1887

Abstract

Mucoid strains of that overproduce alginate are associated with chronic pulmonary disease (e.g. cystic fibrosis). Mutants defective in one of several periplasmic proteins (AlgKGX) for alginate secretion release alginate fragments due to the activity of an alginate lyase (AlgL) in the periplasm, which cleaves the newly formed polymers. However, mutants defective in Alg8 or Alg44 did not secrete polymer or alginate fragments, suggesting that both these membrane proteins have a role in the polymerization reaction. A model for the membrane topology of Alg8, a glycosyltransferase (GT), was constructed using PhoA fusions. This provided evidence for a large cytoplasmic loop containing the active domains predicted for -GTs such as Alg8 and five transmembrane (TM) domains, one of which resembles a cleavable signal peptide. The C-terminal TM domain of Alg8 was critical for the polymerization reaction . Alanine substitution mutagenesis showed that all of the predicted active site residues in the widely spaced D, DxD, D, LxxRW motif were required for polymerization activity , and two of these substitutions also affected Alg8 protein stability. A membrane topology model for Alg44 was also constructed using PhoA fusions, and this showed a central TM domain and predicted an N-terminal TM domain that may be a membrane anchor. An N-terminal PilZ domain in Alg44 for c-di-GMP [bis-(3′,5′)-cyclic dimeric GMP] binding, which is required for alginate synthesis, was localized to the cytoplasmic loop. The long periplasmic C terminus of Alg44 contains a region similar to membrane fusion proteins (MFPs) of multi-drug efflux systems, which predicts the possibility of its interaction with another protein in this compartment. A Western blot analysis of the outer-membrane porin AlgE showed reduced AlgE levels in the mutant, whereas expression of Alg44 restored AlgE within the cell. C-terminal truncations of Alg44 as small as 24 amino acids blocked alginate polymerization , indicating a critical role for the MFP domain. These studies suggest that Alg44 may act as a co-polymerase in concert with Alg8, the major GT, and that both inner-membrane proteins are required for the polymerization reaction leading to alginate production.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/015305-0
2008-06-01
2019-10-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/6/1605.html?itemId=/content/journal/micro/10.1099/mic.0.2007/015305-0&mimeType=html&fmt=ahah

References

  1. Barny, M. A., Schoonejans, E., Economou, A., Johnston, A. W. & Downie, J. A. ( 1996; ). The C-terminal domain of the Rhizobium leguminosarum chitin synthase NodC is important for function and determines the orientation of the N-terminal region in the inner membrane. Mol Microbiol 19, 443–453.[CrossRef]
    [Google Scholar]
  2. Boucher, J. C., Martinez-Salazar, J., Schurr, M. J., Mudd, M. H., Yu, H. & Deretic, V. ( 1996; ). Two distinct loci affecting conversion to mucoidy in Pseudomonas aeruginosa in cystic fibrosis encode homologs of the serine protease HtrA. J Bacteriol 178, 511–523.
    [Google Scholar]
  3. Brickman, E. & Beckwith, J. ( 1975; ). Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and phi80 transducing phages. J Mol Biol 96, 307–316.[CrossRef]
    [Google Scholar]
  4. Campbell, J. A., Davies, G. J., Bulone, V. & Henrissat, B. ( 1997; ). A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities. Biochem J 326, 929–939.
    [Google Scholar]
  5. DeVries, C. A. & Ohman, D. E. ( 1994; ). Mucoid to nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in algT, encoding a putative alternative sigma factor, and shows evidence for autoregulation. J Bacteriol 176, 6677–6687.
    [Google Scholar]
  6. Elkins, C. A. & Nikaido, H. ( 2003; ). Chimeric analysis of AcrA function reveals the importance of its C-terminal domain in its interaction with the AcrB multidrug efflux pump. J Bacteriol 185, 5349–5356.[CrossRef]
    [Google Scholar]
  7. Forsee, W. T., Cartee, R. T. & Yother, J. ( 2000; ). Biosynthesis of type 3 capsular polysaccharide in Streptococcus pneumoniae. Enzymatic chain release by an abortive translocation process. J Biol Chem 275, 25972–25978.[CrossRef]
    [Google Scholar]
  8. Franklin, M. J. & Ohman, D. E. ( 2002; ). Mutant analysis and cellular localization of the AlgI, AlgJ, and AlgF proteins required for O acetylation of alginate in Pseudomonas aeruginosa. J Bacteriol 184, 3000–3007.[CrossRef]
    [Google Scholar]
  9. Franklin, M. J., Chitnis, C. E., Gacesa, P., Sonesson, A., White, D. C. & Ohman, D. E. ( 1994; ). Pseudomonas aeruginosa AlgG is a polymer level alginate C5-mannuronan epimerase. J Bacteriol 176, 1821–1830.
