1887

Abstract

Lipooligosaccharide (LOS) is a critical virulence factor of . A Tn insertion mutant, designated 469, was found to exhibit a markedly truncated LOS of 29 kDa when compared by Tricine/SDS-PAGE to the parental LOS (46 kDa). Electrospray mass spectrometry analysis of 469 LOS revealed that it consisted of the deep rough, heptose-deficient structure, Kdo-lipid A. Sequencing of chromosomal DNA flanking the Tn insertion in mutant 469 revealed that the transposon had inserted into an ORF predicted to encode a 187 aa protein with sequence homology to the histidinol-phosphate phosphatase domain of HisB and to a family of genes of unknown function. The gene, designated , is part of a polycistronic operon () containing two other genes, and encodes a lysophosphatidic-acid acyltransferase and is predicted to encode a -acetyltransferase. Specific polar and non-polar mutations in the parental strain, NMB, exhibited the truncated LOS structure of mutant 469, and repair of mutants by homologous recombination with the wild-type restored the LOS parental phenotype. GmhX mutants demonstrated increased sensitivity to polymyxin B. GmhX mutants and other Kdo-lipid A mutants also demonstrated increased sensitivity to killing by normal human serum but were not as sensitive as inner-core mutants containing heptose. In the genomes of and , homologues are associated with heptose biosynthesis genes; however, in , was found in a location distinct from that of , , , and . GmhX is a novel enzyme required for the incorporation of L--D--heptose into meningococcal LOS, and is a candidate for the 2-D--heptose phosphatase of the heptose biosynthesis pathway.

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2001-08-01
2019-10-17
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References

  1. Apicella, M. A., Bennett, K. M., Hermerath, C. A. & Roberts, D. E. ( 1981; ). Monoclonal antibody analysis of lipopolysaccharide from Neisseria gonorrhoeae and Neisseria meningitidis. Infect Immun 34, 751-756.
    [Google Scholar]
  2. 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 b chain synthesizing glucosyl transferase from Neisseria gonorrhoeae. Proc Natl Acad Sci USA 95, 10872-10877.[CrossRef]
    [Google Scholar]
  3. Brooke, J. S. & Valvano, M. A. ( 1996a; ). Biosynthesis of inner core lipopolysaccharide in enteric bacteria: identification and characterization of a conserved phosphoheptose isomerase. J Biol Chem 271, 3608-3614.[CrossRef]
    [Google Scholar]
  4. Brooke, J. S. & Valvano, M. A. ( 1996b; ). Molecular cloning of the Haemophilus influenzae gmhA (lpcA) gene encoding a phosphoheptose isomerase required for lipooligosaccharide biosynthesis. J Bacteriol 178, 3339-3341.
    [Google Scholar]
  5. Chen, L. & Coleman, W. G.Jr ( 1993; ). Cloning and characterization of the Escherichia coli K-12 rfa-2 (rfaC) gene, a gene required for lipopolysaccharide inner core synthesis. J Bacteriol 175, 2534-2540.
    [Google Scholar]
  6. Chiariotti, L., Nappo, A. G., Carlomagno, M. S. & Bruni, C. B. ( 1986; ). Gene structure in the hisitidine operon of Escherichia coli. Identification and nucleotide sequence of the hisB gene. Mol Gen Genet 202, 42-47.[CrossRef]
    [Google Scholar]
  7. Chung, C. T., Niemela, S. L. & Miller, R. H. ( 1989; ). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci USA 86, 2172-2175.[CrossRef]
    [Google Scholar]
  8. Coleman, J. ( 1992; ). Characterization of the Escherichia coli gene for 1-acyl-sn-glycerol-3-phosphate acyltransferase. Mol Gen Genet 232, 295-303.
    [Google Scholar]
  9. Coleman, W. G.Jr ( 1983; ). The rfaD gene codes for ADP-l-glycero-d-mannoheptose-6-epimerase. An enzyme required for lipopolysaccharide core biosynthesis. J Biol Chem 258, 1985-1990.
    [Google Scholar]
  10. Dicker, I. B. & Seetharam, S. ( 1992; ). What is known about the structure and function of the Escherichia coli protein FirA? Mol Microbiol 6, 817-823.[CrossRef]
    [Google Scholar]
  11. Drazek, E. S., Stein, D. C. & Deal, C. D. ( 1995; ). A mutation in the Neisseria gonorrhoeae rfaD homolog results in altered lipooligosaccharide expression. J Bacteriol 177, 2321-2327.
