1 Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada, N6A 5C1
2 Department of Medicine, University of Western Ontario, London, Ontario, Canada, N6A 5C1
Correspondence
Miguel A. Valvano
mvalvano{at}uwo.ca
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ABSTRACT |
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INTRODUCTION |
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Two predominant mechanisms for the biosynthesis and assembly of O antigens have been described. One of them involves the synthesis of individual O repeating subunits on the cytosolic side of the plasma membrane followed by their translocation across this membrane (Raetz & Whitfield, 2002; Valvano, 2003
). Once on the periplasmic face of the plasma membrane the subunits become polymerized and subsequently the polysaccharide is ligated en bloc to the lipid A-core oligosaccharide with the release of Und-PP (Marino et al., 1991
; McGrath & Osborn, 1991
; Mulford & Osborn, 1983
). This pathway, which is also referred to as the wzy (polymerase)-dependent pathway, occurs in the synthesis of the majority of O antigens, especially in those made of repeating units of different sugars (heteropolymeric O antigens) (Keenleyside & Whitfield, 1999
), and involves the concerted functions of three gene products: Wzy (O-antigen polymerase), Wzz (O antigen chain regulator) and Wzx (putative O-antigen flippase) (Raetz & Whitfield, 2002
; Valvano, 2003
).
In the other mechanism, the formation of a polymeric O antigen takes place on the cytosolic face of the plasma membrane and is mediated by the sequential action of glycosyltransferases elongating the polysaccharide at the non-reducing end (Raetz & Whitfield, 2002; Valvano, 2003
). The nascent polysaccharide is transported across the plasma membrane by an ATP-binding cassette (ABC) transporter (Bronner et al., 1994
), and is subsequently ligated to lipid A-core oligosaccharide. The proteins Wzm and Wzt function as the permease and ATPase of the ABC transporter, respectively (Keenleyside & Whitfield, 1999
). This pathway predominates in O antigens made of repeating units of the same sugar (homopolymeric O antigens) such as those from Escherichia coli O8 and O9 (Whitfield, 1995
), and in group 2 and 3 exopolysaccharide capsules (Whitfield & Roberts, 1999
).
In both the wzy-dependent and the wzy-independent mechanism, the synthesis of the O subunit is initiated by the formation of a sugar phosphodiester bond with Und-P. The initiating enzyme in most E. coli O-types is a tunicamycin-sensitive UDP-GlcNAc : Und-P GlcNAc-1-P transferase (Alexander & Valvano, 1994; Klena & Schnaitman, 1993
; Rick & Silver, 1996
). This enzyme is also required for the initiation of enterobacterial common antigen synthesis (Rick & Silver, 1996
). Genetic and biochemical evidence strongly suggest that wecA is the structural gene encoding this enzyme (Rick & Silver, 1996
). In most Salmonella O antigens, the initiating sugar is galactose, and the initiation reaction is mediated by the UDP-Gal : Und-P Gal-1-P transferase WbaP (formerly RfbP) (Wang et al., 1996
).
In contrast to the wzy-independent O-antigen systems, no obvious ABC transporters have been identified in wzy-dependent systems. It has been proposed that in these cases, undecaprenol-bound O subunits on the periplasmic face of the membrane arise by a process of transmembrane flipping. All wzy-dependent O-antigen clusters studied to date contain a gene that encodes a predicted cytoplasmic membrane protein designated Wzx (Reeves et al., 1996) that has been postulated as a candidate for the O-unit flippase or translocase (Liu et al., 1996
). Homologues of these proteins are also found in the biosynthetic clusters of some exopolysaccharides and also in the enterobacterial common antigen biosynthesis cluster (Barr et al., 1999
; Rahman et al., 2001
).
The most characteristic feature common to all Wzx proteins is the presence of 10 to 12 predicted transmembrane helices, seven of which are included within a loosely conserved region of approximately 208 amino acids (Polysacc_synt domain, Protein families database of alignments and HMMs; http://pfam.wustl.edu/cgi-bin/getdesc?acc=PF01943). However, protein alignments show relatively low conservation in their primary amino acid sequence. In addition, the wzx genes have very poor nucleotide sequence homology, and they can be used as genetic markers for distinguishing among specific O antigens (Marolda et al., 1999; Wang & Reeves, 1998
). Liu et al. (1996)
reported that a strain carrying a wzx mutation of the Shigella dysenteriae type 1 O-antigen gene cluster accumulates Und-P-P-linked O subunits on the plasma membrane, but the evidence that accumulated O subunits are indeed on the cytoplasmic side of the membrane is less conclusive. Thus, the assignment of Wzx as a flippase is only tentative, and awaits further confirmation as well as the elucidation of the biochemical mechanism involved in the presumed flipping activity.
