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E and has a role in cell envelope integrity and virulence of Salmonella enterica serovar Typhimurium
1 Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK
2 Institute of Molecular Biology, Slovak Academy of Science, Dubravska cesta 21, 845 51 Bratislava, Slovak Republik
3 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
Correspondence
Mark Roberts
m.roberts{at}vet.gla.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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E. An smpA null mutant was constructed in S. Typhimurium SL1344 and was shown to be more sensitive than its wild-type parent to growth at high temperature and in the presence of sodium cholate, SDS plus EDTA, and the hydrophobic antibiotic rifampicin. The lack of SmpA in S. Typhimurium elicits a E-dependent stress response. These findings are indicative of altered outer-membrane integrity in the smpA mutant, probably due to a defect in outer-membrane protein biogenesis. SmpA was not important for entry or survival within murine macrophages; however, the S. Typhimurium smpA mutant was attenuated in mice by both the oral and parenteral routes of infection, and SmpA appeared to be most important for the growth of S. Typhimurium at systemic sites.
Supplementary figures showing a comparison of the smpA promoter regions in the genera of the Enterobacteriales and an amino acid sequence alignment of SmpA/OmlA homologues of representatives of the three phylogenetic classes of proteobacteria are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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E (Rowley et al., 2006
).
In Salmonella and E. coli, the rpoE gene encoding E is located in an operon rpoE, rseA, rseB, rseC. The activity of E and its gene is tightly regulated. Under non-stress conditions, E is inactive because it is sequestered by its specific membrane-bound anti-sigma factor RseA. In response to perturbations in outer-membrane protein (OMP) folding elicited by various stress conditions, 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 to release E (Alba & Gross, 2004). Liberated E can associate with core RNA polymerase to govern the expression of various genes of the E regulon; these include rpoE itself, genes that encode proteins involved in cell envelope biogenesis and homeostasis, as well as many genes of unknown function (Dartigalongue et al., 2001; Kabir et al., 2005; Rezuchova et al., 2003; Rhodius et al., 2005; Skovierova et al., 2006). In E. coli, rpoE is an essential gene, but this is not the case for many other bacteria, including Salmonella.
S. Typhimurium E is required for oxidative stress resistance, stationary phase survival and pathogenicity. The virulence of S. Typhimurium rpoE mutants is highly attenuated in mouse models of infection and E is critical for survival of S. Typhimurium in macrophages (Humphreys et al., 1999; Testerman et al., 2002). Moreover, E is also essential for resistance to non-oxidative mammalian host defence mechanisms such as antimicrobial peptides (Crouch et al., 2005; Humphreys et al., 1999; Kenyon et al., 2002; Testerman et al., 2002). Therefore, some of the genes of the E regulon should also play a role in pathogenicity.
We have recently identified 62 genes dependent upon E in S. Typhimurium (Skovierova et al., 2006). In addition to many genes common to the E. coli E regulon with a known or suspected role in envelope homeostasis, several genes specific to S. Typhimurium were identified. Also, in both organisms, there are still many genes of unknown function (Dartigalongue et al., 2001; Kabir et al., 2005; Rezuchova et al., 2003; Rhodius et al., 2005; Skovierova et al., 2006).
A number of salmonella envelope proteins are involved in pathogenesis; these envelope proteins or their genes could be targeted in the design of new vaccines and therapeutics. Therefore, we have been investigating the functions of these identified members of the E regulon with an emphasis on their role in pathogenesis. In the present report, we focus on small membrane protein A (SmpA) and its role in the cell envelope integrity and virulence of S. Typhimurium. We have previously found that the smpA gene is a member of the E regulon in E. coli (Rezuchova et al., 2003), and this was recently independently confirmed (Rhodius et al., 2005). However, we did not identify smpA using a similar screening procedure for S. Typhimurium E-regulated genes (Skovierova et al., 2006).