    [Google Scholar]
  12. Eidels, L. & Osborn, M. J. ( 1974; ). Phosphoheptose isomerase, first enzyme in the biosynthesis of aldoheptose in Salmonella typhimurium. J Biol Chem 249, 5642-5648.
    [Google Scholar]
  13. Engebrecht, J. & Brent, R. (1996). Minipreps of plasmid DNA: alkaline lysis miniprep. In Current Protocols in Molecular Biology, unit 1.6.3. Edited by F. M. Ausubel and others. New York: Wiley.
  14. Erwin, A. L. & Stephens, D. S. ( 1995; ). Identification and characterization of auxotrophs of Neisseria meningitidis produced by Tn916 mutagenesis. FEMS Microbiol Lett 127, 223-228.[CrossRef]
    [Google Scholar]
  15. Galanos, C., Luderitz, O. & Westphal, O. ( 1969; ). A new method for the extraction of R lipopolysaccharides. Eur J Biochem 9, 245-249.[CrossRef]
    [Google Scholar]
  16. 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]
  17. Hughes, M. J. G. & Andrews, D. W. ( 1996; ). Creation of deletion, insertion and substitution mutations using a single pair of primers and PCR. BioTechniques 20, 188-196.
    [Google Scholar]
  18. Janik, A., Juni, E. & Heym, G. A. ( 1976; ). Genetic transformation as a tool for detection of Neisseria gonorrhoeae. J Clin Microbiol 4, 71-81.
    [Google Scholar]
  19. Jennings, M. P., Hood, D. W., Peak, I. R., 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]
  20. Kadrmas, J. L. & Raetz, C. R. H. ( 1998; ). Enzymatic synthesis of lipopolysaccharide in Escherichia coli: purification and properties of heptosyltransferase I. J Biol Chem 273, 2799-2807.[CrossRef]
    [Google Scholar]
  21. Kahler, C. M. & Stephens, D. S. ( 1998; ). Genetic basis for biosynthesis, structure and function of meningococcal lipooligosaccharide (endotoxin). Crit Rev Microbiol 24, 281-334.
    [Google Scholar]
  22. Kahler, C. M., Carlson, R. W., Rahman, M. M., Martin, L. E. & Stephens, D. S. ( 1996a; ). Inner core biosynthesis of lipooligosaccharide in Neisseria meningitidis serogroup B: identification and role in LOS assembly of the 1,2 N-acetylglucosamine transferase (rfaK). J Bacteriol 178, 1265-1273.
    [Google Scholar]
  23. Kahler, C. M., Carlson, R. W., Rahman, M. M., Martin, L. E. & Stephens, D. S. ( 1996b; ). 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]
  24. Kahler, C. M., Martin, L. E., Shih, G. C., Rahman, M. M., Carlson, R. W. & Stephens, D. S. ( 1998; ). The (α2→8)-linked polysialic acid capsule and lipooligosaccharide structure both contribute to the ability of serogroup B Neisseria meningitidis to resist the bactericidal activity of normal human serum. Infect Immun 66, 5939-5947.
    [Google Scholar]
  25. Lee, F. K. N., Stephens, D. S., Gibson, B. W., Engstrom, J. J., Zhou, D. & Apicella, M. A. ( 1995; ). Microheterogeneity of Neisseria lipooligosaccharide: analysis of a UDP-glucose 4-epimerase mutant of Neisseria meningitidis NMB. Infect Immun 63, 2508-2515.
    [Google Scholar]
  26. Lee, N.-G., Sunshine, M. G. & Apicella, M. A. ( 1995; ). Molecular cloning and characterization of the nontypeable Haemophilus influenzae 2019 rfaE gene required for lipopolysaccharide biosynthesis. Infect Immun 63, 818-824.
    [Google Scholar]
  27. Lesse, A. J., Campagnari, A. A., Bittner, W. E. & Apicella, M. A. ( 1990; ). Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Immunol Methods 126, 109-117.[CrossRef]
    [Google Scholar]
  28. Levin, J. C. & Stein, D. C. ( 1996; ). Cloning, complementation, and characterization of an rfaE homolog from Neisseria gonorrhoeae. J Bacteriol 178, 4571-4575.
