Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Mar;178(5):1265–1273. doi: 10.1128/jb.178.5.1265-1273.1996

Inner core biosynthesis of lipooligosaccharide (LOS) in Neisseria meningitidis serogroup B: identification and role in LOS assembly of the alpha1,2 N-acetylglucosamine transferase (RfaK).

C M Kahler 1, R W Carlson 1, M M Rahman 1, L E Martin 1, D S Stephens 1
PMCID: PMC177798  PMID: 8631701

Abstract

A lipooligosaccharide (LOS) mutant of Neisseria meningitidis serogroup B strain NMB (immunotype L3,7,9) was identified in a Tn916 (tetM) mutant bank by loss of reactivity with monoclonal antibody 3F11, which recognizes the terminal Galbeta1-->4GlcNAc epitope in the lacto-N-neotetraose moiety of the wild-type LOS structure. The mutant, designated 559, was found to express a truncated LOS of 3.0 kDa. Southern and PCR analyses demonstrated that there was a single intact Tn916 insertion (class I) in the mutant 559 chromosome. Linkage of the LOS phenotype and the Tn916 insertion was confirmed by transformation of the wild-type parent. Nucleotide sequence analysis of the region surrounding the transposition site revealed a 1,065-bp open reading frame (ORF). A homology search of the GenBank/EMBL database revealed that the amino acid sequence of this ORF had 46.8% similarity and 21.2% identity with the alpha1,2 N-acetylglucosamine transferase (RfaK) from Salmonella typhimurium. Glycosyl composition and linkage analysis of the LOS produced by mutant 559 revealed that the lacto-N-neotetraose group which is attached to heptose I (HepI) and the N-acetylglucosamine and glucose residues that are attached to HepII in the inner core of the parental LOS were absent. These analyses also showed that the HepII residue in both the parent and the mutant LOS molecules was phosphorylated, presumably by a phosphoethanolamine substituent. The insertion of nonpolar and polar antibiotic resistance cartridges into the parental rfaK gene resulted in the expression of LOS with the same mobility as that produced by mutant 559. This result indicated that the inability to add the lacto-N-neotetraose group to the 559 LOS is not due to a polar effect on a gene(s) downstream of rfaK. Our data indicate that we have identified the meningococcal alpha1,2 N-acetylglucosamine transferase responsible for the addition of N-acetylglucosamine to HepII. We propose that the lack of alpha-chain extension from HepI in the LOS of mutant 559 may be due to structural constraints imposed by the incomplete biosynthesis of the LOS inner core.

Full Text

The Full Text of this article is available as a PDF (323.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brandtzaeg P., Bryn K., Kierulf P., Ovstebø R., Namork E., Aase B., Jantzen E. Meningococcal endotoxin in lethal septic shock plasma studied by gas chromatography, mass-spectrometry, ultracentrifugation, and electron microscopy. J Clin Invest. 1992 Mar;89(3):816–823. doi: 10.1172/JCI115660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chung C. T., Niemela S. L., Miller R. H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2172–2175. doi: 10.1073/pnas.86.7.2172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Drazek E. S., Stein D. C., Deal C. D. A mutation in the Neisseria gonorrhoeae rfaD homolog results in altered lipooligosaccharide expression. J Bacteriol. 1995 May;177(9):2321–2327. doi: 10.1128/jb.177.9.2321-2327.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dudas K. C., Apicella M. A. Selection and immunochemical analysis of lipooligosaccharide mutants of Neisseria gonorrhoeae. Infect Immun. 1988 Feb;56(2):499–504. doi: 10.1128/iai.56.2.499-504.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dunn K. L., Virji M., Moxon E. R. Investigations into the molecular basis of meningococcal toxicity for human endothelial and epithelial cells: the synergistic effect of LPS and pili. Microb Pathog. 1995 Feb;18(2):81–96. doi: 10.1016/s0882-4010(95)90085-3. [DOI] [PubMed] [Google Scholar]
  7. Galanos C., Lüderitz O., Westphal O. A new method for the extraction of R lipopolysaccharides. Eur J Biochem. 1969 Jun;9(2):245–249. doi: 10.1111/j.1432-1033.1969.tb00601.x. [DOI] [PubMed] [Google Scholar]