In the current work, we used a reconstitution system that allows for the biosynthesis of O16 LPS in E. coli K-12 strain W3110 to assess the ability of wzx genes from several different O-antigen biosynthesis clusters to complement a wzx deletion mutant. We demonstrate that Wzx proteins from several different O-antigen systems appear to have specificity for the first sugar attached to Und-P, regardless of the chemical structure of the remainder of the O subunit.
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METHODS |
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LPS analysis.
LPS was prepared as previously described (Marolda et al., 1990) and its concentration was determined by measuring KDO (2-keto-3-deoxyoctulosonic acid, 3-deoxy-D-manno-octulosonic acid) following the procedure of R. E. W. Hancock (http://www.cmdr.ubc.ca/bobh/showmethod.php?methodid=31). Equal amounts of LPS were analysed using Tricine-SDS-PAGE in 14 % acrylamide gel, and the gels were stained with silver nitrate (Marolda et al., 1990
) or transferred to nitrocellulose membranes for immunoblot analysis. The membranes were reacted with O16 polyclonal rabbit antibody (The Gastroenteric Disease Center, Wiley Laboratory, University Park, PA, USA) and specific bands were detected by fluorescence with an Odyssey infrared imaging system (LI-COR Biosciences) using IRDye800CW affinity purified anti-rabbit IgG antibodies (Rockland Immunochemicals).
Protein expression.
Membranes were isolated from bacterial cultures that were grown in LB to an OD600 of 0·7. Arabinose was added to a final concentration of 0·2 % (w/v) and the cultures were incubated for 3 h. Cells were harvested by centrifugation and resuspended in 20 ml 10 mM Tris/HCl (pH 7·4) containing protease inhibitors (Complete Protease Inhibitor Cocktail Tablets, Roche Diagnostics). The bacterial suspension was passed twice through a French pressure cell at 16 000 p.s.i. Cell debris was removed by centrifugation at 27 000 g for 15 min at 4 °C and the resulting supernatant was centrifuged for 2 h at 70 000 g at 4 °C. The pellet containing total membranes was resuspended in Tris buffer and the protein concentration determined by the method of Bradford (Bio-Rad). Loading dye was added and the samples were incubated at 45 °C for 30 min prior to electrophoresis in 12 % SDS-polyacrylamide gels (Amer & Valvano, 2000). Proteins were transferred to nitrocellulose membranes and the fusion proteins were reacted with the FLAG M2 mAb as the primary antibody and horseradish-peroxidase-linked sheep anti-mouse IgG (Amersham Pharmacia Biotechnology) as the secondary antibody. Detection by chemiluminescence was performed using the Chemiluminescence Blotting Substrate (Roche Diagnostics), as recommended by the manufacturer.
Construction of vector pBADNTF and cloning of wzx genes.
The cloning vector pBADNTF was constructed using primers 615 and 616 (Table 2), and pBAD24 DNA as a template. The primers were designed in a manner such that each encoded at its 5' end one half of the eight-amino acid FLAG epitope (Fig. 1
). PCR amplification using the Expand Long Template Kit (Roche Diagnostics) was carried out and the product was treated with DpnI followed by T4 DNA polymerase and heat inactivation. The product was phosphorylated with T4 polynucleotide kinase, self-ligated, and transformed into E. coli DH5
. Clones were screened by PCR, and the incorporation of the oligonucleotide encoding the FLAG epitope was confirmed by DNA sequencing. For the cloning of wzx genes into pBADNTF, sense and antisense primers with SmaI and XbaI restriction sites, respectively, were designed for each gene (Table 2
) and the PCR products were ligated to pBADNTF, which was also digested with SmaI and XbaI. These experiments resulted in the construction of plasmids pCM237, pJV2, pJV4, pJV5, pJV6, pJV7 and pJV9 (Table 1
), which all encoded Wzx proteins N-terminally fused to the FLAG epitope.
