1887

Abstract

is highly resistant to antimicrobial peptides and we hypothesized that the conversion of UDP-glucose to UDP-glucuronic acid, a reaction catalysed by the enzyme UDP-glucose dehydrogenase (Ugd) would be important for this resistance. The genome of contains three predicted genes: , and , all of which were individually inactivated. Only inactivation of resulted in increased sensitivity to polymyxin B and this sensitivity could be overcome when either or but not was expressed from plasmids. The growth of a conditional mutant, created in the Δ background, was significantly impaired under non-permissive conditions. Growth could be rescued by either or expressed , but not by . Biochemical analysis of the purified, recombinant forms of Ugd and Ugd revealed that they are soluble homodimers with similar Ugd activity and comparable kinetic constants for their substrates UDP-glucose and NAD. Purified Ugd showed no Ugd activity. Real-time PCR analysis showed that the expression of was 5.4- and 135-fold greater than that of and , respectively. Together, these data indicate that the combined activity of Ugd and Ugd is essential for the survival of but only the most highly expressed gene, , is required for polymyxin B resistance.

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2009-06-01
2019-10-13
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References

  1. Aaron, S. D., Ferris, W., Henry, D. A., Speert, D. P. & Macdonald, N. E. ( 2000; ). Multiple combination bactericidal antibiotic testing for patients with cystic fibrosis infected with Burkholderia cepacia. Am J Respir Crit Care Med 161, 1206–1212.[CrossRef]
    [Google Scholar]
  2. Aubert, D. F., Flannagan, R. S. & Valvano, M. A. ( 2008; ). A novel sensor kinase-response regulator hybrid controls biofilm formation and type VI secretion system activity in Burkholderia cenocepacia. Infect Immun 76, 1979–1991.[CrossRef]
    [Google Scholar]
  3. Bader, M. W., Sanowar, S., Daley, M. E., Schneider, A. R., Cho, U., Xu, W., Klevit, R. E., Le Moual, H. & Miller, S. I. ( 2005; ). Recognition of antimicrobial peptides by a bacterial sensor kinase. Cell 122, 461–472.[CrossRef]
    [Google Scholar]
  4. Balandreau, J., Viallard, V., Cournoyer, B., Coenye, T., Laevens, S. & Vandamme, P. ( 2001; ). Burkholderia cepacia genomovar III is a common plant-associated bacterium. Appl Environ Microbiol 67, 982–985.[CrossRef]
    [Google Scholar]
  5. Breazeale, S. D., Ribeiro, A. A. & Raetz, C. R. ( 2002; ). Oxidative decarboxylation of UDP-glucuronic acid in extracts of polymyxin-resistant Escherichia coli. Origin of lipid A species modified with 4-amino-4-deoxy-l-arabinose. J Biol Chem 277, 2886–2896.[CrossRef]
    [Google Scholar]
  6. Brogden, K. A. ( 2005; ). Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3, 238–250.[CrossRef]
    [Google Scholar]
  7. Burns, J. L., Wadsworth, C. D., Barry, J. J. & Goodall, C. P. ( 1996; ). Nucleotide sequence analysis of a gene from Burkholderia (Pseudomonas) cepacia encoding an outer membrane lipoprotein involved in multiple antibiotic resistance. Antimicrob Agents Chemother 40, 307–313.
    [Google Scholar]
  8. Campbell, R. E., Mosimann, S. C., van De Rijn, I., Tanner, M. E. & Strynadka, N. C. ( 2000; ). The first structure of UDP-glucose dehydrogenase reveals the catalytic residues necessary for the two-fold oxidation. Biochemistry 39, 7012–7023.[CrossRef]
    [Google Scholar]
  9. De Leon, G. P., Elowe, N. H., Koteva, K. P., Valvano, M. A. & Wright, G. D. ( 2006; ). An in vitro screen of bacterial lipopolysaccharide biosynthetic enzymes identifies an inhibitor of ADP-heptose biosynthesis. Chem Biol 13, 437–441.[CrossRef]
    [Google Scholar]
  10. Ernst, R. K., Yi, E. C., Guo, L., Lim, K. B., Burns, J. L., Hackett, M. & Miller, S. I. ( 1999; ). Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa. Science 286, 1561–1565.[CrossRef]
    [Google Scholar]
  11. Figurski, D. H. & Helinski, D. R. ( 1979; ). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76, 1648–1652.[CrossRef]
    [Google Scholar]
  12. Flannagan, R. S., Aubert, D., Kooi, C., Sokol, P. A. & Valvano, M. A. ( 2007; ). Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo. Infect Immun 75, 1679–1689.[CrossRef]
    [Google Scholar]
  13. Flannagan, R. S., Linn, T. & Valvano, M. A. ( 2008; ). A system for the construction of targeted unmarked gene deletions in the genus Burkholderia. Environ Microbiol 10, 1652–1660.[CrossRef]
    [Google Scholar]
  14. Ganz, T. ( 2003; ). Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 3, 710–720.[CrossRef]
    [Google Scholar]
  15. Ge, X., Penney, L. C., van de Rijn, I. & Tanner, M. E. ( 2004; ). Active site residues and mechanism of UDP-glucose dehydrogenase. Eur J Biochem 271, 14–22.
