| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, PO Box 174 Correo 22, Santiago, Chile
2 Infectious Diseases Research Group, Siebens-Drake Research Institute, Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario N6A 5C1, Canada
Correspondence
Inés Contreras
icontrer{at}uchile.cl
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
rfaH mutant, which synthesized only low-molecular-mass OAg molecules and a small amount of S-OAg. Real-time RT-PCR revealed a drastic reduction of wzy polymerase gene expression in the rfaH mutant. Complementation of this mutant with the wzy gene cloned into a high-copy-number plasmid restored the bimodal OAg distribution, suggesting that cellular levels of Wzy influence not only OAg polymerization but also chain-length distribution. Accordingly, overexpression of wzy in the wild-type strain resulted in production of a large amount of high-molecular-mass OAg molecules. An increased dosage of either wzzB or wzzpHS-2 also altered OAg chain-length distribution. Transcription of wzzB and wzzpHS-2 genes was regulated during bacterial growth but in an RfaH-independent manner. Overall, these findings indicate that expression of the wzy, wzzB and wzzpHS-2 genes is finely regulated to determine an appropriate balance between the proteins responsible for polymerization and chain-length distribution of S. flexneri OAg.
These authors contributed equally to this work.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Sansonetti, 2001). S. flexneri infection requires the expression of several virulence factors encoded in a 220 kb virulence plasmid. This plasmid includes two operons transcribed in opposite directions: the ipa operon encoding the invasion proteins (Ipa) and the mxi/spa genes encoding the type III secretion system for the translocation of Ipa proteins into the epithelial cells (Sansonetti & Egile, 1998).
In addition to invasion proteins, the lipopolysaccharide (LPS) plays a role in Shigella virulence (Morona et al., 2003; Okada et al., 1991; Sandlin et al., 1995; Van den Bosch et al., 1997). LPS, a major component of the outer membrane of Gram-negative bacteria, comprises three domains: the inner hydrophobic lipid A region, the oligosaccharide core and the outer O-polysaccharide chain or O antigen (OAg) that is exposed to the bacterial surface (Raetz & Whitfield, 2002; Valvano, 2003; Whitfield, 1995). Early reports showed that mutants of S. flexneri devoid of OAg or with defects in the core region were less virulent in vivo. Although these mutants invaded and replicated within non-polarized epithelial cells, they failed to spread to adjacent cells in a monolayer (Okada et al., 1991; Sandlin et al., 1995). The altered LPS structures in these mutants led to an incorrect localization and dysfunction of the IcsA protein (Sandlin et al., 1995; Van den Bosch & Morona, 2003; Van den Bosch et al., 1997). Also, adherence to and internalization into polarized intestinal epithelial cells are highly dependent on the length of the LPS, and require both the OAg and core regions (Kohler et al., 2002). In addition to contributing to bacterial invasion, the OAg might, by itself, elicit inflammation and haemorrhage of the intestinal tissue (Zhong, 1999). Moreover, the OAg confers serum resistance by protecting the bacterium from the lytic action of complement (Hong & Payne, 1997).
LPS OAg synthesis is driven by complex biochemical mechanisms (Raetz & Whitfield, 2002; Valvano, 2003; Whitfield, 1995). In S. flexneri, OAg synthesis begins in the cytoplasmic face of the inner membrane with the addition of N-acetylglucosamine (GlcNAc) to the lipid carrier undecaprenyl phosphate. The additional sugars are added to the GlcNAc residue in a sequential manner to form a complete OAg unit that is translocated to the periplasmic side by the Wzx translocase. Then, the Wzy polymerase links the pre-formed OAg units, generating the OAg chain. The complete LPS is formed by the ligation of the OAg chain to pre-formed lipid A-core oligosaccharide by the WaaL ligase, which results in the release of undecaprenyl pyrophosphate. The Wzz protein is essential for generating a non-random OAg LPS structure, resulting in a preferred OAg chain length or modal distribution.
The LPS molecules of S. flexneri 2a have OAg with two preferred OAg chain lengths, a short (S-OAg) composed on average of 17 repeated units (RU) that is regulated by a chromosomally encoded WzzB protein (Morona et al., 1995), and a very long LPS (VL-OAg) of about 90 RU. VL-OAg requires WzzpHS-2, which is encoded in plasmid pHS-2 (Stevenson et al., 1995). The length distribution of the OAg modulates S. flexneri virulence, since mutants affected in wzzpHS-2 are more sensitive to serum killing and less virulent in vivo, while mutants in wzzB are defective in invasiveness and plaque formation (Hong & Payne, 1997; Morona et al., 2003; Van den Bosch et al., 1997).
