1887

Microbiology Society logo

Volume 146, Issue 11

Involvement of the locus in core oligosaccharide and O polysaccharide assembly in Free

Abstract

-Rhamnose (-Rha) is a component of the lipopolysaccharide (LPS) core, several O antigen polysaccharides, and the cell surface surfactant rhamnolipid of . In this study, four contiguous genes () responsible for the synthesis of dTDP--Rha in have been cloned and characterized. Non-polar chromosomal mutants were generated in strains PAO1 (serotype O5) and PAK (serotype O6) and LPS extracted from the mutants was analysed by SDS-PAGE and Western immunoblotting. mutants of both serotype O5 and serotype O6 synthesized a truncated core region which was unable to act as an attachment point for either A-band or B-band O antigen. A PAO1 double mutant (deficient in biosynthesis of both -Rha and -Rha) was constructed to facilitate structural analysis of the mutant core region. This strain has an incomplete core oligosaccharide region and does not produce A-band O antigen. These results provide the genetic and structural evidence that -Rha is the receptor on the LPS core for the attachment of O polysaccharides. This is the first report of a genetically defined mutation that affects the synthesis of a single sugar in the core oligosaccharide region of LPS, and provides further insight into the mechanisms of LPS biosynthesis and assembly in this bacterium.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): Gm, gentamicin , l-rhamnose , lipopolysaccharide , Pseudomonas aeruginosa , Rha, rhamnose , rmd and rml
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-146-11-2803
2000-11-01
2024-04-24
Loading full text...

Full text loading...

/deliver/fulltext/micro/146/11/1462803a.html?itemId=/content/journal/micro/10.1099/00221287-146-11-2803&mimeType=html&fmt=ahah