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RESULTS |
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An association between the specificity of the Wzx proteins and the sugar residue in the first position of the O-antigen subunit
The lack of common features in the primary amino acid sequence of Wzx proteins sharply contrasts with the proposed general function of these proteins in the translocation of O-antigen units across the plasma membrane. In a previous study, we showed that WzxEcO16 restored the O7 antigen synthesis in cells containing a mutated wzxEcO7 gene, to levels that were comparable to those obtained with the parental WzxEcO7 protein (Feldman et al., 1999). In contrast, a plasmid expressing WzxSe from Salmonella enterica only partially complemented O7 LPS production (Feldman et al., 1999
). A comparison of the O-antigen subunit structures of E. coli O16 (Stevenson et al., 1994
), E. coli O7 (L'vov et al., 1984
), and Sal. enterica LT2 (Hellerqvist et al., 1971
) reveals that the biosynthesis of the O-side-chain in both E. coli serotypes is initiated with GlcNAc, while galactose is the first sugar added to the Sal. enterica O unit (Fig. 4
). We hypothesized that the specificity of the Wzx proteins could be associated with the recognition of the first sugar employed for the initiation of the O-antigen subunit biosynthesis.
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Each of the resulting recombinant plasmids was transformed into E. coli K-12 CLM17 (wzxEcO16), and subsequently plasmid pPR1474 was introduced to provide a functional wbbL. These double transformants were examined for production of O antigen. Transformants of CLM17(pPR1474) carrying pMF20 (encoding wzxEcO16) or pMF21 (encoding wzxEcO7) had similar O16 LPS patterns, but they did not attain the amount or distribution of sizes found in the parental W3110(pPR1474) strain (Fig. 5
a). These differences may be attributed to the overexpression of the Wzx proteins, which may alter the stoichiometry of a putative complex involving the Wzy polymerase and the O-chain length-regulator Wzz. A dramatic reduction in the amount and chain length distribution of O16 antigen was detected in exconjugants carrying pCM240 (encoding wzxPa), while no O antigen was detected in those carrying pMF24 (encoding wzxSe) (Fig. 5a
). We could not recover viable CLM17 transformants carrying the high-copy-number plasmid pPR1474. This was presumably due to the accumulation of Und-PP-linked O16 antigen precursors that cannot be translocated across the plasma membrane, which may compromise the synthesis of cell wall peptidoglycan. However, viable colonies were obtained after transformation of CLM17 with plasmid pMF19 (to provide wbbL), which, as expected, did not form O antigen (Fig. 5a
).
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Expression of Wzx proteins from various surface polysaccharide systems and reconstitution of O16 LPS synthesis
To examine the hypothesis that there may be an association between the specificity of Wzx proteins and the first sugar residue of the O subunit, we expanded our studies to additional members of the Wzx family, including proteins from E. coli serotypes O111 and O157, and Shigella flexneri 2a. In a previous study, we showed that at least two Wzx proteins from non-O-antigen systems cannot complement the expression of a wzx transposon mutant in the E. coli O7 antigen cluster (Feldman et al., 1999). Therefore in these studies we included WzxC, a protein implicated in colanic acid capsule synthesis, as a negative control.
A critical aspect of these studies was to avoid differences in gene expression that could affect the interpretation of the complementation results. Thus, the genes encoding these proteins were all cloned in a similar manner using a modified pBAD24 expression vector designated pBADNTF (Fig. 1). In this vector, protein gene expression is under the control of the arabinose-inducible PBAD promoter, and also the gene of interest is cloned in such a manner that the encoded protein becomes N-terminally fused with the FLAG epitope (see Methods). The recombinant fusion proteins were visualized by immunoblots of membrane extracts that were reacted with an anti-FLAG monoclonal antibody. All the proteins were detected by the antibody (Fig. 6
a, b). The differences in the migration of each polypeptide in the gel reflected the predicted molecular masses deduced from the amino acid sequences.
