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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, University of Western Ontario, London, ON N6A 5C1, Canada
3 Infectious Diseases Research Group, Siebens–Drake Research Institute, Department of Medicine, University of Western Ontario, London, ON N6A 5C1, Canada
Correspondence
Miguel A. Valvano
mvalvano{at}uwo.ca
Inés Contreras
icontrer{at}uchile.cl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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wbaP mutants of S. enterica serovars Typhi and Typhimurium. Truncated forms of WbaP lacking the periplasmic loop exhibited altered chain-length distributions in O antigen polymerization, suggesting that this central domain is involved in modulating the chain-length distribution of the O polysaccharide. The N-terminal and periplasmic domains were dispensable for complementation of O antigen synthesis in vivo, suggesting that the C-terminal domain carries the sugar-phosphate transferase activity. However, despite the fact that they complemented the synthesis of O antigen in the wbaP mutant in vivo, membrane extracts containing WbaP derivatives without the N-terminal domain failed to transfer radioactive Gal from UDP-Gal into a lipid-rich fraction. These results suggest that the N-terminal region of WbaP, which contains four transmembrane domains, is essential for the insertion or stability of the protein in the bacterial membrane. We propose that the domain structure of WbaP enables this protein not only to function in the transfer of Gal-1-P to Und-P but also to establish critical interactions with additional proteins required for the correct assembly of O antigen in S. enterica.
These authors contributed equally to this work.
Present address: Departamento de Ciencias Biológicas, Facultad de Ciencias de la Salud, Universidad Nacional Andrés Bello, Santiago, Chile.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Valvano, 2003). The biosynthesis of LPS is a complex process involving a large number of enzyme activities (Raetz & Whitfield, 2002; Valvano, 2003). The core oligosaccharide is assembled on preformed lipid A by the sequential glycosyl transfer of monosaccharides, while the O antigen is assembled onto undecaprenyl phosphate (Und-P) at the cytoplasmic face of the inner membrane. There are two major pathways for the biosynthesis of O antigen, designated the Wzy-dependent and ABC transporter-dependent (Wzy-independent) pathways (Raetz & Whitfield, 2002; Valvano, 2003). In the Wzy-dependent mechanism, Und-PP-linked O repeating units are exported to the periplasmic face of the plasma membrane by an unknown mechanism that requires the protein Wzx. Subsequently, the O units are polymerized by the Wzy polymerase, while the Wzz protein regulates, by an unknown mechanism, the length distribution of the polymers, also referred to as modality (Batchelor et al., 1991). In the most common ABC transporter-dependent mechanism, the O antigen polymer results from the transfer of glycosyl residues to Und-PP at the cytoplasmic face of the plasma membrane, and its export to the periplasmic face by an ABC transporter. An additional ABC-transporter mechanism involving a synthase homologue has also been described (Keenleyside & Whitfield, 1996). In all of these pathways, the Und-PP-linked O antigen polymers are ultimately ligated to the outer core domain of the lipid A core (Raetz & Whitfield, 2002).
The initiation reaction of O antigen biosynthesis involves the formation of a phosphodiester bond between the membrane-associated Und-P and a cytosolic UDP-sugar with the release of UMP. Depending on the specific micro-organism, this reaction is catalysed by two different families of proteins. In Escherichia coli and other members of the Enterobacteriaceae, initiation of the O antigen requires Und-PP-GlcNAc (Alexander & Valvano, 1994; Amer & Valvano, 2000; Feldman et al., 1999; Valvano, 2003), formed by the action of the UDP-GlcNAc : Und-P GlcNAc-1-P transferase encoded by the wecA gene (Amer & Valvano, 2001, 2002; Anderson et al., 2000). WecA belongs to a large family of sugar-phosphate transferases that are widely conserved in both prokaryotes and eukaryotes [the polyisoprenyl-phosphate N-acetylhexosamine-1-phosphate transferase (PNPT) family] (Anderson et al., 2000; Lehrer et al., 2007; Price & Momany, 2005; Valvano, 2003). The other family includes polyisoprenyl-phosphate hexose-1-phosphate transferases (PHPT family), and its prototype member is WbaP from Salmonella enterica serovar Typhimurium (S. Typhimurium) (Valvano, 2003; Wang & Reeves, 1994; Wang et al., 1996). The O antigen repeat in S. Typhimurium consists of a tetrasaccharide made of a linear backbone of galactose, rhamnose and mannose, and a side abequose residue that is linked to the terminal mannose (Reeves, 1993). Upon completion of its synthesis, the tetrasaccharide O unit is translocated across the membrane and polymerized by the Wzy-dependent pathway, and the polymer is incorporated into the lipid A–core oligosaccharide by the O antigen ligase WaaL.
