The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat

During O antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenylpyrophosphate (Und-P-P)-bound O antigen subunits occurs before their polymerization and ligation to the rest of the LPS molecule. Despite the general nature of the translocation process, putative O-antigen translocases display a low level of amino acid sequence similarity. In this work, we investigated whether complete O antigen subunits are required for translocation. We demonstrate that a single sugar, GlcNAc, can be incorporated to LPS of Escherichia coli K-12. This incorporation required the functions of two O antigen synthesis genes, wecA (UDP-GlcNAc:Und-P GlcNAc-1-P transferase) and wzx (O-antigen translocase). Complementation experiments with putative O-antigen translocases from E. coli O7 and Salmonella enterica indicated that translocation of O antigen subunits is independent of the chemical structure of the saccharide moiety. Furthermore, complementation with putative translocases involved in synthesis of exopolysaccharides demonstrated that these proteins could not participate in O antigen assembly. Our data indicate that recognition of a complete Und-P-P-bound O antigen subunit is not required for translocation and suggest a model for O antigen synthesis involving recognition of Und-P-P-linked sugars by a putative complex made of Wzx translocase and other proteins involved in the processing of O antigen.     

Functional characterization of Gne (UDP-N-acetylglucosamine-4-epimerase), Wzz (chain length determinant), and Wzy (O-antigen polymerase) of Yersinia enterocolitica serotype O:8

The lipopolysaccharide (LPS) O-antigen of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repeat units that contain N-acetylgalactosamine (GalNAc), L-fucose (Fuc), D-galactose (Gal), D-mannose (Man), and 6-deoxy-D-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called galE encodes a UDP-N-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed gne. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.     

Diversity of O-Antigen Repeat Unit Structures Can Account for the Substantial Sequence Variation of Wzx Translocases

The most common system for synthesis of cell surface polysaccharides is the Wzx/Wzy-dependent pathway, which involves synthesis, on the cytoplasmic face of the cell membrane, of repeat units, which are then translocated to the periplasmic face by a Wzx translocase and then polymerized by Wzy to generate the polysaccharide. One such polysaccharide is O antigen, which is incorporated into lipopolysaccharide (LPS). The O antigen is extremely variable, with over 186 forms in Escherichia coli. Wzx proteins are also very diverse, but they have been thought to be specific only for the first sugar of the repeat units. However, recent studies demonstrated examples in which Wzx translocases have considerable preference for their native repeat unit, showing that specificity can extend well beyond the first sugar. These results appear to be in conflict with the early conclusions, but they involved specificity for side branch residues and could be a special case. Here we take six Wzx translocases that were critical in the earlier studies on the importance of the first sugar and assess their ability to translocate the Escherichia coli O16 and O111 repeat units. We use gene replacements to optimize maintenance of expression level and show that under these conditions the native translocases are the most effective for their native repeat unit, being, respectively, 64-fold and 4-fold more effective than the next best. We conclude that Wzx translocases are commonly adapted to their native repeat unit, which provides an explanation for the great diversity of wzx genes.     

Gene products required for surface expression of the capsular form of the group 1 K antigen in Escherichia coli (O9a:K30)

The group 1 K30 antigen from Escherichia coli (O9a:K30) is present on the cell surface as both a capsular structure composed of high-molecular-weight K30 polysaccharide and as short K30 oligosaccharides linked to lipid A-core in a lipopolysaccharide molecule (K30LPS). To determine the molecular processes that are responsible for the two forms of K antigen, the 16 kb chromosomal cps region has been characterized. This region encodes 12 gene products required for the synthesis, polymerization and translocation of the K30 antigen. The gene products include four glycosyltransferases responsible for synthesis of the K30 repeat unit; a PST (1) exporter (Wzx), required to transfer lipid-linked K30 units across the plasma membrane to the periplasmic space; and a K30-antigen polymerase (Wzy). These gene products are typical of those seen in O-antigen biosynthesis gene clusters and they interact with the lipopolysaccharide translocation pathway to express K30LPS on the cell surface. The same gene products also provide the biosynthetic intermediates for the capsule assembly pathway, although they are not in themselves sufficient for synthesis of the K30 capsule. Three additional genes, wza, wzb and wzc, encode homologues to proteins that are encoded by gene clusters involved in expression of a variety of bacterial exopolysaccharides. Mutant analysis indicates that Wza and Wzc are required for wild-type surface expression of the capsular structure but are not essential for polymerization and play no role in the translocation of K30LPS. These surface expression components provide the key feature that distinguishes the assembly systems for O antigens and capsules.     