References

  1. Allard S. T., Giraud M. F., Whitfield C., Messner P., Naismith J. H. 2000; The purification, crystallization and structural elucidation of dTDP-d-glucose 4,6-dehydratase (RmlB), the second enzyme of the dTDP-l-rhamnose synthesis pathway from Salmonella enterica serovar Typhimurium. Acta Crystallogr Sect D Biol Crystallogr 56:222–225 [CrossRef]
     [Google Scholar]
  2. Arsenault T. L., Hughes D. W., MacLean D. B., Szarek W. A., Kropinski A. M. B., Lam J. S. 1991; Structural studies on the polysaccharide portion of ‘A-band’ lipopolysaccharide from a mutant (AK1401) of Pseudomonas aeruginosa strain PAO1. Can J Chem 69:1273–1280 [CrossRef]
     [Google Scholar]
  3. Bélanger M., Burrows L. L., Lam J. S. 1999; Functional analysis of genes responsible for the synthesis of B-band O antigen of Pseudomonas aeruginosa serotype O6 lipopolysaccharide. Microbiology 145:3505–3521
     [Google Scholar]
  4. Berry D., Kropinski A. M. 1986; Effect of lipopolysaccharide mutations and temperature on plasmid transformation efficiency in Pseudomonas aeruginosa. Can J Microbiol 32:436–438 [CrossRef]
     [Google Scholar]
  5. Blankenfeldt W., Giraud M. F., Leonard G., Rahim R., Creuzenet C., Lam J. S., Naismith J. H. 2000; The purification, crystallization and preliminary structural characterization of glucose-1-phosphate thymidyltransferase (RmlA), the first enzyme of the dTDP-l-rhamnose synthesis pathway from Pseudomonas aeruginosa. Acta Crystallogr Sect D Biol Crystallogr (in press)
    [Google Scholar]
  6. Burnette W. N. 1981; ‘Western blotting’: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112:195–203 [CrossRef]
     [Google Scholar]
  7. Ciucanu I., Kerek F. 1984; A simple and rapid method for the permethylation of carbohydrates. Carbohydr Res 131:209–217 [CrossRef]
     [Google Scholar]
  8. Fomsgaard A., Freudenberg M. A., Galanos C. 1990; Modification of the silver staining technique to detect lipopolysaccharide in polyacrylamide gels. J Clin Microbiol 28:2627–2631
     [Google Scholar]
  9. Giraud M. F., Gordon F. M., Whitfield C., Messner P., McMahon S. A., Naismith J. H. 1999a; Purification, crystallization and preliminary structural studies of dTDP-6-deoxy-d-xylo-4-hexulose 3,5-epimerase (RmlC), the third enzyme of the dTDP-l-rhamnose synthesis pathway, from Salmonella enterica serovar typhimurium. Acta Crystallogr Sect D Biol Crystallogr 55:706–708 [CrossRef]
     [Google Scholar]
  10. Giraud M. F., McMiken H. J., Leonard G. A., Messner P., Whitfield C., Naismith J. H. 1999b; Overexpression, purification, crystallization and preliminary structural study of dTDP-6-deoxy-l-lyxo-4-hexulose reductase (RmlD), the fourth enzyme of the dTDP-l-rhamnose synthesis pathway, from Salmonella enterica serovar Typhimurium. Acta Crystallogr Sect D Biol Crystallogr 55:2043–2046 [CrossRef]
     [Google Scholar]
  11. Hancock R. E. W., Carey A. M. 1979; Outer membrane of Pseudomonas aeruginosa: heat- and 2-mercaptoethanol-modifiable proteins. J Bacteriol 140:902–910
     [Google Scholar]
  12. Hausman B. S., Williamson J. A., Schreiner R. P., Lakshmidevi P., Gavini N. 1998; The rfb genes in Azotobacter vinelandii are arranged in a rfbFGC gene cluster: a significant deviation to the arrangement of the rfb genes in Enterobacteriaceae. Biochem Biophys Res Commun 245:572–582 [CrossRef]
     [Google Scholar]
  13. Hitchcock P. J., Brown T. M. 1983; Morphological heterogeneity among Salmonella lipopolysaccharide types in silver-stained polyacrylamide gels. J Bacteriol 195:125–142
     [Google Scholar]
  14. Hoang T. T., Karkhoff-Schweizer R. R., Kutchma A. J., Schweizer H. P. 1998; A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86 [CrossRef]
     [Google Scholar]
  15. Huff J. P., Grant B. J., Penning C. A., Sullivan K. F. 1990; Optimization of routine transformation of Escherichia coli with plasmid DNA. Biotechniques 9:570–577
     [Google Scholar]
  16. Jarvis F. G., Johnson M. J. 1949; A glyco-lipid produced by Pseudomonas aeruginosa. J Am Chem Soc 71:4124–4126 [CrossRef]
     [Google Scholar]
  17. Jiang X.-M., Neal B., Santiago F., Lee S. J., Romana L. K., Reeves P. R. 1991; Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar Typhimurium (strain LT2). Mol Microbiol 5:695–713 [CrossRef]
