Polymerase Wzy Research Articles

Genetic and structural elucidation of capsular polysaccharides from Streptococcus pneumoniae serotype 23A and 23B, and comparison to serotype 23F

Streptococcus pneumoniae is a globally important encapsulated human pathogen with approximately 100 different serotypes recognized. Serogroup 23 consists of serotype 23F, present in licensed vaccines, and emerging serotypes 23A and 23B. Here, we report the previously unknown structures of the pneumococcal capsular polysaccharides serotype 23A and 23B determined using genetic analysis, NMR spectroscopy, composition and linkage analysis and Smith degradation (of polysaccharide 23A). The structure of the serotype 23A capsular polysaccharide is: →4)-β-D-Glcp-(1→3)-[[α-L-Rhap-(1→2)]-[Gro-(2→P→3)]-β-D-Galp-(1→4)]-β-L-Rhap-(1→. This structure differs from polysaccharide 23F as it features a disaccharide backbone and the di-substituted β-Gal is linked to β-Rha as a side chain. This is due to the different polymerization position catalysed by the unusually divergent repeat unit polymerase Wzy in the 23A cps biosynthesis locus. Steric crowding in 23A, confirmed by molecular models, causes the NMR signal for H-1 of the di-substituted 2,3-β-Gal to resonate in the α-anomeric region. The structure of the serotype 23B capsular polysaccharide is the same as 23F, but without the terminal α-Rha: →4)-β-D-Glcp-(1→4)-[Gro-(2→P→3)]-β-D-Galp-(1→4)-β-L-Rhap-(1→. The immunodominant terminal α-Rha of 23F is more sterically crowded in 23A and absent in 23B. This may explain the reported typing cross reactions for serotype 23F: slight with 23A and none with 23B.

Acinetobacter baumannii K11 and K83 capsular polysaccharides have the same 6-deoxy-l-talose-containing pentasaccharide K units but different linkages between the K units

Acinetobacter baumannii produces a variety of capsular polysaccharides (CPS) via genes located at the chromosomal K locus and some KL gene clusters include genes for the synthesis of specific sugars. The structures of K11 and K83 CPS produced by isolates LUH5545 and LUH5538, which carry related KL11a and KL83 gene clusters, respectively, were established by sugar analysis and one- and two-dimensional 1H and 13C NMR spectroscopy. Both CPS contain l-rhamnose (l-Rha) and 6-deoxy-l-talose (l-6dTal), and both KL gene clusters include genes for dTDP-l-Rhap synthesis and a tle (talose epimerase) gene encoding an epimerase that converts dTDP-l-Rhap to dTDP-l-6dTalp. The K11 and K83 repeat units are the same pentasaccharide, consisting of d-glucose, l-Rha, l-6dTal, and N-acetyl-d-glucosamine, except that l-6dTal is 2-O-acetylated in K83. However, the K units are linked differently, with l-Rha in the main chain in K11, but as a side-branch in K83. KL11 and KL83 encode unrelated Wzy polymerases that link the K units together and different acetyltransferases, though only Atr8 from KL83 is active. The substrate specificity of each Wzy polymerase was assigned, and the functions of all glycosyltransferases were predicted. The CPS structures produced by three closely related K loci, KL29, KL105 and KL106, were also predicted.

The KL24 gene cluster and a genomic island encoding a Wzy polymerase contribute genes needed for synthesis of the K24 capsular polysaccharide by the multiply antibiotic resistant Acinetobacter baumannii isolate RCH51.

The whole-genome sequence of the multiply antibiotic resistant Acinetobacter baumannii isolate RCH51 belonging to sequence type ST103 (Institut Pasteur scheme) revealed that the set of genes at the capsule locus, KL24, includes four genes predicted to direct the synthesis of 3-acetamido-3,6-dideoxy-d-galactose (d-Fuc3NAc), and this sugar was found in the capsular polysaccharide (CPS). One of these genes, fdtE, encodes a novel bifunctional protein with an N-terminal FdtA 3,4-ketoisomerase domain and a C-terminal acetyltransferase domain. KL24 lacks a gene encoding a Wzy polymerase to link the oligosaccharide K units to form the CPS found associated with isolate RCH51, and a wzy gene was found in a small genomic island (GI) near the cpn60 gene. This GI is in precisely the same location as another GI carrying wzy and atr genes recently found in several A. baumannii isolates, but it does not otherwise resemble it. The CPS isolated from RCH51, studied by sugar analysis and 1D and 2D 1H and 13C NMR spectroscopy, revealed that the K unit has a branched pentasaccharide structure made up of Gal, GalNAc and GlcNAc residues with d-Fuc3NAc as a side branch, and the K units are linked via a β-d-GlcpNAc-(1→3)-β-d-Galp linkage formed by the Wzy encoded by the GI. The functions of the glycosyltransferases encoded by KL24 were assigned to formation of specific bonds. A correspondence between the order of the genes in KL24 and other KL and the order of the linkages they form was noted, and this may be useful in future predictions of glycosyltransferase specificities.