Membrane topology of the Salmonella enterica serovar Typhimurium Group B O-antigen translocase Wzx

The O-antigen translocase, Wzx, is involved in translocation of bacterial polysaccharide repeat units across the cytoplasmic membrane, and is an unusually diverse, highly hydrophobic protein, with high numbers of predicted alpha-helical transmembrane segments (TMS). The Salmonella enterica serovar Typhimurium Group B O-antigen Wzx was an ideal candidate for topological study as the O-antigen gene cluster is one of only a few that have been well characterized. The topology profile prediction for this protein was determined using five programs, with different recognition parameters, which consistently predict that 12 TMS are present. A membrane topology model was constructed by analysis of lacZ and phoA gene fusions at randomly selected and targeted fusion sites within wzx. Enzyme activity of these, and full-length C-terminal fusion proteins, confirmed the 12-TMS topology for this Wzx, and also indicated that the C-terminus was located within the cytoplasm, which is consistent with the predicted topology.     

The Wzx translocases for Salmonella enterica O-antigen processing have unexpected serotype specificity

Most Gram-negative bacteria have an O antigen, a polysaccharide with many repeats of a short oligosaccharide that is a part of the lipopolysaccharide, the major lipid in the outer leaflet of the outer membrane. Lipopolysaccharide is variable with 46 forms in Salmonella enterica that underpin the serotyping scheme. Repeat units are assembled on a lipid carrier that is embedded in the cell membrane, and are then translocated by the Wzx translocase from the cytoplasmic face to the outer face of the cell membrane, followed by polymerization. The O antigen is then incorporated into lipopolysaccharide and exported to the outer membrane. The Wzx translocase is widely thought to be specific only for the first sugar of the repeat unit, despite extensive variation in both O antigens and Wzx translocases. However, we found for S. enterica groups B, D2 and E that Wzx translocation exhibits significant specificity for the repeat-unit structure, as variants with single sugar differences are translocated with lower efficiency and little long-chain O antigen is produced. It appears that Wzx translocases are specific for their O antigen for normal levels of translocation.     

Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides

Translocation of lipid-linked oligosaccharide (LLO) intermediates across membranes is an essential but poorly understood process in eukaryotic and bacterial glycosylation pathways. Membrane proteins defined as translocases or flippases are implicated to mediate the translocation reaction. The membrane protein Wzx has been proposed to mediate the translocation across the plasma membrane of lipopolysaccharide (LPS) O antigen subunits, which are assembled on an undecaprenyl pyrophosphate lipid carrier. Similarly, PglK (formerly WlaB) is a Campylobacter jejuni-encoded ABC-type transporter proposed to mediate the translocation of the undecaprenylpyrophosphate-linked heptasaccharide intermediate involved in the recently identified bacterial N-linked protein glycosylation pathway. A combination of genetic and carbohydrate structural analyses defined and characterized flippase activities in the C. jejuni N-linked protein glycosylation and the Escherichia coli LPS O antigen biosynthesis. PglK displayed relaxed substrate specificity with respect to the oligosaccharide structure of the LLO intermediate and complemented a wzx deficiency in E. coli O-antigen biosynthesis. Our experiments provide strong genetic evidence that LLO translocation across membranes can be catalyzed by two distinct proteins that do not share any sequence similarity.     

Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy)

Lipopolysaccharides (LPS), particularly the O-antigen component, are one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-antigen biosynthesis is determined mostly by genes located in the rfb region of the chromosome. The rfc/wzy gene encodes the O-antigen polymerase, an integral membrane protein, which polymerizes the O-antigen repeat units of the LPS. The wild-type rfc/wzy gene has no detectable ribosome-binding site (RBS) and four rare codons in the translation initiation region (TIR). Site-directed mutagenesis of the rare codons at positions 4, 9 and 23 to those corresponding to more abundant tRNAs and introduction of a RBS allowed detection of the rfc/wzy gene product via a T7 promoter/polymerase expression assay. Complementation studies using the rfc/wzy constructs allowed visualization of a novel LPS with unregulated O-antigen chain length distribution, and a modal chain length could be restored by supplying the gene for the O-antigen chain length regulator (Rol/Wzz) on a low-copy-number plasmid. This suggests that the O-antigen chain length distribution is determined by both Rfc/Wzy and Rol/Wzz proteins. The effect on translation of mutating the rare codons was determined using an Rfc::PhoA fusion protein as a reporter. Alkaline phosphatase enzyme assays showed an approximately twofold increase in expression when three of the rare codons were mutated. Analysis of the Rfc/Wzy amino acid sequence using TM-PREDICT indicated that Rfc/Wzy had 10–13 transmembrane segments. The computer prediction models were tested by genetically fusing C-terminal deletions of Rfc/Wzy to alkaline phosphatase and β-galactosidase. Rfc::PhoA fusion proteins near the amino-terminal end were detected by Coomassie blue staining and Western blotting using anti-PhoA serum. The enzyme activities of cells with the rfc/wzy fusions and the location of the fusions in rfc/wzy indicated that Rfc/Wzy has 12 transmembrane segments with two large periplasmic domains, and that the amino- and carboxy-termini are located on the cytoplasmic face of the membrane.     

