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Journal of Bacteriology, August 2005, p. 5470-5481, Vol. 187, No. 15
0021-9193/05/$08.00+0     doi:10.1128/JB.187.15.5470-5481.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Functional Analysis of Conserved Gene Products Involved in Assembly of Escherichia coli Capsules and Exopolysaccharides: Evidence for Molecular Recognition between Wza and Wzc for Colanic Acid Biosynthesis

Anne N. Reid and Chris Whitfield*

Department of Molecular and Cellular Biology, New Science Complex, University of Guelph, Guelph, Ontario, Canada N1G 2W1

Received 4 February 2005/ Accepted 12 May 2005


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ABSTRACT
 
Group 1 capsular polysaccharides (CPSs) of Escherichia coli and some loosely cell-associated exopolysaccharides (EPSs), such as colanic acid, are assembled by a Wzy-dependent polymerization system. In this biosynthesis pathway, Wza, Wzb, and Wzc homologues are required for surface expression of wild-type CPS or EPS. Multimeric complexes of Wza in the outer membrane are believed to provide a channel for polymer export; Wzc is an inner membrane tyrosine autokinase and Wzb is its cognate phosphatase. This study was performed to determine whether the Wza, Wzb, and Wzc proteins for colanic acid expression in E. coli K-12 could function in the E. coli K30 prototype group 1 capsule system. When expressed together, colanic acid Wza, Wzb, and Wzc could complement a wza-wzb-wzc defect in E. coli K30, suggesting conservation in their collective function in Wzy-dependent CPS and EPS systems. Expressed individually, colanic acid Wza and Wzb could also function in K30 CPS expression. In contrast, the structural requirements for Wzc function were more stringent because colanic acid Wzc could restore translocation of K30 CPS to the cell surface only when expressed with its cognate Wza protein. Chimeric colanic acid-K30 Wzc proteins were constructed to further study this interaction. These proteins could restore K30 biosynthesis but were unable to couple synthesis to export. The chimeric protein comprising the periplasmic domain of colanic acid Wzc was functional for effective K30 CPS surface expression only when coexpressed with colanic acid Wza. These data highlight the importance of Wza-Wzc interactions in group 1 CPS assembly.


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INTRODUCTION
 
Escherichia coli capsules (comprising capsular polysaccharides [CPSs]) are acidic polysaccharide layers surrounding and tightly associated with the surface of the cell. They are recognized virulence determinants and act by increasing adherence to host tissues and conferring resistance to phagocytosis. In some cases, the host immune response against the CPS is impaired because the capsule structure mimics host cell components. More than 80 distinct capsular or K antigens in E. coli have been described, and these are classified into four groups on the basis of genetic and biosynthetic criteria (61). These capsule groups provide prototypes for CPSs and the loosely associated exopolysaccharides (EPSs) produced by many other bacteria.

Group 1 capsules and related polysaccharides are found in pathogens of humans and animals and in plant-associated bacteria, where they enhance virulence or mediate symbiosis. Group 1 CPSs and related EPSs are assembled by a Wzy-dependent polymerization system. The early stages of synthesis are identical to those of lipopolysaccharide biosynthesis, where the action of the Wzy-dependent system is best understood (reviewed in reference 48). In the present model, lipid-linked repeat units are built up on undecaprenol diphosphate at the cytoplasmic face of the inner membrane by the sequential activity of glycosyltransferases. The lipid-linked repeat units are then flipped to the periplasmic face of the inner membrane by the putative flippase (Wzx), where the putative polymerase, Wzy, generates a long-chain polymer. The products of three conserved genes (wza, wzb, and wzc) are required for late steps in the expression of group 1 CPSs and related EPSs, including E. coli colanic acid (reviewed in reference 60). In the absence of Wza and Wzc in the prototype E. coli K30 system, oligosaccharides with low degrees of polymerization are made and ligated to lipid A-core in a glycoform known as KLPS (17, 37, 62).

The functions of Wza, Wzb, and Wzc in E. coli K30 have been studied in detail. Wza is an outer membrane lipoprotein that is essential for the assembly of high-molecular-weight (HMW) CPS on the surfaces of E. coli K30 cells. Wza forms ring-like multimers composed of eight subunits arranged as a tetramer of dimers (3, 4, 17, 43). These complexes are believed to provide a channel through which K30 CPS crosses the outer membrane. Wzc is a tyrosine autokinase that is dephosphorylated by its cognate phosphatase, Wzb (62). Wzc is also essential for HMW-polysaccharide expression in all systems examined to date. The precise role of Wzc is not known, but the presence of homologues in CPS systems in gram-positive bacteria suggests a role in a conserved step of CPS/EPS assembly, perhaps in determining polymer chain length or in coordinating a putative multienzyme complex for CPS/EPS biosynthesis and export (60). While wzb mutants allow formation of some K30 CPS on the cell surface, the Wzb phosphatase is required for a wild-type capsular phenotype (62). The finding that Wzc and Wza interact (43) suggests that the homologues in gram-negative bacteria may have additional roles that are not conserved in the Wzc proteins in gram-positive bacteria.