Related structures of neutral capsular polysaccharides of Acinetobacter baumannii isolates that carry related capsule gene clusters KL43, KL47, and KL88

Capsular polysaccharides were recovered from four Acinetobacter baumannii isolates, and the following related structures of oligosaccharide repeating units were established by sugar analyses along with 1D and 2D 1H and 13C NMR spectroscopy:NIPH 60 and LUH5544 (K43) [Display omitted] NIPH 601 (K47)The K locus for capsule biosynthesis in the genome sequences available for NIPH 60 and LUH5544, designated KL43, was found to be related to gene clusters KL47 in NIPH 601 and KL88 in LUH5548. The three clusters share most gene content differing in only a small portion that includes an additional glycosyltransferase genes in KL47 and KL88, as well as genes encoding distinct Wzy polymerases that were found to form the same α-d-GlcpNAc-(1 → 6)-α-d-GlcpNAc linkage in K43 and K47.

Characterization of Pseudomonas aeruginosa phage K5 genome and identification of its receptor related genes.

Phage genomic information and the nature of host-phage interactions are important for phage applications. In this study, Pseudomonas aeruginosa phage K5 is characterized as a linear double-stranded genomic DNA molecule of 93,754 bp with identical 1182-bp direct terminal repeats. Comparative genomic analysis reveals that phage K5 is highly homologous to the "PaP1-like" phages. Thirteen mutants resistant to phage K5 are screened in a transposon mutant library. The disrupted genetic loci are identified as gene Y880_RS05480 encoding a putative O-antigen polymerase Wzy and gene wapH encoding a glycosyltransferase. The mutants are confirmed by the complementation experiment. The production of biofilm and the profile of lipopolysaccharide (LPS) are further analyzed in the Y880_RS05480 mutant. Our data indicate that LPS is the receptor of phage K5.

The first sugar of the repeat units is essential for the Wzy polymerase activity and elongation of the O-antigen lipopolysaccharide.

In the Wzx/Wzy-dependent assembled pathway, the assembled O-antigen repeat units are translocated from the cytosolic to the periplasmic face of the inner membrane by a Wzx translocase and then polymerized by the integral membrane protein Wzy to form a glycan chain. We demonstrate that the activity of the Escherichia coli O-antigen polymerase (Wzy) is dependent on the first sugar of the O-antigen repeat unit to produce the O-antigen polymerization and therefore, there is a need for a concerted action with the enzyme transferring the initial HexNAc to undecaprenyl phosphate (UDP-HexNAc: polyprenol-P HexNAc-1-P transferase). Furthermore, in the case of Aeromonas hydrophila Wzy-O34 polymerization activity, the enzyme is permissive with the sugar at the nonreducing end of the O-antigen repeat unit.

Chemoenzymatic synthesis of the bacterial polysaccharide repeating unit undecaprenyl pyrophosphate and its analogs.

Polysaccharides are essential and immunologically relevant components of bacterial cell walls. These biomolecules can be found covalently attached to lipids (e.g., O-polysaccharide (PS) contains undecaprenyl and lipopolysaccharide (LPS) contains lipid A) or noncovalently associated with cell wells (e.g., capsular PS (CPS)). Although extensive genetic studies have indicated that the Wzy-dependent biosynthetic pathway is primarily responsible for producing such polysaccharides, in vitro biochemical studies are needed to determine, for example, which gene product is responsible for catalyzing each step in the pathway, and to reveal molecular details about the Wzx translocase, Wzy polymerase and O-PS chain-length determinant. Many of these biochemical studies require access to a structurally well-defined PS repeating unit undecaprenyl pyrophosphate (RU-PP-Und), the key building block in this pathway. We describe herein the chemoenzymatic synthesis of Escherichia coli (serotype O157) RU-PP-Und. This involves (i) chemical synthesis of precursor N-acetyl-D-galactosamine (GalNAc)-PP-Und (2 weeks) and (ii) enzymatic extension of the precursor to produce RU-PP-Und (2 weeks). Undecaprenyl phosphate and peracetylated GalNAc-1-phosphate are prepared from commercially available undecaprenol and peracetylated GalNAc. The chemical coupling of these two products, followed by structural confirmation (mass spectrometry and NMR) and deprotection, generates GalNAc-PP-Und. This compound is then sequentially modified by enzymes in the E. coli serotype O157 (E. coli O157) O-PS biosynthetic pathway. Three glycosyltransferases (GTs) are involved (WbdN, WbdO and WbdP) and they transfer glucose (Glc), L-fucose (L-Fuc) and N-acetylperosamine (PerNAc) onto GalNAc-PP-Und to form the intact RU-PP-Und in a stepwise manner. Final compounds and intermediates are confirmed by mass spectrometry. The procedure can be adapted to the synthesis of analogs with different PS or lipid moieties.

