1887

Abstract

Regulation of the length of the O-antigen (Oag) chain attached to LPS in is important for virulence and is dependent on the inner-membrane protein Wzz. A lack of high-resolution structural data for Wzz has hampered efforts so far to correlate mutations affecting function of Wzz with structure and describe a mechanism for chain length regulation. Here we have used secondary structure prediction to show that the periplasmic domain of the Wzz protein has three regions of significant coiled-coil (CC) potential, two of which lie within an extended helical region. We describe here the first site-directed mutagenesis study to investigate the role of individual predicted CC regions (CCRs) in Wzz function and oligomerization. We found that CCRs 2 and 3 are necessary for wild-type Oag chain length regulation by Wzz. The cross-linking profile of mutants affected in the three CCRs was not altered, indicating that individually each CCR is not required for oligomerization. Interestingly, the CCR3 mutation resulted in a temperature-sensitive phenotype and an inhibitory effect on Oag polymerization. Analysis of Wzz and the mutant constructs in a mutant showed that DegP did not affect the function of wild-type Wzz but its presence influenced the phenotype of the Wzz CCR3 mutant. Additionally, the phenotype of the Wzz CCR3 mutant was suppressed by a mutation near the putative cytoplasmic C-terminus of Wzz.

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2008-04-01
2020-09-24
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References

  1. Bastin D. A., Stevenson G., Brown P. K., Haase A., Reeves P. R.. 1993; Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length. Mol Microbiol7:725–734
    [Google Scholar]
  2. Becker A., Pühler A.. 1998; Specific amino acid substitutions in the proline-rich motif of the Rhizobium meliloti ExoP protein result in enhanced production of low-molecular-weight succinoglycan at the expense of high-molecular-weight succinoglycan. J Bacteriol180:395–399
    [Google Scholar]
  3. Becker A., Niehaus K., Pühler A.. 1995; Low-molecular-weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterized by a periplasmic N-terminal domain and a missing C-terminal domain. Mol Microbiol16:191–203
    [Google Scholar]
  4. Bengoechea J. A., Zhang L., Toivanen P., Skurnik M.. 2002; Regulatory network of lipopolysaccharide O-antigen biosynthesis in Yersinia enterocolitica includes cell envelope-dependent signals. Mol Microbiol44:1045–1062
    [Google Scholar]
  5. Burkhard P., Stetefeld J., Strelkov S. V.. 2001; Coiled coils: a highly versatile protein folding motif. Trends Cell Biol11:82–88
    [Google Scholar]
  6. Carter J. A., Blondel C. J., Zaldívar M., Álvarez S. A., Marolda C. L., Valvano M. A., Contreras I.. 2007; O-antigen modal chain length in Shigella flexneri 2a is growth-regulated through RfaH-mediated transcriptional control of the wzy gene. Microbiology153:3499–3507
    [Google Scholar]
  7. Clausen T., Southan C., Ehrmann M.. 2002; The HtrA family of proteases: implications for protein composition and cell fate. Mol Cell10:443–455
    [Google Scholar]
  8. Collins R. F., Beis K., Clarke B. R., Ford R. C., Hulley M., Naismith J. H., Whitfield C.. 2006; Periplasmic protein-protein contacts in the inner membrane protein Wzc form a tetrameric complex required for the assembly of Escherichia coli group 1 capsules. J Biol Chem281:2144–2150
    [Google Scholar]
  9. Daines D. A., Silver R. P.. 2000; Evidence for multimerization of Neu proteins involved in polysialic acid synthesis in Escherichia coli K1 using improved LexA-based vectors. J Bacteriol182:5267–5270
    [Google Scholar]
  10. Danese P. N., Snyder W. B., Cosma C. L., Davis L. J., Silhavy T. J.. 1995; The Cpx two-component signal transduction pathway of Escherichia coli regulates transcription of the gene specifying the stress-inducible periplasmic protease, DegP. Genes Dev9:387–398
    [Google Scholar]
  11. Daniels C.. 1999; Characterisation of proteins involved in Shigella flexneri O-antigen biosynthesis PhD thesis Department of Microbiology and Immunology, University of Adelaide;
    [Google Scholar]