Little is known about the regulation of OAg chain-length distribution. Hong & Payne (1997) reported that the expression of the very long chain length determinant, WzzpHS-2, is not regulated by a number of different environmental conditions such as iron concentration, temperature, pH and nutrients. More recently, Varela et al. (2001) showed that S. flexneri grown at 30 °C produced increased amounts of long chains relative to short chains, compared to bacteria grown at 37 °C. However, the mechanisms underlying this modulation were not investigated.
We previously demonstrated that production of OAg by Salmonella Typhi Ty2 varies during bacterial growth in direct relationship with the growth-regulated expression of the RfaH transcription elongation factor (Bittner et al., 2002; Rojas et al., 2001). RfaH controls the expression of OAg and core oligosaccharide biosynthesis genes (Bailey et al., 1997; Pradel & Schnaitman, 1991; Wang et al., 1998). Here, we demonstrate that the VL-OAg in S. flexneri increases significantly during growth while the S-OAg distribution remains relatively constant. VL-OAg production correlated with a growth-dependent regulation of the rfaH gene. Our results also indicate that RfaH is essential for expression of the wzy polymerase gene, but not for wzzB or wzzpHS-2, suggesting that the cellular levels of Wzy are critical for VL-OAg production and normal OAg chain-length distribution in S. flexneri 2a.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
summarizes the properties of the bacterial strains and plasmids used in this study. Bacteria were grown aerobically in Luria–Bertani medium (LB) (10 g l–1 Bacto tryptone, 5 g l–1 Bacto yeast extract, 5 g l–1 NaCl). Solid medium contained 1.5 % (w/v) agar. Media were supplemented with 100 µg ampicillin ml–1 or 50 µg kanamycin ml–1 as appropriate.
|
to create chromosomal deletions by homologous recombination using PCR products. Primers were designed according to the DNA sequence information available for the S. flexneri 2a 2457T strain (Wei et al., 2003) and plasmid pHS-2 (NC_002773). To disrupt the genes, S. flexneri 2a 2457T cells were first transformed with the temperature-sensitive plasmid pKD46, which expresses the 
Red recombinase system. These cells were transformed with PCR products that were generated using as template the pKD4 plasmid, which contains the FRT-flanked kanamycin-resistance gene (aph). The primers used carried 40 bases that were homologous to both edges of the gene targeted for disruption. The sequences of the oligonucleotide primers used in this study are available upon request. In the presence of the Red recombinase system, the integration of the amplicons resulted in the targeted replacement of the wild-type gene by the antibiotic-resistance cassette. The kanamycin-resistant transformants were replica-plated in the absence of antibiotic selection at 42 °C and finally assayed for ampicillin sensitivity to confirm the loss of pKD46. To obtain a non-polar deletion of the rfaH gene, the antibiotic-resistance gene was removed by transforming the gene replacement mutant with pCP20, which encodes the FLP recombinase (Cherepanov & Wackernagel, 1995). Transformants were plated on LB agar containing ampicillin and kanamycin at 37 °C. Individual colonies were replica-plated on LB agar, on LB agar containing ampicillin and on LB agar containing kanamycin. The plates were incubated at 42 °C. Transformants that had lost the resistance gene (aph) and plasmid pCP20 were selected as those colonies that were able to grow only on LB agar. Correct insertional gene replacement and the deletion of the antibiotic-resistance gene cassette were confirmed by PCR analysis.
Cloning of the rfaH, wzzB, wzzpHS-2 and wzy genes.
DNA fragments containing the S. flexneri 2457T rfaH (GeneID:1080050), wzzB (GeneID:1077593), wzzpHS-2 (NC_002773) and wzy (GeneID:1078521) genes and their promoter regions were amplified by PCR. The amplicons were cloned into pGEM-T Easy as recommended by the supplier.
Construction of lacZ transcriptional fusions.
