The O antigen (Oag) component of LPS is a major virulence determinant. Oag is polymerized by Wzy, and its modal chain length is determined by Wzz and Wzz. Site-directed mutagenesis was performed on in pWaldo--TEV-GFP to alter Arg residues in Wzy’s two large periplasmic loops (PLs) (PL3 and PL5). Analysis of the LPS profiles conferred by mutated Wzy proteins in the deficient (Δ) strain identified residues that affect Wzy activity. The importance of the guanidium group of the Arg residues was investigated by altering the Arg residues to Lys and Glu, which generated Wzy mutants conferring altered LPS Oag modal chain lengths. The dependence of these Wzy mutants on Wzz was investigated by expressing them in a and deficient (Δ Δ ) strain. Comparison of the LPS profiles identified a role for the Arg residues in the association of Wzy and Wzz during Oag polymerization. Colicin E2 and bacteriophage Sf6c susceptibility supported this conclusion. Comparison of the expression levels of different mutant Wzy-GFPs with the wild-type Wzy-GFP showed that certain Arg residues affected production levels of Wzy in a Wzz-dependent manner. To our knowledge, this is the first report of Wzy mutants having an effect on LPS Oag modal chain length, and identified functionally significant Arg residues in Wzy.


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  1. Allison G. E., Verma N. K.(2000). Serotype-converting bacteriophages and O-antigen modification in Shigella flexneri. Trends Microbiol 8, 1723. [View Article][PubMed] [Google Scholar]
  2. Bardhan P., Faruque A. S., Naheed A., Sack D. A.(2010). Decrease in shigellosis-related deaths without Shigella spp.-specific interventions, Asia. Emerg Infect Dis 16, 17181723. [View Article][PubMed] [Google Scholar]
  3. Daniels C., Vindurampulle C., Morona R.(1998). Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy). Mol Microbiol 28, 12111222. [View Article][PubMed] [Google Scholar]
  4. Islam S. T., Gold A. C., Taylor V. L., Anderson E. M., Ford R. C., Lam J. S.(2011). Dual conserved periplasmic loops possess essential charge characteristics that support a catch-and-release mechanism of O-antigen polymerization by Wzy in Pseudomonas aeruginosa PAO1. J Biol Chem 286, 2060020605. [View Article][PubMed] [Google Scholar]
  5. Islam S. T., Huszczynski S. M., Nugent T., Gold A. C., Lam J. S.(2013). Conserved-residue mutations in Wzy affect O-antigen polymerization and Wzz-mediated chain-length regulation in Pseudomonas aeruginosa PAO1. Sci Rep 3, 3441. [View Article][PubMed] [Google Scholar]
  6. Jennison A. V., Verma N. K.(2004). Shigella flexneri infection: pathogenesis and vaccine development. FEMS Microbiol Rev 28, 4358. [View Article][PubMed] [Google Scholar]
  7. Knirel Y. A., Bystrova O. V., Kocharova N. A., Zähringer U., Pier G. B.(2006). Conserved and variable structural features in the lipopolysaccharide of Pseudomonas aeruginosa. J Endotoxin Res 12, 324336. [View Article][PubMed] [Google Scholar]
  8. Lindberg A. A., Wollin R., Gemski P., Wohlhieter J. A.(1978). Interaction between bacteriophage Sf6 and Shigella flexneri. J Virol 27, 3844.[PubMed] [Google Scholar]
  9. Liu D., Cole R. A., Reeves P. R.(1996). An O-antigen processing function for Wzx (RfbX): a promising candidate for O-unit flippase. J Bacteriol 178, 21022107.[PubMed] [Google Scholar]
  10. Marolda C. L., Tatar L. D., Alaimo C., Aebi M., Valvano M. A.(2006). Interplay of the Wzx translocase and the corresponding polymerase and chain length regulator proteins in the translocation and periplasmic assembly of lipopolysaccharide O antigen. J Bacteriol 188, 51245135. [View Article][PubMed] [Google Scholar]
  11. Mavris M., Manning P. A., Morona R.(1997). Mechanism of bacteriophage SfII-mediated serotype conversion in Shigella flexneri. Mol Microbiol 26, 939950. [View Article][PubMed] [Google Scholar]
  12. McKinney J., Guerrier-Takada C., Galán J., Altman S.(2002). Tightly regulated gene expression system in Salmonella enterica serovar Typhimurium. J Bacteriol 184, 60566059. [View Article][PubMed] [Google Scholar]
  13. Morona R., Van Den Bosch L.(2003). Lipopolysaccharide O antigen chains mask IcsA (VirG) in Shigella flexneri. FEMS Microbiol Lett 221, 173180. [View Article][PubMed] [Google Scholar]
  14. Morona R., Mavris M., Fallarino A., Manning P. A.(1994). Characterization of the rfc region of Shigella flexneri. J Bacteriol 176, 733747.[PubMed] [Google Scholar]
