in this cluster appear to be transcribed from two constitutive promoters, one immediately upstream of ybgC and the other upstream of tolB, and producing two transcripts: ybgC, tolQRAB, pal, ybgF and tolB, pal, ybgF (Vianney et al., 1996Down). The tolQRAB and pal genes are conserved in most Gram-negative bacteria and encode proteins of the Tol–Pal system that are implicated in the maintenance of cell envelope integrity and in the transport of newly synthesized components through the periplasm. This system has also been found to facilitate the uptake of filamentous phage DNA and group A colicins. No obvious phenotypes have been assigned to ybgC and ybgF, which encode cytoplasmic and periplasmic proteins, respectively (Lazzaroni et al., 1999Down; Cascales & Lloubes, 2004Down). Recently, it has been shown that the TolA protein is required for the correct surface expression of the E. coli O7 antigen, thus demonstrating a role of the Tol–Pal system in LPS biogenesis. Interestingly, E. coli tolA and pal mutants, which are associated with defects in the bacterial cell envelope, elicit a specific {sigma}E-mediated ESR that in turn reduces wzy-dependent O antigen polymerization (Vines et al., 2005Down). Increased expression of the mainly periplasmic component of the Tol–Pal system from the internal {sigma}E-dependent tolRp promoter may help S. Typhimurium to cope with extracytoplasmic stress by upregulating the production of proteins that are essential for cell envelope integrity. Interestingly, except for tolR, all other genes encoding the Tol–Pal system have been found recently to be dependent upon {sigma}E in E. coli (Kabir et al., 2005Down). This indicates a different {sigma}E-dependent regulation of this system in the two organisms. Moreover, an S. Typhimurium tolB mutant exhibits increased sensitivity to antimicrobial peptides and is less virulent than its wild-type parent as a consequence of the loss of outer membrane stability (Tamayo et al., 2002Down).

A {sigma}E-dependent promoter has been located upstream of the S. Typhimurium oppA gene. The oppABCDF operon encodes proteins of the major oligopeptide permease (Opp) that belongs to the ABC transporter superfamily. Opp is the major peptide transport system of enteric bacteria, essential for the uptake of oligopeptides from growth medium and for the uptake and recycling of cell-wall peptides for synthesis of peptidoglycan. In addition to nutrient acquisition, peptide transporters have been shown to play an important role in a diverse array of other functions, including chemotaxis, quorum sensing and conjugation (Detmers et al., 2001Down; Higgins, 1992Down). Interestingly, OppA of E. coli also has a chaperone-like function, indicating that OppA, together with some other periplasmic substrate-binding proteins, might be involved in protein folding and protection from stress in the periplasm (Richarme & Caldas, 1997Down). Thus Opp seems to fall into the functional categories of the {sigma}E regulon and its increased production may be needed under conditions of envelope stress. Expression of the opp operon has been suggested to be constitutive in S. Typhimurium, but the OppA protein intriguingly accumulates in the periplasm as cells reach stationary phase (Hiles et al., 1987Down). Analysis of the genomic sequence of S. Typhimurium has revealed that, in contrast to E. coli, the S. Typhimurium opp operon is followed by a potentially cotranscribed gene, stm1741, which encodes a putative membrane transport protein similar to voltage-gated ion channels. In E. coli two promoters, P2 and P3, directing expression of the opp operon have been identified and localized, and there is an additional promoter, P1, which originates in the IS2 sequence present in some E. coli strains (Igarashi et al., 1997Down). In addition to the {sigma}E-dependent oppAp promoter (Fig. 1Up), we have identified a {sigma}E-independent promoter in S. Typhimurium which has an identical TSP to the E. coli P3 promoter (Fig. 1Up). Comparison of the E. coli and S. Typhimurium oppA promoter regions revealed similarity from the ATG codon up to the P3 promoter and in the sequence upstream of the E. coli P2 promoter. However, there was no similarity around the S. Typhimurium {sigma}E-dependent oppAp promoter, indicating that the E. coli oppA operon is probably not {sigma}E-regulated.