Wzc homologues are integral membrane proteins harboring two transmembrane domains flanking a large periplasmic loop and have a cytoplasmic C-terminal region with a Walker A ATP-binding motif and a tyrosine-rich C terminus (2, 15, 16, 20, 40, 58, 62). Phosphorylation of Wzc occurs at the tyrosine-rich C terminus, at the expense of ATP (5, 20, 39, 40, 42, 44, 58, 62). It appears that phosphorylation of Wzc is essential for capsule expression in E. coli K30 (62) and that the level of Wzc phosphorylation rather than the phosphorylation of specific residues is important in this system (46). Interestingly, in the colanic acid system (59) and in Streptococcus pneumoniae type 19F CPS (40), phosphorylation of Wzc is reported to be a negative regulator of EPS/CPS expression. However, recent data revealed contrasting phenotypes for phosphatase mutants of two different S. pneumoniae strains, suggesting that discrepancies in this example potentially reflect the involvement of other unidentified cellular factors (5). Thus, it is unclear whether Wzc proteins have identical functions in all systems.

In E. coli, colanic acid can be coexpressed with the prevalent group 2 capsules but not with group 1 capsules (30). This is because the group 1 cps cluster and the colanic acid cps cluster occupy the same genetic location, reflecting genetic insertions and rearrangements that have resulted in the acquisition of the group 1 cps cluster and the loss of the colanic acid cluster (50). Despite the similar arrangements in the group 1 and colanic acid cps operons and the shared biosynthetic mechanisms of these systems, important differences exist in the regulation of colanic acid and K30 CPS expression. Colanic acid is not produced in significant amounts at physiological temperatures and apparently plays no role in virulence (1, 25, 35, 52). However, this polymer provides protection against desiccation (45) and heat and acid (33, 38), as well as osmotic and oxidative stress (10), and has been implicated in biofilm formation by E. coli K-12 (13, 18, 47). Colanic acid expression is transcriptionally regulated by the RcsC-YojN-RcsB phosphorelay system (11, 19, 55, and 56 and references therein), a system now known to regulate a range of functions (18, 24). In contrast, E. coli group 1 capsule cps loci are constitutively expressed (51) and the polymers produced are important virulence determinants (8, 9, 23, 28, 41, 53, 54).

An important distinction between CPS and EPS polymers is their degrees of attachment to the cell surface. While EPSs are generally loosely associated with the cells, CPSs tend to be more firmly anchored. The method of attachment of the group 1 capsule to the surface is not known, but surface distribution is influenced by the outer membrane protein Wzi, which is confined to group 1 CPS systems (49). The absence of Wzi from EPS systems (including the colanic acid system) may be a factor in the loose arrangement of most EPS polymers. However, it is unclear whether Wza, Wzb, and Wzc also contribute to the features that distinguish CPS and EPS expression. The predicted protein sequences for Wza, Wzb, and Wzc from E. coli K30 and E. coli K-12 (strain MG1655) are well conserved (Fig. 1). While some homologues of Wza, Wzb, and Wzc have been identified and some of the Wzb and Wzc homologues have had phosphatase and kinase activities confirmed in vitro, it has not been determined whether they perform identical functions in CPS and EPS synthesis. The goal of this study was to address these questions by examining the function of E. coli K-12 colanic acid biosynthesis proteins in the prototype E. coli K30 group 1 CPS system.



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  		FIG. 1. Alignment of amino acid sequences of Wza, Wzb, and Wzc from E. coli K30 and K-12. WzaK30 and WzaCA share 65% identity and 90% similarity at the amino acid level (A), the respective Wzb proteins are 51% identical and 80% similar (B), and the Wzc proteins share 51% identity and 85% similarity (C). Identical amino acids are highlighted in black and similar amino acids are highlighted in gray. In panel C, the putative coiled-coil domains are indicated by black bars above or below the sequences. This alignment was generated using Clustal W (http://npsa-pbil.ibcp.fr) (57). Coiled-coil predictions were generated using the COILS program (http://www.ch.embnet.org) with an unweighted matrix and a 21-residue window (36).


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were grown at 37°C in Luria-Bertani (LB) medium (Invitrogen Life Technologies, Burlington, Ontario, Canada) supplemented with ampicillin (100 µg ml–1), kanamycin (50 µg ml–1), or chloramphenicol (30 µg ml–1) where appropriate. Complementation experiments were carried out with derivatives of E. coli E69 (O9a:K30:H12), the prototype group 1 capsule strain. Most E. coli isolates (like E69) contain two chromosomal copies of the wza-wzb-wzc cluster, one in the cps locus and the other at a position corresponding to 22 min on the E. coli K-12 linkage map (17, 29, 31). To simplify the analyses, all mutants studied here were constructed by using strain CWG258 as a background. CWG258 is an E69 derivative with a polar aadA insertion in the 22-min copy of wza that eliminates expression of the 22-min copy of the wza-wzb-wzc cluster (17). The polarity of this insertion and the phenotypes resulting from individual chromosomal mutations in the cps locus versions of wza, wzb, and wzc in this background are well established (17, 62). For induction of gene expression from pBAD (22) derivatives, cells were grown in LB to mid-exponential phase prior to the addition of a final concentration of L-arabinose of 0.002% (for expression of Wza derivatives) or 0.02% (for expression of Wzb and Wzc derivatives). The cultures were then grown for an additional 2.5 h prior to preparation of cell membranes or lysates.