  8. Gamian A., Beurret M., Michon F., Brisson J. R., Jennings H. J. Structure of the L2 lipopolysaccharide core oligosaccharides of Neisseria meningitidis. J Biol Chem. 1992 Jan 15;267(2):922–925. [PubMed] [Google Scholar]
  9. Gotschlich E. C. Genetic locus for the biosynthesis of the variable portion of Neisseria gonorrhoeae lipooligosaccharide. J Exp Med. 1994 Dec 1;180(6):2181–2190. doi: 10.1084/jem.180.6.2181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hanish J., McClelland M. Activity of DNA modification and restriction enzymes in KGB, a potassium glutamate buffer. Gene Anal Tech. 1988 Sep-Oct;5(5):105–107. doi: 10.1016/0735-0651(88)90005-2. [DOI] [PubMed] [Google Scholar]
  11. Helander I. M., Kilpeläinen I., Vaara M. 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. 1994 Feb;11(3):481–487. doi: 10.1111/j.1365-2958.1994.tb00329.x. [DOI] [PubMed] [Google Scholar]
  12. Helander I. M., Lindner B., Brade H., Altmann K., Lindberg A. A., Rietschel E. T., Zähringer U. Chemical structure of the lipopolysaccharide of Haemophilus influenzae strain I-69 Rd-/b+. Description of a novel deep-rough chemotype. Eur J Biochem. 1988 Nov 15;177(3):483–492. doi: 10.1111/j.1432-1033.1988.tb14398.x. [DOI] [PubMed] [Google Scholar]
  13. Hitchcock P. J., Brown T. M. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol. 1983 Apr;154(1):269–277. doi: 10.1128/jb.154.1.269-277.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Janik A., Juni E., Heym G. A. Genetic Transformation as a tool for detection of Neisseria gonorrhoeae. J Clin Microbiol. 1976 Jul;4(1):71–81. doi: 10.1128/jcm.4.1.71-81.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jennings H. J., Bhattacharjee A. K., Kenne L., Kenny C. P., Calver G. The R-type lipopolysaccharides of Neisseria meningitidis. Can J Biochem. 1980 Feb;58(2):128–136. doi: 10.1139/o80-018. [DOI] [PubMed] [Google Scholar]
  16. Jennings H. J., Johnson K. G., Kenne L. The structure of an R-type oligosaccharide core obtained from some lipopolysaccharides of Neisseria meningitidis. Carbohydr Res. 1983 Sep 16;121:233–241. doi: 10.1016/0008-6215(83)84020-8. [DOI] [PubMed] [Google Scholar]
  17. Jennings M. P., van der Ley P., Wilks K. E., Maskell D. J., Poolman J. T., Moxon E. R. Cloning and molecular analysis of the galE gene of Neisseria meningitidis and its role in lipopolysaccharide biosynthesis. Mol Microbiol. 1993 Oct;10(2):361–369. [PubMed] [Google Scholar]
  18. Jones D. M., Borrow R., Fox A. J., Gray S., Cartwright K. A., Poolman J. T. The lipooligosaccharide immunotype as a virulence determinant in Neisseria meningitidis. Microb Pathog. 1992 Sep;13(3):219–224. doi: 10.1016/0882-4010(92)90022-g. [DOI] [PubMed] [Google Scholar]
  19. Kathariou S., Stephens D. S., Spellman P., Morse S. A. Transposition of Tn916 to different sites in the chromosome of Neisseria meningitidis: a genetic tool for meningococcal mutagenesis. Mol Microbiol. 1990 May;4(5):729–735. doi: 10.1111/j.1365-2958.1990.tb00643.x. [DOI] [PubMed] [Google Scholar]
  20. Klena J. D., Ashford R. S., 2nd, Schnaitman C. A. Role of Escherichia coli K-12 rfa genes and the rfp gene of Shigella dysenteriae 1 in generation of lipopolysaccharide core heterogeneity and attachment of O antigen. J Bacteriol. 1992 Nov;174(22):7297–7307. doi: 10.1128/jb.174.22.7297-7307.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Klena J. D., Pradel E., Schnaitman C. A. Comparison of lipopolysaccharide biosynthesis genes rfaK, rfaL, rfaY, and rfaZ of Escherichia coli K-12 and Salmonella typhimurium. J Bacteriol. 1992 Jul;174(14):4746–4752. doi: 10.1128/jb.174.14.4746-4752.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kulshin V. A., Zähringer U., Lindner B., Frasch C. E., Tsai C. M., Dmitriev B. A., Rietschel E. T. Structural characterization of the lipid A component of pathogenic Neisseria meningitidis. J Bacteriol. 1992 Mar;174(6):1793–1800. doi: 10.1128/jb.174.6.1793-1800.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee F. K., Stephens D. S., Gibson B. W., Engstrom J. J., Zhou D., Apicella M. A. Microheterogeneity of Neisseria lipooligosaccharide: analysis of a UDP-glucose 4-epimerase mutant of Neisseria meningitidis NMB. Infect Immun. 