Wzx flippase

The O-antigen, a long polysaccharide that constitutes the distal part of the outer membrane-anchored lipopolysaccharide, is one of the critical components in the protective outer membrane of Gram-negative bacteria. Most species produce one of the structurally diverse O-antigens, with nearly all the polysaccharide components having complex structures made by the Wzx/Wzy pathway. This pathway produces repeat-units of mostly 3-8 sugars on the cytosolic face of the cytoplasmic membrane that is translocated by Wzx flippase to the periplasmic face and polymerized by Wzy polymerase to give long-chain polysaccharides...

Salmonella enterica subsp. enterica (S. enterica) is a significant public health concern and is estimated to cause more than 300,000 deaths annually. Nowadays, the vaccines available for human Salmonellosis prevention are all targeting just one serovar, i.e., S. Typhi, leaving a huge potential risk of Salmonella disease epidemiology change. In this study, we explored the strategy of multiple immunodominant O-epitopes co-expression in S. enterica serovars and evaluated their immunogenicity to induce cross-immune responses and cross-protections against S...

Bacteriophages and phage polysaccharide-degrading enzymes (depolymerases) are garnering attention as possible alternatives to antibiotics. Here, we describe the antimicrobial properties of bacteriophage KpV74 and phage depolymerase Dep_kpv74 specific to the hypervirulent Klebsiella pneumoniae of the K2 capsular type. The depolymerase Dep_kpv74 was identified as a specific glucosidase that cleaved the K2 type capsular polysaccharides of the K. pneumoniae by a hydrolytic mechanism. This depolymerase was effective against thigh soft tissue K...

Bacterial cell envelopes play a critical role in host-pathogen interactions. Macromolecular components of these structures have been closely linked to the virulence of pathogens. Piscirickettsia salmonis is a relevant salmonid pathogen with a worldwide distribution. This bacterium is the etiological agent of piscirickettsiosis, a septicemic disease that causes a high economic burden, especially for the Chilean salmon farming industry. Although P. salmonis has been discovered long ago, its pathogenicity and virulence mechanisms are not completely understood...

MOP (Multidrug/Oligosaccharidyl-lipid/Polysaccharide) family transporters are found in almost all life forms. They are responsible for transporting lipid-linked precursors across the cell membrane to support the synthesis of various glycoconjugates. While significant progress has been made in elucidating their transport mechanism, how these transporters select their substrates remains unclear. Here, we systematically tested the MOP transporters in the Streptococcus pneumoniae capsule pathway for their ability to translocate noncognate capsule precursors...

Proteus mirabilis is a pathogenic, Gram-negative, rod-shaped bacterium that causes ascending urinary tract infections. Swarming motility, urease production, biofilm formation, and the properties of its lipopolysaccharide (LPS) are all factors that contribute to the virulence of this bacterium. Uniquely, members of the O18 serogroup elaborate LPS molecules capped with O antigen polymers built of pentasaccharide repeats; these repeats are modified with a phosphocholine (ChoP) moiety attached to the proximal sugar of each O unit...

Myxococcus xanthus arranges into two morphologically distinct biofilms depending on its nutritional status, i.e., coordinately spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. A secreted polysaccharide, referred to as exopolysaccharide (EPS), is a structural component of both biofilms and is also important for type IV pilus-dependent motility and fruiting body formation. Here, we characterize the biosynthetic machinery responsible for EPS biosynthesis using bioinformatics, genetics, heterologous expression, and biochemical experiments...

The rod-shaped cells of Myxococcus xanthus, a Gram-negative deltaproteobacterium, differentiate to environmentally resistant spores upon starvation or chemical stress. The environmental resistance depends on a spore coat polysaccharide that is synthesized by the ExoA-I proteins, some of which are part of a Wzx/Wzy-dependent pathway for polysaccharide synthesis and export; however, key components of this pathway have remained unidentified. Here, we identify and characterize two additional loci encoding proteins with homology to enzymes involved in polysaccharide synthesis and export, as well as sugar modification, and show that six of the proteins encoded by these loci are essential for the formation of environmentally resistant spores...

The structure of the O-antigen polysaccharide (PS) from the Shiga-toxin producing Escherichia coli O63 has been elucidated using a combination of bioinformatics, component analyses, and NMR spectroscopy. The O-antigen is comprised of tetrasaccharide repeating units with the following structure: →2)-β-d-Quip3N(d-allo-ThrAc)-(1→2)-β-d-Ribf-(1→4)-β-d-Galp-(1→3)-α-d-GlcpNAc-(1→ in which the N-acetylated d-allo-threonine is amide-linked to position 3 of the 3-amino-3-deoxy-d-Quip sugar residue. The presence of a predicted flippase and polymerase encoded in the O63 gene cluster is consistent with the Wzx/Wzy biosynthetic pathway and consequently the biological repeating unit has likely an N-acetyl-d-glucosamine residue at its reducing end...

In lactic acid bacteria, the synthesis of exopolysaccharides (EPS) has been associated with some favorable technological properties as well as health-promoting benefits. Research works have shown the potential of EPS produced by lactobacilli to differentially modulate immune responses. However, most studies were performed in immune cells and few works have concentrated in the immunomodulatory activities of EPS in non-immune cells such as intestinal epithelial cells. In addition, the cellular and molecular mechanisms involved in the immunoregulatory effects of EPS have not been studied in detail...

The Wzx flippase is a critical component of the O-antigen biosynthesis pathway, being responsible for the translocation of oligosaccharide O units across the inner membrane in Gram-negative bacteria. Recent studies have shown that Wzx has a strong preference for its cognate O unit, but the types of O-unit structural variance that a given Wzx can accommodate are poorly understood. In this study, we identified two Yersinia pseudotuberculosis Wzx that can distinguish between different terminal dideoxyhexose sugars on a common O-unit main-chain, despite both being able to translocate several other structurally-divergent O units...

Rhizobia dwell and multiply in the soil and represent a unique group of bacteria able to enter into a symbiotic interaction with plants from the Fabaceae family and fix atmospheric nitrogen inside de novo created plant organs, called nodules. One of the key determinants of the successful interaction between these bacteria and plants are exopolysaccharides, which represent species-specific homo- and heteropolymers of different carbohydrate units frequently decorated by non-carbohydrate substituents. Exopolysaccharides are typically built from repeat units assembled by the Wzx/Wzy-dependent pathway, where individual subunits are synthesized in conjunction with the lipid anchor undecaprenylphosphate (und-PP), due to the activity of glycosyltransferases...

Attachment is essential for microorganisms to establish interactions with both biotic and abiotic surfaces. Stable attachment of Caulobacter crescentus to surfaces requires an adhesive polysaccharide holdfast, but the exact composition of the holdfast is unknown. The holdfast is anchored to the cell envelope by outer membrane proteins HfaA, HfaB, and HfaD. <u>H</u>old<u>f</u>ast <u>a</u>nchor gene mutations result in holdfast shedding and reduced cell adherence. Translocation of HfaA and HfaD to the cell surface requires HfaB...

Translocation of lipid-linked oligosaccharides is a common theme across prokaryotes and eukaryotes. For bacteria, such activity is used in cell wall construction, polysaccharide synthesis, and the relatively recently discovered protein glycosylation. To the best of our knowledge, the Gram-negative inner membrane flippase Wzx was the first protein identified as being involved in oligosaccharide translocation, and yet we still have only a limited understanding of this protein after 3 decades of research. At present, Wzx is known to be a multitransmembrane protein with enormous sequence diversity that flips oligosaccharide substrates with varied degrees of preference...

We have developed a system called the Operon Assembly Protocol (OAP), which takes advantage of the homologous recombination DNA repair pathway in Saccharomyces cerevisiae to assemble full-length operons from a series of overlapping PCR products into a specially engineered yeast-Escherichia coli shuttle vector. This flexible, streamlined system can be used to assemble several operon clones simultaneously, and each clone can be expressed in the same E. coli tester strain to facilitate direct functional comparisons...

A novel lactic acid bacteria strain Lactobacillus kefiranofaciens ZW3 exhibited the characteristics of high production of exopolysaccharide (EPS). The epsN gene, located in the eps gene cluster of this strain, is associated with EPS biosynthesis. Bioinformatics analysis of this gene was performed. The conserved domain analysis showed that the EpsN protein contained MATE-Wzx-like domains. Then the epsN gene was amplified to construct the recombinant expression vector pMG36e-epsN. The results showed that the EPS yields of the recombinants were significantly improved...

Bacterial Wzx flippases translocate (flip) short oligosaccharide repeat units (O units) across the inner membrane into the periplasm, which is a critical step in the assembly of many O antigens, capsules and other surface polysaccharides. There is enormous diversity in O antigens and capsules in particular, even within species. Wzx proteins are similarly diverse, but it has been widely accepted that they have significant specificity only for the first sugar of an O unit. In this study, we analysed the Wzx from the Salmonella enterica group C2 O antigen gene cluster, which is a unique and divergent member of a set of gene clusters that produce galactose-initiated O antigens...

The DNA sequence of the O-antigen gene clusters of Escherichia coli serogroups O62, O68, O131, O140, O142, and O163 was determined, and primers based on the wzx (O-antigen flippase) and/or wzy (O-antigen polymerase) genes within the O-antigen gene clusters were designed and used in PCR assays to identify each serogroup. Specificity was tested with E. coli reference strains, field isolates belonging to the target serogroups, and non-E. coli bacteria. The PCR assays were highly specific for the respective serogroups; however, the PCR assay targeting the O62 wzx gene reacted positively with strains belonging to E...

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...

Several molecular detection marker genes specific for a number of individual Salmonella serogroups have been recently identified in our lab by comparative genomics for the genotyping of diverse serogroups. To further understand the correlation between serotype and genotype, the function of a Salmonella serogroup-C1-specific gene (SC_2092) was analyzed in this study. It was indicated from the topological prediction using the deduced amino acid sequence of SC_2092 that this putative protein was highly similar to the confirmed Wzx flippases...