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  		TABLE 1. Strains and plasmids used in this study

Cloning of wza, wzb, and wzc homologues. The wza, wzb, and wzc genes were amplified from chromosomal DNA of either E. coli E69 (K30 antigen biosynthesis locus, hereafter designated with the subscript K30) or E. coli DH5{alpha} (colanic acid biosynthesis locus, hereafter designated with the subscript CA). Amplification reactions were carried out with primers engineered to include desired restriction sites for cloning into arabinose-inducible pBAD vectors (22). The primers used for PCR amplifications are listed in Table 2. The wza, wzb, and wzc genes were cloned individually and in combination to evaluate the need for interacting partners. Plasmid sequences were verified and inserts deemed to be free of PCR-induced errors by sequencing at the Guelph Molecular Supercenter (University of Guelph, Guelph, Ontario, Canada).


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  		TABLE 2. Primers used in this study

Construction of a chromosomal deletion in the (wza-wzb-wzc)K30 cluster. Strain CWG655 [wza22 min::aadA {Delta}(wza-wzb-wzc)K30::aphA3] was constructed by chromosomal deletion in strain CWG258. Briefly, the region spanning the (wza-wzb-wzc)K30 cluster was amplified using primers EB6/EB7 (each containing a BamHI site to facilitate cloning). The PCR product was cloned into the BamHI-digested suicide vector pWQ173 (51). An internal fragment of about 1,800 bp was removed by digestion of the plasmid with BglII/BclI. This removed most of the wza and wzc genes and all of wzb. The BamHI-digested nonpolar aphA3 kanamycin resistance cassette was ligated into BglII/BclI-digested plasmid (these are compatible ends), generating pWQ96. E. coli CWG258 was electrotransformed (6) with pWQ96, and gene replacement occurred through homologous recombination, as described elsewhere (51). Replacement of the wild-type genes by allelic exchange was confirmed by PCR using primers internal and external to the mutation site.

Construction of chimeric Wzc proteins. Chimeric Wzc proteins were constructed by using a gene splicing overlap procedure (27, 34). Construction of pWQ87 involved PCR amplification of the 5' portion (corresponding to amino acids 1 to 455) of wzcK30 by using a forward primer (AP66) designed to introduce an EcoRI site and a reverse primer (AP67) that yields a product with a 25-bp overhang of the wzcCA sequence. The 3' portion (corresponding to amino acids 454 to 721) of wzcCA was then amplified using a forward primer (AP68) that generates a 25-bp wzcK30 overhang in the product and a reverse primer (AP35) designed to introduce a PstI site. The products of the first two PCRs were used as templates for a third PCR that involved extension of the partly complementary templates and amplification using the AP66/AP35 primer pair. This PCR product was digested with EcoRI/PstI and ligated into pBAD24. Plasmid pWQ88 (N-terminal amino acids 1 to 453 of WzcCA) was constructed using a similar approach.

SDS-PAGE and Western immunoblotting. Whole-cell lysates were prepared in 2x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (32) and heated to 100°C for 10 min. Proteins from whole-cell lysates were separated on 14% resolving SDS-PAGE gels and transferred onto BioTrace NT nitrocellulose membranes (Gelman Laboratory) for Wza expression analysis. Wzc expression and phosphorylation were assessed by Western immunoblotting of isolated cell envelopes in order to detect the low signal intensities that were difficult to visualize in whole-cell lysates. Briefly, cells were grown in 200-ml cultures, and expression of plasmid-carried proteins was induced as described above. Cells were harvested, resuspended in 15 ml of 10 mM Tris-HCl, pH 8.0, and lysed by ultrasonication. Following a low-speed centrifugation step to remove unbroken cells and cell debris, cell envelopes were collected by ultracentrifugation (100,000 x g; 1 h) and resuspended in 2 ml of 10 mM Tris-HCl, pH 8.0. A sample of isolated cell membranes corresponding to 5 µg of total protein was subjected to electrophoresis using 10% resolving SDS-PAGE gels. Proteins were either stained with Coomassie brilliant blue or transferred onto Westran polyvinylidene difluoride membranes (Schleicher and Schuell) or nitrocellulose membranes for Western immunoblot detection of tyrosine-phosphorylated proteins or Wzc, respectively. The transfer buffer contained 25 mM Tris, 192 mM glycine, and 20% (vol/vol) methanol. Wzc and Wza were detected by using polyclonal antisera from rabbits immunized with the E. coli K30 proteins (17, 62). Detection was achieved by using an alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (Caltag, Burlingame, CA). Phosphorylation of Wzc was detected using the PY20 antiphosphotyrosine monoclonal antibody (Transduction Laboratories, Lexington, KY) and an alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate were used for detection.

SDS-PAGE analysis of K30 polysaccharide. Total cellular polysaccharide samples were prepared by the method of Hitchcock and Brown (26). Cells were grown as described above, harvested, and washed prior to preparation of whole-cell lysates in 1x SDS-PAGE sample buffer without ß-mercaptoethanol. Proteins in the lysate were digested with proteinase K (0.5 mg ml–1) overnight at 55°C. Polysaccharide preparations were separated by electrophoresis on NuPAGE 4 to 12% bis-Tris gels (Invitrogen Life Technologies Inc., Burlington, Ontario) and subsequently transferred onto nitrocellulose membranes. A rabbit polyclonal anti-K30 polysaccharide antiserum (14) and an alkaline phosphatase-conjugated goat anti-rabbit secondary antibody were used to detect K30 polysaccharide.