K19 capsular polysaccharide of Acinetobacter baumannii is produced via a Wzy polymerase encoded in a small genomic island rather than the KL19 capsule gene cluster

Polymerization of the oligosaccharides (K units) of complex capsular polysaccharides (CPSs) requires a Wzy polymerase, which is usually encoded in the gene cluster that directs K unit synthesis. Here, a gene cluster at the Acinetobacter K locus (KL) that lacks a wzy gene, KL19, was found in Acinetobacter baumannii ST111 isolates 28 and RBH2 recovered from hospitals in the Russian Federation and Australia, respectively. However, these isolates produced long-chain capsule, and a wzy gene was found in a 6.1 kb genomic island (GI) located adjacent to the cpn60 gene. The GI also includes an acetyltransferase gene, atr25, which is interrupted by an insertion sequence (IS) in RBH2. The capsule structure from both strains was →3)-α-d-GalpNAc-(1→4)-α-d-GalpNAcA-(1→3)-β-d-QuipNAc4NAc-(1→, determined using NMR spectroscopy. Biosynthesis of the K unit was inferred to be initiated with QuiNAc4NAc, and hence the Wzy forms the β-(1→3) linkage between QuipNAc4NAc and GalpNAc. The GalpNAc residue is 6-O-acetylated in isolate 28 only, showing that atr25 is responsible for this acetylation. The same GI with or without an IS in atr25 was found in draft genomes of other KL19 isolates, as well as ones carrying a closely related CPS gene cluster, KL39, which differs from KL19 only in a gene for an acyltransferase in the QuiNAc4NR synthesis pathway. Isolates carrying a KL1 variant with the wzy and atr genes each interrupted by an ISAba125 also have this GI. To our knowledge, this study is the first report of genes involved in capsule biosynthesis normally found at the KL located elsewhere in A. baumannii genomes.

Genetic Evidence for O-Specific Antigen as Receptor of Pseudomonas aeruginosa Phage K8 and Its Genomic Analysis.

Phage therapy requires the comprehensive understanding of the mechanisms underlying the host-phage interactions. In this work, to identify the genes related to Pseudomonas aeruginosa phage K8 receptor synthesis, 16 phage-resistant mutants were selected from a Tn5G transposon mutant library of strain PAK. The disrupted genetic loci were identified and they were related to O-specific antigen (OSA) synthesis, including gene wbpR, ssg, wbpV, wbpO, and Y880_RS05480, which encoded a putative O-antigen polymerase Wzy. The Lipopolysaccharide profile of the Y880_RS05480 mutant was analyzed and shown to lack the O-antigen. Therefore, the data from characterization of Y880_RS05480 by TMHMM and SDS-PAGE silver staining analysis suggest that this locus might encode Wzy. The complete phage K8 genome was characterized as 93879 bp in length and contained identical 1188-bp terminal direct repeats. Comparative genomic analysis showed that phage K8 was highly homologous to members of the genus PaP1-like phages. On the basis of our genetic findings, OSA of P. aeruginosa PAK is proven to be the receptor of phage K8. The highly conserved structural proteins among the genetic closely related phages suggest that they may recognize the same receptor.

Acinetobacter baumannii K27 and K44 capsular polysaccharides have the same K unit but different structures due to the presence of distinct wzy genes in otherwise closely related K gene clusters.

