Localization of the terminal steps of O-antigen synthesis in Salmonella typhimurium.

Previous immunoelectron microscopic studies have shown that both the final intermediate in O-antigen synthesis, undecaprenol-linked O polymer, and newly synthesized O-antigenic lipopolysaccharide are localized to the periplasmic face of the inner membrane (C. A. Mulford and M. J. Osborn, Proc. Natl. Acad. Sci. USA 80:1159-1163, 1983). In vivo pulse-chase experiments now provide further evidence that attachment of O antigen to core lipopolysaccharide, as well as polymerization of O-specific polysaccharide chains, takes place at the periplasmic face of the membrane. Mutants doubly conditional in lipopolysaccharide synthesis [kdsA(Ts) pmi] were constructed in which synthesis of core lipopolysaccharide and O antigen are temperature sensitive and mannose dependent, respectively. Periplasmic orientation of O antigen:core lipopolysaccharide ligase was established by experiments showing rapid chase of undecaprenol-linked O polymer, previously accumulated at 42 degrees C in the absence of core synthesis, into lipopolysaccharide following resumption of core formation at 30 degrees C. In addition, chase of the monomeric O-specific tetrasaccharide unit into lipopolysaccharide was found in similar experiments in an O-polymerase-negative [rfc kdsA(Ts) pmi] mutant, suggesting that polymerization of O chains also occurs at the external face of the inner membrane.     

Functional characterization of WaaL, a ligase associated with linking O-antigen polysaccharide to the core of Pseudomonas aeruginosa lipopolysaccharide

The O antigen of Pseudomonas aeruginosa B-band lipopolysaccharide is synthesized by assembling O-antigen-repeat units at the cytoplasmic face of the inner membrane by nonprocessive glycosyltransferases, followed by polymerization on the periplasmic face. The completed chains are covalently attached to lipid A core by the O-antigen ligase, WaaL. In P. aeruginosa the process of ligating these O-antigen molecules to lipid A core is not clearly defined, and an O-antigen ligase has not been identified until this study. Using the sequence of waaL from Salmonella enterica as a template in a BLAST search, a putative waaL gene was identified in the P. aeruginosa genome. The candidate gene was amplified and cloned, and a chromosomal knockout of PAO1 waaL was generated. Lipopolysaccharide (LPS) from this mutant is devoid of B-band O-polysaccharides and semirough (SR-LPS, or core-plus-one O-antigen). The mutant PAO1waaL is also deficient in the production of A-band polysaccharide, a homopolymer of D-rhamnose. Complementation of the mutant with pPAJL4 containing waaL restored the production of both A-band and B-band O antigens as well as SR-LPS, indicating that the knockout was nonpolar and waaL is required for the attachment of O-antigen repeat units to the core. Mutation of waaL in PAO1 and PA14, respectively, could be complemented with waaL from either strain to restore wild-type LPS production. The waaL mutation also drastically affected the swimming and twitching motilities of the bacteria. These results demonstrate that waaL in P. aeruginosa encodes a functional O-antigen ligase that is important for cell wall integrity and motility of the bacteria.     

Energy dependence of O-antigen synthesis in Salmonella typhimurium.

The uncoupler 2,4-dinitrophenol prevents in vivo synthesis of O antigen in Salmonella typhimurium by inhibiting the first reaction of the pathway, formation of galactosyl-pyrophosphoryl-undecaprenol. Inhibition was observed only in intact cells; dinitrophenol had no effect on activity of the synthase enzyme in isolated membrane fractions. In vivo inhibition could not be explained by changes in intracellular nucleotide pools or a shift in the equilibrium of the reaction and appeared to be specific for the first step in the pathway. Neither the subsequent mannosyl transferase, which catalyzes formation of the trisaccharide-lipid intermediate, mannosyl-rhamnosyl-galactosyl-pyrophosphoryl-undecaprenol, nor O-antigen polymerase was inhibited. In addition, incorporation of galactose into core lipopolysaccharide was only modestly inhibited under conditions in which O-antigen synthesis was abolished. The results suggest that maintenance of proton motive force is required for access of substrate, UDP-galactose and/or undecaprenyl phosphate, to the active site of the galactosyl-pyrophosphoryl-undecaprenol synthase enzyme.     

Role of Escherichia coli K-12 rfa genes and the rfp gene of Shigella dysenteriae 1 in generation of lipopolysaccharide core heterogeneity and attachment of O antigen.

