O-antigen expression in Salmonella enterica serovar Typhi is regulated by nitrogen availability through RpoN-mediated transcriptional control of the rfaH gene

Mauricio Bittner1, Soledad Saldías1, Claudia Estévez1, Mercedes Zaldívar1, Cristina L. Marolda2, Miguel A. Valvano2 and Inés Contreras1

Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, PO Box 174, Correo 22, Santiago, Chile1
Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C12

Author for correspondence:Inés Contreras. Tel: +56 2 678 1658. Fax: +56 2 2227900. e-mail: inescon{at}ciq.uchile.cl


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The authors previously reported increased expression of the Salmonella enterica serovar Typhi (S. typhi) rfaH gene when the bacterial cells reach stationary phase. In this study, using a lacZ fusion to the rfaH promoter region, they demonstrate that growth-dependent regulation of rfaH expression occurs at the level of transcription initiation. It was also observed that production of the lipopolysaccharide (LPS) O-antigen by S. typhi Ty2 correlated with the differential expression of rfaH during bacterial growth. This was probably due to the increased cellular levels of RfaH, since expression of the distal gene in the O-antigen gene cluster of S. typhi Ty2, wbaP, was also increased during stationary growth, as demonstrated by RT-PCR analysis. Examination of the sequences upstream of the rfaH coding region revealed homologies to potential binding sites for the RcsB/RcsA dimer of the RcsC/YopJ/RcsB phosphorelay regulatory system and for the RpoN alternative sigma factor. The expression of the rfaH gene in rpoN and rcsB mutants of S. typhi Ty2 was measured. The results indicate that inactivation of rpoN, but not of rcsB, suppresses the growth-phase-dependent induction of rfaH expression. Furthermore, production of ß-galactosidase mediated by the rfaH–lacZ fusion increased approximately fourfold when bacteria were grown in a nitrogen-limited medium. Nitrogen limitation was also shown to increase the expression of the O-antigen by the wild-type S. typhi Ty2, as demonstrated by a similar electrophoretic profile to that observed during the stationary phase of growth in rich media. It is therefore concluded that the relationship between LPS production and nitrogen limitation parallels the pattern of rfaH regulation under the control of RpoN and is consistent with the idea that RpoN modulates LPS formation via its effect on rfaH gene expression during bacterial growth.

Keywords: lipopolysaccharide, regulation, transcription, sigma factor


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Salmonella entericaserovar Typhi (herein abbreviated as S. typhi) causes typhoid fever in humans, a disease of great public health concern in many developing countries (Pang et al., 1998 ). The detailed molecular mechanisms specifically involved in the pathogenesis of S. typhi are poorly understood due to the lack of an animal model. However, a great deal of information has been obtained from studies using S. entericaserovar Typhimurium, which infects a wide spectrum of animal hosts and causes a typhoid-like disease in the mouse (Jones & Falkow, 1996 ). We are interested in the analysis of the biosynthesis and regulation of S. typhisurface polysaccharides, especially the lipopolysaccharide (LPS). This glycolipid surface molecule is an abundant component of the bacterial outer membrane and plays a role in pathogenesis by protecting micro-organisms from the lytic action of serum complement (Joiner, 1988 ). In S. typhi, LPS also interacts with intestinal epithelial cells during the initial stages of infection (Pier et al., 1998 ; Lyczak et al., 2001 ).

LPS has a tripartite structure that includes lipid A, a core oligosaccharide and the O-specific polysaccharide or O-antigen (Schnaitman & Klena, 1993 ; Raetz, 1996 ). The O-antigen is the most surface-exposed LPS component and displays enormous structural variability, resulting in a large variety of serotypes (Reeves, 1993 ). S. typhi also produces a group I exopolysaccharide known as the Vi antigen, which is made of a homopolymer of high molecular mass (Virlogeux et al., 1996 ) and forms a capsular structure. The Vi antigen is found in virtually all clinical isolates from patients with acute typhoid infection. It protects S. typhi against complement-mediated lysis as well as phagocytosis (Kossack et al., 1981 ).

