1887

Microbiology Society logo

Volume 152, Issue 1

Genetic and functional analyses of the gene, a member of the -1,4-galactosyltransferase gene family in the genus Free

Abstract

Lipooligosaccharide (LOS) is a major virulence factor of the pathogenic . Three galactosyltransferase genes, , and , responsible for the biosynthesis of LOS oligosaccharide chains, were analysed in five species. The function of in 6275 was determined by mutagenesis and chemical characterization of the parent and mutant LOS chains. The chemical characterization included SDS-PAGE, immunoblot, hexose and mass spectrometry analyses. Compared with the parent LOS, the mutant LOS lacked galactose, and its oligosaccharide decreased by three or four sugar units in matrix-assisted laser desorption ionization (MALDI)-MS analysis. The results show that encodes a -1,4-galactosyltransferase, and that the glucose moiety linked to heptose (Hep) in the chain is the acceptor site in the biosynthesis of LOS. To understand the sequence diversity and relationships of , and , the entire locus was further sequenced in three strains and three commensal strains, and compared with the previously reported genes from species. Comparison of the protein sequences of the three enzymes LgtB, LgtE and LgtH showed a conserved N-terminal region, and a highly variable C-terminal region, suggesting functional constraint for substrate and acceptor specificity, respectively. The analyses of allelic variation and evolution of 23 , 12 and 14 sequences revealed a distinct evolutionary history of these genes in . For example, the splits graph of displayed a network evolution, indicating frequent DNA recombination, whereas splits graphs of and displayed star-tree-like evolution, indicating the accumulation of point mutations. The data presented here represent examples of the evolution and variation of prokaryotic glycosyltransferase gene families. These imply the existence of multiple enzyme isoforms for biosynthesis of a great diversity of oligosaccharides in nature.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): ACTH, adrenocorticotropic hormone , DHB, 2,5-dihydroxybenzoic acid , Gal, galactose , Glc, glucose , GlcN, glucosamine , GlcNAc, N-acetylglucosamine , Hep, heptose , Kdo, 2-keto-3-deoxyoctulosonic acid , LOS, lipooligosaccharide , MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight , NeuNAc, N-acetylneuramic acid , OS, oligosaccharide and PEA, phosphorylethanolamine
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28327-0
2006-01-01
2024-04-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/152/1/123.html?itemId=/content/journal/micro/10.1099/mic.0.28327-0&mimeType=html&fmt=ahah

References

  1. Amado M, Almeida R, Schwientek T, Clausen H. 1999; Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions. Biochim Biophys Acta 1473:35–53 [CrossRef]
     [Google Scholar]
  2. Arking D, Tong Y, Stein D. C. 2001; Analysis of lipooligosaccharide biosynthesis in the Neisseriaceae. J Bacteriol 183:934–941 [CrossRef]
     [Google Scholar]
  3. Ashton F. E, Ryan J. A, Borczyk A, Caugant D. A, Mancino L, Huang D. 1991; Emergence of a virulent clone of Neisseria meningitidis serotype 2a that is associated with meningococcal group C disease in Canada. J Clin Microbiol 29:2489–2493
     [Google Scholar]
  4. Banerjee A, Wang R, Uljon S. N, Rice P. A, Gotschlich E. C, Stein D. C. 1998; Identification of the gene (lgtG) encoding the lipooligosaccharide β chain synthesizing glucosyl transferase from Neisseria meningitidis . Proc Natl Acad Sci U S A 95:10872–10877 [CrossRef]
     [Google Scholar]
  5. Breton C, Snajdrova L, Jeanneau C, Koea J, Imberty A. 2005; Structures and mechanisms of glycosyltransferases superfamily. Glycobiology (in press)
    [Google Scholar]
  6. Burch C. L, Danaher R. J, Stein D. C. 1997; Antigenic variation in Neisseria gonorrhoeae : production of multiple lipooligosaccharides. J Bacteriol 179:982–986
     [Google Scholar]
  7. Busch C, Hofmann F, Selzer J, Munro S, Jeckel D, Aktories K. 1998; A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins. J Biol Chem 273:19566–19572 [CrossRef]
     [Google Scholar]
  8. 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]
  9. Coutinho P. M, Deleury E, Davies G. J, Henrissat B. 2003; An evolving hierarchical family classification for glycosyltransferases. J Mol Biol 328:307–317 [CrossRef]
     [Google Scholar]
  10. Cox A. D, Li J, Richards J. C. 2002; Identification and localization of glycine in the inner core lipopolysaccharide of Neisseria meningitidis . Eur J Biochem 269:4169–4175 [CrossRef]
     [Google Scholar]
  11. Danaher R. J, Levin J. C, Arking D, Burch C. L, Sandlin R, Stein D. C. 1995; Genetic basis of Neisseria gonorrhoeae lipooligosaccharide antigenic variation. J Bacteriol 177:7275–7279
     [Google Scholar]
  12. Devereux J, Haeberli P, Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395 [CrossRef]
     [Google Scholar]
  13. Elizabeth A. S, Seifert H. S. 2001; The recX gene potentiates homologous recombination in Neisseria gonorrhoeae . Mol Microbiol 40:1301–1310 [CrossRef]
     [Google Scholar]
  14. Erwin A. L, Haynes P. A, Rice P. A, Gotschlich E. C. 1996; Conservation of the lipooligosaccharide synthesis locus lgt among strains of Neisseria gonorrhoeae : requirement for lgtE in synthesis of the 2C7 epitope and of the β chain of strain 15253. J Exp Med 184:1233–1241 [CrossRef]
     [Google Scholar]
  15. Gibson B. W, Melaugh W, Phillips N. J, Apicella M. A, Campagnari A. A, Griffiss J. M. 1993; Investigation of the structural heterogeneity of lipooligosaccharides of pathogenic Haemophilus and Neisseria species and of R-type lipopolysaccharides from Salmonella typhimurium by electrospray mass spectrometry. J Bacteriol 175:2702–2712
     [Google Scholar]
  16. Gibson B. W, Engstrom J. J, John C. M, Hines W, Falick A. M. 1997; Characterization of bacterial lipooligosaccharides by delayed extraction matrix-assisted laser inonization time-of-flight mass spectrometry. J Am Soc Mass Spectrom 8:645–658 [CrossRef]
     [Google Scholar]
  17. Gilbert M, Watson D. C, Cunningham A. M, Jennings M. P, Young N. M, Wakarchuk W. W. 1996; Cloning of the lipooligosaccharide alpha-2,3-sialyltransferase from the bacterial pathogens Neisseria meningitidis and Neisseria gonorrhoeae . J Biol Chem 271:28271–28276 [CrossRef]
     [Google Scholar]
  18. Gotschlich E. C. 1994; Genetic locus for the biosynthesis of the variable portion of Neisseria gonorrhoeae lipooligosaccharide. J Exp Med 180:2181–2190 [CrossRef]
     [Google Scholar]
  19. Hardy M. R, Townsend R. R, Lee Y. C. 1988; Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection. Anal Biochem 170:54–62 [CrossRef]
     [Google Scholar]
  20. Harvey H. A, Porat N, Campbell C. A, Jennings M, Gibson B. W, Phillips N. J, Apicella M. A, Blake M. S. 2000; Gonococcal lipooligosaccharide is a ligand for the asialoglycoprotein receptor on human sperm. Mol Microbiol 36:1059–1070 [CrossRef]
     [Google Scholar]
  21. Heinrichs D. E, Yethon J. A, Amor P. A, Whitfield C. 1998; The assembly system for the outer core portion of R1- and R4-type lipopolysaccharides of Escherichia coli . The R1 core-specific beta-glucosyltransferase provides a novel attachment site for O -polysaccharides. J Biol Chem 273:29497–29505 [CrossRef]
     [Google Scholar]
  22. Helander I. M, Lindner B, Brade H, Altmann K, Lindberg A. A, Rietschel E. T, Zahringe U. 1988; Chemical structure of Haemophilus influenzae strain I-69Rd/b+. Eur J Biochem 177:483–492 [CrossRef]
     [Google Scholar]
  23. Hu Y, Chen L, Ha S, Gross B, Falcone B, Walker D, Mokhtarzadeh M, Walker S. 2003; Crystal structure of the MurG : UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases. Proc Natl Acad Sci U S A 100:845–849 [CrossRef]
     [Google Scholar]
  24. Huson D. H. 1998; SplitsTree: analyzing and visualizing evolutionary data. Bioinformatics 14:68–73 [CrossRef]
     [Google Scholar]
  25. Jennings H. J, Bhattacharjee A. K, Kenne L, Kenny C. P, Calver G. 1980; The R-type lipopolysaccharides of Neisseria meningitidis . Can J Biochem 52:128–136
     [Google Scholar]
  26. Jennings M. P, Hood D. W, Peak I. R. A, Virji M, Moxon E. R. 1995; Molecular analysis of a locus for the biosynthesis and phase-variable expression of the lacto- N -neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis . Mol Microbiol 18:729–740 [CrossRef]
     [Google Scholar]
  27. Jennings M. P, Srikhanta Y. N, Moxon E. R, Kramer M, Poolman J. T, Kuipers B, van der Ley P. 1999; The genetic basis of the phase variation repertoire of lipopolysaccharide immunotypes in Neisseria meningitidis . Microbiology 145:3013–3021
     [Google Scholar]
  28. Kahler C. M, Stephens D. S. 1998; Genetic bases for biosynthesis, structure and function of meningococcal lipooligosaccharide. Crit Rev Microbiol 24:281–334
     [Google Scholar]
  29. Kahler C. M, Carlson R. W, Rahman M. M, Martin L. E, Stephens D. S. 1996a; Two glycosyltransferase genes, lgtF and rfaK , constitute the lipooligosaccharide ice (inner core extension) biosynthesis operon of Neisseria meningitidis . J Bacteriol 178:6677–6684
     [Google Scholar]
  30. Kahler C. M, Carlson R. W, Rahman M. M, Martin L. E, Stephens D. S. 1996b; Inner core biosynthesis of lipooligosaccharide (LOS) in Neisseria meningitidis serogroup B: identification and role in LOS assembly of the α 1,2 N -acetylglucosamine transferase (RfaK). J Bacteriol 178:1265–1273
     [Google Scholar]
  31. Kogan G, Uhrin D, Brisson J. R, Jennings H. J. 1997; Structural basis of the Neisseria meningitidis immunotypes including the L4 and L7 immunotypes. Carbohydr Res 298:191–199 [CrossRef]
     [Google Scholar]
  32. Kulshin V. A, Zahringer U, Lindner B, Frasch C. E, Tsai C. M, Dmitriev B. A, Rietschel E. T. 1992; Structural characterization of the lipid A component of pathogenic Neisseria meningitidis . J Bacteriol 174:1793–1800
     [Google Scholar]
  33. Kumar S, Tamura K, Nei M. 1994; mega: Molecular Evolutionary Genetics Analysis software for microcomputers. Comput Appl Biosci 10:189–191
     [Google Scholar]
  34. Lewis L. A, Gillaspy A. F, McLaughlin R. E. 22 other authors 2003; The gonococcal genome sequencing project. http://www.genome.ou.edu/gono.html
  35. Lupas A. 1996; Coiled coils: new structures and new functions. Trends Biochem Sci 21:375–382 [CrossRef]
     [Google Scholar]
  36. Lupas A, Van Dyke M, Stock J. 1991; Predicting coiled coils from protein sequences. Science 252:1162–1164 [CrossRef]
     [Google Scholar]
  37. Maynard Smith J, Smith N. H. 1998; Detecting recombination from gene trees. Mol Biol Evol 15:590–599 [CrossRef]
     [Google Scholar]
  38. Parkhill J, Achtman M, James K. D. 25 other authors 2000; Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 404:502–506 [CrossRef]
     [Google Scholar]
  39. Persson K, Ly H. D, Dieckelmann M, Wakarchuk W. W, Withers S. G, Strynadka N. C. 2001; Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs. Nat Struct Biol 8:166–175 [CrossRef]
     [Google Scholar]
  40. Preston A, Mandrell R. E, Gibson B. W, Apicella M. A. 1996; The lipooligosaccharides of pathogenic gram-negative bacteria. Crit Rev Microbiol 22:139–180 [CrossRef]
     [Google Scholar]
  41. Quakyi E. K, Hochstein H. D, Tsai C. M. 1997; Modulation of the biological activities of meningococcal endotoxins by association with outer membrane proteins is not inevitably linked to toxicity. Infect Immun 65:1972–1979
     [Google Scholar]
  42. Sarkari J, Pandit N, Moxon E. R, Achtman M. 1994; Variable expression of the Opc outer membrane protein in Neisseria meningitidis is caused by size variation of promoter containing poly-cytidine. Mol Microbiol 13:207–217 [CrossRef]