The rfaH, wzzB and wzzpHS-2 promoter regions were amplified by PCR. The corresponding fragments were cloned into the pGEM-T Easy vector and then subcloned into plasmid pFZY1. This is a single-copy-number vector designed for the construction of transcriptional fusions to the lac operon (Koop et al., 1987). The resulting plasmids, pCB315, pCB326 and pCB280, respectively, were transformed into S. flexneri 2457T. Also, a 261 bp PCR fragment of an intragenic region of the rfaH gene was cloned into pFZY1 to generate plasmid pCB261, which was used as negative control.
RNA extraction.
Bacterial cells grown to early exponential (OD600 0.1) and stationary phase (OD600 1.5) were incubated with lysozyme (1 mg ml–1) for 10 min at 4 °C, then total RNA was isolated using the TRIzol reagent (Invitrogen) according to the manufacturer's recommendations. Genomic DNA contamination from RNA samples was removed by treatment with Turbo DNase from Ambion. The integrity and purity of the RNA was assessed by denaturing agarose/formaldehyde gel electrophoresis and by nucleic acid/protein ratio (A260/A280). A ratio A260/A280 >1.90 was obtained for all samples.
Real-time quantitative RT-PCR.
Expression of the wzy gene was examined by qRT-PCR. Five micrograms of total RNA was treated with 200 units of Superscript II Reverse Transcriptase (Invitrogen) by using gene-specific primers according to the manufacturer's recommendations. Quantitative PCR was performed using an Opticon 2 Thermal cycler PT (MJ Research) and SYBR Green technology (Platinum SYBR Green qPCR SuperMix-UDG, Invitrogen). Reaction mixtures containing no template and reaction mixtures containing DNase-treated RNA were included in each real-time PCR experiment to assess primer-dimers formation and residual chromosomal DNA, respectively. The identities of the amplicons resulting from the reactions were checked after amplification by melting curve analysis and amplicon DNA gel electrophoresis. The relative expression of wzy was normalized to the transcript levels of the hisG gene, whose expression remains constant throughout the bacterial growth (unpublished results), using the Relative Standard Curve method (Applied Biosystems). The statistical significance of differences in the data was determined using an unpaired Student's t-test.
LPS analysis.
LPS was prepared as described elsewhere (Marolda et al., 2006). Briefly, culture samples obtained at different times during growth were adjusted to OD600 2.0 in a final volume of 1.5 ml LB. Cells were centrifuged and the pellets were suspended in 100 µl lysis buffer containing proteinase K, followed by hot phenol extraction and a subsequent extraction of the aqueous phase with ether. LPS was separated on 14 % (w/v) acrylamide gels using a Tricine-SDS buffer system (Lesse et al., 1990) and visualized by silver staining (Marolda et al., 2006). The concentration of LPS was determined by measuring 2-keto-3-deoxyoctulosonic acid (KDO) using the Purpald assay (Marolda et al., 2006). Densitometry analysis was performed using the UN-SCANT-IT gel software (Silk Scientific). The ratio of the relative intensity of the lipid A-core band to the average intensity of the bands corresponding to the S-OAg and VL-OAg was calculated by quantifying the pixels in a narrow window across the centre of each lane. The densitometry analysis was calibrated by determining the ratio of the relative intensity of the lipid A-core to the average intensity of the O-antigen bands using a range of loading volumes of S. flexneri 2a 2457T LPS. The statistical significance of differences in the data was determined using the one-way ANOVA test and the Tukey post test.
Western blot analysis was performed as described by Marolda et al. (2006). Briefly, the gel was transferred to a PVDF membrane for 75 min at 250 mA and blocked for 90 min in 5 % (w/v) skim milk at room temperature. The membrane was incubated with a polyclonal rabbit antiserum against S. flexneri (Probac do Brasil, Produtos Bacteriológicos) as a primary antibody, and goat anti-rabbit HRP conjugated (Pierce) as a secondary antibody. Detection was performed using the SuperSignal West Pico chemiluminiscent substrate (Pierce).
β-Galactosidase assays.