  12. Daniels C., Morona R.. 1999; Analysis of Shigella flexneri Wzz (Rol) function by mutagenesis and cross-linking: Wzz is able to oligomerize. Mol Microbiol34:181–194
    [Google Scholar]
  13. Daniels C., Vindurampulle C., Morona R.. 1998; Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy. Mol Microbiol28:1211–1222
    [Google Scholar]
  14. Daniels C., Griffiths C., Cowles B., Lam J. S.. 2002; Pseudomonas aeruginosa O-antigen chain length is determined before ligation to lipid A core. Environ Microbiol4:883–897
    [Google Scholar]
  15. Datsenko K. A., Wanner B. L.. 2000; One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A97:6640–6645
    [Google Scholar]
  16. Dmitrova M., Younès-Cauet G., Oertel-Buchheit P., Porte D., Schnarr M., Granger-Schnarr M.. 1998; A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli. Mol Gen Genet257:205–212
    [Google Scholar]
  17. Donnenberg M. S., Kaper J. B.. 1991; Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun59:4310–4317
    [Google Scholar]
  18. Franco A. V., Liu D., Reeves P. R.. 1998; The Wzz (Cld) protein in Escherichia coli: amino acid sequence variation determines O-antigen chain length specificity. J Bacteriol180:2670–2675
    [Google Scholar]
  19. Guo H., Lokko K., Zhang Y., Yi W., Wu Z., Wang P. G.. 2006; Overexpression and characterization of Wzz of Escherichia coli O86 : H2. Protein Expr Purif48:49–55
    [Google Scholar]
  20. Hong M., Payne S. M.. 1997; Effect of mutations in Shigella flexneri chromosomal and plasmid-encoded lipopolysaccharide genes on invasion and serum resistance. Mol Microbiol24:779–791
    [Google Scholar]
  21. Karimova G., Pidoux J., Ullmann A., Ladant D.. 1998; A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A95:5752–5756
    [Google Scholar]
  22. Klee S. R., Tzschaschel B. D., Timmis K. N., Guzman C. A.. 1997; Influence of different rol gene products on the chain length of Shigella dysenteriae type 1 lipopolysaccharide O antigen expressed by Shigella flexneri carrier strains. J Bacteriol179:2421–2425
    [Google Scholar]
  23. Lipińska B., Sharma S., Georgopoulos C.. 1988; Sequence analysis and regulation of the htrA gene of Escherichia coli: a σ32-independent mechanism of heat-inducible transcription. Nucleic Acids Res16:10053–10067
    [Google Scholar]
  24. Lipinska B., Fayet O., Baird L., Georgopoulos C.. 1989; Identification, characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures. J Bacteriol171:1574–1584
    [Google Scholar]
  25. Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L.. 1975; Electrophoretic resolution of the “major outer membrane protein” of Escherichia coli K12 into four bands. FEBS Lett58:254–258
    [Google Scholar]
  26. Lupas A.. 1996a; Coiled coils: new structures and new functions. Trends Biochem Sci21:375–382
    [Google Scholar]
  27. Lupas A.. 1996b; Prediction and analysis of coiled-coil structures. Methods Enzymol266:513–525
    [Google Scholar]
  28. Lupas A., Van Dyke M., Stock J.. 1991; Predicting coiled coils from protein sequences. Science252:1162–1164
    [Google Scholar]
  29. 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 Bacteriol188:5124–5135
    [Google Scholar]
  30. 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 Bacteriol177:1059–1068
    [Google Scholar]
  31. Morona J. K., Paton J. C., Miller D. C., Morona R.. 2000a; Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in Streptococcus pneumoniae. Mol Microbiol35:1431–1442
    [Google Scholar]
  32. Morona R., Van Den Bosch L., Daniels C.. 2000b; Evaluation of Wzz/MPA1/MPA2 proteins based on the presence of coiled-coil regions. Microbiology146:1–4
    [Google Scholar]
  33. 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. Microbiology149:925–939
    [Google Scholar]
  34. 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 Microbiol47:1395–1406
    [Google Scholar]
  35. Murray G. L., Attridge S. R., Morona R.. 2006; Altering the length of the lipopolysaccharide O antigen has an impact on the interaction of Salmonella enterica serovar Typhimurium with macrophages and complement. J Bacteriol188:2735–2739
    [Google Scholar]