WbaP is the initiating UDP-Gal : Und-P Gal-1-P transferase. It has no known homologues in eukaryotic cells and lacks amino acid sequence similarity with members of the PNPT family. WbaP shows high sequence similarity with proteins involved in the synthesis of capsule exopolysaccharides in Xanthomonas campestris (Katzen et al., 1998), Erwinia amylovora (Bugert & Geider, 1995), Streptococcus pneumoniae (Cartee et al., 2005), Klebsiella pneumoniae (Arakawa et al., 1995; Drummelsmith & Whitfield, 1999) and E. coli K-12 (Stevenson et al., 1996) among others, and also with proteins involved in S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus (Steiner et al., 2007). The WbaP protein is a large hydrophobic and basic protein with a predicted mass of 56 kDa and a pI of 9. Wang et al. (1996) have proposed that the S. Typhimurium WbaP is a bifunctional protein that possesses two domains, the C-terminal portion, responsible for the Gal-1-P transferase activity, and the N-terminal region, required for the release of Und-PP-Gal from WbaP (Wang & Reeves, 1994). According to these authors, the two domains are required for the synthesis of the O antigen. Their conclusions are based on the analysis of two wbaP(T) mutations, wbaP4451(T) and wbaP4452(T), mapping to the 5' half of the wbaP gene, which result in an enzyme retaining Gal-1-P transferase activity but unable to mediate synthesis of O antigen (Wang et al., 1996). Here, we propose that WbaP possesses three major domains and assign functional roles to each predicted domain. Our results suggest that the N-terminal domain of WbaP is required for the insertion or stability of the enzyme in the bacterial membrane, that the large periplasmic region is probably involved directly or indirectly in modulating O antigen chain length, and that the C-terminal domain carries the Gal-1-P transferase activity.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Bacteria were grown aerobically at 37 °C in Luria–Bertani (LB) medium (Difco). Media were supplemented as appropriate with ampicillin, chloramphenicol or spectinomycin at final concentrations of 100, 30 and 80 µg ml–1, respectively.
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. DNA sequences were determined using an automated sequencer at the DNA Sequencing Facility, Robarts Research Institute (London, ON, Canada).
Topological model for WbaP and sequence analysis.
To analyse WbaP topology, four topological prediction methods were used: TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), MEMSAT (http://bioinf.cs.ucl.ac.uk/psipred/), TopPred 2.0 (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html) and PHD 2.1 (www.predictprotein.org/). PSI-BLAST searches were conducted using resources available at NCBI (http://www.ncbi.nlm.nih.gov/BLAST/). Searches were done using the Salmonella enterica serovar Typhi (herein S. Typhi) WbaP C-terminal region, periplasmic loop and N-terminal domain sequences as a query. Multiple-sequence alignments of WbaP homologues were determined with the program CLUSTAL W (http://www.ebi.ac.uk/clustalw/). Protein secondary structure was predicted with PSI-PRED (http://bioinf.cs.ucl.ac.uk/psipred/).
Cloning of WbaP domains.
PCR was carried out with PwoI DNA polymerase (Roche Diagnostics) in a Perkin Elmer 2400 GeneAmp PCR system. Plasmid pSM10 was constructed by PCR amplification of a 2.9 kb fragment containing the wbaP and manB genes using primers manB3 (5'-CTGGATTTTCGAAGGAGTGGACTAA-3') and 592 (5'-TCGGATATCTTAATACGCACCATCTCGCC-3') with S. Typhi Ty2 DNA as template. This fragment was ligated into the SmaI site of pAA8 (Table 1
). pSM13 is a pSM10 derivative in which the manB gene was deleted by digestion with SacI and the plasmid self-ligated. pSM28 was constructed by PCR amplification of a 610 bp fragment from the 5' end of wbaP using pSM13 as a DNA template, which was obtained using primers 591 (5'-CTCCCCGGGAATGGATAATATTGATAATAAG-3') and 1136 (5'-CTCCCCGGGTTAAATCCCAAATAGTCCCAGTG-3'), each incorporating SmaI sites (underlined). The resulting amplicon was digested with SmaI and ligated into the SmaI site of pAA8. pSM30 was constructed by PCR amplification of a 630 bp fragment from the 3' end of wbaP using primers 592 (see above) and 897 (5'-CTCCCCGGGTTTAGCCATGAAGTTATGTTATTAAGG-3') with S. Typhi Ty2 DNA as template. The fragment was digested with SmaI and ligated into the SmaI site of pAA8. Plasmids pSM31 and pSM18 were constructed by inverse PCR using pSM13 as template and primers 1156 (5'-TTACCCGGGAGTAAAATAGACACATCCTAATGAAAGCC-3') and 1155 (5'-ATGCCCGGGAAAGTTACTCGAGATGGTGGTCCG-3'), and primers 897 (see above) and 900 (5'-CTCCCCGGGGATACCTAGCTTGTTCAATAAATG-3'), respectively. Plasmids pKP10, pKP12, pKP13 and pKP14 were constructed in pBADNTF from wbaP derivatives of plasmids pSM18, pSM30, pSM31 and pSM28, respectively, by PCR using primers 1151 (5'-CTCCCCGGGGATAATATTGATAATAAGTATAATCCACAGC-3'; SmaI site underlined), and 1152 (5'-CTCTCTAGATTAATACGCACCATCTCGCCG-3'). The resulting amplicons were digested with SmaI, treated with polynucleotide kinase and ligated to SmaI-digested and alkaline phosphatase-treated pBADNTF. Plasmid pKP1 was constructed from S. Typhimurium LT2 DNA by PCR using primers 2158 (5'-CTCCCCGGGTTGGCTCTGATAGCGTTTAC-3'; SmaI site underlined) and 2159 (5'-GACTGTCGACTCCCTGGCAATAGGATCGTTAG-3'; SalI site underlined). The resulting amplicon was digested with SmaI and SalI and ligated to the corresponding sites on pBADFLAG.
Disruption of the wbaP gene.
Mutagenesis was performed according to the method described by Datsenko & Wanner (2000) to disrupt specific chromosomal genes using PCR products. Thus, S. Typhi Ty2 and S. Typhimurium LT2 cells carrying pKD46 were transformed by electroporation with a PCR product that was generated using plasmid pKD3 as template and primers 613 (5'-ATGGATAATATTGATAATAAGTATAATCCACAGCTATGTAAGTGTAGGCTGGAGCTGCTTCG-3') and 614 (5'-ATCACTGCCATACCGACGACGCCGATCTGTTGCTTGGACATATGAATATCCTCCTATG-3'). Transformants were plated on LB agar plates containing chloramphenicol, for the selection of wbaP mutants.
Oligonucleotide-directed mutagenesis of wbaP.
Site-directed mutagenesis was performed using the QuikChange Site-Directed Mutagenesis kit from Stratagene, as recommended by the supplier. Plasmid pSM18 was used as template in the PCR reactions for the construction of pSM22 and pSM23, as indicated in Table 1. Plasmid pSM23 was constructed using primers 886 (5'-CTTTTGCCATAATCCTGGTGCTTTTTTTCGCGCACTTACAAAG-3') and 887 (5'-CTTTGTAAGTGCGCGAAAAAAAGCACCAGGATTATGGCAAAAG-3'). Two consecutive site-directed mutagenesis experiments were performed to construct plasmid pSM22. Primers 882 (5'-GGTTTTGGATTCGTTTGAGATATTATACATACCGCAAGCC-3') and 883 (5'-GGCTTGCGGTATGTATAATATCGCAAACGAATCCAAAACC-3') were used for the first PCR amplification, and primers 884 (5'-CGTTTGCGATATTATACATACCGCGAGCCATTTTGGTATGAGTTAAAAG-3') and 885 (5'-CTTTTAACTCATACCAAAATGGCTCGCGGTATGTATAATATCGCAAACG-3') for the second. The resulting plasmids were transformed into E. coli DH5
by the calcium chloride method (Hanahan, 1983). Replacement mutants were confirmed by sequencing the entire wbaP gene.
Growth curves.
For growth curves, overnight cultures of E. coli strains containing the appropriate plasmids were diluted to OD600 0.03 (corresponding to a bacterial density of 
104 c.f.u. ml–1), and growth was monitored every 30 min in a 100-well microtitre plate using a Bioscreen C automated microbiology growth curve analysis system (MTX Lab Systems). Growth rates were also compared between cultures with and without antibiotic selection to rule out antibiotic effects. Bioscreen C is a computerized bacterial incubator that measures growth continuously by optical density (Löwdin et al., 1993). The results were analysed statistically by one-way ANOVA and the Tukey post-test using the Prism 4 software package (GraphPad Software).
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 previously (Marolda et al., 1990, 2006) and LPS was separated on 14 % acrylamide gels using a Tricine–SDS buffer system (Marolda et al., 2006). 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 as described previously (Marolda et al., 2006). Specific detection of Salmonella group D1 (for S. Typhi) and group B (for S. Typhimurium) O antigens was carried out by Western blotting. Group D1 and B rabbit antisera (Difco) were used at a 1 : 500 dilution. The reacting O antigen polysaccharides were detected by fluorescence with an Odyssey infrared imaging system (Li-cor Biosciences) using Rdye800-conjugated anti-rabbit affinity-purified secondary antibodies (Rockland). For estimation of the variation in the length of O antigen polysaccharide chains, a Tris–glycine system was used as described previously (Marolda et al., 1990). The O antigen polysaccharide bands in all gels were quantified by densitometry and the pixel densities were measured using ImageJ (Abramoff et al., 2004).
In vitro transferase assay.
Plasmids encoding the different WbaP recombinant proteins (pSM13, pSM18, pSM30 and pJD132) were transformed by electroporation into S. Typhimurium MSS2, which carries a wbaP deletion (Table 1). Membranes (containing enzymes and endogenous Und-P) were isolated from these transformants as described by Osborn et al. (1972). The reaction mixture for the transferase assay contained the membrane fraction (40 µg total protein) and 0.5 µCi (18.5 kBq) radiolabelled UDP-[3H]Gal (Sigma) in 250 µl buffer (5 mM Tris/acetate, pH 8.5, 0.1 mM EDTA, 3 mM MgCl2). After incubation at 37 °C for 30 min, the lipid-associated material was extracted twice with 250 µl 1-butanol. The combined 1-butanol extracts were washed once with 500 µl distilled water and the radioactivity counts of the 1-butanol fraction were determined with a Beckman liquid scintillation counter. Radioactivity counts were normalized with respect to the amount of total protein in the membrane fraction used for the enzymic assay. Enzyme activity was expressed as pmol [3H]Gal incorporated (mg membrane protein)–1. For comparisons among the various preparations, relative enzymic activities were expressed as a percentage of parental WbaP activity at 1 mM MgCl2.
Membrane preparation and Western blotting.
For visualization of WbaP constructs containing the FLAG epitope, bacterial cultures were grown overnight in 5 ml LB, diluted to an initial OD600 of 0.02, and incubated at 37 °C for 2 h until cultures reached OD600 0.5. At this point, arabinose was added to a final concentration of 0.2 % (w/v), and cells were incubated for an additional 3 h until reaching OD600 0.8–1.0. Cells were then harvested by centrifugation at 10 000 g for 10 min at 4 °C. The bacterial pellet was suspended in Tris/NaCl (20 mM Tris/HCl, pH 8.5, 300 mM NaCl) and the suspension lysed using a French Press cell. Cell debris was removed by centrifugation (15 000 g for 15 min at 4 °C), and the clear supernatant was centrifuged at 30 000 g for 30 min at 4 °C. The pellet, containing total membranes, was suspended in Tris/NaCl and frozen at –80 °C until required. The protein concentration was determined by the Bradford assay (Bio-Rad). SDS-PAGE, protein transfer to nitrocellulose membranes and immunoblots with the FLAG M2 mAb were performed as described previously (Amer & Valvano, 2002), except that the reacting bands were detected by fluorescence with the Odyssey infrared imaging system (Li-cor Biosciences) using Alexa Fluor 680 goat anti-mouse secondary antibody (Molecular Probes). An affinity-purified rabbit antiserum was used for detection of the Wzz protein in similar preparations to those described above, using Alexa Fluor IRDye800 CW affinity-purified anti-rabbit IgG antibody (Rockland) as the secondary antibody for the detection of specific bands by fluorescence.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Nilsson et al., 2000), we established that the Salmonella WbaP polypeptide (476 aa) possesses three predicted domains: an N-terminal region of 133 aa containing transmembrane helices I–IV (NWbaP), a large central periplasmic loop of 150 aa (PWbaP), and the remaining 193 aa which include transmembrane helix V and a large C-terminal cytosolic tail (CWbaP; Fig. 1a). To determine the relative conservation of each predicted domain we performed PSI-BLAST searches using the amino acid sequences of the NWbaP, PWbaP, and CWbaP regions of the S. Typhi WbaP as queries. The WbaP proteins in S. Typhi and S. Typhimurium are virtually identical (Jiang et al., 1991), except for four non-conservative changes (WbaPSTyphi/WbaPSTyphimurium) at positions 31 (Leu/Phe), 39 (Thr/Ile), 198 (Gly/Glu) and 199 (Thr/Ile). Multiple-sequence alignments of WbaP homologues revealed that the CWbaP domain is highly conserved in a large number of different bacterial sugar transferases (pfam02397) involved in the biosynthesis of exopolysaccharides and LPSs from a broad spectrum of Gram-negative, Gram-positive and archaeal species (data not shown). In contrast, the NWbaP and PWbaP domains had amino acid sequence conservation with proteins predominantly involved in O antigen LPS biosynthesis from only a reduced number of species, which included, among others, Salmonella, E. coli, Haemophilus influenzae and Actinobacillus. WbaP did not show any sequence homology with members of the WecA family. Therefore, WbaP clearly falls within a different family of transferases that is restricted to prokaryotes only (Valvano, 2003).
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wbaP : : cat) and MSS2 (S. Typhimurium wbaP : : cat) produced a lipid A–core oligosaccharide band devoid of O antigen that did not react with group B (S. Typhimurium) or group D1 (S. Typhi) O antigen-specific antisera (Fig. 2, second lane in all panels). The wbaP gene is the last gene of the S. enterica O antigen gene cluster and does not appear to contain a promoter immediately upstream (Jiang et al., 1991; Reeves, 1993; Wang & Reeves, 1994; Wang et al., 1996). Therefore, we attempted to clone this gene as a single ORF downstream of the lac promoter region provided by the pAA8 plasmid vector (Amer & Valvano, 2000) (Table 1). Restriction enzyme analysis and DNA sequencing of the recombinant plasmids obtained from these experiments revealed that none of the plasmids carried an intact wbaP gene. On a few occasions, an apparently correct clone was obtained, but DNA sequencing revealed gene deletions and/or frame-shift mutations in the coding region of wbaP. Failure to clone wbaP could not be explained by technical difficulties or PCR polymerase errors, since several cloning experiments on other unrelated Salmonella genes done in parallel with the same reagents were successful (data not shown).
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, pSM10), and subsequently deleted most of the manB gene (see Methods). The resulting plasmid, pSM13, restored O antigen expression in the wbaP mutants MSS2 (S. Typhimurium) and MSS1 (S. Typhi) (Fig. 2, third lane in all panels), and was selected for further study. However, less O antigen was produced by both the wbaP mutant strains that contained pSM13, and the O antigen had a different migration pattern from that of the respective parental strains (Fig. 2). Transformation of MSS1 and MSS2 with pJD132, carrying an E. coli O9 : K30 wbaP gene homologue and flanking sequences (Schäffer et al., 2002), restored O antigen production in both mutant strains (Fig. 2, fourth lane in all panels). The migration pattern and length of O antigen polymers mediated by pJD132 were identical to those of each respective parental strain. Therefore, the abnormal O antigen expressed by MSS1 (pSM13) and MSS2 (pSM13) mutants is not due to an intrinsic defect caused during the deletion mutagenesis procedure used to remove the wbaP gene in the Ty2 and LT2 strains, suggesting that the WbaP protein encoded by pSM13 is not functionally identical to wild-type WbaP (see next section below). However, the complemented mutants produced O antigens that were immunochemically identical to those of the respective parental strains, as detected by Western blots with group B- and group D1-specific antibodies (Fig. 2, third lane in right panels). Conservation of the immunoreactive epitopes indicates that the complete O antigen subunit was properly synthesized in both mutants. Therefore, the differences in O antigen profile are probably due to different processing of the O antigen and not to a defect in the initiation of its synthesis (see below).
The difficulties in obtaining a recombinant clone encoding a full-length WbaP protein in E. coli K-12 suggested that the expression of this protein could be deleterious to the host strain. Indeed, the growth rate of E. coli DH5 (pSM13) was significantly slower (doubling time 106±3 min, n=7) than that of E. coli DH5 carrying the vector control (doubling time 76±3.4 min; P<0.001, n=7). We have previously demonstrated that the E. coli Wzx flippase does not recognize the product of the reaction catalysed by the Salmonella WbaP protein (Und-PP-Gal) (Feldman et al., 1999; Marolda et al., 2004). Therefore, the growth defect caused by WbaP expression in E. coli could be due to accumulation of Und-PP-Gal in the cytoplasmic side of the plasma membrane, which could presumably block the recycling of Und-P and thus compromise cell wall biosynthesis. To test this possibility, we transformed E. coli DH5 (pSM13) with plasmid pMF24, carrying the S. Typhimurium wzx flippase gene. This strain had a doubling time of 67±1 min (n=7), which was not significantly different from that of DH5 (pAA8), confirming that introducing the S. Typhimurium wzx gene relieves the growth delay in E. coli DH5 (pSM13).
The N-terminal domain of WbaP is dispensable for in vivo enzymic activity
The sequence of the DNA inserts in pSM10 and pSM13 revealed a one-base deletion at position 583, producing a frame shift in the wbaP reading frame with a stop codon at base 601, and a two-base deletion at position 645 that restored the wbaP reading frame (Fig. 3). These mutations suggested that the WbaP protein expressed by pSM13 was a truncated form containing the N-terminal domain and part of the predicted periplasmic loop. However, this conclusion was not supported by previous studies demonstrating that S. Typhimurium WbaP possesses two functional domains: the C-terminal portion (GT domain) involved in Gal-1-P transferase function, and the N-terminal region (T domain) proposed to act in the release of the Und-PP-Gal from WbaP, prior to the flipping step (Wang & Reeves, 1994; Wang et al., 1996). Therefore, various recombinant proteins were constructed to ascertain the functions of these two putative WbaP domains (Fig. 1), which could explain our complementation results with pSM13. Plasmid pSM28 contains a 609 bp insert corresponding to a truncated wbaP gene encoding the first 198 aa of WbaP (spanning four transmembrane domains and 63 aa of the periplasmic loop), designated WbaPM1–L203 (Fig. 1b; Table 1). This corresponds to the same end point of the frame-shift mutation in pSM13 (Fig. 3). Plasmid pSM30 encoded WbaPY250–Y476 (Fig. 1b), a derivative of WbaP containing 19 aa of the periplasmic loop, TM-V, and the cytoplasmic C-terminal region. Plasmids pSM28 and pSM30 were separately transformed into MSS2 and the LPS profiles were analysed. The MSS2 (pSM30) strain expressing WbaPY250–Y476 produced O antigen while MSS2 (pSM28) expressing WbaPM1–L203 did not (Fig. 4). These results indicate that the enzymic activity of WbaP resides in its C-terminal region, and suggest that this region must also be expressed in pSM13, most likely from an internal translation start site within the 3' half of the wbaP gene (Wang et al., 1996).
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). To determine whether this transmembrane segment is indeed required for enzyme activity, the predicted C-terminal cytoplasmic domain was fused to the first transmembrane domain of WbaP to yield WbaPM1–T39/K305–Y476 (encoded by pSM31; Fig. 1b; Table 1). Fig. 4 shows that pSM31 (WbaPM1–T39/K305–Y476) did not restore O antigen production in the MSS2 mutant, suggesting that the predicted transmembrane helix V is required for WbaP activity. Failure to complement O antigen production was not due to lack of protein expression or membrane localization of the mutated WbaP protein, since an N-terminal FLAG-fusion derivative of WbaPM1–T39/K305–Y476 (expressed by plasmid pKP13) was detected by Western blot analysis of total membrane preparations (see below).
The results described above suggested that the N-terminal region of WbaP is not required for in vivo O antigen synthesis in S. enterica. However, earlier work based on the analysis of wbaP(T) mutants concluded that the N terminus of WbaP is also required for normal O antigen expression (Wang et al., 1996). To clarify the contribution of the wbaP(T) mutations in WbaP function we performed site-directed mutagenesis of the wbaP gene cloned into pSM18 (encoding WbaPM1–I144/F258–Y476) to recreate the wbaP(T) mutations wbaP4451(T) and wbaP4452(T) described before (Wang et al., 1996). Plasmid pSM23 carries a deletion at position 386 of the wbaP gene resulting in a frame-shift mutation that recreates wbaP4451(T), whereas pSM22 carries a wbaP gene with two point mutations within its coding region that result in His76Tyr and Lys81Glu replacements and recreate wbaP4452(T). Fig. 5 shows that the LPS profiles of MSS2 transformed with each of these plasmids were similar to the LPS profile of MSS2 (pSM18) encoding WbaPM1–I144/F258–Y476 (Fig. 1b), which carries a deleted periplasmic loop that is not required for O antigen production (see below). In addition, we transformed the original wbaP(T) mutant strains of S. Typhimurium (SL1196 and SL1197; Table 1) with pJD132 that carries the E. coli K-30 wbaP gene (WbaPEcK30). This plasmid complemented O antigen synthesis in MSS2 (Fig. 2, fourth lanes) but not in the T mutants (Fig. 5). Together, these results demonstrate that the mutations in the wbaP(T) genes do not affect O antigen synthesis, and suggest that the lack of O antigen production in these strains is probably due to additional mutations in another gene or genes of the Salmonella O antigen cluster.
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reveals that although strain MSS2 expressing WbaPY250–Y476 (pSM30) produces O antigen in similar amounts to LT2, the chain-length distribution of the O antigen polysaccharide is altered in the mutant. Densitometric quantification demonstrated an eightfold increased average intensity of the bands within the region corresponding to 10–20 O-unit repeats (Fig. 4, dashed rectangle; data not shown). These results were confirmed using a different gel separation system, based on Tris–glycine, that provides higher resolution of the O antigen polymeric bands (Fig. 6a). Since WbaPY250–Y476 lacks most of the predicted periplasmic loop of WbaP (Fig. 1), we speculated that this region could be involved in O antigen chain-length distribution, possibly by interacting with the Wzz O chain-length regulator. To confirm this hypothesis we constructed WbaPM1–I144/F258–Y476, a deletion derivative of WbaP protein (encoded by pSM18) that only contained 24 aa of the periplasmic loop. MSS2 expressing WbaPM1–I144/F258–Y476 produced a smooth LPS, but the O antigen chain-length distribution was different from the wild-type profile (third lane in Figs 5 and 6a). This result suggests that although the periplasmic region of Salmonella WbaP is dispensable for the transferase activity, it is required for normal O antigen chain-length distribution.
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-helices and β-strands (Fig. 7). Most of the residues that are conserved in the alignments with WbaP homologues from other bacterial species are also present within the predicted -helices and β-strands (Fig. 7; data not shown), supporting the idea that the periplasmic loop of WbaP could interact with other proteins involved in O antigen synthesis, with the O antigen itself, or with both. Therefore, we investigated whether expression of a truncated and non-functional WbaP protein with an intact predicted periplasmic loop could affect the function of the parental WbaP. We constructed plasmid pKP1 encoding a non-functional WbaP derivative containing most of the amino acids of the periplasmic loop, transmembrane helix V, and a truncated cytoplasmic tail fused to the FLAG epitope under the control of the arabinose-inducible PBAD promoter (Fig. 1b). The polypeptide expressed by pKP1 (WbaPM170–R354–FLAG) was detected in membrane fractions by immunoblotting with a FLAG mAb (see following section). After introduction of pKP1 into S. Typhimurium LT2 and incubation with arabinose, we observed reduced production of high-molecular-mass O antigens (Fig. 8a). In contrast, a control experiment on LT2 cells carrying the vector pBADFLAG showed no effect on O antigen production or chain-length distribution (Fig. 8b). As an additional control, we used plasmid pKP12 encoding a truncated WbaP protein (WbaPF258–Y476) that contains only the last 24 aa of the periplasmic loop, transmembrane helix V, and the entire cytoplasmic tail (Fig. 1; Table 1). Introduction of pKP12 into strain LT2 (parental wbaP+) resulted in an O antigen profile with altered chain-length distribution (Fig. 8b), similar to that found in the wbaP : : cat mutant MSS2 complemented with pSM18 (which encodes a WbaP protein derivative with a deletion of most of the periplasmic loop). Together, these results suggest that the WbaP periplasmic loop is to some extent involved in modulating the length of the O antigen chains, thereby affecting the distribution of the O antigen polysaccharide chains. Alternatively, expression of truncated or mutated WbaP proteins could induce stress responses that act non-specifically on Wzz, resulting in reduced production or stability of this protein. To address this possibility we determined the relative amount of Wzz expressed from its chromosomal gene in strains carrying the various recombinant plasmids. The immunoblot was quantitatively analysed by double labelling using FLAG- and Wzz-specific antibodies, to take advantage of an anti-FLAG reacting protein in Salmonella that served as an internal control (Fig. 6b, lower panel). The results of this experiment clearly indicate that, compared to the wild-type, Wzz production is not reduced by expressing the various recombinant WbaP proteins (Fig. 6b; data not shown). Also, there was no correlation between the levels of WbaP recombinant protein expression and the O antigen banding phenotype (see following section). Therefore, stress on wzz gene expression or Wzz protein stability cannot explain the abnormal O antigen chain distribution observed with the WbaP recombinant proteins. We conclude that the periplasmic loop of WbaP has a function that is not directly related to Gal-1-P transferase activity and that may involve protein–protein interactions with one or more additional proteins involved in the assembly of the O antigen chain.
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. It should be noted that the doublet present in all the lanes corresponds to minor outer-membrane proteins that cross-react with the FLAG epitope and are usually detected in total membrane preparations from E. coli. Heating the protein sample from E. coli DH5 (pKP1) at 100 °C for 5 min prior to loading resulted in aggregation, as determined by a new large protein band that barely penetrated the gel matrix (Fig. 9a, lane 2, black arrow) and in a polypeptide with an apparent mass of 26 kDa, which was consistent with the predicted mass of WbaPM170–R354–FLAG (23 kDa) (Fig. 9a, lane 2, asterisk). Also, a 20 kDa polypeptide was detected (Fig. 9a, lane 2, white arrow), which was interpreted as a WbaPM170–R354–FLAG degradation product. Protein aggregates that barely penetrate the gel matrix and anomalous migration in the gel are commonly observed with membrane proteins upon heat denaturation, particularly in proteins with high pIs (Kashino, 2003). To avoid this problem, the protein samples were incubated at 45 °C for 30 min, as we have done in previous studies with the integral membrane proteins WecA and Wzx (Amer & Valvano, 2000; Lehrer et al., 2007; Marolda et al., 2004). In this case, we also detected a polypeptide with an apparent mass of 26 kDa corresponding to WbaPM170–R354–FLAG (Fig. 9a, lane 3, asterisk), and several larger oligomeric forms, most likely due to the mild denaturation conditions (Fig. 9a, lane 3, black arrows), as well as the putative degradation product of 20 kDa (Fig. 9a, lane 3, white arrow). Because of the difficulty in obtaining C-terminally FLAG-tagged derivatives of WbaP, each of the deleted versions of the wbaP gene in pSM18, pSM30, pSM31 and pSM28 was reconstructed as a FLAG N-terminal fusion derivative of WbaP, giving rise to pKP10, pKP12, PKP13 and pKP14, respectively (Fig. 1b; Table 1). All of these plasmids were derivatives of the vector pBADNTF, such that protein expression was under the control of the arabinose-inducible PBAD promoter. The chimeric proteins were detectable by immunoblotting of total membranes prepared from S. Typhimurium MSS2 containing each of the recombinant plasmids, although the levels of each WbaP fusion protein varied from construct to construct (Fig. 9b, arrows). Additional bands were also found in total membranes of MSS2 carrying the pBADNTF vector control (Fig. 9b), and correspond to polypeptides that cross-react with the M2 mAb. The presence of the FLAG epitope did not alter the results of complementation experiments done with the corresponding untagged proteins, as demonstrated in experiments in which the O antigen expression mediated by tagged and untagged versions of WbaP was assessed side by side (data not shown). Also, the genetic complementation did not correlate with the levels of protein expression, demonstrating that the levels of expressed protein did not cause the phenotypes observed with the various WbaP forms.
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), probably reflecting background levels of enzyme activity most likely due to additional transferases, such as those involved in the terminal steps of lipid A–core biosynthesis, which can also utilize UDP-Gal as a donor substrate. While, as discussed above, pSM13 probably expresses the WbaP N- and C-terminal domains independently as separate polypeptides, pSM30 can only produce the C-terminal region. In contrast, membranes prepared from MSS2 containing pSM18 showed a similar transferase activity to that of membranes from MSS2 (pJD132). Therefore, the results of the enzymic assays suggest that the mutated forms of WbaP that lack the N-terminal domain are enzymically inactive in vitro, despite being able to complement the synthesis of O antigen in vivo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), and also in the biosynthesis of the glycan moiety attached to bacterial glycoproteins (Steiner et al., 2007). The predicted topology of WbaP revealed important differences from WecA, which belongs to another large family of initiating enzymes in prokaryotes and eukaryotes that catalyse the transfer of N-acetylhexosamine-phosphate onto isoprenoid lipid intermediates (Valvano, 2003). WecA consists of 11 transmembrane helices, five external and five internal loops (Amer & Valvano, 2001, 2002; Anderson et al., 2000; Lehrer et al., 2007). Whereas specific amino acid residues of functional importance in the transfer of GlcNAc-1-P to Und-P have been identified in cytosolic loops II, III and V of WecA, a domain structure in this protein is not obvious (Amer & Valvano, 2001, 2002; Lehrer et al., 2007). In contrast, the topological model for WbaP revealed three predicted domains that could be clearly delineated: an N-terminal region containing a cluster of four transmembrane helices, a large central periplasmic loop, and a C-terminal cytosolic tail. Previous work by Wang et al. (1996) has shown that the C-terminal half of WbaP carries the Gal-1-P transferase activity. The results of the present study, based on the expression of truncated forms of WbaP, strongly agree with this conclusion and refine the boundaries of the predicted Gal-1-P transferase domain, which contains the last transmembrane helix and a large cytoplasmic tail. The C-terminal domain of WbaP is highly conserved in a broad spectrum of bacterial species, while the N-terminal domain and the periplasmic loop show homology only with a reduced number of proteins, the majority of which are involved in O antigen synthesis.
The unequal conservation of each domain in the bacterial homologues of WbaP prompted us to investigate the potential functional roles of each predicted domain. Despite numerous attempts with various different cloning strategies we could not clone the intact wbaP gene from S. Typhimurium or S. Typhi. The only clone we obtained (plasmid pSM13) had mutations causing a frame shift that would yield a truncated WbaP protein. However, this plasmid could partially complement O antigen synthesis in the S. Typhimurium strain carrying a deletion of wbaP. Given that the predicted Gal-1-P transferase domain is in the C terminus of WbaP, we concluded that one or more translation start sites within the wbaP gene could be responsible for the expression of a polypeptide with Gal-P transferase activity. This is in agreement with earlier observations from in vivo radiolabelling showing that the 3' half of wbaP can indeed initiate translation to synthesize a 25 kDa protein with Gal-P transferase activity (Wang et al., 1996). Several proteins maintain tertiary structure when the peptide backbone is cleaved, such that substrate binding and/or catalytic activity may be retained. For example, two independently cloned fragments containing pieces of the lacY and mdfA genes in the same plasmid lead to membrane insertion of functional proteins and the synthesis of polypeptides that correspond to the N- and C-terminal portions of the LacY and MdfA proteins (Adler & Bibi, 2004; Bibi & Kaback, 1990). Therefore, although we could not demonstrate it directly, the functional data on WbaP strongly suggest that pSM13 encodes a split functional protein. Bacteria containing pSM13 displayed lower O antigen levels than those containing pSM18 and pSM30, despite all three plasmids having apparently intact C-terminal domains. These differences can be attributed to the split nature of the protein encoded by pSM13, in contrast to the WbaP forms encoded by the other plasmids from optimized promoters in the cloning vectors.
Sequence alignments of WbaP N-terminal domain homologues showed a highly conserved region of 14 aa between helices II and III, which are predicted to face the periplasm. Considering that O antigen polymerization and assembly occur at the periplasmic side of the plasma membrane, this region may be involved in protein–protein interactions with other components of O antigen biosynthetic machinery. In earlier work (Wang & Reeves, 1994; Wang et al., 1996), mutations of S. Typhimurium LT2 affecting the ligation of polymerized O antigen to core–lipid A were mapped to the 3' half of wbaP. The authors concluded that these mutations, referred to as wbaP(T), caused a possible block either in the flipping of the Und-PP-linked O antigen subunits across the plasma membrane or in the release of Und-P-linked Gal from WbaP. One of the O antigen-defective S. Typhimurium mutants described earlier (Wang & Reeves, 1994; Wang et al., 1996) carries two amino acid substitutions (His76Tyr and Lys81Glu) within the 14 aa conserved region of WbaP. We recreated these mutations in our cloned wbaP genes and showed that they have no effect on O antigen production. However, O antigen synthesis in the original T mutants could not be complemented by our plasmids that encode the various forms of WbaP and the WbaP homologue from E. coli K-30. We thus conclude that the T mutants of S. Typhimurium LT2 carry other defects outside the wbaP gene that prevent the synthesis of O antigen. Genomic sequencing of the T mutants would be required to locate small mutations that may explain the defect in O antigen synthesis.
We also demonstrated that the N-terminal region of WbaP is not required for in vivo sugar-phosphate transferase activity and complementation of O antigen synthesis, but is essential for in vitro enzymic activity using crude cell-membrane extracts. The combined results of our experiments suggest that the N-terminal domain of WbaP plays a role in the stability or correct folding of WbaP in the plasma membrane. Further studies, currently under way in our laboratories, are required to determine the precise function of the N-terminal domain of WbaP. The domain corresponding to a predicted periplasmic loop of WbaP was also dispensable for Gal-P transferase activity, since it can be deleted without compromising the function of the protein as determined in vivo by complementation of O antigen synthesis and in vitro by examining the transfer of radioactive Gal to the endogenous Und-P acceptor. Nevertheless, this region appears to be involved in regulating O antigen polymerization, because mutant proteins that lack the periplasmic loop exhibit an altered chain-length distribution of the O antigen. The chain-length distribution of the O antigen polysaccharide is determined by the Wzz protein (Morona et al., 2000). This protein can form multimers, and is proposed to interact with the Wzy polymerase and the WaaL ligase in the periplasmic space, although the detailed mechanism that controls the chain-length distribution of O antigens is not well understood. Furthermore, wzz gene expression in Salmonella is subject to control by several global two-component regulators (Delgado et al., 2006), and we have evidence that the stability of the Wzz protein in E. coli is modulated by the extracytoplasmic stress response (C. L. Marolda and others, unpublished data). However, we could not detect differences in Wzz production in any of the strains that contained the various WbaP-expressing recombinant plasmids, ruling out stress as a mechanism to explain the variations in O antigen length distribution that we observed in this study. Therefore, our results suggest that the periplasmic loop of WbaP also interacts with Wzz and/or other proteins involved in O antigen processing, including the Wzy polymerase. This is supported by experiments in which a non-functional WbaP protein with an intact periplasmic domain altered O antigen production in the presence of the parental WbaP protein. We are currently mapping the specific residues from this region in WbaP that are required for the chain-length distribution defect, which may reveal possible sites of contact with Wzz.
Together, the experiments described in this paper demonstrate that WbaP possesses domains with different functional roles. These predicted structural domains carry out several functions that are required not only for the expected transfer of Gal-1-P from UDP-Gal to Und-P, but also for the proper chain-length distribution of the O antigen. We propose that WbaP acts as a scaffold protein that facilitates the completion of the O antigen subunit on the cytosolic side of the membrane and the downstream steps of O antigen assembly leading to the polymerization of the O antigen.
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ACKNOWLEDGEMENTS |
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for strains and plasmids, and K. E. Sanderson, University of Calgary, for supplying strains SL1196 and SL1197. This work was supported by grants from the Canadian Institutes of Health Research (to M. A. V.) and grant 1040562 from Fondecyt (to I. C.). M. S. S. was supported by a fellowship from Conicyt and fellowships PG/108/02 and PG/92/2003 from Departamento de Postgrado y Postítulo, Universidad de Chile, and M. B. was supported by Beca de Tesis Doctoral Conicyt. M. A. V. holds a Canada Research Chair in Infectious Diseases and Microbial Pathogenesis. Edited by: P. H. Everest
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Received 10 September 2007;
revised 11 October 2007;
accepted 19 October 2007.
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