Visualization of CPS by electron microscopy. Cells were grown to mid-exponential phase in 5 ml of LB broth containing the appropriate antibiotics. Where appropriate, L-arabinose was added to induce expression of plasmid-carried proteins. Cultures were standardized such that the optical densities at 600 nm were 0.5, and cells from 0.5 ml of culture were collected by centrifugation. All centrifugation steps were carried out at 5,600 x g for 5 min to minimize adverse effects on the integrity of the capsule. Cells were washed twice with 0.5 ml of 100 mM HEPES, pH 7.5. The cell pellets were then incubated with 20 µl of cationized ferritin solution (Sigma, St. Louis, MO) for 30 min at room temperature with gentle agitation. Cationized ferritin binds to the negatively charged capsule, stabilizing it against dehydration for analysis by electron microscopy. Cells were collected by centrifugation and washed twice with 0.5 ml of 100 mM HEPES, pH 7.5, to remove unbound ferritin. The cells were then processed for electron microscopy (Natural Sciences and Engineering Research Council Guelph Regional Scanning Transmission Electron Microscopy Facility, Guelph, Ontario, Canada).

Visualization of K30 CPS by immunofluorescence microscopy. Cells were grown overnight in 5 ml of LB broth in the presence of antibiotics and 0.02% L-arabinose (final concentration) and collected by centrifugation. All centrifugation steps were carried out at 5,600 x g. Cell pellets were washed with phosphate-buffered saline (PBS) and incubated on circular glass coverslips coated with poly-L-lysine for 5 min at room temperature. Cells were fixed to the coverslips by overnight incubation in 3.7% (vol/vol) formaldehyde. Following two PBS wash steps, free aldehydes were quenched with 50 mM NH4Cl. Where indicated, cells were then permeabilized by incubation with EDTA, lysozyme, and Triton X-100, as described previously (12). Coverslips were blocked in 1% (wt/vol) bovine serum albumin in PBS for 30 min prior to overnight incubation with polyclonal anti-K30 polysaccharide antiserum (14), used at a 1/500 dilution. Coverslips were washed by three successive transfers into PBS before incubation with rhodamine red-conjugated goat anti-rabbit antibodies (1/50; Jackson Immunoresearch) for 60 min at 37°C in the dark. Coverslips were mounted with Vectashield fluorescence mounting medium (Vector Laboratories) and examined using a Zeiss Axiovert 200 microscope at a total magnification of x1,000. OpenLab software (Improvision) was used for image processing.


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RESULTS
 
Wza, Wzb, and Wzc from the colanic acid system can complement a wza-wzb-wzc defect in E. coli K30. Initial experiments examined the ability of the wza, wzb, and wzc genes from the colanic acid locus to complement the corresponding defect in E. coli CWG655 [wza22 min::aadA {Delta}(wza-wzb-wzc)K30::aphA3]. For all experiments reported in this study, the restoration of K30 polysaccharide synthesis was assessed by Western immunoblotting of cellular polysaccharides probed with anti-K30 polysaccharide antiserum. As polysaccharide detected in this manner can be either intracellular or surface expressed, cell surface polysaccharides were either stabilized with cationized ferritin and visualized using transmission electron microscopy or labeled with anti-K30 polysaccharide antiserum for analysis by immunofluorescence microscopy. As expected based on the phenotypes of wzc and wza mutants (see below), CWG655 was acapsular and produced no immunoreactive K30 polysaccharide (Fig. 2). The defect in CWG655 was complemented with the respective K30 genes (pWQ91) in trans, restoring K30 polymer synthesis and surface expression of CPS in a phenotype indistinguishable from that of the parent (Fig. 2). This defect was also complemented by the (wza-wzb-wzc)CA cluster (pWQ92), with the restoration of synthesis and surface expression of HMW K30 CPS (Fig. 2). There was a noticeable difference in the migration patterns of the HMW polysaccharides in the gel when the two complemented strains were compared, suggesting that the average size distribution of the product resulting from expression of the colanic acid proteins was significantly larger (Fig. 2). This could also reflect a difference in the amounts of material produced. Given the complex nature of the complementation, it is impossible to establish the precise reason for the difference in PAGE migration patterns. However, despite this difference in phenotype, the Wza-Wzb-Wzc proteins for colanic acid expression can effectively interface with the remaining K30 polymer synthesis and export machinery in CWG655 to produce a capsule.



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  		FIG. 2. WzaCA, WzbCA, and WzcCA can restore K30 CPS synthesis and surface expression in an E. coli K30 {Delta}(wza-wzb-wzc)K30 strain. Synthesis of K30 polysaccharide was detected by Western immunoblotting of polysaccharide preparations probed with a polyclonal anti-K30 polysaccharide antiserum (anti-K30 PS), and surface-expressed K30 CPS was evident as electron-dense material on the surface of cationized ferritin-stained whole cells examined by electron microscopy. Size bars = 0.5 µm.

Wza shows no specificity towards the translocated polymer. Wza proteins involved in the assembly of group 1 CPSs are highly conserved among different serotypes (50). This conservation makes it unlikely that Wza proteins recognize the polymer being transported but does not preclude the possibility that differences between a CPS and an EPS polymer could affect Wza function or that Wza function is influenced by the presence or absence of Wzi. To begin to address these questions, WzaK30 and WzaCA were expressed in an E. coli K30 wza mutant (CWG281), and complementation of the defect was assessed. Complementation with either WzaK30 or WzaCA restored K30 CPS synthesis and surface expression (Fig. 3). The higher apparent Mrs of the polysaccharide products seen upon complementation of the CWG655 [{Delta}(wza-wzb-wzc)K30] mutant with the colanic acid genes (Fig. 2) were not evident in complementation of the wza single mutant (Fig. 3).