1995 Jul;63(7):2508–2515. doi: 10.1128/iai.63.7.2508-2515.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. MacLachlan P. R., Kadam S. K., Sanderson K. E. Cloning, characterization, and DNA sequence of the rfaLK region for lipopolysaccharide synthesis in Salmonella typhimurium LT2. J Bacteriol. 1991 Nov;173(22):7151–7163. doi: 10.1128/jb.173.22.7151-7163.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mackinnon F. G., Borrow R., Gorringe A. R., Fox A. J., Jones D. M., Robinson A. Demonstration of lipooligosaccharide immunotype and capsule as virulence factors for Neisseria meningitidis using an infant mouse intranasal infection model. Microb Pathog. 1993 Nov;15(5):359–366. doi: 10.1006/mpat.1993.1085. [DOI] [PubMed] [Google Scholar]
  26. Mandrell R. E., Griffiss J. M., Macher B. A. Lipooligosaccharides (LOS) of Neisseria gonorrhoeae and Neisseria meningitidis have components that are immunochemically similar to precursors of human blood group antigens. Carbohydrate sequence specificity of the mouse monoclonal antibodies that recognize crossreacting antigens on LOS and human erythrocytes. J Exp Med. 1988 Jul 1;168(1):107–126. doi: 10.1084/jem.168.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mandrell R. E., Griffiss J. M., Smith H., Cole J. A. Distribution of a lipooligosaccharide-specific sialyltransferase in pathogenic and non-pathogenic Neisseria. Microb Pathog. 1993 Apr;14(4):315–327. doi: 10.1006/mpat.1993.1031. [DOI] [PubMed] [Google Scholar]
  28. Michon F., Beurret M., Gamian A., Brisson J. R., Jennings H. J. Structure of the L5 lipopolysaccharide core oligosaccharides of Neisseria meningitidis. J Biol Chem. 1990 May 5;265(13):7243–7247. [PubMed] [Google Scholar]
  29. Morse S. A., Bartenstein L. Factors affecting autolysis of Neisseria gonorrhoeae. Proc Soc Exp Biol Med. 1974 Apr;145(4):1418–1421. doi: 10.3181/00379727-145-38025. [DOI] [PubMed] [Google Scholar]
  30. Ménard R., Sansonetti P. J., Parsot C. Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J Bacteriol. 1993 Sep;175(18):5899–5906. doi: 10.1128/jb.175.18.5899-5906.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nath K. A rapid DNA isolation procedure from petri dish grown clinical bacterial isolates. Nucleic Acids Res. 1990 Nov 11;18(21):6462–6462. doi: 10.1093/nar/18.21.6462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nummila K., Kilpeläinen I., Zähringer U., Vaara M., Helander I. M. Lipopolysaccharides of polymyxin B-resistant mutants of Escherichia coli are extensively substituted by 2-aminoethyl pyrophosphate and contain aminoarabinose in lipid A. Mol Microbiol. 1995 Apr;16(2):271–278. doi: 10.1111/j.1365-2958.1995.tb02299.x. [DOI] [PubMed] [Google Scholar]
  33. Pavliak V., Brisson J. R., Michon F., Uhrín D., Jennings H. J. Structure of the sialylated L3 lipopolysaccharide of Neisseria meningitidis. J Biol Chem. 1993 Jul 5;268(19):14146–14152. [PubMed] [Google Scholar]
  34. Porat N., Apicella M. A., Blake M. S. A lipooligosaccharide-binding site on HepG2 cells similar to the gonococcal opacity-associated surface protein Opa. Infect Immun. 1995 Jun;63(6):2164–2172. doi: 10.1128/iai.63.6.2164-2172.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Porat N., Apicella M. A., Blake M. S. Neisseria gonorrhoeae utilizes and enhances the biosynthesis of the asialoglycoprotein receptor expressed on the surface of the hepatic HepG2 cell line. Infect Immun. 1995 Apr;63(4):1498–1506. doi: 10.1128/iai.63.4.1498-1506.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  37. Quigley N. B., Reeves P. R. Chloramphenicol resistance cloning vector based on pUC9. Plasmid. 1987 Jan;17(1):54–57. doi: 10.1016/0147-619x(87)90008-4. [DOI] [PubMed] [Google Scholar]
  38. Raetz C. R. Biochemistry of endotoxins. Annu Rev Biochem. 1990;59:129–170. doi: 10.1146/annurev.bi.59.070190.001021. [DOI] [PubMed] [Google Scholar]
  39. Robertson B. D., Frosch M., van Putten J. P. The role of galE in the biosynthesis and function of gonococcal lipopolysaccharide. Mol Microbiol. 1993 May;8(5):891–901. doi: 10.1111/j.1365-2958.1993.tb01635.x. [DOI] [PubMed] [Google Scholar]