Capsular polysaccharides (CPSs), from Acinetobacter baumannii isolates 1432, 4190 and NIPH 70, which have related gene content at the K locus, were examined, and the chemical structures established using 2D(1)H and(13)C NMR spectroscopy. The three isolates produce the same pentasaccharide repeat unit, which consists of 5-N-acetyl-7-N-[(S)-3-hydroxybutanoyl] (major) or 5,7-di-N-acetyl (minor) derivatives of 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic (legionaminic) acid (Leg5Ac7R), D-galactose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. However, the linkage between repeat units in NIPH 70 was different to that in 1432 and 4190, and this significantly alters the CPS structure. The KL27 gene cluster in 4190 and KL44 gene cluster in NIPH 70 are organized identically and contain lga genes for Leg5Ac7R synthesis, genes for the synthesis of the common sugars, as well as anitrA2 initiating transferase and four glycosyltransferases genes. They share high-level nucleotide sequence identity for corresponding genes, but differ in the wzy gene encoding the Wzy polymerase. The Wzy proteins, which have different lengths and share no similarity, would form the unrelated linkages in the K27 and K44 structures. The linkages formed by the four shared glycosyltransferases were predicted by comparison with gene clusters that synthesize related structures. These findings unambiguously identify the linkages formed by WzyK27 and WzyK44, and show that the presence of different wzy genes in otherwise closely related K gene clusters changes the structure of the CPS. This may affect its capacity as a protective barrier for A. baumannii.

Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types of Klebsiella spp.

A total of 79 capsular types have been reported in Klebsiella spp., whereas capsular polysaccharide synthesis (cps) regions were available in only 22 types. Due to the limitations of serotyping, complete repertoire of cps will be helpful for capsular genotyping. We therefore resolved the rest 57 cps and conducted comparative analysis. Clustering results of 1,515 predicted proteins from cps loci categorized proteins which share similarity into homology groups (HGs) revealing that 77 Wzy polymerases were classified into 56 HGs, which indicate the high specificity of wzy between different types. Accordingly, wzy-based capsular genotyping could differentiate capsule types except for those lacking wzy (K29 and K50), those sharing identical wzy (K22 vs. K37); and should be carefully applied in those exhibited high similarity (K12 vs. K41, K2 vs. K13, K74 vs. K80, K79 vs. KN1 and K30 vs. K69). Comparison of CPS structures in several capsular types that shared similarity in their gene contents implies possible functions of glycosyltransferases. Therefore, our results provide complete set of cps in various types of Klebsiella spp., which enable the understandings of relationship between genes and CPS structures and are useful for identification of documented or new capsular types.

Structural determination of the K14 capsular polysaccharide from an ST25 Acinetobacter baumannii isolate, D46

The structure of the capsular polysaccharide (CPS) recovered from D46, an extensively antibiotic resistant ST25 Acinetobacter baumannii clinical isolate, was elucidated. The structure was resolved on the basis of NMR spectroscopy and chemical analyses, and was found to contain a branched neutral pentasaccharide with a backbone composed of GalpNAc and Galp residues, all d configured, and a d-Glcp side group. The KL14 gene cluster found in the D46 genome includes genes for four glycosyltransferases but no modules for synthesis of complex sugars, and this is consistent with the structure of K14. The K14 structure and KL14 sequence clarify the relationship between the structure and K locus sequence for A. nosocomialis isolate LUH5541. The identity of the first sugar of the K14 repeat unit (K unit), and the functions of the four encoded glycosyltransferases and Wzy polymerase were predicted.

Three Wzy polymerases are specific for particular forms of an internal linkage in otherwise identical O units

The Wzx/Wzy-dependent pathway is the predominant pathway for O-antigen production in Gram-negative bacteria. The O-antigen repeat unit (O unit) is an oligosaccharide that is assembled at the cytoplasmic face of the membrane on undecaprenyl pyrophosphate. Wzx then flips it to the periplasmic face for polymerization by Wzy, which adds an O unit to the reducing end of a growing O-unit polymer in each round of polymerization. Wzx and Wzy both exhibit enormous sequence diversity. It has recently been shown that, contrary to earlier reports, the efficiency of diverse Wzx forms can be significantly reduced by minor structural variations to their native O-unit substrate. However, details of Wzy substrate specificity remain unexplored. The closely related galactose-initiated Salmonella O antigens present a rare opportunity to address these matters. The D1 and D2 O units differ only in an internal mannose-rhamnose linkage, and D3 expresses both in the same chain. D1 and D2 polymerases were shown to be specific for O units with their respective α or β configuration for the internal mannose-rhamnose linkage. The Wzy encoded by D3 gene cluster polymerizes only D1 O units, and deleting the gene does not eliminate polymeric O antigen, both observations indicating the presence of an additional wzy gene. The levels of Wzx and Wzy substrate specificity will affect the ease with which new O units can evolve, and also our ability to modify O antigens, capsules or secreted polysaccharides by glyco-engineering, to generate novel polysaccharides, as the Wzx/Wzy-dependent pathway is responsible for much of the diversity.

Structure of the K12 capsule containing 5,7-di-N-acetylacinetaminic acid from Acinetobacter baumannii isolate D36.

The repeat unit of the K12 capsular polysaccharide isolated from the Acinetobacter baumannii global clone 1 clinical isolate, D36, was elucidated by means of chemical and spectroscopical methods. The structure was shown to contain N-acetyl-d-galactosamine (d-GalpNAc), N-acetyl-d-fucosamine and N-acetyl-l-fucosamine linked together in the main chain, with the novel sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-altro-non-2-ulosonic acid (5,7-di-N-acetylacinetaminic acid or Aci5Ac7Ac), attached to d-GalpNAc as a side branch. This matched the sugar composition of the K12 capsule and the genetic content of the KL12 capsule gene cluster reported previously. d-FucpNAc was predicted to be the substrate for the initiating transferase, ItrB3, with the Wzy polymerase making a α-d-FucpNAc-(1 → 3)-d-GalpNAc linkage between the repeat units. The three glycosyltransferases encoded by KL12 are all retaining glycosyltransferases and were predicted to form specific linkages between the sugars in the K12 repeat unit.

Structure of the K6 capsular polysaccharide from Acinetobacter baumannii isolate RBH4

The structure of the capsular polysaccharide (CPS) from an Acinetobacter baumannii global clone 2 (GC2) clinical isolate RBH4 that carries the KL6 gene cluster was elucidated by means of chemical and spectroscopical methods. The repeating unit of K6 CPS is linear and contains N-acetyl-d-galactosamine (d-GalpNAc), two d-galactose (d-Galp) residues and 5,7-di-N-acetylpseudaminic acid (Pse5Ac7Ac). The synthesis of these sugars could be attributed to genes in the KL6 capsule biosynthesis gene cluster, and the formation of the linkages between the sugars were assigned to glycosyltransferases or the Wzy polymerase encoded in KL6.

Synthesis of bacterial polysaccharides via the Wzx/Wzy-dependent pathway.

The surfaces of bacteria mediate a multitude of functions in the environment and in an infected host, including adhesion to both biotic and abiotic substrata, motility, immune system interaction and (or) activation, biofilm formation, and cell-cell communication, with many of these features directly influenced by cell-surface glycans. In both Gram-negative and Gram-positive bacteria, the majority of cell-surface polysaccharides are produced via the Wzx/Wzy-dependent assembly pathway; these glycans include heteropolymeric O-antigen, enterobacterial common antigen, exopolysaccharide, spore coat, and capsule in diverse bacteria. The key components of this assembly pathway are the integral inner membrane Wzx flippase, Wzy polymerase, and Wzz chain-length regulator proteins, which until recently have resisted detailed structural and functional characterization. In this review, we have provided a comprehensive synthesis of the latest structural and mechanistic data for each protein, as well as an examination of substrate specificity for each assembly step and complex formation between the constituent proteins. To complement the unprecedented explosion of genomic-sequencing data for bacteria, we have also highlighted both classical and state-of-the-art methods by which encoded Wzx, Wzy, and Wzz proteins can be reliably identified and annotated, using the model Gram-negative bacterium Pseudomonas aeruginosa as an example data set. Lastly, we outline future avenues of research, with the aim of stimulating researchers to take the next steps in investigating the function of, and interplay between, the constituents of this widespread assembly scheme.

Structure of the K2 capsule associated with the KL2 gene cluster of Acinetobacter baumannii

The repeat unit structure of the K2 capsule from an extensively antibiotic-resistant Acinetobacter baumannii global clone 2 (GC2) strain was determined. The oligosaccharide contains three simple sugars, d-glucopyranose, d-galatopyranose and N-acetyl-d-galactosamine, and the complex sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (Pse5Ac7Ac or pseudaminic acid), which has not previously been reported in any A. baumannii capsule. The strain was found to carry all the genes required for the synthesis of the sugars and construction of the K2 structure. The linkages catalyzed by the initiating transferase, three glycosyltransferases and the Wzy polymerase were also predicted. Examination of publicly available A. baumannii genome sequences revealed that the same gene cluster, KL2, often occurs in extensively antibiotic-resistant GC2 isolates and in further strain types. The gene module responsible for the synthesis of pseudaminic acid was also detected in four other K loci. A related module including genes for an acylated relative of pseudaminic acid was also found in two new KL types. A polymerase chain reaction scheme was developed to detect all modules containing genes for sugars based on pseudaminic acid and to specifically detect KL2.

The Wzy O-antigen polymerase ofYersinia pseudotuberculosisO:2a has a dependence on the Wzz chain-length determinant for efficient polymerization

Lipopolysaccharide is a major immunogenic structure for the pathogen Yersinia pseudotuberculosis, which contains the O-specific polysaccharide (OPS) that is presented on the cell surface. The OPS contains many repeats of the oligosaccharide O-unit and exhibits a preferred modal chain length that has been shown to be crucial for cell protection in Yersinia. It is well established that the Wzz protein determines the preferred chain length of the OPS, and in its absence, the polymerization of O units by the Wzy polymerase is uncontrolled. However, for Y.pseudotuberculosis, a wzz mutation has never been described. In this study, we examine the effect of Wzz loss in Y.pseudotuberculosis serotype O:2a and compare the lipopolysaccharide chain-length profile to that of Escherichia coli serotype O111. In the absence of Wzz, the lipopolysaccharides of the two species showed significant differences in Wzy polymerization. Yersinia pseudotuberculosis O:2a exhibited only OPS with very short chain lengths, which is atypical of wzz-mutant phenotypes that have been observed for other species. We hypothesise that the Wzy polymerase of Y.pseudotuberculosis O:2a has a unique default activity in the absence of the Wzz, revealing the requirement of Wzz to drive O-unit polymerization to greater lengths.

Twenty-eight divergent polysaccharide loci specifying within- and amongst-strain capsule diversity in three strains of Bacteroides fragilis.

Comparison of the complete genome sequence of Bacteroides fragilis 638R, originally isolated in the USA, was made with two previously sequenced strains isolated in the UK (NCTC 9343) and Japan (YCH46). The presence of 10 loci containing genes associated with polysaccharide (PS) biosynthesis, each including a putative Wzx flippase and Wzy polymerase, was confirmed in all three strains, despite a lack of cross-reactivity between NCTC 9343 and 638R surface PS-specific antibodies by immunolabelling and microscopy. Genomic comparisons revealed an exceptional level of PS biosynthesis locus diversity. Of the 10 divergent PS-associated loci apparent in each strain, none is similar between NCTC 9343 and 638R. YCH46 shares one locus with NCTC 9343, confirmed by mAb labelling, and a second different locus with 638R, making a total of 28 divergent PS biosynthesis loci amongst the three strains. The lack of expression of the phase-variable large capsule (LC) in strain 638R, observed in NCTC 9343, is likely to be due to a point mutation that generates a stop codon within a putative initiating glycosyltransferase, necessary for the expression of the LC in NCTC 9343. Other major sequence differences were observed to arise from different numbers and variety of inserted extra-chromosomal elements, in particular prophages. Extensive horizontal gene transfer has occurred within these strains, despite the presence of a significant number of divergent DNA restriction and modification systems that act to prevent acquisition of foreign DNA. The level of amongst-strain diversity in PS biosynthesis loci is unprecedented.

In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz

Polysaccharides constitute a major component of bacterial cell surfaces and play critical roles in bacteria/host interactions. The biosynthesis of such molecules, however, has mainly been characterized through in vivo genetic studies, thus precluding discernment of the details of this pathway. Accordingly, we present a chemical approach which enabled reconstitution of the E. coli O-polysaccharide biosynthetic pathway in vitro. Starting with chemically prepared N-Acetyl-D-galactosamine-diphospho-undecaprenyl, the E. coli O86 oligosaccharide repeating unit was assembled via sequential enzymatic glycosylation. Successful expression of the putative polymerase Wzy via a chaperone co-expression system then allowed demonstration of polymerization in vitro using this substrate. Analysis of additional substrates revealed a defined mode of recognition for Wzy towards the lipid moiety. Specific polysaccharide chain length modality was furthermore demonstrated to result from the action of Wzz. Collectively, polysaccharide biosynthesis was chemically reconstituted in vitro, providing a well-defined system for further underpinning molecular details of this biosynthetic pathway.