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  		FIG. 3. WzaCA can functionally replace WzaK30. Plasmids carrying either wzaK30 or wzaCA were used to transform CWG281 (wza22 min::aadA wzaK30::aacC1). K30 polysaccharide synthesis was assessed by Western immunoblotting of polysaccharide preparations probed with anti-K30 polysaccharide antiserum (anti-K30 PS), and surface expression of K30 CPS was determined by examination of cationized ferritin-stained whole cells by electron microscopy. Size bars = 0.5 µm.

A Wzb phosphatase for EPS synthesis functions in a CPS system. To determine whether the Wzb phosphatase involved in colanic acid assembly could functionally replace that of E. coli K30, WzbK30 and WzbCA were individually expressed in CWG343 (wza22 min::aadA wzbK30::aphA3). In CWG343, residual HMW K30 polysaccharide of higher apparent Mr than the wild type was detected in Western immunoblots probed with anti-K30 polysaccharide antiserum, and a small amount of electron-dense material was seen on the surfaces of cells in micrographs (Fig. 4). Expression of either WzbK30 or WzbCA in CWG343 restored synthesis of K30 CPS (with comparable apparent Mrs in PAGE analysis) and surface expression to a phenotype indistinguishable from that of the parent strain (Fig. 4).



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  		FIG. 4. WzbCA is a functional homologue of WzbK30. Complementation of CWG343 (wza22 min::aadA wzbK30::aphA3) was achieved using plasmids carrying either wzbK30 or wzbCA. A Western immunoblot of polysaccharide preparations was used to assess K30 polysaccharide synthesis, while K30 CPS surface expression was visualized by electron microscopy. Size bars = 0.5 µm. Anti-K30 PS, anti-K30 polysaccharide antiserum.

WzcCA must be coexpressed with WzaCA to function in K30 CPS assembly. In E. coli K30, complementation of CWG285 (wza22 min::aadA wzcK30::aacC1) could be achieved by using plasmids expressing either WzcK30 alone (62), WzaK30-WzbK30-WzcK30 (Fig. 5), or WzbK30-WzcK30 (Fig. 6). In all cases, the polymer was detectable by probing Western immunoblots with anti-K30 polysaccharide antiserum and formed an electron-dense layer on the cell surface reminiscent of the wild-type capsule. In contrast, expression of WzcCA alone in this background did not restore K30 CPS synthesis, despite detectable, albeit low, levels of WzcCA expression and phosphorylation (Fig. 5). The low level of WzcCA produced may reflect proteolysis, as a smaller tyrosine-phosphorylated polypeptide was evident in this sample. Analysis of culture supernatants revealed that no polysaccharide was secreted from the cell and released into the growth medium (data not shown), consistent with the inability of WzcCA to function in K30 CPS synthesis rather than with a loss of the polymer's surface association. Simultaneous expression of WzaCA, WzbCA, and WzcCA restored K30 CPS synthesis and surface expression in CWG285(pWQ92) (Fig. 5). As seen when E. coli CWG655 [{Delta}(wza-wzb-wzc)K30] was complemented with the colanic acid derivatives (Fig. 2), the polymer had an altered migration pattern in SDS-PAGE, suggesting an increased average size or a reduction in the amount of K30 polysaccharide produced (Fig. 5). The amount of WzcCA detected was also greatly increased upon coexpression with WzaCA and WzbCA, although the level of phosphorylation was low (Fig. 5). This increase in WzcCA could reflect altered transcript stability in the constructs or protection from the proteolytic degradation of WzcCA observed when it was expressed alone. While the former cannot be ruled out, it is certainly not the only explanation since the phosphorylated Wzc degradation product was no longer detected in CWG285(pWQ92) (Fig. 5).



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  		FIG. 5. Coexpression of WzaCA, WzbCA, and WzcCA is required to restore K30 CPS synthesis and surface expression in a wzc mutant strain. WzcK30 or WzcCA was expressed alone and in combination with its cognate Wza and Wzb proteins in CWG285 (wza22 min::aadA wzcK30::aacC1). Expression of Wzc and Wzc~P in membrane fractions was detected by using Western immunoblots probed with anti-Wzc and antiphosphotyrosine (anti-Tyr~P) antibodies, respectively. Synthesis of K30 polysaccharide was assessed by Western immunoblotting of polysaccharide preparations probed with anti-K30 polysaccharide antiserum (anti-K30 PS), and surface-expressed K30 CPS was visualized by electron microscopy. Size bars = 0.5 µm.



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  		FIG. 6. Coexpression of WzaCA and WzcCA is required to restore K30 CPS synthesis and surface expression in a wzcK30::aacC1 strain. WzcK30 or WzcCA was expressed with its cognate Wza or Wzb protein in CWG285 (wza22 min::aadA wzcK30::aacC1). Expression of Wza in cell lysates and Wzc and Wzc~P in membrane fractions was assessed by using Western immunoblots probed with the relevant antibodies. The two anti-Wza immunoreactive bands apparent upon overexpression of Wza result from inefficient processing of the precursor lipoprotein (17). In addition, degradation products of WzaK30 are often detected under high-level-expression conditions (unpublished results). K30 polysaccharide synthesis was assessed by Western immunoblotting of polysaccharides probed with anti-K30 polysaccharide antiserum (anti-K30 PS), and K30 CPS surface expression was detected by electron microscopy. Size bars = 0.5 µm. Anti-Tyr~P, antiphosphotyrosine.