     [Google Scholar]
  43. Saxena I. M, Geremia R. A, Henrissat B, Brown R. M., Jr, Fevre M. 1995; Multidomain architecture of beta-glycosyl transferases: implications for mechanism of action. J Bacteriol 177:1419–1424
     [Google Scholar]
  44. Scholten R. J, Kuipers B, Valkenburg H. A, Dankert J, Zollinger W. D, Poolman J. T. 1994; Lipo-oligosaccharide immunotyping of Neisseria meningitidis by a whole-cell ELISA with monoclonal antibodies. J Med Microbiol 41:236–243 [CrossRef]
     [Google Scholar]
  45. Tettelin H, Saunders N. J, Heidelberg J. 39 other authors 2000; Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 278:1809–1815
     [Google Scholar]
  46. 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]
  47. Tsai C. M, Boykins R, Frasch C. E. 1983; Heterogeneity and variation among Neisseria meningitidis lipopolysaccharides. J Bacteriol 155:498–504
     [Google Scholar]
  48. Tsai C. M, Kao G, Zhu P. 2002; Influence of the length of the lipooligosaccharide alpha chain on its sialylation in Neisseria meningitidis . Infect Immun 70:407–411 [CrossRef]
     [Google Scholar]
  49. Tsang R. S, Tsai C. M, Zhu P, Ringuette L, Lorange M, Law D. K. 2004; Phenotypic and genetic characterization of a unique variant of serogroup C ET-15 meningococci (with the antigenic formula C : 2a : P1.7,1) causing invasive meningococcal disease in Quebec, Canada. J Clin Microbiol 42:1460–1465 [CrossRef]
     [Google Scholar]
  50. van der Ley P, Kramer M, Martin A, Richards J. C, Poolman J. T. 1997; Analysis of the icsBA locus required for biosynthesis of the inner core region from Neisseria meningitidis lipopolysaccharide. FEMS Microbiol Lett 146:247–253 [CrossRef]
     [Google Scholar]
  51. Verheul A. F. M, Snippe H, Poolman J. T. 1993; Meningococcal lipopolysaccharides: virulence factor and potential vaccine component. Microbiol Rev 57:34–39
     [Google Scholar]
  52. Wakarchuk W, Martin A, Jennings M. P, Moxon E. R, Richards J. C. 1996; Functional relationships of the genetic locus encoding the glycosyltransferase enzymes involved in expression of the lacto- N -neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis . J Biol Chem 271:19166–19173 [CrossRef]
     [Google Scholar]
  53. Wakarchuk W. W, Cunningham A, Watson D. C, Young N. M. 1998; Role of paired basic residues in the expression of active recombinant galactosyltransferases from the bacterial pathogen Neisseria meningitidis . Protein Eng 11:295–302 [CrossRef]
     [Google Scholar]
  54. Westphal O, Jann K. 1965; Bacterial lipopolysaccharides. Extraction with phenol-water and further application of the procedure. Methods Carbohydr Chem 5:83–91
     [Google Scholar]
  55. Wiggins C. A, Munro S. 1998; Activity of the yeast MNN1 α -1,3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. Proc Natl Acad Sci U S A 95:7945–7950 [CrossRef]
     [Google Scholar]
  56. Yamasaki R, Griffiss J. M, Quinn K. P, Mandrell R. E. 1993; Neuraminic acid is alpha 2→3 linked in the lipooligosaccharide of Neisseria meningitidis serogroup B strain 6275. J Bacteriol 175:4565–4568
     [Google Scholar]
  57. Yang Q. L, Gotschlich E. C. 1996; Variation of gonococcal lipooligosaccharide structure is due to alterations in poly-G tracts in lgt genes encoding glycosyltransferases. J Exp Med 183:323–327 [CrossRef]
     [Google Scholar]
  58. Zheng Y, Roberts R. J, Kasif S. 2004; Segmentally variable genes: a new perspective on adaptation. PLoS Biol 2:452–464
     [Google Scholar]
  59. Zhu P, Klutch M. J, Tsai C. M. 2001; Genetic analysis of conservation and variation of lipooligosaccharide expression in two L8-immunotype strains of Neisseria meningitidis . FEMS Microbiol Lett 203:173–177 [CrossRef]
     [Google Scholar]
  60. Zhu P, Klutch M. J, Bash M. J, Tsang R. S. W, Ng L. K, Tsai C. M. 2002; Genetic diversity of three lgt loci for biosynthesis of lipooligosaccharide (LOS) in Neisseria species. Microbiology 148:1833–1844
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.28327-0
Loading
Genetic and functional analyses of the lgtH gene, a member of the β-1,4-galactosyltransferase gene family in the genus Neisseria
Microbiology 152, 123 (2006); https://doi.org/10.1099/mic.0.28327-0
/content/journal/micro/10.1099/mic.0.28327-0
/content/journal/micro/10.1099/mic.0.28327-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

PDF

Supplementary material 3

PDF

Supplementary material 4

PDF

Supplementary material 5

PDF

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