The Modality of Enterobacterial Common Antigen Polysaccharide Chain Lengths Is Regulated by o349 of the wec Gene Cluster of Escherichia coli K-12

The assembly of the phosphoglyceride-linked form of enterobacterial common antigen (ECA(PG)) occurs by a mechanism that involves modulation of polysaccharide chain length. However, the genetic determinant of this modulation has not been identified. Site-directed mutagenesis of o349 of the Escherichia coli K-12 wec gene cluster revealed that this locus encodes a Wzz protein that specifically modulates the chain length of ECA(PG) polysaccharides, and we have designated this locus wzz(ECA). The Wzz(ECA)-mediated modulation of ECA(PG) polysaccharide chains is the first demonstrated example of Wzz regulation involving a polysaccharide that is not linked to the core-lipid A structure of lipopolysaccharide.     

Evidence that the wzxE gene of Escherichia coli K-12 encodes a protein involved in the transbilayer movement of a trisaccharide-lipid intermediate in the assembly of enterobacterial common antigen

The assembly of many bacterial cell surface polysaccharides requires the transbilayer movement of polyisoprenoid-linked saccharide intermediates across the cytoplasmic membrane. It is generally believed that transverse diffusion of glycolipid intermediates is mediated by integral membrane proteins called translocases or "flippases." The bacterial genes proposed to encode these translocases have been collectively designated wzx genes. The wzxE gene of Escherichia coli K-12 has been implicated in the transbilayer movement of Fuc4NAc-ManNAcA-GlcNAc-P-P-undecaprenol (lipid III), the donor of the trisaccharide repeat unit in the biosynthesis of enterobacterial common antigen (ECA). Previous studies (Feldman, M. F., Marolda, C. L., Monteiro, M. A., Perry, M. B., Parodi, A. J., and Valvano, M. (1999) J. Biol. Chem. 274, 35129-35138) provided indirect evidence that the wzx(016) gene product of E. coli K-12 encoded a translocase capable of mediating the transbilayer movement of N-acetylglucosaminylpyrophosphorylundecaprenol (GlcNAc-P-P-Und), an early intermediate in the synthesis of ECA and many lipopolysaccharide O antigens. Therefore, genetic and biochemical studies were conducted to determine if the putative Wzx(O16) translocase was capable of mediating the transport of N-acetylglucosaminylpyrophosphorylnerol (GlcNAc-P-P-Ner), a water-soluble analogue of GlcNAc-P-P-Und. [(3)H]GlcNAc-P-P-Ner was transported into sealed, everted cytoplasmic membrane vesicles of E. coli K-12 as well as a deletion mutant lacking both the wzx(016) and wzxC genes. In contrast, [(3)H]GlcNAc-P-P-Ner was not transported into membrane vesicles prepared from a wzxE-null mutant, and metabolic radiolabeling experiments revealed the accumulation of lipid III in this mutant. The WzxE transport system exhibited substrate specificity by recognizing both a pyrophosphoryl-linked saccharide and an unsaturated alpha-isoprene unit in the carrier lipid. These results support the conclusion that the wzxE gene encodes a membrane protein involved in the transbilayer movement of lipid III in E. coli.     

Structural and molecular characterization of Shigella boydii type 16 O antigen

Shigella is a well-known human pathogen causing dysentery and their typing is solely based on the O antigens. We investigated the chemical structure and gene cluster of Shigella boydii type 16 O antigen. As judged by sugar and methylation analyses along with NMR spectroscopy data, the O antigen has an O-acetylated branched pentasaccharide repeating O unit, which consists of two D-mannose residues (D-Man), one residue each of d-glucuronic acid (D-GlcA), N-acetylglucosamine (D-GlcNAc) and D-galactose (D-Gal), and the structure of the O unit was established. The O antigen gene cluster of S. boydii type 16 was identified and shown to contain putative genes for the synthesis of GDP-D-Man, genes encoding sugar transferases, O unit flippase (Wzx) and O antigen polymerase (Wzy) as expected. The function of the wzy gene was characterized by mutation test. Genes specific to S. boydii type 16 O antigen gene cluster were identified by screening 186 Escherichia coli and Shigella type strains, and can be used to develop PCR assays for detection of type 16 strains.     

Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (Wzx_EcO157) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletion-fusions to green fluorescent protein and alkaline phosphatase. We conclude that Wzx_EcO157 consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PP-linked saccharide substrate across the membrane.Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, plays critical roles in bacterial cell physiology (36) and in disease (53). The structure of LPS is complex and consists at a minimum of lipid A and core oligosaccharide (OS) (42). Many Gram-negative bacteria also have an O-specific antigen polysaccharide (or O antigen) attached to one of the terminal residues of the core OS (42). The O antigen is the most variable portion of the LPS molecule and arises from the polymerization of discrete oligosaccharide units (42, 54).The biosynthesis of LPS requires many enzymes and assembly proteins and generally involves two separate pathways. One pathway results in the synthesis of the lipid A-core OS (42), which is translocated across the inner membrane by the lipid A flippase MsbA, an ABC transporter (14, 15, 60). The other pathway involves the synthesis and assembly of the O-antigen polysaccharide, which also begins at the cytosolic side of the inner membrane resulting in the formation of a lipid-linked molecule that is further translocated across the inner membrane. The formation of a complete LPS molecule containing O antigen is catalyzed by the O-antigen ligase WaaL (41). LPS molecules are further translocated to the outer leaflet of the outer membrane by the Lpt transport system involving a number of inner membrane, periplasmic, and outer membrane proteins (44, 45, 48, 49).There are at least three known mechanisms for the assembly and translocation of lipid-linked O antigens (42, 54). One of them involves a synthase protein that is homologous to processive glycosyltransferases for the synthesis of cellulose and chitin (24, 42). The other mechanism requires ATP hydrolysis for the translocation step, which is mediated by a two-component ABC transporter. This mechanism was initially described for homopolymeric O antigens (42) but also occurs with heteropolymeric O antigens (38). The third mechanism, known as the Wzy-dependent pathway (42, 54), requires three proteins: Wzx (O-antigen translocase), Wzy (O-antigen polymerase), and Wzz (regulator of O-antigen chain-length distribution). This mechanism, used primarily for the synthesis of heteropolymeric O antigens, differs from the other two in that each O unit is separately synthesized and individually translocated across the inner membrane, while the polymerization takes place at the periplasmic side of the membrane (42, 54). The O-antigen precursors are always synthesized as oligosaccharides covalently attached by a phospho-anhydride linkage to an isoprenoid lipid known as undecaprenyl phosphate (Und-P). The formation of the phospho-anhydride linkage is the first committed step toward the synthesis of O antigens and is catalyzed by two classes of membrane enzymes whose prototypes are WecA and WbaP (3, 26, 39, 46, 54). Remarkably, the involvement of an isoprenoid phosphate lipid for these reactions is a common theme in nature and also appears in the synthesis of glycan precursors for cell wall peptidoglycan and in protein glycosylation in bacteria and eukaryotic cells (9, 10). Furthermore, the Wzy-dependent pathway is functionally analogous to the initial steps of dolichol-PP-linked glycans at the endoplasmic reticulum, which are involved in protein N glycosylation (21, 54). Indeed, a membrane protein with roughly similar features as Wzx has been identified in eukaryotic cells as the dolichol-PP-linked glycan flippase and named Rft1 (22).Our laboratory focuses on the characterization of the Wzy-dependent pathway, and we have previously shown that a single Und-PP-sugar is the minimal substrate for translocation (19, 33). Consistent with this notion, Wzx proteins appear to recognize the Und-PP-bound sugar of the O-antigen unit, irrespective of the composition and structure of the remainder O unit (19, 33). Based on these observations, Wzx proteins can be loosely separated among those that can function with Und-PP-linked N-acetylhexosamines versus those that can function with Und-PP-linked N-hexoses (33). However, comparisons among Wzx primary amino acid sequences do not provide any hints on putative functional residues conserved across the members of this family. It is generally accepted that the translocation process mediated by members of Wzx and Rft1 families does not involve ATP hydrolysis (21, 54), which agrees with the absence of features in the protein that are characteristic of ATP binding or hydrolysis domains. Another complication to investigate functionally the members of these families is the lack of solid topological models that accurately predict transmembrane helices and solvent-exposed loops. Currently, experimentally based topological models have only been established for the Salmonella enterica serovar Typhimurium group B Wzx protein (12), and the Wzx-like protein PssL from Rhizobium leguminosarum (35), which is involved in exopolysaccharide capsule production. However, these studies did not identify any regions or specific amino acids from the protein that could play a functional role in the translocation process. In the present study, we have experimentally characterized the topology of the Wzx protein from Escherichia coli O157 (Wzx_EcO157) and subjected this protein to extensive mutagenesis by alanine and serine replacements targeting native cysteines and most of the aspartic acid, arginine, and lysine residues. Complementation experiments measuring the ability of each mutant protein to restore O-antigen synthesis in an E. coli K-12 Δwzx mutant resulted in the identification of four charged residues that are required for function, two of which occur in transmembrane helices. Additional replacement mutagenesis revealed that charge but not the nature of the residue is important for Wzx function.     