    [Google Scholar]
  29. Nath, K. ( 1990; ). A rapid DNA isolation procedure from petri dish grown clinical bacterial isolates. Nucleic Acids Res 18, 6462.[CrossRef]
    [Google Scholar]
  30. Nichols, W. E., Gibson, B. W., Melaugh, W., Lee, N.-G., Sunshine, M. & Apicella, M. A. ( 1997; ). Identification of the ADP-l-glycero-d-manno-heptose-6-epimerase (rfaD) and heptosyltransferase II (rfaF) biosynthetic genes from nontypeable Haemophilus influenzae 2019. Infect Immun 65, 1377-1386.
    [Google Scholar]
  31. Pegues, J. C., Chen, L. S., Gordon, A. W., Ding, L. & Coleman, W. G.Jr ( 1990; ). Cloning, expression, and characterization of the Escherichia coli K-12 rfaD gene. J Bacteriol 172, 4652-4660.
    [Google Scholar]
  32. Peschke, U., Schmidt, H., Zhang, H. Z. & Piepersberg, W. ( 1995; ). Molecular characterization of the lincomycin-production gene cluster of Streptomyces lincolnensis 78–11. Mol Microbiol 16, 1137-1156.[CrossRef]
    [Google Scholar]
  33. Polissi, A. & Georgopoulos, C. ( 1996; ). Mutational analysis and properties of the msbA gene of Escherichia coli, coding for an essential ABC family transporter. Mol Microbiol 20, 1221-1233.[CrossRef]
    [Google Scholar]
  34. Prentki, P. & Krisch, H. M. ( 1984; ). In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29, 303-313.[CrossRef]
    [Google Scholar]
  35. Price, N. P. J., Jeyaretnam, B., Carlson, R. W., Kadrmas, J. L., Raetz, C. R. H. & Brozek, K. A. ( 1995; ). Lipid A biosynthesis in Rhizobium leguminosarum: role of a 2-keto-3-deoxyoctulosonate-activated 4′ phosphatase. Proc Natl Acad Sci USA 92, 7352-7356.[CrossRef]
    [Google Scholar]
  36. Raetz, C. R. H. (1996). Bacterial lipopolysaccharides: a remarkable family of bioreactive macroamphiphiles. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, pp. 1035–1063. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
  37. Rahman, M. M., Stephens, D. S., Kahler, C. M., Glushka, J. & Carlson, R. W. ( 1998; ). The lipooligosaccharide (LOS) of Neisseria meningitidis serogroup B strain NMB contains L2, L3, and novel oligosaccharides, and lacks the lipid-A 4′-phosphate substituent. Carbohydr Res 307, 311-324.[CrossRef]
    [Google Scholar]
  38. Robbins, A. & Freeman, P. ( 1988; ). Obstacles to developing vaccines for the third world. Sci Am 259, 90-95.
    [Google Scholar]
  39. Schagger, H. & von Jagow, G. ( 1987; ). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 368-379.[CrossRef]
    [Google Scholar]
  40. Schnaitman, C. A. & Klena, J. D. ( 1993; ). Genetics of lipopolysaccharide biosynthesis in enteric bacteria. Microbiol Rev 57, 655-682.
    [Google Scholar]
  41. Shafer, W. M., Guymon, L. F., Lind, I. & Sparling, P. F. ( 1984; ). Identification of an envelope mutation (env-10) resulting in increased antibiotic susceptibility and pyocin resistance in a clinical isolate of Neisseria gonorrhoeae. Antimicrob Agents Chemother 25, 767-769.[CrossRef]
    [Google Scholar]
  42. Shih, G., Kahler, C., Swartley, J., Coleman, J., Rahman, M., Carlson, R. & Stephens, D. ( 1999; ). Multiple lysophosphatidic acid acyltransferases in Neisseria meningitidis. Mol Microbiol 32, 942-952.[CrossRef]
    [Google Scholar]
  43. Shyamala, V. & Ames, G. F.-L. ( 1989; ). Genome walking by single-specific-primer polymerase chain reaction: SSP-PCR. Gene 84, 1-8.[CrossRef]
    [Google Scholar]
  44. Sirisena, D. M., Brozek, K. A., MacLachlan, P. R., Sanderson, K. E. & Raetz, C. R. ( 1992; ). The rfaC gene of Salmonella typhimurium: cloning, sequencing, and enzymatic function in heptose transfer to lipopolysaccharide. J Biol Chem 267, 18874-18884.