The rfp gene of Shigella dysenteriae 1 and the rfa genes of Escherichia coli K-12 and Salmonella typhimurium LT2 have been studied to determine their relationship to lipopolysaccharide (LPS) core heterogeneity and their role in the attachment of O antigen to LPS. It has been inferred from the nucleotide sequence that the rfp gene encodes a protein of 41,864 Da which has a structure similar to that of RfaG protein. Expression of this gene in E. coli K-12 results in the loss of one of the three bands seen in gel analysis of the LPS and in the appearance of a new, more slowly migrating band. This is consistent with the hypothesis that Rfp is a sugar transferase which modifies a subset of core molecules so that they become substrates for attachment of S. dysenteriae O antigen. A shift in gel migration of the bands carrying S. dysenteriae O antigen and disappearance of the Rfp-modified band in strains producing O antigen suggest that the core may be trimmed or modified further before attachment of O antigen. Mutation of rfaL results in a loss of the rough LPS band which appears to be modified by Rfp and prevents the appearance of the Rfp-modified band. Thus, RfaL protein is involved in core modification and is more than just a component of the O-antigen ligase. The products of rfaK and rfaQ also appear to be involved in modification of the core prior to attachment of O antigen, and the sites of rfaK modification are different in E. coli K-12 and S. typhimurium. In contrast, mutations in rfaS and rfaZ result in changes in the LPS core but do not affect the attachment of O antigen. We propose that these genes are involved in an alternative pathway for the synthesis of rough LPS species which are similar to lipooligosaccharides of other species and which are not substrates for O-antigen attachment. All of these studies indicate that the apparent heterogeneity of E. coli K-12 LPS observed on gels is not an artifact but instead a reflection of functional differences among LPS species.     

肠道沙门氏菌O-抗原多糖的研究进展

O-抗原是由多糖重复单元组成的多聚糖,表达于细菌的外膜,具有多样性,是划分沙门菌血清型的重要依据。O-抗原多糖由多基因协同作用而合成,这些基因在沙门菌基因组上成簇存在,形成O-抗原基因簇。O-抗原多糖也是重要的毒力因子,在沙门菌入侵宿主、体内存活、定殖等致病过程中均发挥着重要的作用。此外,O-抗原还是沙门菌主要的保护性抗原,能激发宿主产生高水平抗体并发挥免疫保护作用,成为疫苗研究的靶点。本文综述O-抗原多糖的基因结构和合成、生物学功能及其在疫苗研制中的应用与前景。     

Serological cross-reaction between the lipopolysaccharide O-polysaccharaide antigens of Escherichia coli O157:H7 and strains of Citrobcter freundii and Citrobacter sedlakii

A strain of Citrobacter sedlakii showing serological cross-reaction with Escherichia coli O157 antisera was demonstrated to produce a lipopolysaccharide O-antigen having an identical structure with that of the E. coli O157 O-antigen. A strain of Citrobacter freunndii showing similar cross-reaction with E. coli O157 specific monoclonal antibody was shown to produce a lipopolysaccharide O-antigen composed of a trisaccharide repeating unit having the structure [ 2)-alpha-D Rhap-(1-3)-beta-D-Rhap-(1-4)-beta-D-Glcp-(1-]. This O-antigen differs from that of the E. coli O157 O-antigen and also lacks a component 2-substituted 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residue implicated as the common epitope in the lipopolysaccharide O-antigens of previously investigated bacterial species showing serological cross-reactivity with E. coli O157 antisera. The C freundii O-antigen presents an interesting example of structural mimicry within a bacterial polysaccharide antigen.     

Investigation of the structural requirements in the lipopolysaccharide core acceptor for ligation of O antigens in the genus Salmonella: WaaL "ligase" is not the sole determinant of acceptor specificity

The ligation of O antigen polysaccharide to lipid A-core oligosaccharide is a late step in the formation of the complex glycolipid known as lipopolysaccharide. Although the process has been localized to the periplasmic face of the inner membrane, details of the ligation mechanism have not been resolved. To date, there is only one gene product (WaaL, often referred to as "ligase") known to be required. There exists a requirement for a specific lipid A-core oligosaccharide acceptor structure for ligation activity, and it has been proposed that the WaaL protein imparts this acceptor specificity. Here the structural requirements in the core oligosaccharide acceptor for O antigen ligation are investigated in prototype serovars of Salmonella enterica. Complementation experiments in mutants with defined core oligosaccharide structure indicate that the specificity of the ligation reaction for a particular core oligosaccharide structure is not dependent on the WaaL protein alone. The data provide the first indication of a more complicated recognition process involving additional cellular components.     

