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Wzx protein

Nicolas Hegerle, Janhabi Bose, Girish Ramachandran, James E Galen, Myron M Levine, Raphael Simon, Sharon M Tennant
AIMS: O-polysaccharide (OPS) molecules are protective antigens for several bacterial pathogens, and have broad utility as components of glycoconjugate vaccines. Variability in the OPS chain length is one obstacle towards further development of these vaaccines. Introduction of sizing steps during purification of OPS molecules of sub-optimal or of mixed lengths introduces additional costs and complexity while decreasing the final yield. The overall goal of this study was to demonstrate the utility of engineering Gram-negative bacteria to produce homogenous O-polysaccharide populations that can be used as the basis of carbohydrate vaccines by over-expressing O-polysaccharide chain length regulators of the Wzx/Wzy-dependent pathway...
March 30, 2018: Journal of Applied Microbiology
Jochen Schmid
The distinct biosynthesis pathways for microbial exopolysaccharide production provide different engineering strategies to tailor the chemical structures of the final polymers. This review focuses on the latest insights in the various pathways and identifies bottlenecks as well as promising targets for tailoring microbial polysaccharide production. The main engineering strategies includes the combinatorial assembly of glycosyltransferases and engineering of the Wzx and Wzy proteins for flipping of repeating units as well as polymerization...
January 17, 2018: Current Opinion in Biotechnology
Małgorzata Marczak, Andrzej Mazur, Piotr Koper, Kamil Żebracki, Anna Skorupska
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...
December 1, 2017: Genes
Gail G Hardy, Evelyn Toh, Cécile Berne, Yves V Brun
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...
February 1, 2018: Journal of Bacteriology
Yaoqin Hong, Michael A Liu, Peter R Reeves
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...
January 1, 2018: Journal of Bacteriology
Mark T Anderson, Lindsay A Mitchell, Lili Zhao, Harry L T Mobley
Serratia marcescens is an opportunistic pathogen that causes a range of human infections, including bacteremia, keratitis, wound infections, and urinary tract infections. Compared to other members of the Enterobacteriaceae family, the genetic factors that facilitate Serratia proliferation within the mammalian host are less well defined. An in vivo screen of transposon insertion mutants identified 212 S. marcescens fitness genes that contribute to bacterial survival in a murine model of bloodstream infection...
May 23, 2017: MBio
Susana Merino, Victor Gonzalez, Juan M Tomás
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)...
July 2016: Future Microbiology
Jingrui Wang, Wei Tang, Yongna Zheng, Zhuqing Xing, Yanping Wang
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...
September 2016: Archives of Microbiology
Véronique L Taylor, Jesse F J Hoage, Sandra Wingaard Thrane, Steven M Huszczynski, Lars Jelsbak, Joseph S Lam
Pseudomonas aeruginosa is a Gram-negative bacterium that produces highly varied lipopolysaccharide (LPS) structures. The O antigen (O-Ag) in the LPS is synthesized through the Wzx/Wzy-dependent pathway where lipid-linked O-Ag repeats are polymerized by Wzy. Horizontal-gene transfer has been associated with O-Ag diversity. The O-Ag present on the surface of serotypes O5 and O16, differ in the intra-molecular bonds, α and β, respectively; the latter arose from the action of three genes in a serotype converting unit acquired from bacteriophage D3, including a β-polymerase (Wzyβ)...
2016: Frontiers in Microbiology
Véronique L Taylor, Steven M Huszczynski, Joseph S Lam
Bacterial polysaccharides play an essential role in cell viability, virulence, and evasion of host defenses. Although the polysaccharides themselves are highly diverse, the pathways by which bacteria synthesize these essential polymers are conserved in both Gram-negative and Gram-positive organisms. By utilizing a lipid linker, a series of glycosyltransferases and integral membrane proteins act in concert to synthesize capsular polysaccharide, teichoic acid, and teichuronic acid. The pathways used to produce these molecules are the Wzx/Wzy-dependent, the ABC-transporter-dependent, and the synthase-dependent pathways...
2017: Current Topics in Microbiology and Immunology
Perrin Baker, Gregory B Whitfield, Preston J Hill, Dustin J Little, Matthew J Pestrak, Howard Robinson, Daniel J Wozniak, P Lynne Howell
A key component of colonization, biofilm formation, and protection of the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharide Psl. Composed of a pentameric repeating unit of mannose, glucose, and rhamnose, the biosynthesis of Psl is proposed to occur via a Wzx/Wzy-dependent mechanism. Previous genetic studies have shown that the putative glycoside hydrolase PslG is essential for Psl biosynthesis. To understand the function of this protein, the apo-structure of the periplasmic domain of PslG (PslG(31-442)) and its complex with mannose were determined to 2...