     [Google Scholar]
  18. Kent J. L., Osborn M. J. 1968; Properties of the O-specific hapten formed in vivo by mutant strains of Salmonella typhimurium. Biochemistry 7:4396–4408 [CrossRef]
     [Google Scholar]
  19. de Kievit T. R., Lam J. S. 1994; Monoclonal antibodies that distinguish inner core, outer core and lipid A regions of Pseudomonas aeruginosa lipopolysaccharides. J Bacteriol 176:7129–7139
     [Google Scholar]
  20. Klena J. D., Schnaitman C. A. 1993; Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1. Mol Microbiol 9:393–402 [CrossRef]
     [Google Scholar]
  21. Knirel Y. A., Kochetkov N. K. 1994; The structure of lipopolysaccharides of Gram-negative bacteria. III. The structure of O-antigens: a review. Biochemistry (Moscow) 12:1325–1383
     [Google Scholar]
  22. Kornfeld S., Glaser L. 1961; The enzymatic synthesis of thymidine-linked sugars. J Biol Chem 236:1791–1794
     [Google Scholar]
  23. Lam J. S., MacDonald L. A., Lam M. Y., Duchesne L. G., Southam G. G. 1987; Production and characterization of monoclonal antibodies against serotype strains of Pseudomonas aeruginosa. Infect Immun 55:1051–1057
     [Google Scholar]
  24. Lam M. Y. C., McGroarty E. J., Kropinski A. M., MacDonald L. A., Peterson S. S., Høiby N., Lam J. S. 1989; Occurrence of a common lipopolysaccharide antigen in standard and clinical strains of Pseudomonas aeruginosa. J Clin Microbiol 27:962–967
     [Google Scholar]
  25. Lightfoot J., Lam J. S. 1991; Molecular cloning of genes involved with expression of A-band lipopolysaccharide, an antigenically conserved form, in Pseudomonas aeruginosa. J Bacteriol 173:5624–5630
     [Google Scholar]
  26. Lindquist L., Kaiser R., Reeves P. R., Lindberg A. A. 1993; Purification, characterization and HPLC assay of Salmonella glucose-1-phosphate thymidyltransferase from the cloned rfbA gene. Eur J Biochem 211:763–770 [CrossRef]
     [Google Scholar]
  27. Liu D., Reeves P. R. 1994; Escherichia coli K-12 regains its O antigen. Microbiology 140:49–57 [CrossRef]
     [Google Scholar]
  28. Ma S., Selvaraj U., Ohman D. E., Quarless R., Hassett D. J., Wozniak D. J. 1998; Phosphorylation-independent activity of the response regulators AlgB and AlgR in promoting alginate biosynthesis in mucoid Pseudomonas aeruginosa. J Bacteriol 180:956–968
     [Google Scholar]
  29. Macpherson D. F., Manning P. A., Morona R. 1994; Characterization of the dTDP-rhamnose biosynthetic genes encoded in the rfb locus of Shigella flexneri. Mol Microbiol 11:281–292 [CrossRef]
     [Google Scholar]
  30. Masoud H. I., Sadovskaya I., de Kievit T., Altman E., Richards J. C., Lam J. S. 1995; Structural elucidation of the lipopolysaccharide core region of the O-chain-deficient mutant strain A28 from Pseudomonas aeruginosa serotype O6 (International antigenic typing scheme). J Bacteriol 177:6718–6726
     [Google Scholar]
  31. Matewish M., Rocchetta H. L., Burrows L. L., Rahim R., Pigeon K., Lam J. S. 1998; Genomics of Pseudomonas aeruginosa: identification of multiple homologues of proteins required for the biosynthesis of cell-surface polysaccharides. Twelfth Annual North American Cystic Fibrosis Conference15–18 OctoberMontreal, Quebec, Canada In Pediatric Pulmonology Supplement 17 p. 309 abstract 374. Wiley-Liss
    [Google Scholar]
  32. Palleroni N. J. 1984; Pseudomonas. In Bergey’s Manual of Systematic Bacteriology vol. 1 pp. 141–198Edited by Krieg N. R., Holt J. G. Baltimore: Williams & Wilkins;
     [Google Scholar]
  33. Pier G. B., Grout M., Zaidi T. S., Goldberg J. B. 1996a; How mutant CFTR may contribute to Pseudomonas aeruginosa infection in cystic fibrosis. Am J Respir Crit Care Med 154:S157–S182
     [Google Scholar]
  34. Pier G. B., Grout M., Zaidi T. S., Olsen J. C., Johnson L. G., Yankasakas J. R., Goldberg J. B. 1996b; Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections. Science 271:64–67 [CrossRef]
     [Google Scholar]
  35. Rajakumar K., Jost B. H., Sasakawa C., Okada N., Yoshikawa M., Adler B. 1994; Nucleotide sequence of the rhamnose biosynthetic operon of Shigella flexneri 2a and role of lipopolysaccharide in virulence. J Bacteriol 176:2362–2373