Molecular Dynamics and NMR Spectroscopy Studies of E. coli Lipopolysaccharide Structure and Dynamics

Lipopolysaccharide (LPS), a component of Gram-negative bacterial outer membranes, comprises three regions: lipid A, core oligosaccharide, and O-antigen polysaccharide. Using the CHARMM36 lipid and carbohydrate force fields, we have constructed a model of an Escherichia coli R1 (core) O6 (antigen) LPS molecule. Several all-atom bilayers are built and simulated with lipid A only (LIPA) and varying lengths of 0 (LPS0), 5 (LPS5), and 10 (LPS10) O6 antigen repeating units; a single unit of O6 antigen contains five sugar residues. From ¹H,¹H-NOESY experiments, cross-relaxation rates are obtained from an O-antigen polysaccharide sample. Although some experimental deviations are due to spin-diffusion, the remaining effective proton-proton distances show generally very good agreement between NMR experiments and molecular dynamics simulations. The simulation results show that increasing the LPS molecular length has an impact on LPS structure and dynamics and also on LPS bilayer properties. Terminal residues in a LPS bilayer are more flexible and extended along the membrane normal. As the core and O-antigen are added, per-lipid area increases and lipid bilayer order decreases. In addition, results from mixed LPS0/5 and LPS0/10 bilayer simulations show that the LPS O-antigen conformations at a higher concentration of LPS5 and LPS10 are more orthogonal to the membrane and less flexible. The O-antigen concentration of mixed LPS bilayers does not have a significant effect on per-lipid area and hydrophobic thickness. Analysis of ion and water penetration shows that water molecules can penetrate inside the inner core region, and hydration is critical to maintain the integrity of the bilayer structure.     

Molecular Dynamics and NMR Spectroscopy Studies of E.?coli Lipopolysaccharide Structure and Dynamics

Lipopolysaccharide (LPS), a component of Gram-negative bacterial outer membranes, comprises three regions: lipid A, core oligosaccharide, and O-antigen polysaccharide. Using the CHARMM36 lipid and carbohydrate force fields, we have constructed a model of an Escherichia coli R1 (core) O6 (antigen) LPS molecule. Several all-atom bilayers are built and simulated with lipid A only (LIPA) and varying lengths of 0 (LPS0), 5 (LPS5), and 10 (LPS10) O6 antigen repeating units; a single unit of O6 antigen contains five sugar residues. From ¹H,¹H-NOESY experiments, cross-relaxation rates are obtained from an O-antigen polysaccharide sample. Although some experimental deviations are due to spin-diffusion, the remaining effective proton-proton distances show generally very good agreement between NMR experiments and molecular dynamics simulations. The simulation results show that increasing the LPS molecular length has an impact on LPS structure and dynamics and also on LPS bilayer properties. Terminal residues in a LPS bilayer are more flexible and extended along the membrane normal. As the core and O-antigen are added, per-lipid area increases and lipid bilayer order decreases. In addition, results from mixed LPS0/5 and LPS0/10 bilayer simulations show that the LPS O-antigen conformations at a higher concentration of LPS5 and LPS10 are more orthogonal to the membrane and less flexible. The O-antigen concentration of mixed LPS bilayers does not have a significant effect on per-lipid area and hydrophobic thickness. Analysis of ion and water penetration shows that water molecules can penetrate inside the inner core region, and hydration is critical to maintain the integrity of the bilayer structure.     

Interplay of the Wzx translocase and the corresponding polymerase and chain length regulator proteins in the translocation and periplasmic assembly of lipopolysaccharide o antigen