November 20, 2015: Journal of Biological Chemistry
Michael A Liu, Thomas L Stent, Yaoqin Hong, Peter R Reeves
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...
May 2015: FEMS Microbiology Letters
Salim T Islam, Joseph S Lam
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...
November 2014: Canadian Journal of Microbiology
Małgorzata Marczak, Paulina Matysiak, Jolanta Kutkowska, Anna Skorupska
Production of extracellular polysaccharides is a complex process engaging proteins localized in different subcellular compartments, yet communicating with each other or even directly interacting in multicomponent complexes. Proteins involved in polymerization and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum are encoded within the chromosomal Pss-I cluster. However, genes implicated in polysaccharide synthesis are common in rhizobia, with several homologues of pss genes identified in other regions of the R...
2014: PloS One
Xiujuan Zhou, Bin Liu, Chunlei Shi, Xianming Shi
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...
2014: PloS One
K O Mironov, A E Platonov, E A Dunaeva, V I Kuseva, G A Shipulin
AIM: Development and application of real-time PCR (RT-PCR) procedure for determination of Streptococcus pneumoniae serotypes. MATERIALS AND METHODS: S. pneumoniae cps-locus wzx, wzy, wzz, wcwV and galU genes were chosen as PCR targets to select serotype-specific oligonucleotide primers and fluorescent labeled probes. 89 samples of cerebrospinal fluid (CSF) obtained in 2007 - 2010 from patients with pneumococcal meningitis diagnosis undergoing therapy in the Infectious Clinical Hospital No...
January 2014: Zhurnal Mikrobiologii, Epidemiologii, i Immunobiologii
Yaoqin Hong, Peter R Reeves
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...
May 2014: Journal of Bacteriology
Salim T Islam, Steven M Huszczynski, Timothy Nugent, Alexander C Gold, Joseph S Lam
O antigen (O-Ag) in many bacteria is synthesized via the Wzx/Wzy-dependent pathway in which Wzy polymerizes lipid-linked O-Ag subunits to modal lengths regulated by Wzz. Characterization of 83 site-directed mutants of Wzy from Pseudomonas aeruginosa PAO1 (WzyPa) in topologically-mapped periplasmic (PL) and cytoplasmic loops (CL) verified the functional importance of PL3 and PL5, with the former shown to require overall cationic properties. Essential Arg residues in the RX10G motifs of PL3 and PL5 were found to be conserved in putative homologues of WzyPa, as was the overall sequence homology between these two periplasmic loops in each protein...
2013: Scientific Reports
Zac D Paddock, David G Renter, Charley A Cull, Xiarong Shi, Jianfa Bai, Tiruvoor G Nagaraja
Escherichia coli O26 is second only to O157 in causing foodborne, Shiga toxin-producing E. coli (STEC) infections. Our objectives were to determine fecal prevalence and characteristics of E. coli O26 in commercial feedlot cattle (17,148) that were enrolled in a study to evaluate an E. coli O157:H7 siderophore receptor and porin (SRP(®)) vaccine (VAC) and a direct-fed microbial (DFM; 10(6) colony-forming units [CFU]/animal/day of Lactobacillus acidophilus and 10(9) CFU/animal/day of Propionibacterium freudenreichii)...
March 2014: Foodborne Pathogens and Disease
Salim T Islam, Paul D W Eckford, Michelle L Jones, Timothy Nugent, Christine E Bear, Christian Vogel, Joseph S Lam
Wzx flippases are crucial for bacterial cell surface polysaccharide assembly as they transport undecaprenyl pyrophosphate-linked sugar repeat units from the cytoplasmic to the periplasmic leaflets of the inner membrane (IM) for final assembly. Our recently reported three-dimensional (3D) model structure of Wzx from Pseudomonas aeruginosa PAO1 (WzxPa) displayed a cationic internal vestibule and functionally essential acidic amino acids within transmembrane segment bundles. Herein, we examined the intrinsic transport function of WzxPa following its purification and reconstitution in phospholipid liposomes...
September 10, 2013: MBio
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