     [Google Scholar]
  36. Rivera M., Bryan L. E., Hancock R. E. W., McGroarty E. J. 1988; Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length. J Bacteriol 170:512–521
     [Google Scholar]
  37. Robertson B. D., Frosch M., van Putten J. P. M. 1994; The identification of cryptic rhamnose biosynthesis genes in Neisseria gonorrhoeae and their relationship to lipopolysaccharide biosynthesis. J Bacteriol 176:6915–6920
     [Google Scholar]
  38. Rocchetta H. L., Lam J. S. 1997; Identification and functional characterization of an ABC transport system involved in polysaccharide export of A-band lipopolysaccharide in Pseudomonas aeruginosa. J Bacteriol 179:4713–4724
     [Google Scholar]
  39. Rocchetta H. L., Pacan J. C., Lam J. S. 1998; Synthesis of the A-band polysaccharide sugar d-rhamnose requires Rmd and WbpW: identification of multiple AlgA homologues, WbpW and ORF488, in Pseudomonas aeruginosa. Mol Microbiol 29:1419–1434 [CrossRef]
     [Google Scholar]
  40. Rocchetta H. L., Burrows L. L., Lam J. S. 1999; The genetics of O-antigen biosynthesis in Pseudomonas aeruginosa. Microbiol Mol Biol Rev 63:523–553
     [Google Scholar]
  41. Sadovskaya I., Brisson J.-R., Lam J. S., Richards J. C., Altman E. 1998; Structural elucidation of the lipopolysaccharide core regions of the wild-type strain PAO1 and O-chain-deficient mutant strains AK1401 and AK1012 from Pseudomonas aeruginosa. Eur J Biochem 255:673–684 [CrossRef]
     [Google Scholar]
  42. Sadovskaya I., Brisson J.-R., Thibault P., Richards J. C., Lam J. S., Altman E. 2000; Structural characterization of the outer core and O-antigen linkage region of lipopolysaccharide from Pseudomonas aeruginosa O5. Eur J Biochem 267:1640–1650 [CrossRef]
     [Google Scholar]
  43. Saigı́ F., Climent N., Piqué N., Sanchez C., Merino S., Rubirés X., Aguilar A., Tomas J. M., Regue M. 1999; Genetic analysis of the Serratia marcescens N28b O4 antigen gene cluster. J Bacteriol 181:1883–1891
     [Google Scholar]
  44. Sawardeker J. S., Sloneker J. H., Jeanes A. 1967; Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Anal Chem 37:1602–1604
     [Google Scholar]
  45. Schweizer H. P., Hoang T. T. 1995; An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 158:15–22 [CrossRef]
     [Google Scholar]
  46. Simon R., Priefer U., Pühler A. 1983; A broad-host-range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1:784–791 [CrossRef]
     [Google Scholar]
  47. Stevenson G., Neal B., Liu D., Hobbs M., Packer N. H., Batley M., Redmond J. W., Lindquist L., Reeves P. 1994; Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster. J Bacteriol 176:4144–4156
     [Google Scholar]
  48. Sturm S., Jann B., Jann K., Fortnagel P., Timmis K. N. 1986; Genetic analysis of Shigella dysenteriae 1 O antigen polysaccharide biosynthesis in Escherichia coli K-12: structure and functions of the rfb gene cluster. Microb Pathog 1:307–324 [CrossRef]
     [Google Scholar]
  49. Tsai C. M., Frasch C. E. 1982; A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 119:115–119 [CrossRef]
     [Google Scholar]
  50. West S. E., Schweizer H. P., Dall C., Sample A. K., Runyen-Janecky L. J. 1994; Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148:81–86 [CrossRef]
     [Google Scholar]
  51. Westphal O., Jann K. 1965; Bacterial lipopolysaccharides: extraction with phenol-water and further applications of the procedure. Methods Carbohydr Chem 5:83–91
     [Google Scholar]
  52. Woods D. E., Lam J. S., Paranchych W., Speert D. P., Campbell M., Godfrey A. J. 1997; Correlation of Pseudomonas aeruginosa virulence factors from clinical and environmental isolates with pathogenicity in the neutropenic mouse. Can J Microbiol 43:541–551 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-146-11-2803
Loading
Involvement of the rml locus in core oligosaccharide and O polysaccharide assembly in Pseudomonas aeruginosa
Microbiology 146, 2803 (2000); https://doi.org/10.1099/00221287-146-11-2803
/content/journal/micro/10.1099/00221287-146-11-2803
/content/journal/micro/10.1099/00221287-146-11-2803
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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