A cationic lumen in the Wzx flippase mediates anionic O-antigen subunit translocation in Pseudomonas aeruginosa PAO1

Heteropolymeric B-band O-antigen (O-Ag) biosynthesis in Pseudomonas aeruginosa PAO1 follows the Wzy-dependent pathway, beginning with translocation of undecaprenyl pyrophosphate-linked anionic O-Ag subunits (O units) from the inner to the outer leaflets of the inner membrane (IM). This translocation is mediated by the integral IM flippase Wzx. Through experimentally based and unbiased topological mapping, our group previously observed that Wzx possesses many charged and aromatic amino acid residues within its 12 transmembrane segments (TMS). Herein, site-directed mutagenesis targeting 102 residues was carried out on the TMS and loops of Wzx, followed by assessment of each construct's ability to restore B-band O-Ag production, identifying eight residues important for flippase function. The importance of various charged and aromatic residues was highlighted, predominantly within the TMS of the protein, revealing functional 'hotspots' within the flippase, particularly within TMS2 and TMS8. Construction of a tertiary structure homology model for Wzx indicated that TMS2 and TMS8 line a central cationic lumen. This is the first report to describe a charged flippase lumen for mediating anionic O-unit translocation across the hydrophobic IM.     

Defective O-antigen polymerization in tolA and pal mutants of Escherichia coli in response to extracytoplasmic stress

We have previously shown that the TolA protein is required for the correct surface expression of the Escherichia coli O7 antigen lipopolysaccharide (LPS). In this work, delta tolA and delta pal mutants of E. coli K-12 W3110 were transformed with pMF19 (encoding a rhamnosyltransferase that reconstitutes the expression of O16-specific LPS), pWQ5 (encoding the Klebsiella pneumoniae O1 LPS gene cluster), or pWQ802 (encoding the genes necessary for the synthesis of Salmonella enterica O:54). Both DeltatolA and delta pal mutants exhibited reduced surface expression of O16 LPS as compared to parental W3110, but no significant differences were observed in the expression of K. pneumoniae O1 LPS and S. enterica O:54 LPS. Therefore, TolA and Pal are required for the correct surface expression of O antigens that are assembled in a wzy (polymerase)-dependent manner (like those of E. coli O7 and O16) but not for O antigens assembled by wzy-independent pathways (like K. pneumoniae O1 and S. enterica O:54). Furthermore, we show that the reduced surface expression of O16 LPS in delta tolA and delta pal mutants was associated with a partial defect in O-antigen polymerization and it was corrected by complementation with intact tolA and pal genes, respectively. Using derivatives of W3110 delta tolA and W3110 delta pal containing lacZ reporter fusions to fkpA and degP, we also demonstrate that the RpoE-mediated extracytoplasmic stress response is upregulated in these mutants. Moreover, an altered O16 polymerization was also detected under conditions that stimulate RpoE-mediated extracytoplasmic stress responses in tol+ and pal+ genetic backgrounds. A Wzy derivative with an epitope tag at the C-terminal end of the protein was stable in all the mutants, ruling out stress-mediated proteolysis of Wzy. We conclude that the absence of TolA and Pal elicits a sustained extracytoplasmic stress response that in turn reduces O-antigen polymerization but does not affect the stability of the Wzy O-antigen polymerase.     

A Wzz (Cld) protein determines the chain length of K lipopolysaccharide in Escherichia coli O8 and O9 strains.

