Wzx proteins involved in biosynthesis of O antigen function in association with the first sugar of the O-specific lipopolysaccharide subunit

Cristina L. Marolda1, Jessica Vicarioli1 and Miguel A. Valvano1,2

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
One of the most common pathways for the export of O-specific lipopolysaccharide (LPS) across the plasma membrane requires the participation of the Wzx protein. Wzx belongs to a family of integral membrane proteins that share little conservation in their primary amino acid sequence, making it difficult to delineate functional domains. This paper reports the cloning and expression in Escherichia coli K-12 of various Wzx homologues from different bacteria as FLAG epitope-tagged protein fusions. A reconstitution system for O16 LPS synthesis was used to assess the ability of each Wzx protein to complement an E. coli K-12 {Delta}wzx mutant. The results demonstrate that Wzx proteins from O-antigen systems that use N-acetylglucosamine or N-acetylgalactosamine for the initiation of the biosynthesis of the O repeat can fully complement the formation of O16 LPS. Partial complementation was seen with Wzx from Pseudomonas aeruginosa, a system that uses N-acetylfucosamine in the initiation reaction. In contrast, there was negligible complementation with the Wzx protein from Salmonella enterica, a system in which galactose is the initiating sugar. These results support a model whereby the first sugar of the O repeat can be recognized by the O-antigen translocation machinery.


Abbreviations: KDO, 2-keto-3-deoxyoctulosonic acid (3-deoxy-D-manno-octulosonic acid); Und-P, undecaprenol-phosphate


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Lipopolysaccharide (LPS) is a major constituent of the bacterial outer membrane (Nikaido, 1996). It consists of lipid A, core oligosaccharide and in some micro-organisms an O-specific polysaccharide (or O antigen) that is made of repeating oligosaccharide subunits (Raetz & Whitfield, 2002; Valvano, 2003). The biogenesis of LPS is a complex multi-step process initiated on the cytosolic face of the plasma membrane that culminates on the bacterial cell surface (for recent reviews see Raetz & Whitfield, 2002; Valvano, 2003). The core oligosaccharide is assembled on preformed lipid A by the sequential glycosyl transfer of each monosaccharide, while the O antigen is assembled on undecaprenol-phosphate (Und-P). These pathways eventually converge by the ligation of the O antigen onto the outer core domain of the lipid A-core oligosaccharide acceptor, with the concomitant release of Und-PP (Valvano, 2003). Und-P is also required as a lipid intermediate for the biosynthesis of other cell-surface glycans, including the enterobacterial common antigen and the cell-wall peptidoglycan (Rick & Silver, 1996; van Heijenoort, 2001).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and reagents.
Strains and plasmids used in this study are described in Table 1. Bacteria were cultured in Luria broth (LB) supplemented with antibiotics at the following final concentrations: 100 µg ampicillin ml–1, 40 µg kanamycin ml–1 and 80 µg spectinomycin ml–1. Chemicals and antibiotics were purchased from Sigma Aldrich and Roche Diagnostics. Oligonucleotide primers were purchased from Invitrogen and are listed in Table 2. Plasmids were introduced into electrocompetent cells by electroporation (Dower et al., 1988).


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Table 1. Strains and plasmids

 

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Table 2. Primers used for mutagenesis experiments and for the plasmid construction

 
Construction of the wzxEcO16 deletion mutant.
The wzx chromosomal gene was deleted as described by Datsenko & Wanner (2000). We generated primers composed of 40 to 45 nucleotides corresponding to regions adjacent to the gene targeted for deletion. The primers also contained 20 additional nucleotides that annealed to the template DNA from plasmid pKD4, which carries a kanamycin-resistance gene flanked by FRT (FLP recognition target) sites (Table 2). Competent cells were prepared by growing E. coli W3110 carrying pKD46 in LB containing 0·5 % (w/v) arabinose and the PCR products were introduced by electroporation. The plasmid pKD46 encodes the Red recombinase of the {lambda} phage, which was placed under the control of the arabinose-inducible promoter PBAD. Kanamycin-resistant colonies were screened by PCR using primers annealing to regions outside of the mutated gene. Next, the antibiotic gene was excised by introducing the plasmid pCP20 encoding the FLP recombinase. Plasmids pKD46 and pCP20 are both thermosensitive for replication and they were cured at 42 °C.