The failure of WzcCA to restore CPS expression in CWG285 unless expressed with its cognate Wza and Wzb proteins raised the question of whether both the cognate outer membrane protein and the phosphatase were required for WzcCA activity or whether the requirement was specifically for one of these components. To address this, wzbCA and wzcCA were cloned into pBAD24 (pWQ90) and expressed in CWG285. The resulting strain produced significant amounts of K30 antigen, but this material was not present on the cell surface when the cells were examined by electron microscopy (Fig. 6). The accumulation of intracellular K30 polysaccharide in CWG285(pWQ90) represents only the second situation where polymer synthesis occurs in the absence of its surface assembly. While Wza and Wzc are thought to act late in the capsule assembly pathway, a characteristic feature of wza and wzc mutants is the absence of extensive polymer synthesis. In the case of a wza mutation, this is proposed to result from a feedback mechanism whereby cells unable to export polymers turn down polymer synthesis. A nonacylated Wza derivative that forms unstable outer membrane multimers lacks this regulatory control and produces a periplasmic polymer (43).

To examine the influence of WzaCA coexpression on WzcCA function, CWG285 was transformed with pWQ98 (WzaCA) and pWQ85 (WzcCA); this restored both K30 CPS synthesis and surface expression (Fig. 6). While the amounts of full-length Wzc and Wzc~P (Fig. 6) and the susceptibility to degradation (data not shown) were not altered drastically by coexpression with WzaCA, synthesis (albeit in small amounts with higher apparent Mrs) and surface expression of K30 CPS were restored. To test whether WzcCA specifically required its cognate Wza protein, WzaK30 was coexpressed with WzcCA. K30 CPS synthesis was not restored in this strain (Fig. 6). In summary, while coexpression of WzcCA with either WzbCA or WzaCA was sufficient to restore K30 polysaccharide synthesis, only coexpression with WzaCA allowed expression of detectable levels of K30 CPS on the cell surface (Fig. 6).

Activity of chimeric Wzc proteins in K30 antigen synthesis and surface expression. Interactions between WzaK30 and WzcK30 have been previously shown by using chemical cross-linking (43). Thus, the requirement for coexpression of WzaCA and WzcCA to complement a wzc mutation could reflect specificity in interacting domains of these colanic acid biosynthesis proteins.

In an attempt to determine which domains of WzcCA were responsible for the specific requirement for WzaCA, chimeric Wzc proteins were constructed that expressed portions of both the K30 and CA proteins. The goal was to generate chimeric Wzc proteins that could interface productively with WzaK30. A region of six identical residues located immediately C-terminal to the predicted end of the second transmembrane domain of Wzc was chosen as the breakpoint in order to isolate the functions of the periplasmic domain from those of the C-terminal kinase domain. A derivative [WzcK30(1-455)CA (pWQ87)] was constructed that expressed the N-terminal 455 amino acids of WzcK30 (this includes both transmembrane domains) and the C-terminal 268 amino acids of WzcCA (including Walker motifs A and B and the tyrosine-rich C terminus) (Fig. 7). The second derivative [WzcCA(1-453)K30 (pWQ88)] was the reciprocal chimera, containing the N-terminal portion of the colanic acid protein and the C-terminal portion of the K30 protein (Fig. 7).



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  		FIG. 7. Composition of chimeric Wzc proteins. Full-length WzcK30 (white) and WzcCA (gray) are illustrated, with the positions of the transmembrane domains (TM), coiled-coil motifs (COILS), and Walker A and tyrosine (Y)-rich motifs indicated. The fusion points of the chimeric Wzc proteins are indicated, with gray shading to indicate portions of the colanic acid derivative and white to indicate regions of WzcK30.

The amounts of the Wzc chimeras produced in CWG285 (wza22 min::aadA wzcK30::aacC1) differed dramatically. While the WzcK30(1-455)CA (pWQ87) derivative was evident in significant amounts in both Coomassie-stained gels and Western immunoblots, the corresponding amount of WzcCA(1-453)K30 (pWQ88) was much lower (Fig. 8). This reflected the extensive degradation of WzcCA(1-453)K30, consistent with the proteolytic susceptibility of WzcCA described above. The phosphorylation levels of these chimeric proteins appeared to correlate with amounts of full-length Wzc (Fig. 8). While both chimeric Wzc proteins were able to restore polymer synthesis in CWG285, K30 CPS was not detected on the cell surface (Fig. 8), suggesting that K30 polysaccharide was accumulating within the cells. To confirm this, cells of CWG285 expressing the chimeric proteins were permeabilized prior to antibody labeling for immunofluorescence microscopy. Unlike the intact cells, the permeabilized cells showed a strong fluorescent signal (Fig. 8). Cells expressing WzcCA(1-453)K30 (pWQ88) were also elongated, suggesting that expression of this chimeric protein had additional detrimental effects on cellular physiology. Given that intracellular polysaccharide was detected in strains expressing either chimera, the altered cellular morphology observed upon expression of WzcCA(1-453)K30 (pWQ88) did not appear to be a simple consequence of intracellular polymer accumulation. Derivatives with the elongated cell shapes still produced smooth serotype O9a lipopolysaccharide (data not shown).