Genetic evidence suggests that a family of bacterial and eukaryotic integral membrane proteins (referred to as Wzx and Rft1, respectively) mediates the transbilayer movement of isoprenoid lipid-linked glycans. Recent work in our laboratory has shown that Wzx proteins involved in O-antigen lipopolysaccharide (LPS) assembly have relaxed specificity for the carbohydrate structure of the O-antigen subunit. Furthermore, the proximal sugar bound to the isoprenoid lipid carrier, undecaprenyl-phosphate (Und-P), is the minimal structure required for translocation. In Escherichia coli K-12, N-acetylglucosamine (GlcNAc) is the proximal sugar of the O16 and enterobacterial common antigen (ECA) subunits. Both O16 and ECA systems have their respective translocases, WzxO16 and WzxE, and also corresponding polymerases (WzyO16 and WzyE) and O-antigen chain-length regulators (WzzO16 and WzzE), respectively. In this study, we show that the E. coli wzxE gene can fully complement a wzxO16 translocase deletion mutant only if the majority of the ECA gene cluster is deleted. In addition, we demonstrate that introduction of plasmids expressing either the WzyE polymerase or the WzzE chain-length regulator proteins drastically reduces the O16 LPS-complementing activity of WzxE. We also show that this property is not unique to WzxE, since WzxO16 and WzxO7 can cross-complement translocase defects in the O16 and O7 antigen clusters only in the absence of their corresponding Wzz and Wzy proteins. These genetic data are consistent with the notion that the translocation of O-antigen and ECA subunits across the plasma membrane and the subsequent assembly of periplasmic O-antigen and ECA Und-PP-linked polymers depend on interactions among Wzx, Wzz, and Wzy, which presumably form a multiprotein complex.     

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.     

Escherichia coli Clone Sonnei (Shigella sonnei) Had a Chromosomal O-Antigen Gene Cluster Prior to Gaining Its Current Plasmid-Borne O-Antigen Genes

O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria. The surface-exposed O antigen is subject to selection by the host immune system, which may account for the maintenance of many different O-antigen forms. Characteristically, all genes specific to O-antigen synthesis are clustered in a region close to the his and gnd genes on the chromosome of Escherichia coli and related species. Shigella sonnei, essentially a clone of E. coli (E. coli clone Sonnei), is an important human pathogen and is unusual in that its O-antigen gene cluster is located on a plasmid. Our results suggest that it once had a normal chromosomal O-antigen gene cluster which has been largely deleted. We suggest that the O antigen encoded by the plasmid-borne genes offered a selective advantage in adapting to a new environment and that the chromosomal O-antigen genes were eventually inactivated. We also identified, by PCR and sequencing, a potential ancestor of E. coli Sonnei among the 166 known E. coli serotype strains.     

Mechanism of O-antigen distribution in lipopolysaccharide.

O-antigen units are nonuniformly distributed among lipid A-core molecules in lipopolysaccharide (LPS) from gram-negative bacteria, as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the actual distribution patterns are complex, multimodal, and strain specific. Although the basic biochemical steps involved in synthesis and polymerization of O-antigen monomers and their subsequent attachment to lipid A-core are known, the mechanism by which specific multimodal distribution patterns are attained in mature LPS has not been previously considered theoretically or experimentally. We have developed probability equations which completely describe O-antigen distribution among lipid A-core molecules in terms of the probability of finding a nascent polymer (O antigen linked to carrier lipid) of length k (Tk) and the probability that a nascent polymer of length k will be extended to k + 1 by polymerase (pk) or transferred to lipid A-core by ligase (qk). These equations were used to show that multimodal distribution patterns in mature LPS cannot be produced if all pk are equal to p and all qk are equal to q, conditions which indicate a lack of selectivity of polymerase and ligase, respectively, for nascent O-antigen chain lengths. A completely stochastic model (pk = p, qk = q) of O-antigen polymerization and transfer to lipid A-core was also inconsistent with observed effects of mutations which resulted in partial inhibition of O-antigen monomer synthesis, lipid A-core synthesis, or ligase activity. The simplest explanation compatible with experimental observations is that polymerase or ligase, or perhaps both, have specificity for certain O-antigen chain lengths during biosynthesis of LPS. Our mathematical model indicates selectively probably was associated with the polymerase reaction. Although one may argue for a multimodal distribution pattern based on a kinetic mechanism i.e., varying reaction parameters in space or in time during cell growth, such a model requires complex sensory and regulatory mechanisms to explain the mutant data and mechanisms for sequestering specific components of LPS biosynthesis to explain the distribution pattern in normal cells. We favor the simple alternative of enzyme specificity and present generalized equations which should be useful in analysis of other analogous biochemical systems.     