Until recently, almost nothing was known about the function of SmpA in E. coli and related bacteria. Homologous genes are present in several currently sequenced genomes of Gram-negative bacteria (see below). The SmpA homologue OmlA is an outer-membrane lipoprotein in the opportunistic Gram-negative pathogen Pseudomonas aeruginosa, where it is proposed to have a structural role in maintaining cell envelope integrity (Ochsner et al., 1999). Very recently, a comprehensive genetic and biochemical analysis revealed a role for SmpA as a novel structurally and functionally important, but non-essential, member of the multi-component YaeT OMP assembly complex (Sklar et al., 2007). In the present study we characterize the regulation of smpA in S. Typhimurium, construct an S. Typhimurium smpA null mutant, and investigate the role of SmpA in cell envelope integrity and S. Typhimurium virulence.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Bacteria were grown in Luria–Bertani (LB), minimal M9 medium or on LB agar (LA) plates (Miller, 1972). M9 medium was supplemented with 0.2 % glucose, 1 mM MgSO4 and 0.2 mM histidine. When required, the media were supplemented with 100 µg ampicillin ml–1, 40 µg chloramphenicol ml–1, 50 µg gentamicin ml–1 or 50 µg kanamycin ml–1.
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). The MIC of compounds towards the strains was determined as described by Sklar et al. (2007).
Isolation of RNA and S1-nuclease mapping.
RNA was isolated and S1-mapping was carried out essentially as previously described (Kormanec, 2001; Miticka et al., 2003; Skovierova et al., 2006). Briefly, an overnight culture was diluted 500-fold into fresh LB and incubated at 37 °C with aeration to the exponential (OD600=0.5) and stationary phases (OD600=1.7). Heat-shock- and cold-shock-stressed cells were grown to exponential phase and subjected to 30 min at 45 °C or 60 min at 10 °C, respectively. For artificial rpoE expression, S. Typhimurium SL1344 containing pAC-rpoEST4 or pAC7 was grown in LB with chloramphenicol to early exponential phase (OD600=0.24) and expression of rpoE was induced for 3 h with 0.2 % arabinose.
At the appropriate time point, the S. Typhimurium culture was chilled, washed with diethylpyrocarbonate (DEPC)-treated ice-cold 0.15 M NaCl, and total RNA was prepared essentially as described previously (Kormanec, 2001). High-resolution S1-nuclease mapping was performed according to Kormanec (2001). RNA samples (40 µg) were hybridized to 0.02 pmol of appropriate DNA probe labelled at the 5' end with [
-32P]ATP and treated with 120 U S1-nuclease (Promega). The probe (a 487 bp DNA fragment) was prepared by PCR amplification from the chromosomal DNA of S. Typhimurium SL1344 using the 5' end-labelled reverse primer smpARev (5'-GCGGTCAACATCAGGAGTACTGC-3') and the direct primer smpADir (5'-CGAGGTCGATGTTGGCATCAGC-3'). The SmpARev primer was labelled at the 5' end with [-32P]ATP [ICN; 4500 Ci mmol–1 (167 TBq)] and T4 polynucleotide kinase (Promega), as described in Ausubel et al. (1995). The protected DNA fragments were analysed on DNA sequencing gels together with G+A and T+C chemical sequencing ladders derived from the end-labelled fragment (Maxam & Gilbert, 1980).
β-Galactosidase assays.
Overnight cultures of the S. Typhimurium strains with plasmid prpoEP3 containing the E-dependent rpoEp3 promoter upstream of the lacZ reporter gene in pTL61T (Miticka et al., 2003) were diluted 500-fold into 50 ml fresh LB medium with ampicillin, grown at 37 °C with aeration and assayed at hourly intervals. β-Galactosidase assays were performed in triplicate (Miller, 1972).
Construction of an smpA mutant.
An smpA mutant was constructed in S. Typhimurium SL1344 using a 
Red mutagenesis system (Datsenko & Wanner, 2000; Rowley et al., 2005). To decrease background, the template plasmid was digested with HindIII and an agarose gel-purified 1635 bp DNA fragment was used for PCR with mutagenesis primers smpAFw (5'-ATCACTATGCGCTGTAAAACGCTGACTGCTGCCGCAGCAGGTGTAGGCTGGAGCTGCTTC-3') and smpARv (5'-ACAAAATTACTTCGTCAACGCCGGTTTGTTATCAATATTGCATATGAATATCCTCCTTAG-3') to amplify the kanamycin-resistance gene cassette with homologous flanking regions from the start and end of the smpA gene. The amplified DNA fragment was column-purified (Qiagen) and electroporated into S. Typhimurium SL1344 pKD46. Deletion of smpA was confirmed by PCR using primers which are external to the site of mutagenesis, smpADir (5'-CGAGGTCGATGTTGGCATCAGC-3') and smpARev2 (5'-GTGTGAAAGCCGTACCACACC-3'), as well as primers k1 (5'-CAGTCATAGCCGAATAGCCT-3') and k2 (5'-CGGTGCCCTGAATGAACTGC-3') that amplify the kanamycin cassette in order to confirm the orientation of the marker (Datsenko & Wanner, 2000). P22 HT transduction was used to transfer the smpA mutation into a clean SL1344 background.
The S. Typhimurium SL1344 smpA mutant strain was named GVB1741. To complement the smpA mutation, S. Typhimurium GVB1741 was transformed with plasmid pAC-smpA2 containing the S. Typhimurium smpA gene including both promoters. The pAC-smpA2 plasmid was constructed by cloning a 780 bp DNA fragment, prepared by PCR amplification using the primers smpADir (5'-CGAGGTCGATGTTGGCATCAGC-3') and SmpAHind (5'-GGGGGAAGCTTCACAAAATTACTTCGTCAAC-3') and S. Typhimurium SL1344 chromosomal DNA as a template, blunt-ended by the Klenow fragment of DNA polymerase and cut with HindIII, in a low-copy-number plasmid pACYC184 digested with HindIII and EcoRV.
Infection of murine macrophages.
The ability of S. Typhimurium strains to invade, and survive and replicate within, the murine macrophage cell line Raw 264.7 was determined using a gentamicin protection assay performed essentially as previously described (Elsinghorst, 1994; Humphreys et al., 1999; Sydenham et al., 2000). Briefly, an m.o.i. of 
1 : 1 was used, and the number of bacteria inside infected cells was determined at 3 and 24 h post-infection using a gentamicin protection assay.
Analysis of virulence.
For all in vivo studies, strains were grown statically overnight at 37 °C, centrifuged, washed and resuspended to the appropriate concentration in sterile PBS (pH 7.2), and administered to mice in doses of 200 µl. Female BALB/c mice (6–8 weeks old; Harlan) were used throughout. Virulence was assessed initially by intraperitoneal (i.p.) infection using a competition index assay, as previously described (Beuzon & Holden, 2001; Humphreys et al., 2003; Rowley et al., 2005) using three mice per group. For oral infection, five mice per group were used, the inoculum was administered via oral gavage and mice were culled 7 days later. Organs (livers, spleens, Peyer's patches and mesenteric lymph nodes) were isolated and homogenized, and the number of bacteria present was determined by viable counting.
Statistical analysis.
The statistical significance of datasets was analysed by two-tailed Student's t test or ANOVA, as appropriate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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E in S. TyphimuriumE, we have recently identified 34 E-dependent promoters directing the expression of 62 genes that are members of the E regulon in S. Typhimurium (Skovierova et al., 2006). However, several E-dependent promoters previously identified in E. coli using the same system were not found by this screen in S. Typhimurium, among them smpAp (Rezuchova et al., 2003).
Therefore, in order to investigate the expression of the S. Typhimurium smpA gene and to determine whether it is regulated by E, high-resolution S1-nuclease mapping was performed using the 5'-labelled S1 probe (Fig. 1a). RNA was isolated from S. Typhimurium SL1344 and its isogenic rpoE mutant S. Typhimurium GVB311 from different growth phases and under stress conditions that have previously been shown to induce the expression of E-dependent promoters (Miticka et al., 2003). RNA was also isolated from S. Typhimurium SL1344 containing pAC-rpoEST4 (which has the S. Typhimurium rpoE gene under the control of the arabinose-inducible PBAD promoter) induced with arabinose. As shown in Fig. 1(b), an RNA-protected fragment was identified that corresponded to the smpAp1 promoter with a transcriptional start point (TSP) in a position identical to that of the E-dependent E. coli smpAp promoter (Rezuchova et al., 2003; Rhodius et al., 2005). An identical RNA-protected fragment was also identified using RNA isolated from S. Typhimurium SL1344 pAC-rpoEST4 (Fig. 1b, lane 5) but not RNA from S. Typhimurium SL1344 pAC7 (the control vector) (Fig. 1, lane 6) or with control tRNA (Fig. 1b, lane C). The smpAp1 promoter was induced in stationary phase and by cold shock, conditions previously shown to induce E-dependent promoters in S. Typhimurium (Miticka et al., 2003). However, when RNA from the same conditions was prepared from the S. Typhimurium rpoE mutant, no RNA-protected fragment was identified that corresponded to the smpAp1 promoter (Fig. 1b). These results clearly indicated that this promoter is dependent upon E in S. Typhimurium. The sequence of the smpAp1 promoter (Fig. 1c) was highly similar to the E consensus sequence GGAACTT–N15–GTCTAA (Skovierova et al., 2006). Downstream of the E-dependent smpAp1 promoter (and upstream of the smpA gene), we identified a second, E-independent, promoter, smpAp2, expression of which in both the wild-type (WT) and rpoE strains was almost constitutive during exponential growth, with a partial decrease after heat shock and in stationary phase (Fig. 1b). Based on its sequence (Fig. 1c), it is probably recognized by RNA polymerase containing the principal sigma factor 70, as it is similar to its consensus sequence, TTGACA–N16–18–TATAAT (Pribnow, 1975). In E. coli, in addition to the E-dependent smpAp1 promoter that we previously identified (Rezuchova et al., 2003), we also identified a E-independent promoter with a TSP in a position similar to that of S. Typhimurium smpAp2 (data not shown). Thus, expression of smpA in both S. Typhimurium and E. coli is governed by two promoters, of which one, smpAp1, is dependent upon E.
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E consensus sequence for the E-dependent promoter (one mismatch in the –10 region), and identical –10 and –35 regions for the E-independent promoters (Supplementary Fig. S1). Thus, it is likely that regulation of smpA is similar in both strains.
Characterization of SmpA, and its gene and promoter, in other bacteria
SmpA belongs to the bacterial outer-membrane lipoprotein family SmpA/OmlA (pfam04355). Using a BLAST search of all databases and currently sequenced genomes (759 bacterial, 41 archaeal and 135 eukaryotic) we found homologues in Gram-negative proteobacteria only, and then only in the 
, β and classes. Comparison of S. Typhimurium SmpA with several selected homologues revealed the highest similarity to members of the -proteobacteria. As for S. Typhimurium SmpA, all the homologues contain a lipobox motif with a conserved Cys residue at position 1 that is covalently modified with a lipid moiety. Based on the rules for amino acids in second and third positions (Narita et al., 2004), they should all be outer-membrane lipoproteins (Supplementary Fig. S2). Interestingly, although SmpA homologues are present in the -, β- and -proteobacteria, the chromosomal context of the Salmonella and E. coli gene order recN, smpA, yfjF, yfjG (Fig. 1a
) is conserved in -proteobacterial orders Enterobacteriales and Vibrionales only.
The smpA promoter region is identical in all six currently sequenced S. enterica serovars. There are only four mismatches in the non-essential promoter regions in Salmonella bongori. Comparison of the smpA promoter regions among members of Enterobacteriales (Supplementary Fig. S1) has revealed that they all contain a sequence highly similar to the E-dependent smpAp1 promoter, indicating that in all the strains smpA is likely under the control of E. However, the similarity of the E-independent smpAp2 promoter is lower and is mainly conserved in the closely related Enterobacteriales genera Salmonella, Escherichia and Shigella.
Construction of an S. Typhimurium smpA mutant and its phenotypic analysis
Using Red mutagenesis (Datsenko & Wanner, 2000), we replaced the coding region of the smpA gene in S. Typhimurium SL1344 with a kanamycin-resistance cassette to generate strain GVB1741. The mutation did not affect colony morphology and growth in liquid media at temperatures ranging from 25 to 42 °C (data not shown). However, the smpA mutant grew less well than the WT strain at high temperature (46 °C) (Fig. 2a). The means of the optical densities of the two strains were significantly different (P<0.05) from 7 h onwards. This effect was complemented by plasmid pAC-smpA2, which encodes a copy of the S. Typhimurium smpA gene with both promoters (Fig. 2a).
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). We therefore investigated the sensitivity of the S. Typhimurium smpA mutant to a variety of small toxic compounds and detergents by a similar disc diffusion method on solid agar medium. As shown in Table 2, at the concentrations tested, none of the detergents tested inhibited the growth of either the WT or smpA strains, which is in contrast to the phenotype of a P. aeruginosa omlA mutant (Ochsner et al., 1999). The smpA mutant was not more sensitive than the WT strain to chloramphenicol, gentamicin or polymyxin B, but it was slightly more sensitive to H2O2. However, compared to the WT strain, the smpA mutant was more sensitive to the hydrophobic antibiotic rifampicin (Table 2). The MIC of rifampicin for the smpA mutant (12.5 µg ml–1) was half that of the WT strain and the smpA mutant harbouring the complementing plasmid pAC-smpA2 (25 µg ml–1). This is in agreement with the increased rifampicin sensitivity of an E. coli smpA mutant (Sklar et al., 2007). The E. coli smpA mutant also exhibits increased sensitivity to cholate, and it is unable to grow on medium containing 0.5 % SDS plus 1 mM EDTA (Sklar et al., 2007). Similarly, the S. Typhimurium smpA mutant exhibited increased sensitivity to sodium cholate (MIC 12.5 mg ml–1 for WT and smpA pAC-smpA2, and 10 mg ml–1 for the smpA mutant), although not to such an extent as the E. coli mutant. However, in contrast to the results in E. coli, the smpA mutation had little effect on the growth of S. Typhimurium in LB medium with 0.5 % SDS and 1 mM EDTA. When a higher concentration of SDS (1.0 %) and 1 mM EDTA were used, the smpA mutant grew slightly slower and lysed more rapidly than the WT strain after prolonged cultivation (Fig. 2b). The differences in the means of the optical densities of the smpA cultures versus the WT and smpA pAC-smpA2 cultures were statistically significant (P<0.05) at all time points post 4 h.
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; Fig. 2a, b).
Loss of SmpA activates the E-mediated envelope stress response
Defects in OMP folding and localization have been found to trigger the E stress response (Alba & Gross, 2004; Rowley et al., 2006). As SmpA has recently been found to be a part of the YaeT complex essential for assembly of OMPs (Sklar et al., 2007), we tested whether the absence of SmpA from this complex elicits a E stress response in S. Typhimurium. We have previously shown that expression of the auto-regulated E-dependent promoter rpoEp3 is dramatically induced at stationary phase in S. Typhimurium, and that its activity corresponds to the level of E, thus representing a sensitive marker for the E stress response (Miticka et al., 2003). Therefore, the rpoEp3–lacZ transcriptional fusion plasmid prpoEP3 (Miticka et al., 2003) was transformed into the S. Typhimurium WT and smpA mutant strains, and the level of β-galactosidase was measured during growth in LB medium. As shown in Fig. 2(c), the activity of the E-dependent rpoEp3 promoter was more than twofold higher in the smpA mutant than the WT strain at early exponential phase, and this increase persisted at all stages of growth. These results indicated that the lack of SmpA elicited a E-dependent envelope stress response in S. Typhimurium.
Effect of smpA mutation upon S. Typhimurium invasion of and survival within a murine macrophage cell line
To investigate whether smpA is important for entry and/or survival within macrophages, the S. Typhimurium smpA mutant and its isogenic WT and rpoE strains were used to infect the macrophage-like cell line RAW264.7, and the number of viable salmonellae present at 3 and 24 h after infection was determined; the results are presented in Fig. 3(a). At both the 3 and 24 h time points the smpA mutant was present in macrophages at slightly lower levels than the WT strain; however, these differences were not statistically significant. In contrast, as reported previously, S. Typhimurium rpoE mutants have a severe defect in intramacrophage survival (Humphreys et al., 1999; Testerman et al., 2002). This suggests that at least under the conditions tested, smpA is not important for invasion of or survival and replication within macrophages.
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1x103 c.f.u. of both the WT and smpA strains was given by the i.p. route to mice. A competitive index (CI) of 1 indicates that the strains are of equal virulence. The assay was performed twice and the CIs ranged from 0.08 to 0.12. In all cases the smpA mutant was recovered from the liver and spleens in significantly lower numbers compared to the input inoculum than the WT strain (P<0.05). We also investigated the role of SmpA during infection via the oral route. Mice were inoculated orally with either 5x105 c.f.u. of the WT strain or 1.2x107 c.f.u. of the smpA mutant, and the number of bacteria present was determined 7 days later (Fig. 3b). We gave a higher dose of the smpA mutant because we were concerned that we would not recover c.f.u. of the smpA bacteria from the tissues if a dose similar to that of the WT strain was used. The oral LD50 of SL1344 is 1x105 to 1x106 c.f.u. (Humphreys et al., 1999; Monack et al., 2000). Therefore, if the mice were infected with SL1344 at the dose used for the smpA mutant, most of the mice would be dead or dying at 7 days. Similar numbers of c.f.u. of the WT and smpA strains were found in the Peyer's patches and mesenteric lymph nodes of infected mice. If the numbers of c.f.u. present in the mesenteric lymph nodes and Peyer's patches were normalized for the c.f.u. in the inoculum, then the result for the smpA mutant was significantly lower (P<0.05) than that of the WT strain. Even though the dose of the smpA mutant was much higher than that of the WT strain, there were significantly fewer smpA than WT bacteria in the liver and spleen (P<0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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E is critically important for the oxidative stress resistance, stationary-phase survival and pathogenicity of S. Typhimurium (Crouch et al., 2005; Humphreys et al., 1999; Kenyon et al., 2002; Testerman et al., 2002), indicating that some members of the E regulon may have a role in the virulence of S. Typhimurium. We are investigating the E regulon with respect to salmonella virulence. Although smpA was not identified in our previous genetic screen for members of the E regulon in S. Typhimurium (Skovierova et al., 2006), the present results clearly show that smpA belongs to the S. Typhimurium E regulon. Expression of smpA in S. Typhimurium is directed by two promoters. The distal promoter, smpAp1, is dependent upon E, and the proximal promoter, smpAp2, is probably recognized by 70. Similar regulation has also been found for its counterpart in E. coli, reflecting the high conservation of the smpA promoter region in both strains (Supplementary Fig. S1). Interestingly, although SmpA homologues are present in three classes of proteobacteria (, β and ), the smpA promoter region is conserved only in the -proteobacterial order Enterobacteriales. The highest similarity was in the region covering the E-dependent promoter. Thus, it is likely that smpA is dependent upon E and regulated in a similar way in all these bacteria.
As with E. coli, SmpA is not essential for S. Typhimurium under standard growth conditions. The growth rate of the smpA mutant was reduced relative to the WT strain at high temperature (46 °C), although this growth defect was smaller than for an S. Typhimurium rpoE mutant (Rowley et al., 2005). The S. Typhimurium smpA mutant showed increased sensitivity to sodium cholate, SDS plus EDTA, and the hydrophobic antibiotic rifampicin. P. aeruginosa omlA and E. coli smpA mutants also exhibit increased sensitivity to detergents, although there are differences between the strains regarding the magnitude of this sensitivity. The P. aeruginosa omlA mutant exhibits a much higher susceptibility to anionic detergents. The omlA mutant almost stops growing in 0.1 % SDS (Ochsner et al., 1999). Likewise, an E. coli smpA mutant does not grow in the presence of 0.5 % SDS plus 1 mM EDTA (Sklar et al., 2007). In contrast, the growth of the S. Typhimurium smpA mutant was not so strongly affected by detergents. In fact, both WT and smpA S. Typhimurium strains grew in the presence of 5 % SDS (data not shown). This may reflect the high resistance of Salmonella strains to detergents. Despite differences in the magnitude of the effects of loss of SmpA/OmlA, the in vitro phenotypes of E. coli, P. aeruginosa and S. Typhimurium mutants are similar, indicating a role for SmpA/OmlA homologues in maintaining the cell envelope integrity in Gram-negative bacteria. This is presumed to be due to their function as a component of the YaeT OMP assembly complex. SmpA has been shown to associate with YfiO and NlpB in the complex, and this interaction is independent of another component, YfgL (Sklar et al., 2007). In E. coli, mutations in nlpB and yfgL also increase sensitivity to hydrophobic antibiotics, bile salts and detergents (Sklar et al., 2007). The two other genes that encode members of the complex, yaeT and yfiO, are essential in E. coli (Wu et al., 2005).
The function or activity of the YaeT complex seems to vary in Gram-negative bacteria. While homologues of the essential YaeT are present in all Gram-negative bacteria (Gentle et al., 2005), the other components of the complex are not so conserved. Examination of available bacterial genomes revealed that YfiO, which is essential in E. coli, has no homologues in Chlamydiae, Chlorobi, Fusobacteria, Planctomycetes, Thermotogae, Deinococcus–Thermus and Spirochaetes; homologues of the non-essential YfgL are absent in Chlamydiae, Spirochaetes and 
-Proteobacteria; and, as for SmpA, homologues of the non-essential NlpB are present only in -, β- and -Proteobacteria.
The E-dependent ESR is induced by OMP folding perturbations that are elicited by various stress conditions. The members of the E regulon produced following these stress conditions are involved in the correction of these cell envelope defects (Alba & Gross, 2004; Rowley et al., 2006). As the YaeT complex is essential for assembly of OMPs, flaws in this complex should result in OMP assembly defects which should elicit a E-dependent ESR. We found that this was the case in S. Typhimurium using the E-dependent rpoEp3 promoter fusion as a reporter. In an E. coli smpA mutant, production of the E-regulated protein HtrA (DegP) is increased 1.5–2-fold relative to the WT strain, indicating that a E response is activated in this strain (Sklar et al., 2007). Interestingly, all the components of the YaeT complex belong to the E regulon, both in E. coli and S. Typhimurium (Rhodius et al., 2005; Skovierova et al., 2006; M. Roberts, unpublished results). Likewise, lack of another component of the YaeT complex, YfgL, increases E activity in E. coli and S. enterica serovar Enteritidis (Fardini et al., 2007; Onufryk et al., 2005).
In contrast to the S. Typhimurium rpoE mutant, the smpA mutation did not significantly affect the ability of S. Typhimurium to enter or replicate within macrophages in vitro (Fig. 3a). However, the mutant was significantly attenuated in mice by both the oral and parenteral routes of infection. Compared to the WT strain, approximately 10-fold fewer smpA bacteria could be isolated from the liver and spleen after parenteral infection of mice, indicating that the smpA mutant survived significantly less than the WT strain. Comparison of the ability of the smpA mutant and the WT strain to infect mice via the oral route revealed that the smpA mutation affected growth/survival of S. Typhimurium at systemic sites (spleen and liver) more than at gut-associated sites (Peyer's patches and mesenteric lymph nodes). This could reflect defective replication/survival in systemic tissues or defective translocation of the smpA mutant from the mesenteric lymph nodes. Intriguingly, a similar effect has also been found for S. Typhimurium strains with mutation in rpoE, in the rpoE-regulated genes htrA and surA, and in degS, which encodes a protease that regulates E (Humphreys et al., 1999; Rowley et al., 2005; Sydenham et al., 2000). This possibly indicates that members of the E regulon are more important for systemic than enteric S. Typhimurium infection.
Although the S. Typhimurium smpA mutant exhibited relatively mild phenotypes in vitro (as does an E. coli smpA mutant), it exhibited a more severe phenotype in vivo and was essential for full salmonella virulence. Therefore, it is possible that the stresses to which the S. Typhimurium smpA mutant was exposed in vitro did not replicate and/or were not as severe as those encountered in vivo during murine infection. This shows that the relevance of stress-response genes may not be revealed by in vitro studies alone. Interestingly, smpA was one of the genes (along with rpoE) found to be activated in S. Typhimurium during infection of macrophages in vitro (Eriksson et al., 2003). Also, smpA was identified as one of the most highly expressed genes in the spleens of S. Typhimurium-infected mice (Rollenhagen et al., 2004).
Recently, another member of the E regulon and the YaeT complex, YfgL, has been characterized in S. Enteritidis. As for the S. Typhimurium smpA mutant, the S. Enteritidis yfgL mutant is more sensitive to rifampicin and is greatly attenuated in mice (Fardini et al., 2007). By an unknown mechanism, the yfgL mutation negatively affects the expression of genes that encode proteins of the two S. Enteritidis type III secretion systems (TTSS-1 and TTSS-2) (Charlson et al., 2006). We found no difference in the production and secretion of the TTSS-1 protein SipC and the TTSS-2 protein SseC by the WT and smpA mutant strains (data not shown).
These E-dependent YaeT complex proteins may be good targets for anti-salmonella therapy, and Salmonella strains with mutations in these genes may be live vaccine candidates. Experiments investigating the role of other YaeT complex genes in salmonella virulence are in progress.
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ACKNOWLEDGEMENTS |
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Edited by: D. L. Gally
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REFERENCES |
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Received 24 July 2007;
revised 29 October 2007;
accepted 23 November 2007.
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