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  		FIG. 8. Chimeric Wzc proteins cannot restore K30 CPS surface expression in E. coli CWG285. Chimeric Wzc proteins from pWQ87 and pWQ88 were expressed in CWG285 (wza22 min::aadA wzcK30::aacC1). The expression levels of Wzc and Wzc~P in membrane fractions were assessed by using anti-Wzc and antiphosphotyrosine (anti-Tyr~P) antibody-probed Western immunoblots, respectively. The synthesis of K30 polysaccharide was assessed by Western immunoblotting of polysaccharide samples probed with anti-K30 polysaccharide antiserum (anti-K30 PS), and the presence of polymers on the surface was assessed by immunofluorescence microscopy (where indicated, cells were permeabilized prior to antibody labeling).

Chimeric Wzc proteins also require coexpression with Wza for proper function. The failure of the WzcK30(1-455)CA and WzcCA(1-453)K30 chimeras to complement a wzc defect may indicate a requirement for features of both the N- and C-terminal domains for function in a given system or be indicative of improper protein folding. However, given that coexpression of WzcCA with WzaCA was necessary to restore CPS expression in CWG285, it was of interest to determine if coexpression of the chimeras with elevated levels of their cognate Wza protein could restore surface expression of K30 CPS. Coexpression of WzcK30(1-455)CA (pWQ87) with either of the Wza proteins restored surface expression of K30 CPS (Fig. 9 and 10). It is unclear why this chimera was active only when Wza was overexpressed since the wild-type WzcK30 functions effectively in the presence of chromosomal levels of Wza expression (Fig. 5). One possible explanation is that this Wzc chimera has lower affinity for WzaK30, which could be overcome by higher-level expression of the outer membrane component. This was in contrast to the results obtained with WzcCA(1-453)K30 (pWQ88). Strong fluorescent signals for surface-expressed CPS were detected only when the chimera was coexpressed with WzaCA (Fig. 9 and 10). These complemented cells had a morphology that more closely resembled that of the parent strain. In contrast, the cells expressing WzcCA(1-453)K30 (pWQ88) and WzaK30 still appeared elongated and indistinguishable from cells expressing WzcCA(1-453)K30 alone (Fig. 10). Small amounts of very faint and poorly distributed fluorescence were detected on the surfaces of some of the elongated cells. Given that the chimera alone did not yield any surface fluorescence, this is not likely a result of "leaky" cells but rather of an extremely low efficiency of export.



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  		FIG. 9. Coexpression of chimeric Wzc proteins with WzaCA restores surface expression of K30 CPS. WzaCA (from pWQ98) was coexpressed in CWG285 with chimeric Wzc proteins expressed from pWQ87 and pWQ88. Expression of Wza in cell lysates and Wzc and Wzc~P in membrane fractions was assessed by using Western immunoblots probed with the relevant antibodies. Synthesis of K30 polysaccharide was determined by Western immunoblotting of polysaccharide preparations probed with anti-K30 polysaccharide antiserum (anti-K30 PS), and surface-expressed K30 CPS was detected by immunofluorescence microscopy. Anti-Tyr~P, antiphosphotyrosine.



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  		FIG. 10. Coexpression of WzcK30(1-455)CA with high levels of WzaK30 restores surface expression of K30 CPS. The amount of WzaK30 in CWG285 was increased through the introduction of plasmid-expressed WzaK30 from pWQ99. Chimeric Wzc proteins were introduced into this strain, and Western immunoblots were used to determine the levels of Wza in cell lysates and Wzc and Wzc~P in membrane fractions. A Western immunoblot of polysaccharides was used to assess K30 polysaccharide synthesis, and immunofluorescence microscopy was used to detect K30 CPS on the cell surface. Anti-Tyr~P, antiphosphotyrosine; anti-K30 PS, anti-K30 polysaccharide antiserum.


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DISCUSSION
 
The presence of homologues of Wza, Wzb, and Wzc in assembly systems for group 1 CPSs and group 1-like EPSs, as well as homologues of Wzc in CPS systems in gram-positive bacteria, raises the question of whether these proteins are functionally conserved. WzaCA, WzbCA, and WzcCA could collectively function to restore K30 CPS synthesis and surface expression in a {Delta}(wza-wzb-wzc)K30 strain, suggesting that the overall function of these proteins is conserved between the K30 CPS and the colanic acid EPS systems.

The ability of WzaCA and WzbCA to restore K30 CPS synthesis and surface expression in E. coli K30 mutant strains is consistent with a shared function of these proteins in CPS and EPS systems. Thus, any essential protein-protein interactions involving components of the K30 assembly system were not compromised by the involvement of the colanic acid homologues. Restoration of K30 CPS surface expression in the wza mutant supports the idea that the outer membrane channel does not recognize the polymer being transported and is consistent with the high conservation of wza sequence in isolates with different group 1 serotypes (50). To the extent that the phenotypes can be examined with the available methods, the Wza homologues have indistinguishable functions in assembling capsules comprising CPSs with similar size distributions. The finding that WzbCA could function in the K30 system was also not surprising, given that Wzb proteins from several organisms have been shown to dephosphorylate a number of exogenous substrates (7, 21, 42, 58).