    [Google Scholar]
  10. Garinot-Schneider, C., Lellouch, A. C. & Geremia, R. A. ( 2000; ). Identification of essential amino acid residues in the Sinorhizobium meliloti glucosyltransferase ExoM. J Biol Chem 275, 31407–31413.[CrossRef]
    [Google Scholar]
  11. Gilligan, P. H. ( 1991; ). Microbiology of airway disease in patients with cystic fibrosis. Clin Microbiol Rev 4, 35–51.
    [Google Scholar]
  12. Gutierrez, C. & Devedjian, J. C. ( 1989; ). A plasmid facilitating in vitro construction of phoA gene fusions in Escherichia coli. Nucleic Acids Res 17, 3999 [CrossRef]
    [Google Scholar]
  13. Haardt, M. & Bremer, E. ( 1996; ). Use of phoA and lacZ fusions to study the membrane topology of ProW, a component of the osmoregulated ProU transport system of Escherichia coli. J Bacteriol 178, 5370–5381.
    [Google Scholar]
  14. Jain, S. & Ohman, D. E. ( 1998; ). Deletion of algK in mucoid Pseudomonas aeruginosa blocks alginate polymer formation and results in uronic acid secretion. J Bacteriol 180, 634–641.
    [Google Scholar]
  15. Jain, S. & Ohman, D. E. ( 2004; ). Alginate biosynthesis. In Pseudomonas, pp. 53–81. Edited by J.-L. Ramos. New York: Kluwer Academic/Plenum Publishers.
  16. Jain, S. & Ohman, D. E. ( 2005; ). Role of an alginate lyase for alginate transport in mucoid Pseudomonas aeruginosa. Infect Immun 73, 6429–6436.[CrossRef]
    [Google Scholar]
  17. Jain, S., Franklin, M. J., Ertesvag, H., Valla, S. & Ohman, D. E. ( 2003; ). The dual roles of AlgG in C-5-epimerization and secretion of alginate polymers in Pseudomonas aeruginosa. Mol Microbiol 47, 1123–1133.[CrossRef]
    [Google Scholar]
  18. Johnson, J. M. & Church, G. M. ( 1999; ). Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efflux pumps. J Mol Biol 287, 695–715.[CrossRef]
    [Google Scholar]
  19. Keenleyside, W. J., Clarke, A. J. & Whitfield, C. ( 2001; ). Identification of residues involved in catalytic activity of the inverting glycosyl transferase WbbE from Salmonella enterica serovar borreze. J Bacteriol 183, 77–85.[CrossRef]
    [Google Scholar]
  20. Knutson, C. A. & Jeanes, A. ( 1968; ). A new modification of the carbazole analysis: application to heteropolysaccharides. Anal Biochem 24, 470–481.[CrossRef]
    [Google Scholar]
  21. Lobsanov, Y. D., Romero, P. A., Sleno, B., Yu, B., Yip, P., Herscovics, A. & Howell, P. L. ( 2004; ). Structure of Kre2p/Mnt1p: a yeast α1,2-mannosyltransferase involved in mannoprotein biosynthesis. J Biol Chem 279, 17921–17931.[CrossRef]
    [Google Scholar]
  22. Maharaj, R., May, T. B., Wang, S. K. & Chakrabarty, A. M. ( 1993; ). Sequence of the alg8 and alg44 genes involved in the synthesis of alginate by Pseudomonas aeruginosa. Gene 136, 267–269.[CrossRef]
    [Google Scholar]
  23. Manoil, C., Boyd, D. & Beckwith, J. ( 1988; ). Molecular genetic analysis of membrane protein topology. Trends Genet 4, 223–226.[CrossRef]
    [Google Scholar]
  24. Mejia-Ruiz, H., Guzman, J., Moreno, S., Soberon-Chavez, G. & Espin, G. ( 1997; ). The Azotobacter vinelandii alg8 and alg44 genes are essential for alginate synthesis and can be transcribed from an algD-independent promoter. Gene 199, 271–277.[CrossRef]
    [Google Scholar]
  25. Merighi, M., Lee, V. T., Hyodo, M., Hayakawa, Y. & Lory, S. ( 2007; ). The second messenger bis-(3′-5′)-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa. Mol Microbiol 65, 876–895.[CrossRef]
    [Google Scholar]
  26. Nehme, D., Li, X. Z., Elliot, R. & Poole, K. ( 2004; ). Assembly of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa: identification and characterization of mutations in mexA compromising MexA multimerization and interaction with MexB. J Bacteriol 186, 2973–2983.[CrossRef]
    [Google Scholar]
  27. Ohman, D. E. & Chakrabarty, A. M. ( 1981; ). Genetic mapping of chromosomal determinants for the production of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate. Infect Immun 33, 142–148.