    [Google Scholar]
  45. Sirisena, D. M., MacLachlan, P. R., Liu, S.-l., Hessel, A. & Sanderson, K. E. ( 1994; ). Molecular analysis of the rfaD gene, for heptose synthesis, and the rfaF gene, for heptose transfer, in lipopolysaccharide synthesis in Salmonella typhimurium. J Bacteriol 176, 2379-2385.
    [Google Scholar]
  46. Steeghs, L., Jennings, M. P., Poolman, J. T. & van der Ley, P. ( 1997; ). Isolation and characterization of the Neisseria meningitidis lpxD-fabZ-lpxA gene cluster involved in lipid A biosynthesis. Gene 190, 263-270.[CrossRef]
    [Google Scholar]
  47. Stephens, D. S., Swartley, J. S., Kathariou, S. & Morse, S. A. ( 1991; ). Insertion of Tn916 in Neisseria meningitidis resulting in loss of group B capsular polysaccharide. Infect Immun 59, 4097-4102.
    [Google Scholar]
  48. Stephens, D. S., McAllister, C. F., Zhou, D., Lee, F. K. & Apicella, M. A. ( 1994; ). Tn916-generated, lipooligosaccharide mutants of Neisseria meningitidis and Neisseria gonorrhoeae. Infect Immun 62, 2947-2952.
    [Google Scholar]
  49. Swartley, J. S., McAllister, C. F., Hajjeh, R. A., Heinrich, D. W. & Stephens, D. S. ( 1993; ). Deletions of Tn916-like transposons are implicated in tetM-mediated resistance in pathogenic Neisseria. Mol Microbiol 10, 299-310.[CrossRef]
    [Google Scholar]
  50. Takahashi, K., Fukada, M., Kawai, M. & Yokochi, T. ( 1992; ). Detection of lipopolysaccharide (LPS) and identification of its serotype by an enzyme-linked immunosorbent assay (ELISA) using poly-l-lysine. J Immunol Methods 153, 67-71.[CrossRef]
    [Google Scholar]
  51. Tikhomirov, E., Santamaria, M. & Esteves, K. ( 1997; ). Meningococcal disease: public health burden and control. World Health Stat Q 50, 170-177.
    [Google Scholar]
  52. Tunkel, A. R. & Scheld, W. M. ( 1993; ). Pathogenesis and pathophysiology of bacterial meningitis. Clin Microbiol Rev 6, 118-136.
    [Google Scholar]
  53. Valvano, M. ( 1999; ). Biosynthesis and genetics of ADP-heptose. J Endotox Res 5, 90-95.[CrossRef]
    [Google Scholar]
  54. Valvano, M., Marolda, C. L., Bittner, M., Glaskin-Clay, M., Simon, T. L. & Lkena, J. D. ( 2000; ). The rfaE gene from Escherichia coli encodes a biofunctional protein involved in biosynthesis of the lipopolysaccharide core precursor ADP-l-glycero-d-manno-heptose. J Bacteriol 182, 488-497.[CrossRef]
    [Google Scholar]
  55. Vuorio, R., Harkonen, T., Tolvane, M. & Vaara, M. ( 1994; ). The novel hexapeptide motif found in the acyltransferases LpxA and LpxD of lipid A biosynthesis is conserved in various bacteria. FEBS Lett 337, 289-292.[CrossRef]
    [Google Scholar]
  56. Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. ( 1982; ). Distantly related sequences in the α- and β-subunits of ATP synthase, myosin, kinases, and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1, 945-951.
    [Google Scholar]
  57. Wilkinson, R. G., Gemski, P.Jr & Stocker, B. A. ( 1972; ). Non-smooth mutants of Salmonella typhimurium: differentiation by phage sensitivity and genetic mapping. J Gen Microbiol 70, 527-554.[CrossRef]
    [Google Scholar]
  58. Zhou, D., Stephens, D. S., Gibson, B. W., Engstrom, J. J., McAllister, C. F., Lee, F. K. N. & Apicella, M. A. ( 1994; ). Lipooligosaccharide biosynthesis in pathogenic Neisseria: cloning, identification, and characterization of the phosphoglucomutase gene. J Biol Chem 269, 11162-11169.
    [Google Scholar]
  59. Zhou, Z., White, K. A., Polissi, A., Georgopoulos, C. & Raetz, C. R. ( 1998; ). Function of Escherichia coli MsbA, an essential ABC family transporter, in lipid A and phospholipid biosynthesis. J Biol Chem 273, 12466-12475.[CrossRef]
    [Google Scholar]
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