The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

Shigella flexneri causes bacillary dysentery in humans. Essential to the establishment of the disease is the invasion of the colonic epithelial cells. Here we investigated the role of the lipopolysaccharide (LPS) O antigen in the ability of S. flexneri to adhere to and invade polarized Caco-2 cells. The S. flexneri 2a O antigen has two preferred chain lengths: a short O antigen (S-OAg) regulated by the WzzB protein and a very long O antigen (VL-OAg) regulated by Wzz pHS2. Mutants with defined deletions of the genes required for O-antigen assembly and polymerization were constructed and assayed for their abilities to adhere to and enter cultured epithelial cells. The results show that both VL- and S-OAg are required for invasion through the basolateral cell membrane. In contrast, the absence of O antigen does not impair adhesion. Purified LPS does not act as a competitor for the invasion of Caco-2 cells by the wild-type strain, suggesting that LPS is not directly involved in the internalization process by epithelial cells.     

The biosynthesis and biological role of 6-deoxyheptose in the lipopolysaccharide O-antigen of Yersinia pseudotuberculosis

Yersinia pseudotuberculosis O:2a harbours 6-deoxy- d - manno -heptose in its O-antigen. The biological function of 6-deoxyheptose and its role in virulence is unknown and its biosynthetic pathway has not been demonstrated experimentally. Here, we show that dmhA and dmhB are necessary for 6-deoxyheptose biosynthesis in Y. pseudotuberculosis . Their disruption resulted in the lack of 6-deoxyheptose in the O-unit and its replacement by d - glycero - d - manno -heptose, thus indicating relaxed specificity of the glycosyltransferases, polymerase and ligase involved in lipopolysaccharide synthesis. The dmhB mutant exhibited a lower content in ketooctonic acid (Ko)-containing core molecules and reduced ligation and polymerization of the O-unit. We also show that Tyr128 is essential for activity of DmhB, and that DmhB functions as an oligomer, based on the dominant negative effect of overexpression of DmhB Y128F in dmhA . Moreover, we demonstrate that 6-deoxyheptose is important for virulence-related functions of the outer membrane and its appendages in vitro , such as barrier function against bile salts, polymyxin and novobiocin, and flagella-mediated motility. Although both mutants colonized the mouse ceacum as well as the wild type, the dmhB mutant was impaired for colonization of the liver, suggesting that DmhB represents a potential therapeutic target.     

The Escherichia coli O111 and Salmonella enterica O35 gene clusters: gene clusters encoding the same colitose-containing O antigen are highly conserved

O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria. Escherichia coli and Salmonella enterica each have many forms of O antigen, but only three are common to the two species. It has been found that, in general, O-antigen genes are of low GC content. This deviation in GC content from that of typical S. enterica or E. coli genes (51%) is thought to indicate that the O-antigen DNA originated in species other than S. enterica or E. coli and was captured by lateral transfer. The O-antigen structure of Salmonella enterica O35 is identical to that of E. coli O111, commonly found in enteropathogenic E. coli strains. This O antigen, which has been shown to be a virulence factor in E. coli, contains colitose, a 3,6-dideoxyhexose found only rarely in the Enterobacteriaceae. Sequencing of the O35-antigen gene cluster of S. enterica serovar Adelaide revealed the same gene order and flanking genes as in E. coli O111. The divergence between corresponding genes of these two gene clusters at the nucleotide level ranges from 21.8 to 11.7%, within the normal range of divergence between S. enterica and E. coli. We conclude that the ancestor of E. coli and S. enterica had an O antigen identical to the O111 and O35 antigens, respectively, of these species and that the gene cluster encoding it has survived in both species.     

大肠杆菌O141 O-抗原基因簇的破译及进化分析

对大肠杆菌O141 O-抗原基因簇进行测序,序列全长15601bp,用生物信息学的方法进行序列分析,共发现12个基因：鼠李糖合成酶基因(rmlB,rmlD,rmlA,rmlC)、甘露糖合成酶基因(manB,manC),糖基转移酶基因(orf6,orf7,orf9,orf10)、O-抗原转运酶基因(wzx)和O-抗原聚合酶基因(wzy)。用PCR的方法筛选出了针对大肠杆菌O141的特异基因,可以用于基因芯片或PCR方法对大肠杆菌O141的快速检测。通过对大肠杆菌O141的O-抗原基因簇及甘露糖和鼠李糖合成酶基因的进化分析发现：大肠杆菌O141 O-抗原基因簇是低GC含量的片段,仅O-抗原特异的基因才出现在O-抗原基因簇;并且这些基因可能介导了O-抗原基因簇间的重组及以O141 O-抗原基因簇的形成。     

PagP activation in the outer membrane triggers R3 core oligosaccharide truncation in the cytoplasm of Escherichia coli O157:H7