The biosynthesis of exopolysaccharides is modulated by environmental factors through several regulatory systems (Virlogeux et al., 1996 ; Arricau et al., 1998 ; Whitfield & Roberts, 1999 ). One of these regulatory components is the RcsC/RcsB two-component system, which consists of a cytoplasmic sensor kinase (RcsC) and cytosolic response regulator (RcsB). More recently, a phosphorelay system involving the phosphotransmitter YojN was shown to be essential for transducing the signal from RcsC to RcsB in Escherichia coli (Takeda et al., 2001 ). It is quite possible that the same mechanism operates in S. typhi, since a homologue of YojN is also present in this bacterium. In E. coli, stimuli like osmotic shock (Sledjeski & Gottesman, 1996 ) or growth at a low temperature (Whitfield & Roberts, 1999 ) result in the phosphorylation of RcsB, which associates with the proteolytically labile protein RcsA forming a heterodimer that acts as a positive transcriptional regulator (Stout & Gottesman, 1990 ). The activation of RcsC/YojN/RcsB-responsive promoters in E. coli and in other Enterobacteriaceae involves the recognition by the RcsB/RcsA dimer of a relatively conserved DNA sequence known as the ‘RcsAB box’ (Wehland & Bernhard, 2000 ). This box, consisting of 14 base pairs (TaAGaatatTCctA), has been found in the upstream region of the promoter sequences of the colanic acid biosynthesis cluster of E. coli K-12, the K2 antigen cluster of Klebsiella pneumoniae, and the Vi antigen cluster of S. typhi (Wehland & Bernhard, 2000 ).

The synthesis of LPS in S. typhiinvolves a large number of genes, the majority of which are organized in various clusters located on separate regions of the bacterial chromosome. It is conceivable that LPS gene expression at all of these various sites must be coordinated to ensure that all necessary components are available at any given time. Yet the regulation of LPS synthesis is not well understood. In E. coliand S. entericaserovar Typhimurium, a covalent substitution of lipid A with 4-amino-4-deoxy-L-arabinose is regulated by the pmrA gene (Gunn & Miller, 1996 ), which encodes a transcription factor that is activated during growth under mildly acidic conditions, in a PhoP/PhoQ-dependent manner during Mg2+ limitation, or by exposure to Fe3+ ions (Guo et al., 1997 ; Gunn et al., 1998 ; Ernst et al., 2001 ). This LPS modification reduces the net negative charge of the molecule, thus contributing to bacterial resistance to cationic peptides and presumably enhancing intracellular survival within phagosomes. Another level of regulation involves the regulation of gene expression of the core biosynthetic cluster by the RfaH protein (Farewell et al., 1991 ; Pradel & Schnaitman, 1991 ) and also by the heat-shock response (Karow et al., 1991 ). RfaH is a homologue of the NusG factor that regulates gene expression of the haemolysin operon (Bailey et al., 1992 ; Leeds & Welch, 1996 , 1997 ), polysaccharide capsule genes (Stevens et al., 1997 ), the F plasmid traoperon (Beutin & Achtman, 1979 ), and a gene involved in iron acquisition (Nagy et al., 2001 ). RfaH regulation occurs during transcript elongation and depends on a 5'-proximal, transcribed nucleic acid sequence known as ops(for operon polarity suppressor; Nieto et al., 1996 ; Bailey et al., 1997 ) that induces transcriptional pausing in vitro (Artsimovitch & Landick, 2000 ) and in vivo(Leeds & Welch, 1997 ). It has been recently demonstrated that RfaH recognizes RNA-polymerase transcribing RfaH-regulated operons by interacting with the opssequence in the exposed nontemplate DNA strand of ops-paused transcription complexes (Artsimovitch & Landick, 2002 ).

5'-proximally transcribed sequences containing ops elements exist in the O-polysaccharide gene clusters of many enteric bacteria (Hobbs & Reeves, 1994 ), suggesting that RfaH also plays a role in the regulation of the transcription elongation of O-antigen genes. In a previous study, Marolda & Valvano (1998) conducted a detailed analysis of the promoter region of the O7-specific genes in E. coli. Using single-copy-number fusions to a reporter gene, these authors did not observe any detectable regulation of the O7-specific promoter at the level of initiation of transcription, concluding that regulation only occurs at the level of mRNA elongation in an RfaH-dependent manner.

Therefore, modulation of the cellular levels of RfaH may contribute to coordinate expression of O-antigen and core LPS biosynthetic enzymes. Not much is known, however, about the regulation of the rfaHgene itself. In a previous study, we isolated the rfaHgene from the S. typhistrain Ty2 and confirmed that its function is essential for LPS expression (Rojas et al., 2001 ). More importantly, we demonstrated that rfaH gene expression varies with the growth phase, with the highest expression during stationary phase (Rojas et al., 2001 ). In this study, we provide evidence showing that regulation of rfaH depends, at least in part, on the activity of the RpoN alternative sigma factor and that differential rfaH expression influences a similar pattern of O-antigen production during the bacterial growth cycle.


   METHODS
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INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
 
Bacterial strains, plasmids, media and growth conditions.
Table 1 summarizes the properties of the bacterial strains and plasmids used in this study. Bacteria were grown in Luria–Bertani medium (LB: Bacto tryptone, 10 g l-1; Bacto yeast extract, 5 g l-1; NaCl, 5 g l-1) or in minimal E medium (MgSO4.7H2O, 0·2 g l-1; citric acid monohydrate, 2 g l-1; K2HPO4.3H2O, 13·1 g l-1; NaNH4HPO4.4H2O, 3·3 g l-1) containing 0·2% glucose as carbon source. Low-nitrogen E medium contained 1% of the amount of nitrogen in E medium. Minimal media were supplemented with 50 µg cysteine ml-1 and 50 µg tryptophan ml-1. Media were supplemented with 100 µg ampicillin ml-1, 20 µg chloramphenicol ml-1 or 50 µg kanamycin ml-1 as appropriate.


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Table 1. S. typhi strains and plasmids used in this study

 
Construction of a lacZ transcriptional fusion to the rfaH gene.
The S. typhi Ty2 rfaH promoter region was amplified by PCR using the rfaH2 and rfaHint2 primers (Table 2), which were designed according to the DNA sequence information available for the S. typhi strain CT18 (Parkhill et al., 2001 ). A 334 bp amplicon was cloned into the vector pGEM-T Easy, to yield plasmid pSM334. The 334 bp insert in pSM334 was then transferred into the plasmid pFZY1. This is a single-copy-number vector designed for the construction of transcriptional fusions to the lacoperon (Koop et al., 1987 ). The resulting plasmid, pCE334, was transformed into S. typhi Ty2. The pFZY1 vector was also transformed as a negative control.


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Table 2. Primers used in this study

 
Mutagenesis of S. typhi rcsB and rpoN genes.
Mutagenesis was performed according to the method described by Datsenko & Wanner (2000) to disrupt specific chromosomal genes using PCR products. For this purpose, S. typhi Ty2 was transformed with pKD46, a temperature-sensitive, low-copy-number plasmid that expresses the phage {lambda} Red recombinase system under the control of the arabinose-inducible ParaB promoter. The Red-mediated recombination is required to replace the targeted chromosomal sequence with an antibiotic-resistance gene that is generated by PCR. S. typhi Ty2 cells carrying pKD46 were transformed by electroporation with a PCR product that was generated using either plasmid pKD3 or pKD4 as templates. The plasmid pKD3 carries a chloramphenicol acetyltransferase gene flanked by FRT sites while pKD4 carries a gene encoding kanamycin resistance, also flanked by FRT sites. Primers WrpoN1 and WrpoN2 were used with the pKD3 template to obtain a product for disruption of rpoN.Primers WrcsB1 and WrcsB2 were used with the pKD4 template to obtain a product for disruption of rcsB. The sequences of the primers are shown in Table 2. Transformants were plated on LB agar plates containing chloramphenicol, for the selection of rpoNmutants, or kanamycin, for the selection of rcsBmutants. Transformants were replica plated in the absence of antibiotic selection at 43 °C and then assayed for ampicillin sensitivity, to confirm the loss of pKD46.

RT-PCR.
For expression analysis, each strain was grown to the selected OD600 in 50 ml LB. RNA was extracted using the standard TRIzol procedure. After DNase I treatment, RNA was reverse transcribed using SuperScript II (200 U µl-1) and antisense primers for hisG and wbaP (HisG2 and WbaP592, respectively). Single-stranded DNA was then amplified using the primers for genes: hisG (HisG1 and HisG2) and wbaP (WbaP591 and WbaP592). The sequences of the primers are indicated in Table 2. The PCR products were analysed by electrophoresis on 1·5% agarose gels.

LPS analysis.
Culture samples were adjusted to OD600 2·0 in a final volume of 100 µl. Then, proteinase-K-digested whole-cell lysates were prepared as described by Hitchcock & Brown (1983) and LPS was separated on 14% acrylamide gels using a Tricine/SDS buffer system (Lesse et al., 1990 ). Gel loadings were normalized so that each sample represented the same number of cells. Each well was loaded with approximately 1x108 c.f.u. Gels were silver stained by a modification of the procedure of Tsai & Frasch (1982) . Densitometric analyses of the gels were performed using the UN-SCAN-IT gel software (Silk Scientific).

ß-Galactosidase assays.
Bacteria were grown overnight in LB or minimal E medium, subcultured and grown in 100 ml of the same medium on an orbital shaker. Every 30 min, a 2 ml sample was withdrawn to measure the bacterial growth (OD600 and c.f.u. ml-1) and the ß-galactosidase activity according to Miller (1972) . Enzyme activities (Miller units), normalized for cell density (OD600), were calculated using the equation [(A420-1·75A550)x1000]/[reaction time (min)xculture volume (ml)xOD600]. Each sample was analysed in triplicate during at least three independent experiments.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Expression of S. typhi LPS during growth correlates with rfaH transcription
It is well documented that the RfaH elongation factor positively regulates the LPS core and O-polysaccharide genes. The environmental signals, however, that modulate the expression of the rfaH gene are largely unknown. In a previous study, we used RT-PCR analysis to test the effects of various growth conditions on the transcription of rfaH (Rojas et al., 2001 ). We demonstrated that the transcription of rfaH in S. typhi Ty2 displays a growth-phase-dependent regulation, with maximal expression during late-exponential and stationary phases. In contrast, we found that environmental factors that are known to be important for LPS regulation in other systems, such as growth temperature (Al-Hendy et al., 1991 ) and osmolarity (Aguilar et al., 1997 ), do not affect transcription of the rfaHgene in cells grown to late exponential phase (Rojas et al., 2001 ). To determine whether the transcriptional regulation of the rfaH gene is exerted at the promoter level we constructed a transcriptional fusion of the rfaHpromoter with a promoterless lacoperon in a single-copy-number plasmid (pFZY1). The resulting plasmid, pCE334, was transformed into S. typhi Ty2. The pFZY1 vector was also transformed as a negative control. The production of ß-galactosidase in strain Ty2/pCE334 was assayed during the various stages of growth in rich medium at 37 °C. The units of enzyme activity were corrected for the background levels determined in strain Ty2/pFZY1, which ranged between 0·5 and 5·0 Miller units during growth in LB medium. Fig. 1(a) shows that ß-galactosidase production, driven by the rfaH promoter region, increases at the late exponential growth phase, reaching maximal expression during stationary phase. These results are consistent with the idea that growth-regulated expression of rfaH is exerted at the level of transcription. To rule out the possibility that the upregulation of the rfaHgene was a general growth-phase-dependent phenomenon, we assayed the production of ß-galactosidase in S. typhistrain MEI120, which contains a MudJ(kan, lac) insertion in the hisD gene (Lyczak et al., 2001 ). Fig. 1(a) shows that the hisD–lacZexpression does not change in response to the bacterial growth phase.



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Fig. 1. Expression of rfaH and production of LPS in S. typhi Ty2/pCE334 at different points of the growth curve in LB at 37 °C. (a) Growth curve of S. typhi Ty2/pCE334 and expression of rfaH–lacZ transcriptional fusion. {circ}, Growth curve (OD600); {bullet}, ß-galactosidase activity of the rfaH–lacZ fusion; {square}, growth of strain MEI120 (hisD–lacZ fusion) used as control; {blacksquare}, ß-galactosidase activity of the hisD–lacZ fusion. Data are the mean±SD of three independent assays. (b) LPS profiles of cells grown to the following OD600: lane 1, 0·157; lane 2, 0·353; lane 3, 0·462; lane 4, 0·572; lane 5, 0·775; lane 6, 1·004; lane 7, 1·239. All samples were adjusted to an OD600 of 2·0 in a final volume of 100 µl for LPS preparation. LPS was analysed by Tricine/SDS-PAGE on a 14% acrylamide gel. Each well was loaded with approximately 1x108 c.f.u.