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{alpha}. 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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Fig. 1. Relevant features of the vector pBADNTF. The DNA sequence containing the multiple cloning site and encoding the FLAG oligopeptide epitope (boxed) is shown. araC, transcriptional regulator; bla, {beta}-lactamase; MCS, multiple cloning site; ori, ColE1 origin of replication; PBAD, arabinose-inducible promoter.

 

   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Reconstitution of O16 LPS synthesis in E. coli K-12
The E. coli K-12/O16 strain W3110 cannot form O antigen because of an IS5 element inserted in wbbL, the last gene of the wbEcK12/O16 cluster (Fig. 2; Liu & Reeves, 1994). Since this gene encodes a rhamnosyltransferase involved in the transfer of rhamnose to the Und-PP-GlcNAc acceptor molecule (Fig. 2), the mutation effectively blocks the synthesis of the complete O16 subunit (Fig. 3a, W3110). However, all the other genes of the wbEcK12/O16 cluster are functional in strain W3110, and complementation of the wbbL function in trans with pMF19, a plasmid that encodes a functional wbbL gene under the control of the Ptac promoter (Fig. 2; Feldman et al., 1999; Liu & Reeves, 1994), restores the formation of a complete O16-specific polysaccharide [Fig. 3a, W3110(pMF19)].



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Fig. 2. Gene organization and structure of the wbEcO16 cluster of E. coli K-12. The galF and gnd genes flanking the wbEcO16 are shown. The wbEcO16 genes are as follows: rmlBDAC (dTDP-rhamnose biosynthesis), wzx (O-antigen translocase), glf (UDP-galactopyranose mutase), wzy (O-antigen polymerase), wbbI (putative galactofuranosyl transferase), wbbJ (O-acetyl transferase), wbbK (putative glucosyl transferase), wbbL (rhamnosyl transferase). The flag between galF and rmlB indicates the location of the wbEcO16 promoter region. The DNA inserts of plasmids pMF19 and pPR1474, and the location of the IS5 insertion element within wbbL are indicated. Chemical structure of the O16 subunit: D-Galf, D-galactofuranose; D-Glc, D-glucose; D-GlcNAc, D-N-acetylglucosamine; L-Rha, L-rhamnose; OAc, O-acetyl; P-P-Und, undecaprenol pyrophosphate. The synthesis of O16 is initiated by the formation of a phosphodiester bond between Und-P and GlcNAc-1P (donated from UDP-GlcNAc), a reaction catalysed by the WecA protein (Amer & Valvano, 2002). The X indicates the block in the elongation of the O16 subunit as a consequence of the wbbL : : IS5 mutation.

 


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Fig. 3 (a) Production of O16 LPS by strains W3110 and the isogenic mutant CLM17 (W3110{Delta}wzx). LPS samples (10 µl) were run on 14 % Tricine SDS-PAGE and stained with silver nitrate (see Methods). Regions corresponding to core-lipid A, core plus one O unit and highly polymerized O antigen are indicated. (b) Regulation of Wzx expression in the presence of arabinose. Cells were grown in the presence of no arabinose or 0·002 %, 0·02 %, 0·2 %, 0·5 % arabinose (w/v), as indicated.