Two hundred microlitres of an overnight culture in LB was inoculated into 100 ml of the same medium and grown in an orbital shaker. At different times during growth, a 1 ml sample was withdrawn to measure the bacterial growth (OD600) and the β-galactosidase activity according to Miller (1972). Enzyme activities are expressed as Miller units. Each sample was analysed in triplicate in two independent experiments.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) revealed a differential growth-phase-dependent regulation of OAg synthesis. While the amount of VL-OAg increased throughout the bacterial growth, the S-OAg chains remained relatively constant. The densitometric quantification of the lanes in the gel showed a significant increase in the ratio of VL-OAg to the lipid A-core region in the samples grown to mid-exponential and stationary phases, compared to the sample grown to early exponential phase. In contrast, no significant differences in the amount of S-OAg relative to the lipid A-core during growth were observed (Fig. 1b). The differences in OAg expression were confirmed by Western blot analysis. As shown in Fig. 1(c), a higher amount of VL-OAg was detected at stationary phase (lane 2) compared to early exponential phase (lane 1). LPS obtained from a mutant lacking the VL-OAg (strain MSF107, Table 1
) was also analysed (Fig. 1c, lane 3).
|
). To examine whether RfaH plays any role in growth-regulated OAg synthesis in S. flexneri, we constructed an rfaH–lacZ transcriptional fusion in a single-copy-number plasmid (pFZY1). The resulting plasmid, pCB315, was transformed into S. flexneri 2a 2457T. Plasmid pCB261, containing an intragenic region of the rfaH gene cloned in pFZY1, was also transformed as a negative control. The production of β-galactosidase by S. flexneri/pCB315 and S. flexneri/pCB261 was assayed at various stages of growth. Fig. 2 shows that β-galactosidase production driven by the rfaH promoter increases at late exponential phase, reaching maximal expression during stationary phase. Thus, the pattern of VL-OAg production reflects the differential rfaH expression during the bacterial growth cycle.
|
rfaH mutant. As shown in Fig. 3(a), MSF487 lacked LPS with VL-OAg chains at both exponential and stationary phases of growth (lanes 2 and 3). The absence of VL-OAg was confirmed in a gel loaded with a 10-fold excess of LPS sample (lane 4). Also, strain MSF487 produced a small amount of low-molecular-mass OAg chains (1–4 RU) and fewer S-OAg molecules migrating slightly faster than the wild-type S-OAg. In addition, greater amounts of unsubstituted core-lipid A molecules than in the wild-type strain were detected. When MSF487 was transformed with plasmid pJC75, harbouring the rfaH gene in a high-copy-number plasmid, the wild-type LPS phenotype was restored (Fig. 3b).
|
) that encodes the majority of the genes required for OAg synthesis. RfaH recognizes RNA polymerase by interacting with the ops sequence, allowing transcript elongation in long operons (Artsimovitch & Landick, 2002). Hence, the RfaH function affects particularly transcription of distal genes. In S. flexneri, the wzy gene, which encodes the OAg polymerase, is the last gene in the wba operon (Morona et al., 1994). Thus, we reasoned that absence of RfaH in strain MSF487 could result in low levels of transcription of wzy and impaired OAg polymerization. To investigate this hypothesis, we examined by qRT-PCR the transcript levels of wzy in the wild-type and rfaH strains. The results showed a drastic reduction (120-fold) of wzy expression in the rfaH mutant compared to the wild-type (P<0.0022). When strain MSF487 was transformed with pJC75, a significant increase in the expression of wzy was observed (P<0.0009). The transcript levels of wzy in the complemented mutant were approximately 30 % of those obtained in the wild-type strain.
Overexpression of Wzy affects O-antigen chain-length distribution
The results described above indicated that the function of the rfaH gene is essential for wzy expression and normal polymerization of OAg in S. flexneri 2a. To investigate this phenomenon further, we transformed the rfaH mutant with plasmid pJC114, which carries the wzy gene in a multicopy plasmid. Analysis of the LPS patterns showed that both S-OAg and VL-OAg were produced at exponential and stationary phases of growth, while very small amounts of low-molecular-mass OAg molecules were detected (Fig. 4a, lanes 3 and 4). Thus, increased expression of wzy complements the defective LPS phenotype of the rfaH mutant. However, close examination of the LPS profile exhibited by strain MSF487/pJC114 showed an altered LPS pattern compared to the wild-type: a higher amount of high-molecular-mass OAg molecules relative to low-molecular-mass OAg chains was observed. This result suggested that cellular levels of Wzy could influence not only OAg polymerization but also the chain-length distribution of OAg chains. To test this notion, we transformed the wild-type strain with pJC114 and analysed the LPS profiles during bacterial growth. As shown in Fig. 4(b), the LPS molecules had lower numbers of OAg repeat units of low molecular mass (1–3 RU) and fewer S-OAg chains than the wild-type LPS. In contrast, a large amount of high-molecular-mass OAg molecules were produced. These data indicate that levels of Wzy are important for VL-OAg expression.
|
). S. flexneri 2457T/pJC139 produced greater numbers of S-OAg chains than the wild-type, but did not synthesize VL-OAg molecules. In this strain production of S-OAg was growth-phase-regulated, increasing at stationary phase. On the other hand, overexpression of the wzzpHS-2 gene (2457T/pJC144) resulted in higher production of VL-OAg than the wild-type, particularly during stationary phase. In contrast, this strain produced a small amount of S-OAg chains at both exponential and stationary phases of growth (Fig. 5). These results indicate that not only the levels of Wzy, but also those of the Wzz chain-length regulators are critical to determine the chain-length distribution of OAg in S. flexneri 2a. To investigate whether expression of the wzz genes is under growth-phase control, we constructed transcriptional fusions of the wzzB and wzzpHS-2 promoter regions to lacZ in plasmid pFZY1. The resulting plasmids, pCB326 and pCB280, respectively, were transformed into the wild-type and rfaH strains and β-galactosidase activity was assayed during growth (Fig. 6). Expression of both wzzB and wzzpHS-2 genes increased during the growth cycle, reaching maximal activities at stationary phase. However, no differences in expression levels were observed in the rfaH genetic background compared to those obtained in the wild-type strain. From these data, we conclude that the differential effect of growth phase on OAg chain-length distribution is not due to an rfaH-dependent regulation of either chain length determinant.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Morona et al., 2003; Sandlin et al., 1995; Van den Bosch et al., 1997). It has been reported that not only the presence of OAg side chains, but also the number and proper length distribution of OAg molecules are important for full virulence of S. flexneri 2a (Hong & Payne, 1997; Morona et al., 2003). Yet, in contrast to the wealth of information available on the regulatory mechanisms that control expression of Shigella invasion proteins (Dorman & Porter, 1998; Sansonetti, 2001), little is known about environmental regulation of OAg synthesis and chain-length distribution.
In this study, we demonstrate a differential growth-phase regulation of OAg production in S. flexneri 2a. Our results showed that the production of VL-OAg correlates with the growth-dependent expression of the RfaH transcription elongation factor. LPS production was severely impaired in a rfaH mutant which, while it was able to synthesize a small amount of low-molecular-mass OAg chains and a few S-OAg molecules, was totally devoid of VL-OAg. The LPS phenotype exhibited by the rfaH mutant was not attributable to a deficit in the expression of either wzzB or wzzpHS-2 chain length regulators, but rather it was the result of diminished transcription of wzy. Quantitative RT-PCR results showed over 100-fold reduction in the transcript levels of wzy in the rfaH mutant compared to the wild-type.
Interestingly, complementation of the rfaH mutant with a high-copy-number plasmid carrying the rfaH gene could not restore wild-type levels of wzy transcription. Since RfaH controls the expression of an important number of membrane components (Bailey et al., 1997) it is plausible that the deletion or overexpression of this regulator could generate a membrane stress response in order to maintain cellular homeostasis. Two recent studies support this notion. Nagy et al. (2006) showed that loss of RfaH not only had an impact on genes involved in LPS synthesis but also had an indirect and marked effect on a number of membrane components such as the flagellum/chemotaxis complex and type III secretion system. In addition, Bengoechea et al. (2002) demonstrated that overexpression of Wzz in Yersinia enterocolitica causes a membrane stress response that activates the CpxAR two-component signal transduction system, which in turn downregulates the expression of the OAg biosynthetic machinery.
The results discussed above suggested that an augmentation in RfaH levels increases Wzy expression and OAg polymerization during stationary phase. In support of this hypothesis, overexpression of wzy in the wild-type strain results in an altered pattern of OAg synthesis displaced towards the production of high-molecular-mass chains. Our findings are in accordance with results obtained by Daniels et al. (1998) showing that complementation of a wzy mutant with the wzy gene cloned on a high-copy-number plasmid produces LPS with an increased OAg chain length.
The demonstration that overexpression of the chain length determinants can alter the OAg modal distribution further underlines the importance of a specific balance between Wzy, WzzB and WzzpHS-2 in determining the OAg modal distribution, as proposed by Daniels et al. (1998). In addition, our results showing that overexpression of WzzB completely shifts the OAg distribution from VL-OAg to S-OAg, but that overexpression of WzzpHS-2 can not do the opposite as efficiently, support the hypothesis of Stevenson et al. (1995), who proposed that WzzpHS-2 does not compete efficiently with WzzB in influencing the OAg chain-length distribution in S. flexneri.
Altogether, our data suggest that regulation of Wzy levels is not only important for normal OAg polymerization but is also essential in defining the OAg modal distribution for the following reasons. First, a rfaH mutant is unable to produce VL-OAg even though RfaH has no impact on wzzpHS-2 expression. Second, overexpression of wzy in a rfaH background can restore the OAg bimodal distribution despite global OAg synthesis deficiency. Third, overexpression of wzy in the wild-type strain results in an altered pattern of OAg synthesis shifted towards the production of high-molecular-mass chains. And fourth, overexpression studies of the chain length regulators support the notion that there is a competition for the available pool of Wzy in order to determine a specific OAg modal distribution in S. flexneri.
The experiments with lacZ fusions demonstrated that transcription of both wzzB and wzzpHS-2 also increases upon entry into stationary phase, but in an RfaH-independent manner. Although at the moment we can only speculate how environmental conditions could modulate the OAg modal distribution in Shigella, several reports have begun to unravel a variety of mechanisms involved in OAg chain length regulation. Salmonella Typhimurium possesses two chain length regulators, WzzB and WzzfepE (Murray et al., 2003). It has been shown that while wzzB expression is tightly regulated in response to conditions of low Mg2+ plus Fe3+ through the PmrA/PmrB and RcsC/YojN/RcsB systems (Delgado et al., 2006), wzzfepE expression is regulated by conditions that stimulate swarming motility (Wang et al., 2004). Additional control of wzzfepE expression by the flagellar master regulator FlhDC has been reported in Escherichia coli (Stafford et al., 2005). In addition, genome-wide analysis of the DNA adenine methyltransferase (Dam) regulon in E. coli described wzzB as one of many genes found to be repressed by the presence of this regulator (Robbins-Manke et al., 2005). Since Dam levels are downregulated in stationary phase (Seshasayee, 2007), the increase of wzzB transcription could be a result of diminished levels of Dam.
Unpublished results from our laboratories have implicated environmental signals such as oxygen availability and amino acid deprivation in the expression of rfaH and both chain-length regulators. These conditions are present in the stationary phase of growth and could therefore be responsible, at least in part, for the growth-phase regulation of these genes. How these and other environmental signals converge in order to regulate both LPS production and OAg modal distribution is under current investigation in our laboratories.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: P. van der Ley
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bailey, M. J., Hughes, C. & Koronakis, V. (1997). RfaH and the ops element, components of a novel system controlling bacterial transcription elongation. Mol Microbiol 26, 845–851.[CrossRef][Medline]
Bengoechea, J. A., Zhang, L., Toivanen, P. & Skurnik, M. (2002). Regulatory network of lipopolysaccharide O-antigen biosynthesis in Yersinia enterocolitica includes cell envelope-dependent signals. Mol Microbiol 44, 1045–1062.[CrossRef][Medline]
Bittner, M., Saldias, S., Estevez, C., Zaldivar, M., Marolda, C. L., Valvano, M. A. & Contreras, I. (2002). O-antigen expression in Salmonella enterica serovar Typhi is regulated by nitrogen availability through RpoN-mediated transcriptional control of the rfaH gene. Microbiology 148, 3789–3799.
Cherepanov, P. P. & Wackernagel, W. (1995). Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158, 9–14.[CrossRef][Medline]
Daniels, C., Vindurampulle, C. & Morona, R. (1998). Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy). Mol Microbiol 28, 1211–1222.[CrossRef][Medline]
Datsenko, K. A. & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640–6645.
Delgado, M. A., Mouslim, C. & Groisman, E. A. (2006). The PmrA/PmrB and RcsC/YojN/RcsB systems control expression of the Salmonella O-antigen chain length determinant. Mol Microbiol 60, 39–50.[CrossRef][Medline]
Dorman, C. J. & Porter, M. E. (1998). The Shigella virulence gene regulatory cascade: a paradigm of bacterial gene control mechanisms. Mol Microbiol 29, 677–684.[CrossRef][Medline]
Hong, M. & Payne, S. M. (1997). Effect of mutations in Shigella flexneri chromosomal and plasmid-encoded lipopolysaccharide genes on invasion and serum resistance. Mol Microbiol 24, 779–791.[CrossRef][Medline]
Jennison, A. V. & Verma, N. K. (2004). Shigella flexneri infection: pathogenesis and vaccine development. FEMS Microbiol Rev 28, 43–58.[CrossRef][Medline]
Kohler, H., Rodrigues, S. P. & McCormick, B. A. (2002). Shigella flexneri interactions with the basolateral membrane domain of polarized model intestinal epithelium: role of lipopolysaccharide in cell invasion and in activation of the mitogen-activated protein kinase ERK. Infect Immun 70, 1150–1158.
Koop, A. H., Hartley, M. E. & Bourgeois, S. (1987). A low-copy-number vector utilizing beta-galactosidase for the analysis of gene control elements. Gene 52, 245–256.[CrossRef][Medline]
Lesse, A. J., Campagnari, A. A., Bittner, W. E. & Apicella, M. A. (1990). Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Immunol Methods 126, 109–117.[CrossRef][Medline]
Marolda, C. L., Lahiry, P., Vines, E., Saldias, S. & Valvano, M. A. (2006). Micromethods for the characterization of lipid A-core and O-antigen lipopolysaccharide. Methods Mol Biol 347, 237–252.[Medline]
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Morona, R., Mavris, M., Falarino, A. & Manning, P. A. (1994). Characterization of the rfc region of Shigella flexneri. J Bacteriol 176, 733–747.
Morona, R., Van Den Bosch, L. & Manning, P. A. (1995). Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri. J Bacteriol 177, 1059–1068.
Morona, R., Daniels, C. & Van Den Bosch, L. (2003). Genetic modulation of Shigella flexneri 2a lipopolysaccharide O antigen modal chain length reveals that it has been optimized for virulence. Microbiology 149, 925–939.
Murray, G. L., Attridge, S. R. & Morona, R. (2003). Regulation of Salmonella Typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz. Mol Microbiol 47, 1395–1406.[CrossRef][Medline]
Nagy, G., Danino, V., Dobrindt, U., Pallen, M., Chaudhuri, R., Emody, L., Hinton, J. C. & Hacker, J. (2006). Down-regulation of key virulence factors makes the Salmonella enterica serovar Typhimurium rfaH mutant a promising live-attenuated vaccine candidate. Infect Immun 74, 5914–5925.
Okada, N., Sasakawa, C., Tobe, T., Yamada, M., Nagai, S., Talukder, K. A., Komatsu, K., Kanegasaki, S. & Yoshikawa, M. (1991). Virulence-associated chromosomal loci of Shigella flexneri identified by random Tn5 insertion mutagenesis. Mol Microbiol 5, 187–195.[CrossRef][Medline]
Pradel, E. & Schnaitman, C. A. (1991). Effect of rfaH (sfrB) and temperature on expression of rfa genes of Escherichia coli K-12. J Bacteriol 173, 6428–6431.
Raetz, C. R. & Whitfield, C. (2002). Lipopolysaccharide endotoxins. Annu Rev Biochem 71, 635–700.[CrossRef][Medline]
Robbins-Manke, J. L., Zdarveski, Z. Z., Marinus, M. & Essigmann, J. M. (2005). Analysis of global gene expression and double strand-break formation in DNA adenine methyltransferase and mismatch repair-deficient Escherichia coli. J Bacteriol 187, 7027–7037.
Rojas, G., Saldias, S., Bittner, M., Zaldivar, M. & Contreras, I. (2001). The rfaH gene, which affects lipopolysaccharide synthesis in Salmonella enterica serovar Typhi, is differentially expressed during the bacterial growth phase. FEMS Microbiol Lett 204, 123–128.[CrossRef][Medline]
Sandlin, R. C., Lampel, K. A., Keasler, S. P., Goldberg, M. B., Stolzer, A. L. & Maurelli, A. T. (1995). Avirulence of rough mutants of Shigella flexneri: requirement of O antigen for correct unipolar localization of IcsA in the bacterial outer membrane. Infect Immun 63, 229–237.[Abstract]
Sansonetti, P. J. (2001). Microbes and microbial toxins: paradigms for microbial–mucosal interactions III. Shigellosis: from symptoms to molecular pathogenesis. Am J Physiol Gastrointest Liver Physiol 280, G319–G323.
Sansonetti, P. J. & Egile, C. (1998). Molecular bases of epithelial cell invasion by Shigella flexneri. Antonie Van Leeuwenhoek 74, 191–197.[CrossRef][Medline]
Seshasayee, A. S. (2007). An assessment of the role of DNA adenine methyltransferase on gene expression regulation in E. coli. PLoS ONE 2, e273[CrossRef]
Stafford, G. P., Ogi, T. & Hughes, C. (2005). Binding and transcriptional activation of non-flagellar genes by the Escherichia coli flagellar master regulator. Microbiology 151, 1779–1788.
Stevenson, G., Kessler, A. & Reeves, P. R. (1995). A plasmid-borne O-antigen chain length determinant and its relationship to other chain length determinants. FEMS Microbiol Lett 125, 23–30.[CrossRef][Medline]
Valvano, M. A. (2003). Export of O-specific lipopolysaccharide. Front Biosci 8, s452–s471.[Medline]
Van den Bosch, L. & Morona, R. (2003). The actin-based motility defect of a Shigella flexneri rmlD rough LPS mutant is not due to loss of IcsA polarity. Microb Pathog 35, 11–18.[CrossRef][Medline]
Van den Bosch, L., Manning, P. A. & Morona, R. (1997). Regulation of O-antigen chain length is required for Shigella flexneri virulence. Mol Microbiol 23, 765–775.[CrossRef][Medline]
Varela, G., Schelotto, F., di Conza, J. & Ayala, J. A. (2001). Analysis of the O-antigen chain length distribution during extracellular and intracellular growth of Shigella flexneri. Microb Pathog 31, 21–27.[CrossRef][Medline]
Wang, L., Jensen, S., Hallman, R. & Reeves, P. R. (1998). Expression of the O antigen gene cluster is regulated by RfaH through the JUMPstart sequence. FEMS Microbiol Lett 165, 201–206.[CrossRef][Medline]
Wang, Q., Frye, J. G., McClelland, M. & Harshey, R. M. (2004). Gene expression patterns during swarming in Salmonella typhimurium: genes specific to surface growth and putative new motility and pathogenicity genes. Mol Microbiol 52, 169–187.[CrossRef][Medline]
Wei, J., Goldberg, M. B., Burland, V., Venkatesan, M. M., Deng, W., Fournier, G., Mayhew, G. F., Plunkett, G., III, Rose, D. J. & other authors (2003). Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T. Infect Immun 71, 2775–2786.
Whitfield, C. (1995). Biosynthesis of lipopolysaccharide O antigens. Trends Microbiol 3, 178–185.[CrossRef][Medline]
Zhong, Q. P. (1999). Pathogenic effects of O polysaccharide from Shigella flexneri strain. World J Gastroenterol 5, 245–248.[Medline]
Received 25 May 2007;
revised 22 June 2007;
accepted 3 July 2007.
This article has been cited by other articles:
|
|
 |
|
  W. T. Forsee, R. T. Cartee, and J. Yother A Kinetic Model for Chain Length Modulation of Streptococcus pneumoniae Cellubiuronan Capsular Polysaccharide by Nucleotide Sugar Donor Concentrations J. Biol. Chem., May 1, 2009; 284(18): 11836 - 11844. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Bravo, C. Silva, J. A. Carter, A. Hoare, S. A. Alvarez, C. J. Blondel, M. Zaldivar, M. A. Valvano, and I. Contreras Growth-phase regulation of lipopolysaccharide O-antigen chain length influences serum resistance in serovars of Salmonella J. Med. Microbiol., August 1, 2008; 57(8): 938 - 946. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Kintz, J. M. Scarff, A. DiGiandomenico, and J. B. Goldberg Lipopolysaccharide O-Antigen Chain Length Regulation in Pseudomonas aeruginosa Serogroup O11 Strain PA103 J. Bacteriol., April 15, 2008; 190(8): 2709 - 2716. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Purins, L. Van Den Bosch, V. Richardson, and R. Morona Coiled-coil regions play a role in the function of the Shigella flexneri O-antigen chain length regulator WzzpHS2 Microbiology, April 1, 2008; 154(4): 1104 - 1116. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
) and 2457T/pCB261 (
). The expression of the rfaH–lacZ fusion for strains 2457T/pCB315 (
) and 2457T/pCB261 (
) was measured as β-galactosidase activity. Data are the means±SD of triplicate assays in two independent experiments.
,