The modal distribution of O-antigen chain length is determined by the Wzz (Cld/Rol) protein in those cases in which it has been studied. The system of O-antigen synthesis in Escherichia coli serotypes O8 and O9 is different from that reported for most other bacteria, and chain length distribution is thought not to be determined by a Wzz protein. We report the existence in E. coli O8 and O9 strains of wzz genes which are very similar to and have sequences within the range of variation of those which determine the chain length of typical O antigens. We also find that wzz genes previously identified by their effect on O-antigen chain length, when cloned and transferred to O8 and O9 strains, affect the chain length of a capsule-related form of LPS, K(LPS). We conclude that in at least some O8 and O9 strains there is a wzz gene which controls the chain length of K(LPS) but has no effect on the O8 or O9 antigen.     

Pseudomonas aeruginosa B-band O-antigen chain length is modulated by Wzz (Ro1).

The wbp gene cluster, encoding the B-band lipopolysaccharide O antigen of Pseudomonas aeruginosa serotype O5 strain PAO1, was previously shown to contain a wzy (rfc) gene encoding the O-antigen polymerase. This study describes the molecular characterization of the corresponding wzz (rol) gene, responsible for modulating O-antigen chain length. P. aeruginosa O5 Wzz has 19 to 20% amino acid identity with Wzz of Escherichia coli, Salmonella enterica, and Shigella flexneri. Knockout mutations of the wzz gene in serotypes O5 and O16 (which has an O antigen structurally related to that of O5) yielded mutants expressing O antigens with a distribution of chain lengths differing markedly from that of the parent strains. Unlike enteric wzz mutants, the P. aeruginosa wzz mutants continued to display some chain length modulation. The P. aeruginosa O5 wzz gene complemented both O5 and O16 wzz mutants as well as an E. coli wzz mutant. Coexpression of E. coli and P. aeruginosa wzz genes in a rough strain of E. coli carrying the P. aeruginosa wbp cluster resulted in the expression of two populations of O-antigen chain lengths. Sequence analysis of the region upstream of wzz led to identification of the genes rpsA and himD, encoding 30S ribosomal subunit protein S1 and integration host factor, respectively. This finding places rpsA and himD adjacent to wzz and the wbp cluster at 37 min on the PAO1 chromosomal map and completes the delineation of the O5 serogroup-specific region of the wbp cluster.     

Structural and genetic characterization of the O-antigen of Salmonella enterica O56 containing a novel derivative of 4-amino-4,6-dideoxy-d-glucose

Based on the O-antigens (O-polysaccharides), one of the most variable cell constituents, 46 O-serogroups have been recognized in the Kauffmann-White serotyping scheme for Salmonella enterica. In this work, the structure of the O-polysaccharide and the genetic organization of the O-antigen gene cluster of S. enterica O56 were investigated. As judged by sugar and methylation analyses, along with NMR spectroscopic data, the O-polysaccharide has a linear tetrasaccharide O-unit, which consists of one residue each of d-ribofuranose, N-acetyl-d-glucosamine, N-acetyl-d-galactosamine, and a novel sugar derivative, 4-(N-acetyl-l-seryl)amino-4,6-dideoxy-d-glucose (d-Qui4NSerAc). The following structure of the O-polysaccharide was established:→3)-β-d-Quip4NSerAc-(1→3)-β-d-Ribf-(1→4)-α-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→The O-antigen gene cluster of S. enterica O56 having 12 open reading frames was found between the housekeeping genes galF and gnd. A comparison with databases and using the O-antigen structure data enabled us to ascribe functions to genes for (i) synthesis of d-GalNAc and d-Qui4NSerAc, (ii) sugar transfer, and (iii) O-antigen processing, including genes for O-unit flippase (Wzx) and O-antigen polymerase (Wzy).     

Proper expression of the O-antigen of lipopolysaccharide is essential for the virulence of Yersinia enterocolitica O:8 in experimental oral infection of rabbits

The O-antigen of lipopolysaccharide (LPS) is required for virulence in Yersinia enterocolitica serotype O:8. Here we evaluated the importance of controlling the O-antigen biosynthesis using an in vivo rabbit model of infection. Y. enterocolitica O:8 wild-type strain was compared to three mutants differing in the O-antigen phenotype: (i) the rough strain completely devoid of the O-antigen, (ii) the wzy strain that lacks the O-antigen polymerase (Wzy protein) and expresses LPS with only one repeat unit, and (iii) the wzz strain that lacks the O-antigen chain length determinant (Wzz protein) and expresses LPS without modal distribution of O-antigen chain lengths. The most attenuated strain was the wzz mutant. The wzz bacteria were cleared from the tissues by day 30, the blood parameters were least dramatic and histologically only immunomorphological findings were seen. The level of attenuation of the rough and the wzy strain bacteria was between the wild-type and the wzz strain. Wild-type bacteria were highly resistant to killing by polymorphonuclear leukocytes, the wzz strain bacteria were most sensitive and the rough and wzy strain bacteria were intermediate resistant. These results clearly demonstrated that the presence of O-antigen on the bacterial surface is not alone sufficient for full virulence, but also there is a requirement for its controlled chain length.     