 
To examine whether the growth-regulated expression of rfaH has any biological significance in terms of LPS synthesis, we monitored the formation of S. typhi LPS during growth. Fig. 1(b) shows that the production of O-specific LPS varies during the bacterial growth. During the exponential phase (lanes 1 and 2), a very small amount of O-antigen is observed. As cells reach the late exponential phase, the formation of O-antigen increases (lanes 3, 4 and 5), and it is maximally expressed at the stationary phase (lanes 6 and 7). Densitometric analysis of the LPS gel in Fig. 1(b) revealed that the ratio of O-antigen to the lipid A-core region in the sample grown to OD600 1·239 (Fig. 1b, lane 7) was approximately twofold (183%) higher than that of the sample grown to OD600 0·157 (Fig. 1b, lane 1). The pattern of O-antigen expression parallels the growth-regulated expression of rfaH, suggesting that the formation of O-antigen LPS by the bacterial cells during growth is modulated by the cellular levels of RfaH. In support of this notion, no changes in O-antigen expression during growth were observed when S. typhi Ty2 was transformed with the high-copy-number plasmid pKHT19, harbouring the rfaH gene (data not shown). Similar results were obtained when an rfaH null mutant was transformed with pKHT19.

The first step in the synthesis of the O-antigen in species of Salmonellainvolves the addition of galactose 1-phosphate onto undecaprenol-P to produce a galactose-P-P-undecaprenol intermediate that is strictly required for the assembly of the O-antigen unit (Wang & Reeves, 1994 ; Wang et al., 1996 ). This reaction is catalysed by the product of the wbaPgene, which is the terminal gene in the O-antigen cluster. Thus, the level of transcription of wbaPcan be used to monitor whether RfaH modulates the expression of the entire S. typhiO-antigen gene cluster directly. We examined by RT-PCR the levels of wbaP mRNA in cells grown to mid-exponential phase and stationary phase. As a control, we also determined the mRNA levels of hisG. Fig. 2 shows the mRNA levels of wbaP and hisG genes at different stages of growth as assessed by RT-PCR, in the wild-type Ty2 strain. The densitometric quantification of the lanes demonstrated that the expression of wbaP relative to the expression of hisGis low in mid-exponential phase but increases significantly (over 6·0-fold) when cells reach stationary phase (data not shown). The expression of hisGdoes not change in response to the bacterial growth phase (Fig. 1a and data not shown). The wbaP-specific mRNA levels in strain Ty2/pKHT19, which overexpresses the RfaH protein, remained unchanged during growth (data not shown). No wbaP transcription was detected in the rfaH null mutant. Taken together, our results indicate that the growth-phase regulation of O-antigen expression is associated with the growth-phase expression of RfaH. In contrast, the production of the lipid A-core region was not affected similarly, since a complete lipid A-core band was observed at all different stages of growth (Fig. 1b). This is not completely unexpected, since in a previous study, Marolda & Valvano (1998) have shown that RfaH-mediated regulation of the core operons in E. coli and Salmonellais less tight than the regulation exerted in the O-antigen operon. These differences in RfaH-mediated regulation may be due to structural differences that exist in the regions surrounding the ops elements in the O-antigen and core promoter regions (Marolda & Valvano, 1998 ).



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Fig. 2. Expression of wbaP mRNA in S. typhi Ty2 at the exponential and stationary phases of growth. mRNA levels of wbaP and hisG were examined by RT-PCR in samples obtained at mid-exponential phase (OD600 0·2, lane 1) and stationary phase (OD600 1·0, lane 2). Arrows indicate RT-PCR products using wbaP- and hisG-specific primers. The PCR products were analysed by electrophoresis on a 1·5% agarose gel.

 
Characterization of the rfaH–yigC promoter region
To investigate the factors influencing expression of the rfaHgene, the DNA sequence of the 334 bp insert in pCE334 was determined. The sequence was identical to that reported for the same region in S. typhiCT18 ( www.sanger.ac.uk/Projects/S.typhi/blast_server.shtml). This fragment includes 128 bp from the rfaH coding region and also 46 bp from the coding region of the upstream gene, yigC, which is transcribed in the opposite direction (Fig. 3). Therefore, the 334 bp fragment spans two divergently transcribed promoters.