    [Google Scholar]
  28. Pier, G. B., Coleman, F., Grout, M., Franklin, M. & Ohman, D. E. ( 2001; ). Role of alginate O acetylation in resistance of mucoid Pseudomonas aeruginosa to opsonic phagocytosis. Infect Immun 69, 1895–1901.[CrossRef]
    [Google Scholar]
  29. Pugsley, A. P. ( 1989; ). Early stages in the secretory pathway. In Protein Targeting, chapter III, pp. 45–111. Edited by A. P. Pugsley. San Diego: Academic Press.
  30. Rehm, B. H., Boheim, G., Tommassen, J. & Winkler, U. K. ( 1994; ). Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties. J Bacteriol 176, 5639–5647.
    [Google Scholar]
  31. Remminghorst, U. & Rehm, B. H. ( 2006a; ). In vitro alginate polymerization and the functional role of Alg8 in alginate production by Pseudomonas aeruginosa. Appl Environ Microbiol 72, 298–305.[CrossRef]
    [Google Scholar]
  32. Remminghorst, U. & Rehm, B. H. ( 2006b; ). Alg44, a unique protein required for alginate biosynthesis in Pseudomonas aeruginosa. FEBS Lett 580, 3883–3888.[CrossRef]
    [Google Scholar]
  33. Robles-Price, A., Wong, T. Y., Sletta, H., Valla, S. & Schiller, N. L. ( 2004; ). AlgX is a periplasmic protein required for alginate biosynthesis in Pseudomonas aeruginosa. J Bacteriol 186, 7369–7377.[CrossRef]
    [Google Scholar]
  34. Romling, U., Gomelsky, M. & Galperin, M. Y. ( 2005; ). C-di-GMP: the dawning of a novel bacterial signalling system. Mol Microbiol 57, 629–639.[CrossRef]
    [Google Scholar]
  35. Roychoudhury, S., May, T., Gill, J., Singh, S., Feingold, D. & Chakrabarty, A. ( 1989; ). Purification and characterization of guanosine diphospho-d-mannose dehydrogenase. A key enzyme in the biosynthesis of alginate by Pseudomonas aeruginosa. J Biol Chem 264, 9380–9385.
    [Google Scholar]
  36. Saxena, I. M. & Brown, R. M., Jr ( 1997; ). Identification of cellulose synthase(s) in higher plants: sequence analysis of processive β-glycosyltransferases with the common motif ‘D,D,D35Q(R,Q)XRW’. Cellulose 4, 33–49.[CrossRef]
    [Google Scholar]
  37. Saxena, I. M., Lin, F. C. & Brown, R. M., Jr ( 1990; ). Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum. Plant Mol Biol 15, 673–683.[CrossRef]
    [Google Scholar]
  38. Saxena, I. M., Brown, R. M., Fevre, M., Geremia, R. A. & Henrissat, B. ( 1995; ). Multidomain architecture of β-glycosyl transferases: implications for mechanism of action. J Bacteriol 177, 1419–1424.
    [Google Scholar]
  39. Saxena, I. M., Brown, R. M., Jr & Dandekar, T. ( 2001; ). Structure-function characterization of cellulose synthase: relationship to other glycosyltransferases. Phytochemistry 57, 1135–1148.[CrossRef]
    [Google Scholar]
  40. Schiller, N. L., Monday, S. R., Boyd, C. M., Keen, N. T. & Ohman, D. E. ( 1993; ). Characterization of the Pseudomonas aeruginosa alginate lyase gene (algL): cloning, sequencing, and expression in Escherichia coli. J Bacteriol 175, 4780–4789.
    [Google Scholar]
  41. Schweizer, H. P. ( 1992; ). Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol 6, 1195–1204.[CrossRef]
    [Google Scholar]
  42. Shinabarger, D., Berry, A., May, T. B., Rothmel, R., Fialho, A. & Chakrabarty, A. M. ( 1991; ). Purification and characterization of phosphomannose isomerase-guanosine diphospho-d-mannose pyrophosphorylase – a bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa. J Biol Chem 266, 2080–2088.
    [Google Scholar]
  43. Tikhonova, E. B., Wang, Q. & Zgurskaya, H. I. ( 2002; ). Chimeric analysis of the multicomponent multidrug efflux transporters from gram-negative bacteria. J Bacteriol 184, 6499–6507.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/015305-0
Loading
/content/journal/micro/10.1099/mic.0.2007/015305-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error