  36. Prossnitz E., Nikaido K., Ulbrich S. J., Ames G. F.. 1988; Formaldehyde and photoactivatable cross-linking of the periplasmic binding protein to a membrane component of the histidine transport system of Salmonella typhimurium. J Biol Chem263:17917–17920
    [Google Scholar]
  37. Purdy G. E., Hong M., Payne S. M.. 2002; Shigella flexneri DegP facilitates IcsA surface expression and is required for efficient intercellular spread. Infect Immun70:6355–6364
    [Google Scholar]
  38. Purdy G. E., Fisher C. R., Payne S. M.. 2007; IcsA surface presentation in Shigella flexneri requires the periplasmic chaperones DegP, Skp, and SurA. J Bacteriol189:5566–5573
    [Google Scholar]
  39. Raetz C. R., Whitfield C.. 2002; Lipopolysaccharide endotoxins. Annu Rev Biochem71:635–700
    [Google Scholar]
  40. Rost B., Yachdav G., Liu J.. 2004; The PredictProtein server. Nucleic Acids Res32:W321–W326
    [Google Scholar]
  41. Salamitou S., Lemaire M., Fujino T., Ohayon H., Gounon P., Béguin P., Aubert J. P.. 1994; Subcellular localization of Clostridium thermocellum ORF3p, a protein carrying a receptor for the docking sequence borne by the catalytic components of the cellulosome. J Bacteriol176:2828–2834
    [Google Scholar]
  42. Skórko-Glonek J., Wawrzynów A., Krzewski K., Kurpierz K., Lipińska B.. 1995; Site-directed mutagenesis of the HtrA (DegP) serine protease, whose proteolytic activity is indispensable for Escherichia coli survival at elevated temperatures. Gene163:47–52
    [Google Scholar]
  43. Skórko-Glonek J., Laskowska E., Sobiecka-Szkatula A., Lipińska B.. 2007; Characterization of the chaperone-like activity of HtrA (DegP) protein from Escherichia coli under the conditions of heat shock. Arch Biochem Biophys464:80–89
    [Google Scholar]
  44. Sperandeo P., Cescutti R., Villa R., Di Benedetto C., Candia D., Dehò G., Polissi A.. 2007; Characterization of lptA and lptB, two essential genes implicated in lipopolysaccharide transport to the outer membrane of Escherichia coli. J Bacteriol189:244–253
    [Google Scholar]
  45. Spiess C., Beil A., Ehrmann M.. 1999; A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell97:339–347
    [Google Scholar]
  46. Stevenson G., Kessler A., Reeves P. R.. 1995; A plasmid-borne O-antigen chain length determinant and its relationship to other chain length determinants. FEMS Microbiol Lett125:23–30
    [Google Scholar]
  47. Strauch K. L., Beckwith J.. 1988; An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins. Proc Natl Acad Sci U S A85:1576–1580
    [Google Scholar]
  48. Strauch K. L., Johnson K., Beckwith J.. 1989; Characterization of degP, a gene required for proteolysis in the cell envelope and essential for growth of Escherichia coli at high temperature. J Bacteriol171:2689–2696
    [Google Scholar]
  49. Tang K. H., Guo H., Yi W., Tsai M. D., Wang P. G.. 2007; Investigation of the conformational states of Wzz and the Wzz.O-antigen complex under near-physiological conditions. Biochemistry46:11744–11752
    [Google Scholar]
  50. Valvano M. A.. 2003; Export of O-specific lipopolysaccharide. Front Biosci8:s452–s471
    [Google Scholar]
  51. Vincent C., Doublet P., Grangeasse C., Vaganay E., Cozzone A. J., Duclos B.. 1999; Cells of Escherichia coli contain a protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb. J Bacteriol181:3472–3477
    [Google Scholar]
  52. Whitfield C., Amor P. A., Köplin R.. 1997; Modulation of the surface architecture of Gram-negative bacteria by the action of surface polymer : lipid A-core ligase and by determinants of polymer chain length. Mol Microbiol23:629–638
    [Google Scholar]
  53. Wu T., McCandlish A. C., Gronenberg L. S., Chng S. S., Silhavy T. J., Kahne D.. 2006; Identification of a protein complex that assembles lipopolysaccharide in the outer membrane of Escherichia coli. Proc Natl Acad Sci U S A103:11754–11759
    [Google Scholar]
  54. Wugeditsch T., Paiment A., Hocking J., Drummelsmith J., Forrester C., Whitfield C.. 2001; Phosphorylation of Wzc, a tyrosine autokinase, is essential for assembly of group 1 capsular polysaccharides in Escherichia coli. J Biol Chem276:2361–2371
    [Google Scholar]
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