Vincent and coworkers have proposed that WzcCA~P is a negative regulator of colanic acid expression in E. coli K-12 (59). This conclusion is based on decreased colanic acid production in a wzb mutant strain. Given that colanic acid-producing strains previously used to study WzcCA function (59) overexpressed RcsA (a positive regulator of the colanic acid cps locus), these results could reflect the involvement of other unidentified cellular components. In fact, the Rcs system is now known to have many effects on cellular physiology (18, 24). However, in E. coli K30, both phosphorylation of WzcK30 and expression of WzbK30 are essential for wild-type capsule formation, suggesting that the relationships are more complex and that WzcK30 may need to cycle between phosphorylated and dephosphorylated forms (62). The results presented here suggest that WzcK30 and WzcCA generally function in the same manner within the context of the K30 CPS biosynthesis system, given that at least some phosphorylation of WzcCA was detected in strains competent for K30 CPS synthesis and surface expression. The complex nature of this system and the use of complementation experiments that disturb the natural stoichiometry of the proteins preclude conclusions regarding the "active" form of Wzc or interpretations of differences in the amounts and apparent Mrs of the products formed.

WzcCA showed a specific requirement for its cognate WzaCA protein. In the absence of WzaCA, WzcCA was unable to support surface expression of K30 CPS. These results are consistent with specific interactions between WzcCA and WzaCA; such interactions have been established by chemical cross-linking in the K30 system (43). Coupled with the outer membrane location of Wza, the data from chimeric Wzc proteins implicate the periplasmic domain of WzcCA in specific interactions with WzaCA. Neither WzcCA nor the chimeric Wzc protein harboring the periplasmic domain of WzcCA [WzcCA(1-453)K30]was able to restore surface expression of K30 CPS in CWG285 in the absence of overexpressed WzaCA. Coexpression of WzcCA(1-453)K30 with WzaCA significantly reversed the aberrant cellular morphology (elongation) evident upon expression of this chimera in CWG285. Although it is possible that this reversal in cell morphology reflects in part the relief of intracellular polymer accumulation, it is important that cells showed normal morphology when the reciprocal chimera was expressed alone, despite also producing intracellular K30 polysaccharide. The complexity of cell wall growth and cell division makes it difficult to unequivocally explain the elongation defect. However, the inability to provide a definitive explanation for the elongated shape does not detract from the central conclusion that WzcCA must be coexpressed with its cognate Wza protein in order to function within the K30 CPS biosynthesis and assembly system.

In the absence of additional Wza, the chimeric Wzc proteins both restored K30 polysaccharide biosynthesis but could not restore the surface expression of K30 polysaccharide. Significantly, the expression of WzcCA in CWG285 did not result in detectable polymer synthesis. The collective data therefore suggest that the chimeras are able to uncouple the apparent feedback that turns off polymer synthesis when the final product cannot be translocated to the cell surface. It is known from previous studies that in the absence of Wza, the CPS biosynthesis machinery exhibits reduced output similar to that of the wzc mutant and that some Wza derivatives can overcome this feedback process without facilitating export and capsule assembly on the cell surface (43). The partnership between Wza and Wzc therefore appears to be important in maintaining the feedback control, although the precise mechanism is still unknown. These observations might indicate two roles for Wzc, one involved in interaction with the K30 biosynthesis machinery and the other coupled to the export pathway. Whether phosphorylation of Wzc would be important in both stages remains to be determined.

The large periplasmic loop of Wzc is the logical site of interaction of Wzc with Wza. In Wzc proteins from E. coli K30 and K-12, this region is 374 amino acids long. Interestingly, homologues of Wzc in gram-positive bacteria have much shorter periplasmic loops (e.g., S. pneumoniae CpsC has a predicted 135-amino-acid loop). Given that homologues in gram-positive bacteria do not have to interact with an outer membrane protein, the longer periplasmic loop in gram-negative bacteria may have functional significance. However, it may not be possible to define interacting domains using primary sequence information alone, since Wzc proteins can oligomerize (15, 46) and such higher-order structures may create new biologically relevant domains. Conclusive mapping of the interacting domains of these proteins may require structural elucidation.

Overall, the data presented here reveal a conserved mechanism in the terminal steps of group 1 CPS and EPS expression and reflect the complexity of the system responsible for biosynthesis and assembly of K30 CPS. Furthermore, these data lend support to the existence of essential interactions in a multienzyme complex coordinating polymer biosynthesis and surface expression.


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ACKNOWLEDGMENTS
 
Funding for this research was provided by the Canadian Institutes of Health Research (CIHR). A. N. Reid acknowledges receipt of a CIHR doctoral research award and C. Whitfield is a tier 1 Canada Research Chair in Molecular Biology.

We thank Dianne Moyles for processing samples for electron microscopy and A. Rahn and E. Boyle for construction of CWG655.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular and Cellular Biology, New Science Complex, University of Guelph, Guelph, Ontario, Canada N1G 2W1. Phone: (519) 824-4120, ext. 53361. Fax: (519) 837-1802. E-mail: cwhitfie{at}uoguelph.ca. Back


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Journal of Bacteriology, August 2005, p. 5470-5481, Vol. 187, No. 15
0021-9193/05/$08.00+0     doi:10.1128/JB.187.15.5470-5481.2005
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