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Fig. 3. Sequence analysis of the yigC–rfaH intergenic promoter region using the Natural Network Promoter Prediction program. Arrows indicate the predicted transcription initiation sites for rfaH (C182) and for yigC (T74). Underlined sequences indicate the predicted -35 and -10 promoter regions for yigC (bases 84–105) and for rfaH (bases 148–176). Boxed segments indicate the putative RcsAB and RpoN boxes. Predicted translational start sites for yigC and rfaH are as labelled.

 
The sequence of the intergenetic region was analysed with the Natural Network Promoter Prediction program (www.fruitfly.org/seq_tools/promoter.html) to predict potential promoter sites. The strongest predictions corresponded to positions 84 and 105 for the yigC promoter (score 0·84), and to positions 148 and 176 for the rfaH promoter (score 0·88). The program also predicted the location of the initiation of transcription at bases C182 and T74 for the rfaH and yigC transcripts, respectively (Fig. 3).

A closer examination of the sequences upstream of the rfaHpromoter revealed homologies to potential binding sites of known regulatory proteins. The sequence 5'-TAAGCGCATCATTA-3' (Fig. 3) had similarities with an RcsAB binding box, described in the upstream regions of promoter elements regulated by the RcsB/RcsA dimer (Wehland & Bernhard, 2000 ). Furthermore, sequences containing the conserved motifs 5'-TGCT-3' and 5'-TGGC-3' were also found (Fig. 3). These sequences resemble the -12 and -24 recognition regions for the alternative sigma factor RpoN, which is responsible for gene expression under nitrogen starvation (Shingler, 1996 ; Wang & Gralla, 1998 ).

RpoN regulates rfaH gene expression
To investigate whether RpoN or the RcsC/YojN/RcsB system could regulate the expression of rfaH, we constructed insertional mutations in the rpoNand rcsB genes of S. typhiTy2 as described in Methods. The two mutant strains, named M161 (Ty2 rpoN) and M159 (Ty2 rcsB), were transformed with plasmid pCE334 and the ß-galactosidase activity was measured during the bacterial growth. Fig. 4 shows that inactivation of rpoN abolished the growth-phase-dependent pattern of ß-galactosidase production. In contrast, the rcsB mutant strain displayed a pattern of ß-galactosidase production over the course of the growth experiment that was similar to that of the wild-type S. typhiTy2 (Fig. 1a). These results suggest that the rfaHpromoter activity can be modulated in an RpoN-dependent manner. To support this notion, the production of LPS by the mutant strain was also examined. Fig. 5(a) shows that the LPS profiles of the rcsBmutant remain identical to those of the wild-type strain (Fig. 1b). Densitometric analyses also revealed an approximately twofold increased proportion of O-antigen relative to lipid A-core in the samples obtained from cells grown to stationary phase (data not shown). In contrast, the proportion of O-antigen relative to lipid A-core was constant in the LPS profiles of the rpoNmutant irrespective of the growth stage (Fig. 5b).



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Fig. 4. Growth and expression of rfaH–lacZ transcriptional fusion in S. typhi rpoN/pCE334 and rcsB/pCE334 mutants. {circ}, {square}, Growth (OD600) of S. typhi rcsB/pCE334 ({circ}) and S. typhi rpoN/pCE334 ({square}); {bullet}, {blacksquare}, expression of rfaH–lacZ fusions in S. typhi rcsB ({bullet}) and S. typhi rpoN ({blacksquare}) mutants, measured as ß-galactosidase activity. Data are the mean±SD of three independent assays.

 


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Fig. 5. Production of LPS in S. typhi rcsB/pCE334 and S. typhi rpoN/pCE334 mutants grown to different points of the growth curve in LB. (a) LPS profiles of S. typhi rcsB/pCE334 grown to the following OD600: lane 1, 0·100; lane 2, 0·186; lane 3, 0·445; lane 4, 0·668; lane 5, 1·021. (b) LPS profiles of S. typhi rpoN/pCE334 grown to the following OD600: lane 1, 0·104; lane 2, 0·256; lane 3, 0·401; lane 4, 0·676; lane 5, 1·181. All samples were adjusted to an OD600 of 2·0 in a final volume of 100 µl for LPS preparation. LPS was analysed by Tricine/SDS-PAGE on 14% acrylamide gels. Each well was loaded with approximately 1x108 c.f.u.

 
Nitrogen limitation increases the transcription of the rfaH gene and the production of O-specific LPS
The results of the previous section demonstrate that expression of rfaH in S. typhi Ty2 is regulated, either directly or indirectly, by the alternative sigma factor RpoN. Several cellular functions are regulated by RpoN, including utilization of various nitrogen and carbon sources, energy metabolism, chemotaxis, flagellation, and response to heat shock (reviewed by Buck et al., 2000 ). But to our knowledge, a role for RpoN in regulation of LPS biosynthesis has not been described. Because of our results demonstrating that the growth-dependent regulation of the rfaH gene requires the RpoN function, we reasoned that rfaH expression would also be regulated by nitrogen limitation. To test this hypothesis, we grew S. typhi/pCE334 in low-nitrogen minimal E medium and measured the ß-galactosidase activity in the exponential phase of growth (OD600 0·2). As shown in Table 3, expression of the rfaH–lacZ fusion increased nearly fourfold when bacteria were grown in the low-nitrogen medium. Additionally, we investigated the effect of nitrogen limitation on LPS production by the wild-type Ty2 strain grown to mid-exponential phase (OD600 0·2). Fig. 6 (lane 2) shows that nitrogen limitation increased the expression of the O-antigen by the wild-type S. typhi Ty2, as demonstrated by a similar profile to that observed during the stationary phase of growth in rich medium (Fig. 1b). The relationship between LPS production and nitrogen limitation parallels the pattern of rfaHregulation under the control of RpoN and is consistent with the idea that RpoN modulates LPS formation via its effect on rfaH gene expression during bacterial growth.


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Table 3. Effect of nitrogen availability on the expression of rfaH–lacZ transcriptional fusion

 


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Fig. 6. Effect of nitrogen availability on LPS production by S. typhi Ty2 and S. typhi rpoN, analysed by Tricine/SDS-PAGE. Cultures were grown to mid-exponential phase. Samples were adjusted to an OD600 of 2·0 in a final volume of 100 µl for LPS preparation. LPS was analysed by Tricine/SDS-PAGE on 14% acrylamide gels. Each well was loaded with approximately 1x108 c.f.u. Lane 1, S. typhi Ty2 grown in E medium; lane 2, S. typhi Ty2 grown in low-nitrogen E medium; lane 3, S. typhi rpoN grown in E medium.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Bacterial cells can sense host microenvironments and respond by coordinately regulating gene expression. Invasion of the intestinal epithelium and survival within macrophages are essential properties required for typhoidal pathogenesis. Therefore, Salmonellamust sense the host environment, either at mucosal surfaces or within host cells, and respond by inducing or repressing gene expression. Essential to the adaptation of bacteria during host infection is the remodelling of the bacterial surface (Ernst et al., 2001 ). This involves the modification of outer-membrane proteins and of LPS, the most abundant component of the bacterial envelope and a major pathogenic factor of Salmonella(Joiner, 1988 ; Rosenberger et al., 2000 ; Ernst et al., 2001 ). LPS is required for complement evasion (Joiner, 1988 ) as well as for invasion of intestinal epithelial cells (Pier et al., 1998 ; Lyczak et al., 2001 ) in the pathogenesis of S. typhi.

Evidence from other studies has demonstrated that the transcription of the LPS core and O-polysaccharide gene clusters is subject to positive regulation at the level of mRNA elongation by the RfaH protein (Farewell et al., 1991 ; Pradel & Schnaitman, 1991 ; Bailey et al., 1996 ; Marolda & Valvano, 1998 ; Wang et al., 1998 ). RfaH promotes expression of operons encoding proteins targeted to the cell surface or membrane (Bailey et al., 1997 ) by promoting the efficient elongation of the mRNA (Artsimovitch & Landick, 2002 ). Based on a detailed deletion-fusion analysis of the E. coliO7 LPS promoter region, Marolda & Valvano (1998) have proposed a model involving premature termination of transcription relieved by the RfaH protein that operates to regulate the expression of O-specific polysaccharide genes. In this model, the site for premature transcription termination is located within a relatively long untranslated 5' mRNA, and possibly depends on the formation of a hairpin. A similar 5' untranslated region is present in the case of the Salmonella O-antigen clusters. Therefore, it is reasonable to assume that a similar RfaH-dependent regulation of transcription elongation takes place in the S. typhiO-antigen gene cluster.

