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
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ABSTRACT |
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Keywords: lipopolysaccharide, regulation, transcription, sigma factor
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INTRODUCTION |
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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.
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METHODS |
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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.
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RESULTS |
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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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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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DISCUSSION |
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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
54 (
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.
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ACKNOWLEDGEMENTS |
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Received 22 May 2002;
revised 7 August 2002;
accepted 20 August 2002.
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