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The mild-denaturing conditions used for the preparation of the cell lysates, probably not sufficient to disperse protein aggregates, could explain the presence of larger bands. These bands had a variation in mass that was proportional to the variation found in the respective monomeric proteins (Fig. 6a, b, arrowheads), suggesting that they correspond to either Wzx dimers or aggregates involving Wzx and other proteins, which are reproducibly formed under these experimental conditions. The WzxC polypeptide (encoded by pJV9) was the only protein that could not be resolved as a monomer under the same conditions that worked for the other Wzx proteins (Fig. 6a
). However, a monomeric form of WzxC was detected when the membranes were prepared at pH 8·5 (Fig. 6b
). Overall, we concluded from these experiments that the various bacterial Wzx proteins are expressed at relatively similar levels under our experimental conditions.
The various Wzx-expressing plasmids were introduced into the E. coli strain CLM17(pMF19) and the ability of the different Wzx proteins to complement the formation of O16 LPS was investigated as before. Plasmids pJV2, pJV6 and pJV7, which encoded WzxEcO111FLAG, WzxSfFLAG and WzxEcO157FLAG, respectively, restored the formation of O16 LPS to levels comparable to those found in CLM17(pMF19) transformed with pCLM237, which encoded WzxEcO16FLAG (Fig. 5c). In contrast, pJV4 (encoding WzxSeFLAG) and pJV5 (encoding WzxPaFLAG) complemented O16 LPS expression very poorly, while pJV9 (encoding WzxCFLAG) did not complement at all (Fig. 5c
). The low level of O16 LPS produced in the presence of pJV4 (encoding WzxSe) is probably due to a gene-dosage effect, since the similar construct in the low-copy-number plasmid pMF24 (encoding wzxSe) did not complement the formation of O16 LPS (Fig. 5a
).
The only sugar that is common to the O units of E. coli O16, O111 and Sh. flexneri 2a is a GlcNAc residue (Fig. 4) which is the first sugar attached to Und-P. In the O unit of E. coli O157 there is a GalNAc at the same position (Fig. 4
), suggesting that the WzxEcO157 translocase may function indistinctly with either GalNAc in the natural O157 subunit or GlcNAc in the case of the O16 subunit. In the complementation with WzxPaFLAG a lower level of O16 LPS production may occur because this protein may recognize FucNAc when functioning in P. aeruginosa. A similar conclusion could be drawn for the complementation experiment with the Salmonella WzxSeFLAG protein, which may recognize specifically galactose (Fig. 4
). Therefore, the restoration of O16 LPS production in the wzx-deleted strain CLM17 by the various Wzx protein homologues from different O-antigen systems suggests the possibility that these proteins may recognize the first Und-PP-linked sugar of the O unit, albeit with varying degrees of specificity.
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DISCUSSION |
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Using a genetic reconstitution system in E. coli K-12/O16 for ligation of O-antigen components to the lipid A-core oligosaccharide, we previously discovered that a single GlcNAc residue can be incorporated onto the lipid A-core oligosaccharide in a wzx-dependent manner (Feldman et al., 1999). This process also required the function of WecA, strongly suggesting that the GlcNAc residue was presented to the core oligosaccharide acceptor as an Und-PP-GlcNAc intermediate (Feldman et al., 1999
). Therefore, we concluded that Wzx proteins can recognize, and possibly interact with, the first Und-PP-linked sugar of the O subunits.
Our model also predicted that Wzx proteins from other O-antigen systems containing a GlcNAc residue in the first position should be functionally interchangeable, and therefore capable of restoring O16 LPS production in a wzx mutant of E. coli K-12. The results reported in this work, using Wzx proteins from E. coli O7 and O111, and Sh. flexneri 2a, support this notion since the GlcNAc in the first position is the only shared structural component in the O16, O7, O111 and Sh. flexneri 2a O units (Fig. 4). Moreover, complementation of O16 LPS production was also achieved with WzxEcO157 and, to a lesser extent, with WzxPa. The sugar in the first position in the E. coli O157 and P. aeruginosa O5 antigens is GalNAc and FucNAc, respectively. In contrast, complementation with WzxSe from Sal. enterica, which has a galactose in the first position of the O unit, was negligible and only minimally detectable when the protein was expressed from a high-copy-number plasmid vector.