In this study, we have shown that the synthesis of the S. typhiO-antigen is regulated by RfaH in a growth-phase-dependent manner. Production of the O-antigen correlates with the differential expression of the rfaHgene during the bacterial growth, increasing at the late exponential phase and reaching maximal expression at the stationary phase. As has been shown in other bacteria (Marolda & Valvano, 1998 ; Wang et al., 1998 ), we found that RfaH modulates the O-antigen genes by positively regulating gene transcription, as demonstrated by the increase in the wbaP-specific mRNA levels at the stationary phase of growth.

Previous work in our laboratory has shown that environmental conditions that are known to be important for LPS regulation in other systems, such as growth temperature (Al-Hendy et al., 1991 ) and osmolarity (Aguilar et al., 1997 ), do not affect transcription of the rfaHgene (Rojas et al., 2001 ). In this study, we examined the sequences upstream of the rfaHpromoter and found potential binding sites for the RcsB/RcsA dimer of the RcsC/YojN/RcsB phosphorelay system and for the RpoN alternative sigma factor. We therefore investigated the transcriptional activity of the rfaHgene under growth conditions that activate the RcsC/YojN/RcsB system or rpoN-mediated transcription. No effects were detected with osmotic shock or growth at low temperature, which are known to induce the expression of exopolysaccharides by activating the RcsC/RcsB system (Sledjeski & Gottesman, 1996 ; Whitfield & Roberts, 1999 ). Nor were changes in rfaHexpression resulting from a mutation in the rcsBgene found. In contrast, growth of S. typhiTy2 in a nitrogen-limited medium induced rfaHgene transcription, and the inactivation of the rpoNgene, which encodes the alternative sigma factor {sigma}54 ({sigma}N), abolished the growth-phase-dependent induction of rfaH expression.

Most bacteria possess one copy of rpoN, which generally is constitutively expressed and not essential for survival and growth under favourable conditions (Buck et al., 2000 ). RpoN is a specialized sigma factor that recognizes a subset of promoters in bacteria that control regulation of nitrogen metabolism as well as many other biological activities, transcribing genes with diverse physiological roles, including flagellation, chemotaxis, energy metabolism, RNA modification, electron transport, response to heat shock and expression of alternative sigma factors (Merrick, 1993 ; Buck et al., 2000 ). RpoN-mediated transcription has also been associated with bacterial pathogenicity. Early reports showed that expression of pilin genes in Pseudomonas aeruginosa (Ishimoto & Lory, 1989 ) and in Neisseria gonorrhoeae(Klimpel et al., 1989 ) required RpoN. More recently, Klose & Mekalanos (1998) reported that an rpoNnull mutant of Vibrio cholerae was defective for colonization in an infant mouse model of cholera, and that this defect was distinct from the non-motile and glutamine synthetase phenotypes of the rpoNmutant (Klose & Mekalanos, 1998 ). Other authors have shown that a strain of Proteus mirabiliscarrying a mutation in a gene which is highly homologous to ORF284 of the rpoNoperon has a reduced ability to infect the urinary tract of CBA mice (Zhao et al., 1999 ).

A role for RpoN in the expression of cell-surface polysaccharides has been demonstrated in the case of alginate production by Pseudomonas aeruginosa (Boucher et al., 2000 ), but to our knowledge this is the first observation that RpoN plays a role in modulation of gene expression of O-antigen LPS genes. Our results suggest that RpoN acts directly or indirectly on rfaH gene expression to modulate O-antigen synthesis in an RfaH-mediated fashion. This regulation is manifested not only during the growth cycle but also under conditions of nitrogen limitation. Further studies are under way to characterize in detail the mechanism of RpoN action on the rfaHpromoter region and the possible interrelation between stationary phase and nitrogen limitation in relation to O-antigen synthesis.


   ACKNOWLEDGEMENTS
 
This investigation was supported by grants 1990967 and 7990058 from FONDECYT to I.C. and grant MOP10206 from the Canadian Institutes of Health Research to M.A.V.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 22 May 2002; revised 7 August 2002; accepted 20 August 2002.



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