The eno gene encodes the glycolytic enzyme enolase which implicates the {sigma}E regulon in primary metabolism. Interestingly, enolase has been shown to be a functional part of the membrane-associated RNA degradosome complex essential for mRNA turnover (Bernstein et al., 2004Down; Carpousis, 2002Down). Therefore, another proposed role for the {sigma}E regulon is in specific mRNA turnover during particular conditions of metabolic stress.

Of the remaining S. Typhimurium {sigma}E-dependent genes, 11 (yggT, yggU, yggV, yggW, yjfO, yjfN, yiaD, dedD, ydcG, yfeK, yfeL) have homologues in E. coli and two (stm1250 and stm1251) are specific to S. Typhimurium. An S. Typhimurium {sigma}E-dependent promoter has been localized in the coding region of the yggSTUVW operon (Table 2Up). The proposed {sigma}E-dependent yggTUVW genes encode mainly proteins with unknown function (integral membrane protein, putative cytoplasmic protein, putative xanthosine triphosphate pyrophosphatase and putative oxidase). However, in E. coli, the yggV (rdgB) gene has been shown to have a role in DNA repair during DNA replication, most probably due to its xanthosine triphosphate pyrophosphatase activity which helps in avoiding chromosome fragmentation (Bradshaw & Kuzminov, 2003Down). Thus, this is likely to be a further gene of the {sigma}E regulon in S. Typhimurium, in addition to recB and recD, with a role in DNA repair and recombination.

Seven S. Typhimurium {sigma}E-dependent genes encode putative outer-membrane lipoproteins that contain a signal sequence typical of bacterial lipoproteins followed by a characteristic lipobox containing a Cys residue which could serve as the lipid attachment site. These include four homologues, YfiO, YraP, YeaY and YfeY, to the recently characterized six {sigma}E-dependent lipoproteins from E. coli (Onufryk et al., 2005Down) and three other putative outer-membrane lipoproteins YaiW, YjfO and YiaD (Table 2Up). For the yjfN, dedD and yfeK genes no function could be predicted, although they all encode putative membrane or periplasmic proteins (Table 2Up), thus indicating a possible function in the cell envelope. The yfeK gene is translationally coupled to the yfeL gene encoding a putative membrane carboxypeptidase (penicillin-binding protein), probably involved in cell envelope biogenesis. The ydcG gene encodes a putative periplasmic glucan biosynthesis protein. It is an orthologue of the recently characterized ydcG gene (renamed mdoD) encoding the periplasmic OpgD protein involved in the control of the structural glucose backbone of osmoregulated periplasmic glucans (OPG) in E. coli. Expression of the ydcG/mdoD gene increases during stationary phase (Lequette et al., 2004Down). Interestingly, mutants defective in OPG synthesis were shown to be highly attenuated or avirulent in several pathogenic bacteria, including S. Typhimurium. The OPG seem to be an important component of the cell envelope under extreme environmental conditions and especially during interactions between pathogenic bacteria with their eukaryotic host (Bohin, 2000Down). Moreover, OPG have been shown to be essential for resistance to SDS and other anionic detergents (Rajagopal et al., 2003Down). Hence, all these phenotypes clearly fall into the typical characteristics of the S. Typhimurium {sigma}E regulon.