These results could not be explained merely by differences in protein expression. First, our Wzx-expressing plasmids were all constructed in the same manner, and second, the levels of expression of the various recombinant Wzx proteins, as detected by immunoblotting, were comparable. The incorporation of the FLAG epitope tag in the N terminus of the recombinant Wzx proteins did not compromise their function, as similar results were obtained with proteins lacking the epitope (data not shown). Although Wzx proteins are usually difficult to express, in part due to the low G+C content and the abundance of rare codons in wzx genes, our strategy to express and visualize these proteins was successful.
Our observations strongly suggest the existence of subfamilies of Wzx proteins, some of which may interact with O units that are initiated with N-acetylhexosamines and others that may be better suited to interact with O units initiated with hexoses. This does not preclude a possible interaction of a Wzx protein with the initiating enzyme for the synthesis of the O unit. The notion of subfamilies of Wzx proteins is consistent with an analogous functional classification for the enzymes that catalyse the initiation reaction, which can be clearly separated into two major distinct families according to their specificity for N-acetylhexosamine- or hexose-containing nucleotide phosphates (Valvano, 2003). One of these families corresponds to the polyisoprenyl-phosphate N-acetylhexosamine-1-phosphate transferases (PNPT family; Anderson et al., 2000
; Dal Nogare & Lehrman, 1988
; Lehrman, 1994
), comprising proteins that are present both in prokaryotes and in eukaryotes, such as WecA. The other family corresponds to the polyisoprenyl-phosphate hexose-1-phosphate transferases (PHPT family), none of which has a homologue in eukaryotic cells. The prototype member of this family is WbaP from Sal. enterica (Wang et al., 1996
), which has also been implicated in O-antigen subunit processing (Wang & Reeves, 1994
).
In a previous work, we showed that WzxC (involved in colanic acid capsule synthesis) and ExoT (involved in exopolysaccharide synthesis) from E. coli K-12 and Rhizobium meliloti, respectively, did not complement the synthesis of the O7 antigen subunit (Feldman et al., 1999). These findings may be explained by the model presented above since the initiation of the synthesis of colanic acid and rhizobial exopolysaccharide requires initiating enzymes from the PHTP family. We have previously delineated a region in WecA that is highly conserved in all the proteins of the PNTP family. Analysis of amino acid replacement mutants suggested that the conserved residues are part of a region in the protein that may play a role in forwarding the Und-PP-linked O subunit to the translocation reaction (Amer & Valvano, 2002
). Taking all of this information together, we propose the existence of a complex involving the initiating enzyme and Wzx, whereby the initiating enzyme may also provide a scaffold for the completion of the assembly of the O unit on the cytosolic leaflet of the plasma membrane, but at the site of translocation across the membrane. Further experiments to directly demonstrate a proteinprotein interaction between Wzx and initiating enzymes are currently under way in our laboratory.
E. coli and other enteric bacteria also produce enterobacterial common antigen, a cell-surface glycolipid that requires UDP-GlcNAc and WecA for the initiation reaction (Rick & Silver, 1996). The enterobacterial common antigen biosynthesis gene cluster has a gene encoding a polypeptide with similar characteristics to the Wzx proteins, designated WzxE. We should not observe an O16 LPS defect in W3110(
wzx) if WzxE could fully complement the function of the missing Wzx protein. The lack of complementation by WzxE is not due to a defect in the expression of its gene, since the
wzx mutant produces enterobacterial common antigen (C. L. Marolda & M. A. Valvano, unpublished). Therefore, our model involving a putative WecA-Wzx interaction cannot explain why WzxE cannot complement a
wzx mutant.
Using an in vitro system, Rick et al. (2003) recently showed that the E. coli WzxE protein is involved in the transbilayer movement of a trisaccharide-lipid intermediate used in the assembly of enterobacterial common antigen. These authors could not demonstrate in their model system a similar function for Wzx. These observations contrast with our in vivo findings that a single GlcNAc residue can be translocated and attached to the core-lipid A in a Wzx-dependent fashion (Feldman et al., 1999
). The existence of two Wzx proteins and two cell-surface polysaccharide systems requiring the same initiating enzyme, WecA, provides us with an opportunity to dissect the components of the translocation pathway and the possible interactions of these proteins not only with WecA but also with other proteins putatively involved in the assembly of O16 LPS and enterobacterial common antigen.
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ACKNOWLEDGEMENTS |
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Received 2 July 2004;
revised 12 August 2004;
accepted 17 August 2004.
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