 
We adopted the reconstitution of O16 antigen synthesis in W3110 as a model system to investigate the function of wzx. Fig. 3(a) shows that in contrast to the parental isolate W3110, CLM17 (W3110{Delta}wzx) did not form O16 LPS when transformed with pMF19. The defect in O16 LPS synthesis was corrected by transforming CLM17(pMF19) with pCM223, a plasmid containing wzxEcO16 placed under the control of the arabinose-inducible PBAD (Guzman et al., 1995). Accordingly, the amounts of O16 LPS produced by the complemented strain increased with increasing concentration of arabinose in the growth medium (Fig. 3b). In the absence of arabinose, the promoter activity of PBAD is not completely repressed (Guzman et al., 1995), and consequently very little O16 antigen was detectable in this case, as evidenced by the appearance of a faint band with the same migration as that of lipid A-core plus one O16 subunit (Fig. 3). Also, a lower and fainter band was detected in the LPS preparation from the {Delta}wzx mutant CLM17 (Fig. 3a), which may correspond to a small amount of enterobacterial common antigen bound to core-lipid A (Kiss et al., 1978). Overall, these experiments demonstrated that the deletion of wzx in strain CLM17 is non-polar on the downstream genes of the E. coli K-12/O16 cluster (Fig. 2), and therefore this strain can be used to assess the function of other Wzx proteins by complementation experiments.

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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Fig. 4. Structure of the O-antigen units of E. coli K-12/O16 (Stevenson et al., 1994), O7 (L'vov et al., 1984), O111 (Eklund et al., 1978), O157 (Perry et al., 1986), Sal. enterica LT2 (Hellerqvist et al., 1971), Sh. flexneri 2a (Kenne et al., 1978) and P. aeruginosa O5 (Knirel et al., 1988), all of which require Wzx homologues for biosynthesis. D-Galf, D-galactofuranose; D-Glc, D-glucose; L-Rha, L-rhamnose; D-GlcNAc, D-N-acetylglucosamine; OAc, O-acetyl; VioNAc, 4-acetamido-4,6-dideoxy-D-glucose; D-Gal, D-galactose; D-Col, D-colitose; D-PerNAc, D-N-acetylperosamine; L-Fuc, L-fucose; Abe, 3,6-dideoxy-D-galactose; D-Man(2NAc3N)A, 2-acetamido-3-acetamidino-2,3-dideoxy-D-mannuronic acid; D-Man(2NAc3NAc)A, 2,3-diacetamido-D-mannuronic acid; D-Fuc2NAc, 2-acetamido-2,6-dideoxy-D-galactose; D-Fuc4NAc, 4-acetamido-2,6-dideoxy-D-galactose; D-ManNAc, D-N-acetylmannosamine; D-GlcA, D-glucuronic acid. Sugars in bold type indicate the first sugar of each subunit that is phosphodiester bound to the undecaprenol lipid intermediate.

 
To address this hypothesis we cloned the wzx genes from E. coli O16, E. coli O7 and Sal. enterica LT2 in the low-copy-number vector pEXT21. We also cloned wzxPa from Pseudomonas aeruginosa serotype O5 strain PAO1 as an example of a wzx gene from a non-enteric bacterium. In P. aeruginosa PAO1, the biosynthesis of the O5-specific antigen subunit is initiated with FucNAc (Rocchetta et al., 1998).

Each of the resulting recombinant plasmids was transformed into E. coli K-12 CLM17 ({Delta}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. 5a). 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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Fig. 5. Complementation of O16 LPS in strain CLM17 ({Delta}wzxEcO16) with the different wzx gene homologues. All the samples were separated by Tricine-SDS-PAGE in 14 % polyacrylamide gels. (a) The wzx gene homologues were cloned into pEXT21 and gene expression was induced with 2 mM IPTG. Each lane was loaded with an amount of LPS corresponding to 25 ng KDO and the gel was stained with silver nitrate. (b) The same LPS samples (corresponding to 25 ng KDO per lane except in the last two lanes, where the loading of LPS corresponded to 100 ng KDO) used in (a) were transferred to nitrocellulose paper and the blot was reacted with O16 antibodies. Specific bands were detected by fluorescence with IRDye800-conjugated affinity rabbit IgG antibody. (c) Complementation of CLM17 with wzx genes cloned into the vector pBADNTF. LPS samples were prepared from cultures induced with 0·2 % arabinose. Samples corresponding to 10 ng KDO were loaded into each lane and the gel stained with silver.