    [Google Scholar]
  16. Gold, R., Jin, E., Levison, H., Isles, A. & Fleming, P. C. ( 1983; ). Ceftazidime alone and in combination in patients with cystic fibrosis: lack of efficacy in treatment of severe respiratory infections caused by Pseudomonas cepacia. J Antimicrob Chemother 12 (Suppl A), 331–336.[CrossRef]
    [Google Scholar]
  17. Helander, I. M., Kilpelainen, I. & Vaara, M. ( 1994; ). Increased substitution of phosphate groups in lipopolysaccharides and lipid A of the polymyxin-resistant pmrA mutants of Salmonella typhimurium: a 31P-NMR study. Mol Microbiol 11, 481–487.[CrossRef]
    [Google Scholar]
  18. Hitchcock, P. J. & Brown, T. M. ( 1983; ). Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol 154, 269–277.
    [Google Scholar]
  19. Holden, M. T., Seth-Smith, H. M., Crossman, L. C., Sebaihia, M., Bentley, S. D., Cerdeño-Tárraga, A. M., Thomson, N. R., Bason, N., Quail, M. A. & other authors ( 2009; ). The genome of Burkholderia cenocepacia J2315, an epidemic pathogen of cystic fibrosis patients. J Bacteriol 191, 261–277.[CrossRef]
    [Google Scholar]
  20. Hung, R. J., Chien, H. S., Lin, R. Z., Lin, C. T., Vatsyayan, J., Peng, H. L. & Chang, H. Y. ( 2007; ). Comparative analysis of two UDP-glucose dehydrogenases in Pseudomonas aeruginosa PAO1. J Biol Chem 282, 17738–17748.[CrossRef]
    [Google Scholar]
  21. Isles, A., Maclusky, I., Corey, M., Gold, R., Prober, C., Fleming, P. & Levison, H. ( 1984; ). Pseudomonas cepacia infection in cystic fibrosis: an emerging problem. J Pediatr 104, 206–210.[CrossRef]
    [Google Scholar]
  22. Iwanicka-Nowicka, R., Zielak, A., Cook, A. M., Thomas, M. S. & Hryniewicz, M. M. ( 2007; ). Regulation of sulfur assimilation pathways in Burkholderia cenocepacia: identification of transcription factors CysB and SsuR and their role in control of target genes. J Bacteriol 189, 1675–1688.[CrossRef]
    [Google Scholar]
  23. Kox, L. F., Wösten, M. M. & Groisman, E. A. ( 2000; ). A small protein that mediates the activation of a two-component system by another two-component system. EMBO J 19, 1861–1872.[CrossRef]
    [Google Scholar]
  24. Loutet, S. A., Flannagan, R. S., Kooi, C., Sokol, P. A. & Valvano, M. A. ( 2006; ). A complete lipopolysaccharide inner core oligosaccharide is required for resistance of Burkholderia cenocepacia to antimicrobial peptides and bacterial survival in vivo. J Bacteriol 188, 2073–2080.[CrossRef]
    [Google Scholar]
  25. Mahenthiralingam, E., Coenye, T., Chung, J. W., Speert, D. P., Govan, J. R., Taylor, P. & Vandamme, P. ( 2000; ). Diagnostically and experimentally useful panel of strains from the Burkholderia cepacia complex. J Clin Microbiol 38, 910–913.