  15. Morona R., van den Bosch L., Manning P. A.(1995). Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri. J Bacteriol 177, 10591068.[PubMed] [Google Scholar]
  16. Morona R., Van Den Bosch L., Daniels C.(2000). Evaluation of Wzz/MPA1/MPA2 proteins based on the presence of coiled-coil regions. Microbiology 146, 14.[PubMed] [Google Scholar]
  17. Morona R., Daniels C., Van Den Bosch L.(2003). Genetic modulation of Shigella flexneri 2a lipopolysaccharide O antigen modal chain length reveals that it has been optimized for virulence. Microbiology 149, 925939. [View Article][PubMed] [Google Scholar]
  18. Murray G. L., Attridge S. R., Morona R.(2003). Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz. Mol Microbiol 47, 13951406. [View Article][PubMed] [Google Scholar]
  19. Nath P., Tran E. N., Morona R.(2015). Mutational analysis of the Shigella flexneri O-antigen polymerase Wzy: identification of Wzz-dependent Wzy mutants. J Bacteriol 197, 108119. [View Article][PubMed] [Google Scholar]
  20. Raetz C. R., Whitfield C.(2002). Lipopolysaccharide endotoxins. Annu Rev Biochem 71, 635700. [View Article][PubMed] [Google Scholar]
  21. Ruiz N., Gronenberg L. S., Kahne D., Silhavy T. J.(2008). Identification of two inner-membrane proteins required for the transport of lipopolysaccharide to the outer membrane of Escherichia coli. Proc Natl Acad Sci U S A 105, 55375542. [View Article][PubMed] [Google Scholar]
  22. Sperandeo P., Dehò G., Polissi A.(2009). The lipopolysaccharide transport system of Gram-negative bacteria. Biochim Biophys Acta 1791, 594602. [View Article][PubMed] [Google Scholar]
  23. Stagg R. M., Tang S. S., Carlin N. I., Talukder K. A., Cam P. D., Verma N. K.(2009). A novel glucosyltransferase involved in O-antigen modification of Shigella flexneri serotype 1c. J Bacteriol 191, 66126617. [View Article][PubMed] [Google Scholar]
  24. Sun Q., Knirel Y. A., Lan R., Wang J., Senchenkova S. N., Jin D., Shashkov A. S., Xia S., Perepelov A. V. et al.(2012). A novel plasmid-encoded serotype conversion mechanism through addition of phosphoethanolamine to the O-antigen of Shigella flexneri. PLoS ONE 7, e46095. [View Article][PubMed] [Google Scholar]
  25. Sun Q., Lan R., Wang Y., Wang J., Wang Y., Li P., Du P., Xu J.(2013). Isolation and genomic characterization of SfI, a serotype-converting bacteriophage of Shigella flexneri. BMC Microbiol 13, 39. [View Article][PubMed] [Google Scholar]
  26. Taylor V. L., Udaskin M. L., Islam S. T., Lam J. S.(2013). The D3 bacteriophage α-polymerase inhibitor (Iap) peptide disrupts O-antigen biosynthesis through mimicry of the chain length regulator Wzz in Pseudomonas aeruginosa. J Bacteriol 195, 47354741. [View Article][PubMed] [Google Scholar]
  27. Tocilj A., Munger C., Proteau A., Morona R., Purins L., Ajamian E., Wagner J., Papadopoulos M., Van Den Bosch L. et al.(2008). Bacterial polysaccharide co-polymerases share a common framework for control of polymer length. Nat Struct Mol Biol 15, 130138. [View Article][PubMed] [Google Scholar]
  28. Tran E. N., Papadopoulos M., Morona R.(2014). Relationship between O-antigen chain length and resistance to colicin E2 in Shigella flexneri. Microbiology 160, 589601. [View Article][PubMed] [Google Scholar]
  29. van der Ley P., de Graaff P., Tommassen J.(1986). Shielding of Escherichia coli outer membrane proteins as receptors for bacteriophages and colicins by O-antigenic chains of lipopolysaccharide. J Bacteriol 168, 449451.[PubMed] [Google Scholar]
  30. Waldo G. S., Standish B. M., Berendzen J., Terwilliger T. C.(1999). Rapid protein-folding assay using green fluorescent protein. Nat Biotechnol 17, 691695. [View Article][PubMed] [Google Scholar]
  31. Wang P. G., Guo H., Yi W., Song J. K.(2008). Current understanding on biosynthesis of microbial polysaccharides. Curr Top Med Chem 8, 141151. [View Article][PubMed] [Google Scholar]
  32. Wang X., Zhang C., Shi F., Hu X.(2010). Purification and characterization of lipopolysaccharides. Subcell Biochem 53, 2751. [View Article][PubMed] [Google Scholar]
  33. Woodward R., Yi W., Li L., Zhao G., Eguchi H., Sridhar P. R., Guo H., Song J. K., Motari E. et al.(2010). In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz. Nat Chem Biol 6, 418423. [View Article][PubMed] [Google Scholar]
  34. Zhao G., Wu B., Li L., Wang P. G.(2014). O-antigen polymerase adopts a distributive mechanism for lipopolysaccharide biosynthesis. Appl Microbiol Biotechnol 98, 40754081. [View Article][PubMed] [Google Scholar]

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