 
Fig. 5(b) demonstrates that the O antigen produced in the presence of all the recombinant wzx genes, except for wzxSe (pMF24), reacted with the rabbit polyclonal O16-specific antiserum. Also, CLM17(pMF19) expressed one slow-migrating band with O16 specificity that presumably corresponds to lipid A-core plus one O16 subunit (Fig. 5b). This band could be the result of a partial complementation of the {Delta}wzxEcO16 mutation due to the presence of the wzxE gene of the enterobacterial antigen biosynthesis cluster (Rick et al., 2003). However, if this is the case the complementation must be very poor since no polymerized O16 antigen can be detected. Altogether, these experiments demonstrated that Wzx proteins from different O-antigen systems that contain GlcNAc, and to a lesser extent FucNAc, in the first position, can complement the WzxEcO16 function and restore the synthesis of E. coli O16 antigen in the wzxEcO16 deletion mutant.

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. 6a, 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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Fig. 6. Expression of recombinant Wzx proteins in E. coli DH5{alpha}. Membrane fractions were isolated from cultures induced with 0·2 % arabinose and the protein concentration was adjusted to 7 µg per lane. Proteins were separated on 12 % SDS-PAGE, transferred into a nitrocellulose membrane and the membrane was reacted with monoclonal antibodies against the FLAG epitope (Sigma). Blots were developed with chemiluminescence (Roche Diagnostics). The predicted molecular masses of the recombinant proteins (kDa) are: WzxO16, 49·2; WzxO111, 48·5; WzxSe, 50; WzxPa, 46·6; WzxSf, 47·7; WzxO157, 53·2; and WzxC, 55. The migration of these proteins in the gel is anomalous, probably due to their highly basic nature. Arrows in (a) and (b) indicate probable protein dimers. Sizes (kDa) of pre-stained molecular-mass markers (Bio-Rad) are given on the left of the figure. (a) Membranes prepared with phosphate buffer (pH 7·4). (b) Membranes prepared with Tris/HCl buffer (pH 8·5).

 
Initial experiments in which samples were boiled did not yield any detectable protein band in the immunoblots (data not shown). Similar difficulties with boiling have been previously encountered with other integral membrane proteins (Amer & Valvano, 2000; Dan et al., 1996; Seol & Shatkin, 1993). The best conditions for detection of the Wzx–FLAG polypeptides were obtained by incubating samples at 45 °C for 30 min in a buffer consisting of 0·01 M sodium phosphate, 1 % {beta}-mercaptoethanol, 1 % SDS and 6 M urea.

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 WzxEcO111–FLAG, WzxSf–FLAG and WzxEcO157–FLAG, respectively, restored the formation of O16 LPS to levels comparable to those found in CLM17(pMF19) transformed with pCLM237, which encoded WzxEcO16–FLAG (Fig. 5c). In contrast, pJV4 (encoding WzxSe–FLAG) and pJV5 (encoding WzxPa–FLAG) complemented O16 LPS expression very poorly, while pJV9 (encoding WzxC–FLAG) 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 WzxPa–FLAG 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 WzxSe–FLAG 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.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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The transmembrane ‘flipping’ of polyisoprenol-linked glycans is a common feature in the synthesis of bacterial cell surface polysaccharides and the glycan component for protein glycosylation in eukaryotes (Helenius & Aebi, 2002; Helenius et al., 2002). However, it remains unclear how lipid-linked carbohydrates are translocated from one leaflet of the lipid bilayer. Given that the translocation of Und-PP-linked O-subunits must be a conserved process, the absence of any obvious conserved motifs in the primary amino acid sequences of Wzx proteins, which might provide clues to explain the translocation process, is intriguing. One possible explanation for the considerable variation among Wzx proteins could be the requirement for the recognition of specific O subunits, which are highly variable in terms of structure and sugar composition. However, this model cannot explain the apparently conserved nature of the process of translocation.

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 protein–protein 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({Delta}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 {Delta}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 {Delta}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.


   ACKNOWLEDGEMENTS
 
The authors thank those colleagues referenced or mentioned in Table 1 for strains and plasmids. This work was supported by grant MOP-10206 from the Canadian Institutes of Health Research. M.A.V. holds a Canada Research Chair in Infectious Diseases and Microbial Pathogenesis.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 2 July 2004; revised 12 August 2004; accepted 17 August 2004.



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