Overexpression and characterization of Wzz of Escherichia coli O86:H2

O-Antigen plays a critical role in the bacterium-host interplay, the chain length is an important factor in O-antigen functions. Wzz protein is responsible for O-antigen chain length regulation, but the mechanism is still unknown. Here, we overexpressed the Wzz of Escherichia coli O86:H2 in wzz mutant O86:H2 strain, the yield can achieve 15 mg/L. The recombinant Wzz was purified to 99% purity in dodecylmaltoside by sequential Ni-affinity chromatography and anion-exchange. Size exclusion chromatography and in vivo cross-linking experiments both showed that Wzz formed tetramer. Furthermore, analysis with circular dichroism revealed that the predominant structural composition in Wzz is alpha-helices, and incubation with O-antigen significantly changed Wzz conformation. The results suggested that Wzz protein can interact with O-antigen.     

Mutagenesis and Chemical Cross-Linking Suggest that Wzz Dimer Stability and Oligomerization Affect Lipopolysaccharide O-Antigen Modal Chain Length Control

In Shigella flexneri, the polysaccharide copolymerase (PCP) protein Wzz_SF confers a modal length of 10 to 17 repeat units (RUs) to the O-antigen (Oag) component of lipopolysaccharide (LPS). PCPs form oligomeric structures believed to be related to their function. To identify functionally important regions within Wzz_SF, random in-frame linker mutagenesis was used to create mutants with 5-amino-acid insertions (termed Wzz_i proteins), and DNA sequencing was used to locate the insertions. Analysis of the resulting LPS conferred by Wzz_i proteins identified five mutant classes. The class I mutants were inactive, resulting in nonregulated LPS Oag chains, while classes II and III conferred shorter LPS Oag chains of 2 to 10 and 8 to 14 RUs, respectively. Class IV mutants retained near-wild-type function, and class V mutants increased the LPS Oag chain length to 16 to 25 RUs. In vivo formaldehyde cross-linking indicated class V mutants readily formed high-molecular-mass oligomers; however, class II and III Wzz_i mutants were not effectively cross-linked. Wzz dimer stability was also investigated by heating cross-linked oligomers at 100°C in the presence of SDS. Unlike the Wzz_SF wild type and class IV and V Wzz_i mutants, the class II and III mutant dimers were not detectable. The location of each insertion was mapped onto available PCP three-dimensional (3D) structures, revealing that class V mutations were most likely located within the inner cavity of the PCP oligomer. These data suggest that the ability to produce stable dimers may be important in determining Oag modal chain length.Lipopolysaccharide (LPS) of Shigella flexneri is an important virulence factor, providing protection against host defenses and affecting interaction with host cells. LPS is composed of three regions: the hydrophobic lipid A membrane anchor, the core sugar region, and the O-antigen (Oag) polysaccharide chain (18). The basic Oag repeat unit of S. flexneri is a tetrasaccharide consisting of three rhamnose sugars and one N-acetylglucosamine sugar (19). The contribution of Shigella Oag to establishing virulence has been extensively investigated, and results indicate that regulated Oag modal length is required for virulence (8, 23, 25). Loss of Oag modal chain length regulation affects virulence due to the masking of the outer membrane (OM) protein IcsA (8, 23), and the type III secretion system is also affected by Oag chain length (24).The current model for Oag biogenesis in S. flexneri involves the initiation of Oag repeat unit synthesis on the cytoplasmic face of the inner membrane (IM) and continues with a series of successive sugar transferase reactions. The repeat units are assembled on the lipid carrier undecaprenol phosphate (Und-P), and transported across the IM by the Wzx flippase to the periplasmic face of the IM. Polymerization of Oag repeat units is catalyzed by the Wzy polymerase, linking the individual oligosaccharide repeat units into a chain; the nascent chain is transferred from its lipid carrier to the nonreducing end of the newly flipped oligosaccharide repeat unit. The resulting chain is then ligated to the lipid A core by WaaL ligase (18, 26) to form LPS.The regulation of the chain length of the Oag polysaccharide is controlled by the Wzz protein, a member of the polysaccharide copolymerase 1a (PCP1a) family (13, 21). S. flexneri Wzz (Wzz_SF) confers an average chain modal length of 10 to 17 Oag repeat units. In addition to determining the Oag chain modal length, PCP proteins are involved in enterobacterial common antigen (ECA) modal chain length regulation and biosynthesis and in capsule polysaccharide (CPS) and exopolysaccharide (EPS) biosynthesis (13). The PCP1a proteins are located in the IM and have two transmembrane (TM) regions, TM1 and TM2 (14). TM1 is located close to the N-terminal end, and TM2 is located near the C-terminal end, while the hydrophilic region between TM regions is located in the periplasm (14). PCPs exhibit a conserved motif, proximal to and partly overlapping the TM2 region, rich in proline and glycine residues (2, 3, 13). Site-directed mutagenesis studies targeting a number of these conserved residues, singularly or in combination, indicate that changes to this region have a significant effect on the resulting Oag modal chain length (4). Many mutagenesis studies on residues throughout Wzz indicate that function may be an overall property of the protein and may not be limited to one particular region (4, 6, 21). Despite studies conducted to probe the Wzz structure function relationship, little is known about the mode of action in determining Oag modal chain length. Recently, the periplasmic domain structures of a collection of PCP proteins including Salmonella enterica serovar Typhimurium WzzB (Wzz_ST) and Escherichia coli O157 FepE and WzzE have been solved, and it has been deduced that these structures show marked similarities at the protomer and oligomer levels (21). These protomers are elongated and consist of two structural components: a trapezoidal α/β base domain close to the membrane and an extended α-helical hairpin containing an ∼100-Å-long helix forming anti-parallel coiled-coil interactions with two helices that fold back toward the membrane (21). The protomers self-assemble into bell-shaped oligomers displaying comparable structural features, with Wzz_ST forming pentameric oligomers, WzzE assembling into octameric oligomers, and FepE assembling into nonameric structures (21). In contrast, a recent study from Larue et al. reports that Wzz_ST, FepE, and Wzz_K40 favor hexameric structures (9). A previous study on the oligomeric status of S. flexneri WzzB (Wzz_SF) via in vivo cross-linking with formaldehyde indicated that Wzz_SF has the ability to form hexamers and high-order oligomers, suggesting that oligomerization is important in function (4). Related to this, Marolda et al. have shown that the ECA-associated Wzx can fully complement an LPS Oag-associated Wzx-deficient mutant if the remaining ECA gene cluster is deleted, providing genetic evidence that proteins involved in Oag/ECA biosynthesis and processing may function as a complex (11).Several models of the likely mechanisms of Oag chain regulation have been proposed. Bastin et al. initially suggested that Wzz acts as a molecular timer, allowing polymerization to occur to a particular point, hence increasing the number of repeat units added to the chain (1). An alternative model proposed by Morona et al. suggested that Wzz acts as a molecular chaperone, facilitating the interaction between Wzy and WaaL, and modal length is the result of the ratio of Wzy and WaaL (14). Published data indicated that the ratio of Wzy and Wzz was important in determining Oag modal chain length, which is supportive of the latter model (5). With recent developments in solving the PCP three-dimensional (3D) structure and oligomeric arrangement, a new model has been proposed by Tocilj et al. in which the Wzz oligomers act as molecular scaffolds for multiple Wzy polymerase molecules and the growing Oag chain is transferred from one Wzy to another Wzy molecule (21).In a previous study, site-directed mutagenesis analysis was conducted on Wzz_SF (4). Although mutational alterations targeting the TM regions caused dramatic changes in the resulting LPS Oag chain length, mutations targeting the periplasmic domain generally did not have an obvious effect on the resulting LPS Oag chain length. This was also shown for mutations in FepE (17). Hence, we decided that a more severe approach to Wzz_SF mutagenesis was needed to investigate the relationship between Wzz structure and function by increasing the likelihood of acquiring Wzz mutants displaying phenotypic changes. In this study, we have investigated the structure and function of Wzz_SF by constructing a library of in-frame linker mutants with 5-amino-acid (aa) insertions throughout the Wzz_SF protein. We have identified regions in Wzz_SF which alter the modal length in different ways and present biochemical evidence acquired by in vivo chemical cross-linking that indicates oligomeric differences exist between Wzz mutants and the wild type (WT). We also present evidence that suggests the dimeric form of Wzz_SF is important in establishing modal length.