The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP is normally a latent enzyme, but it can be directly activated in outer membranes by lipid redistribution associated with a breach in the permeability barrier. We now demonstrate that a lipid A myristate deficiency in an E. coli O157:H7 msbB mutant constitutively activates PagP in outer membranes. The lipid A myristate deficiency is associated with hydrophobic antibiotic sensitivity and, unexpectedly, with serum sensitivity, which resulted from O-antigen polysaccharide absence due to a cytoplasmically determined truncation at the first outer core glucose unit of the R3 core oligosaccharide. Mutational inactivation of pagP in the myristate-deficient lipid A background aggravated the hydrophobic antibiotic sensitivity as a result of losing a partially compensatory increase in lipid A palmitoylation while simultaneously restoring serum resistance and O-antigen attachment to intact lipopolysaccharide. Complementation with either wild-type pagP or catalytically inactive pagPSer77Ala alleles restored the R3 core truncation. However, the intact lipopolysaccharide was preserved after complementation with an internal deletion pagPDelta5-14 allele, which mostly eliminates a periplasmic amphipathic alpha-helical domain but fully supports cell surface lipid A palmitoylation. Our findings indicate that activation of PagP not only triggers lipid A palmitoylation in the outer membrane but also separately truncates the R3 core oligosaccharide in the cytoplasm. We discuss the implication that PagP might function as an apical sensory transducer, which can be activated by a breach in the outer membrane permeability barrier.     

Molecular characterization of the Pseudomonas aeruginosa serotype O5 (PAO1) B-band lipopolysaccharide gene cluster

Pseudomonas aeruginosa co-expresses A-band lipopolysaccharide (LPS), a homopolymer of rhamnose, and B-band LPS, a heteropolymer with a repeating unit of 2–5 sugars which is the serotype-specific antigen. The gene clusters for A- and B-band biosynthesis in P. aeruginosa O5 (strain PAO1) have been cloned previously. Here we report the DNA sequence and molecular analysis of the B-band O-antigen biosynthetic cluster. Sixteen open reading frames (ORFs) thought to be involved in synthesis of the O5 O antigen were identified, including wzz ( rol ), wzy ( rfc ), and wbpA – wbpN . A further 3 ORFs not thought to be involved with LPS synthesis were identified ( hisH , hisF , and uvrB ). Most of the wbp genes are found only in serotypes O2, O5, O16, O18, and O20, which form a chemically and structurally related O-antigen serogroup. In contrast, wbpM and wbpN are common to all 20 serotypes of P. aeruginosa. Although wbpM is not serogroup-specific, knockout mutations confirmed it is necessary for O5 O-antigen biosynthesis. A novel insertion sequence, IS 1209 , is present at the junction between the serogroup-specific and non-specific regions. We have predicted the functions of the proteins encoded in the wbp cluster based on their homologies to those in the databases, and provide a proposed pathway of P. aeruginosa O5 O-antigen biosynthesis.     

O-antigen and core carbohydrate of Vibrio fischeri lipopolysaccharide: composition and analysis of their role in Euprymna scolopes light organ colonization

Vibrio fischeri exists in a symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes, where the squid provides a home for the bacteria, and the bacteria in turn provide camouflage that helps protect the squid from night-time predators. Like other gram-negative organisms, V. fischeri expresses lipopolysaccharide (LPS) on its cell surface. The structure of the O-antigen and the core components of the LPS and their possible role in colonization of the squid have not previously been determined. In these studies, an O-antigen ligase mutant, waaL, was utilized to determine the structures of these LPS components and their roles in colonization of the squid. WaaL ligates the O-antigen to the core of the LPS; thus, LPS from waaL mutants lacks O-antigen. Our results show that the V. fischeri waaL mutant has a motility defect, is significantly delayed in colonization, and is unable to compete with the wild-type strain in co-colonization assays. Comparative analyses of the LPS from the wild-type and waaL strains showed that the V. fischeri LPS has a single O-antigen repeat composed of yersiniose, 8-epi-legionaminic acid, and N-acetylfucosamine. In addition, the LPS from the waaL strain showed that the core structure consists of L-glycero-D-manno-heptose, D-glycero-D-manno-heptose, glucose, 3-deoxy-D-manno-octulosonic acid, N-acetylgalactosamine, 8-epi-legionaminic acid, phosphate, and phosphoethanolamine. These studies indicate that the unusual V. fischeri O-antigen sugars play a role in the early phases of bacterial colonization of the squid.