In the case of the stm1250 and stm1251 genes, two S. Typhimurium {sigma}E-dependent promoters, stm1250p and stm1251p, were localized in close proximity, with TSPs just 394 bp apart. The stm1250p promoter is located upstream of stm1250 which encodes a putative cytoplasmic protein, and the stm1251p promoter is located in the stm1250 coding region, directing expression of the downstream stm1251 gene which encodes a putative molecular chaperone or small heat-shock protein. Both proteins appear to be specific for Salmonella species. However, significant similarity (31–42 % aa identity) to STM1251 has been found with several putative molecular chaperones or small heat-shock proteins of the Hsp20 family from Gram-negative bacteria, including the E. coli and S. Typhimurium small heat-shock proteins IbpA and IbpB (32 and 31 % identity, respectively). Based on the S1-nuclease mapping analysis, it is clear that both genes, though separated by a 151 bp intergenic region, form an operon and, interestingly, a further heat-shock-inducible promoter having the {sigma}H consensus sequence has been localized in this intergenic region, directing expression of stm1251 (data not shown). The product of the stm1251 gene has been partially characterized recently in S. Typhimurium. This gene encodes a novel small heat-shock protein named AgsA (the gene has been renamed agsA). Together with the two other small heat-shock proteins, IbpA and IbpB, AgsA has been proved to be an effective chaperone preventing aggregation of non-native cytoplasmic proteins and maintaining them in a state competent for refolding in S. Typhimurium at high temperatures (Tomoyasu et al., 2003Down). Hence, its {sigma}E-dependence indicates a partial overlap of the cytoplasmic stress response ({sigma}H-dependent) and ESR ({sigma}E-dependent) in S. Typhimurium. This overlap has also been described recently in S. Typhimurium, where activation of {sigma}E has been shown to enhance expression of the {sigma}S regulon via {sigma}H and Hfq during stationary phase, indicating that interactions between alternative sigma factors {sigma}E, {sigma}H and {sigma}S permit the integration of various stress signals to produce coordinated responses (Bang et al., 2005Down). This study, which was published during the writing of this manuscript, also provided supplementary material detailing S. Typhimurium DNA microarray data on the expression profile of {sigma}E-dependent genes, based on the different stationary-phase mRNA levels in wild-type and rpoE mutant strains. Comparison of our {sigma}E-dependent genes with this transcriptional profiling data revealed that, although 19 genes were at least twofold down-regulated in the rpoE mutant, many S. Typhimurium {sigma}E-dependent genes that we have identified were unaffected in the rpoE mutant, including such clear {sigma}E-dependent genes like rpoH, fkpA, htrA, surA, yraP and others. This discrepancy may result from the use of overnight cultures for isolation of RNA from stationary-phase cultures for the DNA microarray experiment. Although {sigma}E is clearly induced in stationary phase (Miticka et al., 2003Down; Testerman et al., 2002Down), in our detailed transcriptional experiment of S. Typhimurium {sigma}E activation during growth in LB medium, we found that {sigma}E activity peaked at 7 h and then gradually decreased to a rather low level after 14 h (J. Kormanec, unpublished results). Thus, a lower induction ratio of {sigma}E-dependent genes between wild-type and rpoE mutant strains will be seen in long-term stationary-phase cultures, and differences in expression might not be detected.

In conclusion, in S. Typhimurium, we identified 34 {sigma}E-dependent promoters that can potentially direct the expression of 62 genes; among them, 39 orthologues have been previously shown to be {sigma}E-dependent in E. coli. The identified S. Typhimurium {sigma}E-dependent genes fall into similar functional categories, involved mainly in cell-envelope homeostasis, as previously suggested for the E. coli {sigma}E regulon. However, several new functions have emerged, including a role in DNA repair and recombination, and outer-membrane protein assembly. Recent data on alternative degS-independent induction of {sigma}E during carbon starvation in S. Typhimurium have suggested that members of the {sigma}E regulon, in addition to their function in the repair or elimination of damaged cell-envelope proteins, also have additional functions necessary for the adaptation of cells to new environmental conditions (Kenyon et al., 2005Down). Interestingly, several {sigma}E-dependent genes have been shown to have a critical role in the virulence in S. Typhimurium, thus helping to explain the severe attenuation of S. Typhimurium rpoE mutants. Further work will be needed to characterize the detail of the biochemical function of these {sigma}E-dependent genes and their role in the envelope stress response and virulence in S. Typhimurium. These experiments are in progress.


   ACKNOWLEDGEMENTS
 
We are grateful to M. K. Winson for plasmid pSB40. This work was supported by the Science and Technology Assistance Agency under contract No. APVT-51-012004, a VEGA grant, 2/6010/26, from the Slovak Academy of Sciences, a Wellcome Trust grant, 065027/Z/01/Z, and Wellcome Trust studentships 062631/Z/OO/A and 069099/Z/02/A.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
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