    [Google Scholar]
  26. Mahenthiralingam, E., Urban, T. A. & Goldberg, J. B. ( 2005; ). The multifarious, multireplicon Burkholderia cepacia complex. Nat Rev Microbiol 3, 144–156.[CrossRef]
    [Google Scholar]
  27. McPhee, J. B., Lewenza, S. & Hancock, R. E. ( 2003; ). Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa. Mol Microbiol 50, 205–217.[CrossRef]
    [Google Scholar]
  28. McPhee, J. B., Bains, M., Winsor, G., Lewenza, S., Kwasnicka, A., Brazas, M. D., Brinkman, F. S. L. & Hancock, R. E. W. ( 2006; ). Contribution of the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems to Mg2+-induced gene regulation in Pseudomonas aeruginosa. J Bacteriol 188, 3995–4006.[CrossRef]
    [Google Scholar]
  29. Miller, V. L. & Mekalanos, J. J. ( 1988; ). A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170, 2575–2583.
    [Google Scholar]
  30. Moreira, L. M., Videira, P. A., Sousa, S. A., Leitão, J. H., Cunha, M. V. & Sá-Correia, I. ( 2003; ). Identification and physical organization of the gene cluster involved in the biosynthesis of Burkholderia cepacia complex exopolysaccharide. Biochem Biophys Res Commun 312, 323–333.[CrossRef]
    [Google Scholar]
  31. Mouslim, C. & Groisman, E. A. ( 2003; ). Control of the Salmonella ugd gene by three two-component regulatory systems. Mol Microbiol 47, 335–344.[CrossRef]
    [Google Scholar]
  32. Nummila, K., Kilpelainen, I., Zähringer, U., Vaara, M. & Helander, I. M. ( 1995; ). Lipopolysaccharides of polymyxin B-resistant mutants of Escherichia coli are extensively substituted by 2-aminoethyl pyrophosphate and contain aminoarabinose in lipid A. Mol Microbiol 16, 271–278.[CrossRef]
    [Google Scholar]
  33. Ortega, X. P., Cardona, S. T., Brown, A. R., Loutet, S. A., Flannagan, R. S., Campopiano, D. J., Govan, J. R. & Valvano, M. A. ( 2007; ). A putative gene cluster for aminoarabinose biosynthesis is essential for Burkholderia cenocepacia viability. J Bacteriol 189, 3639–3644.[CrossRef]
    [Google Scholar]
  34. Patrzykat, A., Friedrich, C. L., Zhang, L., Mendoza, V. & Hancock, R. E. ( 2002; ). Sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in Escherichia coli. Antimicrob Agents Chemother 46, 605–614.[CrossRef]
    [Google Scholar]
  35. Raetz, C. R. & Whitfield, C. ( 2002; ). Lipopolysaccharide endotoxins. Annu Rev Biochem 71, 635–700.[CrossRef]
    [Google Scholar]
  36. Rossman, M. G. ( 1981; ). Evolution of glycolytic enzymes. Philos Trans R Soc Lond B Biol Sci 293, 191–203.[CrossRef]
    [Google Scholar]
  37. Silipo, A., Molinaro, A., Cescutti, P., Bedini, E., Rizzo, R., Parrilli, M. & Lanzetta, R. ( 2005; ). Complete structural characterization of the lipid A fraction of a clinical strain of B. cepacia genomovar I lipopolysaccharide. Glycobiology 15, 561–570.
    [Google Scholar]
  38. Strominger, J. L., Maxwell, E. S., Axelrod, J. & Kalckar, H. M. ( 1957; ). Enzymatic formation of uridine diphosphoglucuronic acid. J Biol Chem 224, 79–90.
    [Google Scholar]
  39. Turner, J., Cho, Y., Dinh, N. N., Waring, A. J. & Lehrer, R. I. ( 1998; ). Activities of LL-37, a cathelin-associated antimicrobial peptide of human neutrophils. Antimicrob Agents Chemother 42, 2206–2214.
    [Google Scholar]
  40. Vaara, M., Vaara, T., Jensen, M., Helander, I., Nurminen, M., Rietschel, E. T. & Mäkelä, P. H. ( 1981; ). Characterization of the lipopolysaccharide from the polymyxin-resistant pmrA mutants of Salmonella typhimurium. FEBS Lett 129, 145–149.[CrossRef]
    [Google Scholar]
  41. Wösten, M. M. S. M., Kox, L. F. F., Chamnongpol, S., Soncini, F. C. & Groisman, E. A. ( 2000; ). A signal transduction system that responds to extracellular iron. Cell 103, 113–125.[CrossRef]
    [Google Scholar]
  42. Zanetti, M. ( 2004; ). Cathelicidins, multifunctional peptides of the innate immunity. J Leukoc Biol 75, 39–48.
    [Google Scholar]
  43. Zhang, L., Parente, J., Harris, S. M., Woods, D. E., Hancock, R. E. & Falla, T. J. ( 2005; ). Antimicrobial peptide therapeutics for cystic fibrosis. Antimicrob Agents Chemother 49, 2921–2927.[CrossRef]
    [Google Scholar]
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