  40. Sandlin R. C., Apicella M. A., Stein D. C. Cloning of a gonococcal DNA sequence that complements the lipooligosaccharide defects of Neisseria gonorrhoeae 1291d and 1291e. Infect Immun. 1993 Aug;61(8):3360–3368. doi: 10.1128/iai.61.8.3360-3368.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sandlin R. C., Danaher R. J., Stein D. C. Genetic basis of pyocin resistance in Neisseria gonorrhoeae. J Bacteriol. 1994 Nov;176(22):6869–6876. doi: 10.1128/jb.176.22.6869-6876.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schnaitman C. A., Klena J. D. Genetics of lipopolysaccharide biosynthesis in enteric bacteria. Microbiol Rev. 1993 Sep;57(3):655–682. doi: 10.1128/mr.57.3.655-682.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schneider H., Griffiss J. M., Mandrell R. E., Jarvis G. A. Elaboration of a 3.6-kilodalton lipooligosaccharide, antibody against which is absent from human sera, is associated with serum resistance of Neisseria gonorrhoeae. Infect Immun. 1985 Dec;50(3):672–677. doi: 10.1128/iai.50.3.672-677.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  45. Shyamala V., Ames G. F. Genome walking by single-specific-primer polymerase chain reaction: SSP-PCR. Gene. 1989 Dec 7;84(1):1–8. doi: 10.1016/0378-1119(89)90132-7. [DOI] [PubMed] [Google Scholar]
  46. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  47. Stephens D. S., McAllister C. F., Zhou D., Lee F. K., Apicella M. A. Tn916-generated, lipooligosaccharide mutants of Neisseria meningitidis and Neisseria gonorrhoeae. Infect Immun. 1994 Jul;62(7):2947–2952. doi: 10.1128/iai.62.7.2947-2952.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stephens D. S., Swartley J. S., Kathariou S., Morse S. A. Insertion of Tn916 in Neisseria meningitidis resulting in loss of group B capsular polysaccharide. Infect Immun. 1991 Nov;59(11):4097–4102. doi: 10.1128/iai.59.11.4097-4102.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Swartley J. S., Stephens D. S. Identification of a genetic locus involved in the biosynthesis of N-acetyl-D-mannosamine, a precursor of the (alpha 2-->8)-linked polysialic acid capsule of serogroup B Neisseria meningitidis. J Bacteriol. 1994 Mar;176(5):1530–1534. doi: 10.1128/jb.176.5.1530-1534.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Takeshita S., Sato M., Toba M., Masahashi W., Hashimoto-Gotoh T. High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection. Gene. 1987;61(1):63–74. doi: 10.1016/0378-1119(87)90365-9. [DOI] [PubMed] [Google Scholar]
  51. Tsai C. M., Mocca L. F., Frasch C. E. Immunotype epitopes of Neisseria meningitidis lipooligosaccharide types 1 through 8. Infect Immun. 1987 Jul;55(7):1652–1656. doi: 10.1128/iai.55.7.1652-1656.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tunkel A. R., Scheld W. M. Pathogenesis and pathophysiology of bacterial meningitis. Clin Microbiol Rev. 1993 Apr;6(2):118–136. doi: 10.1128/cmr.6.2.118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Verheul A. F., Snippe H., Poolman J. T. Meningococcal lipopolysaccharides: virulence factor and potential vaccine component. Microbiol Rev. 1993 Mar;57(1):34–49. doi: 10.1128/mr.57.1.34-49.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yamasaki R., Griffiss J. M., Quinn K. P., Mandrell R. E. Neuraminic acid is alpha 2-->3 linked in the lipooligosaccharide of Neisseria meningitidis serogroup B strain 6275. J Bacteriol. 1993 Jul;175(14):4565–4568. doi: 10.1128/jb.175.14.4565-4568.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Zhou D., Lee N. G., Apicella M. A. Lipooligosaccharide biosynthesis in Neisseria gonorrhoeae: cloning, identification and characterization of the alpha 1,5 heptosyltransferase I gene (rfaC) Mol Microbiol. 1994 Nov;14(4):609–618. doi: 10.1111/j.1365-2958.1994.tb01300.x. [DOI] [PubMed] [Google Scholar]
  56. Zhou D., Stephens D. S., Gibson B. W., Engstrom J. J., McAllister C. F., Lee F. K., Apicella M. A. Lipooligosaccharide biosynthesis in pathogenic Neisseria. Cloning, identification, and characterization of the phosphoglucomutase gene. J Biol Chem. 1994 Apr 15;269(15):11162–11169. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES