1887

Abstract

The ∼16·5 kb surface layer (S-layer) glycan biosynthesis () gene cluster of the Gram-positive thermophile NRS 2004/3a has been sequenced. The cluster is located immediately downstream of the S-layer structural gene and consists of 13 ORFs that have been identified by database sequence comparisons. The cluster encodes dTDP--rhamnose biosynthesis ( operon), required for building up the polyrhamnan S-layer glycan, as well as for assembly and export of the elongated glycan chain, and its transfer to the S-layer protein. This is the first report of a gene cluster likely to be involved in the glycosylation of an S-layer protein. There is evidence that this cluster is transcribed as a polycistronic unit, whereas is transcribed monocistronically. To get insights into the regulatory mechanisms underlying glycosylation of the S-layer protein, the influence of growth temperature on the S-layer was investigated in seven closely related strains, of which only strain NRS 2004/3a possessed a glycosylated S-layer. Chromosomal DNA preparations of these strains were screened for the presence of the operon, because -rhamnose is a frequent constituent of S-layer glycans. From -positive strains, flanking regions of the operon were sequenced. Comparison with the gene cluster of NRS 2004/3a revealed sequence homologies between adjacent genes. The temperature inducibility of S-layer protein glycosylation was investigated in those strains by raising the growth temperature from 55 °C to 67 °C; no change of either the protein banding pattern or the glycan staining behaviour was observed on SDS-PAGE gels, although the transcript was several-fold more abundant at 67 °C. Cell-free extracts of the strains were capable of converting dTDP--glucose to dtdp--rhamnose. Taken together, the results indicate that the locus is highly conserved among strains, and that in the investigated containing strains, dTDP--rhamnose is actively synthesized . However, in contrast to previous reports for wild-type strains, an increase in growth temperature did not switch an S-layer protein phenotype to an S-layer glycoprotein phenotype, via the generation of a new S-layer gene sequence.

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2004-04-01
2019-10-19
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References

  1. Altman, E., Schäffer, C., Brisson, J.-R. & Messner, P. ( 1995; ). Characterization of the glycan structure of a major glycopeptide from the surface layer glycoprotein of Clostridium thermosaccharolyticum E207-71. Eur J Biochem 229, 308–315.[CrossRef]
    [Google Scholar]
  2. Becker, A., Rüberg, S., Küster, H., Roxlau, A. A., Keller, M., Ivashina, T., Cheng, H.-P., Walker, G. C. & Pühler, A. ( 1997; ). The 32-kilobase exp gene cluster of Rhizobium meliloti directing the biosynthesis of galactoglucan: genetic organization and properties of the encoded gene products. J Bacteriol 179, 1375–1384.
    [Google Scholar]
  3. Benz, I. & Schmidt, M. A. ( 2001; ). Glycosylation with heptose residues mediated by the aah gene product is essential for adherence of the AIDA-I adhesin. Mol Microbiol 40, 1403–1413.[CrossRef]
    [Google Scholar]
  4. Blatch, G. L. & Lässle, M. ( 1999; ). The tetratricopeptide repeat: a structural motif mediating protein-protein interactions. Bioessays 21, 932–939.[CrossRef]
    [Google Scholar]
  5. Bock, K., Schuster-Kolbe, J., Altman, E., Allmaier, G., Stahl, B., Christian, R., Sleytr, U. B. & Messner, P. ( 1994; ). Primary structure of the O-glycosidically linked glycan chain of the crystalline surface layer glycoprotein of Thermoanaerobacter thermohydrosulfuricus L111-69. Galactosyl tyrosine as a novel linkage unit. J Biol Chem 269, 7137–7144.
    [Google Scholar]
  6. Bradford, M. M. ( 1976; ). A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef]
    [Google Scholar]
  7. Bronner, D., Clarke, B. R. & Whitfield, C. ( 1994; ). Identification of an ATP-binding cassette transport system required for translocation of lipopolysaccharide O-antigen side chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1. Mol Microbiol 14, 505–519.[CrossRef]
    [Google Scholar]
  8. Comstock, L. E., Coyne, M. J., Tzianabos, A. O. & Kasper, D. L. ( 1999; ). Interstrain variation of the polysaccharide B biosynthesis locus of Bacteroides fragilis: characterization of the region from strain 638R. J Bacteriol 181, 6192–6196.
    [Google Scholar]
  9. Deckert, G., Warren, P., Gaasterland, T. & 12 other authors ( 1998; ). The complete genome of the hyperthermophilic bacterium Aquifex aeolicus. Nature 392, 353–358.[CrossRef]
    [Google Scholar]
  10. Drummelsmith, J. & Whitfield, C. ( 1999; ). Gene products required for surface expression of the capsular form of the group 1 K antigen in Escherichia coli (O9a : K30). Mol Microbiol 31, 1321–1332.[CrossRef]
    [Google Scholar]
  11. Egelseer, E. M., Idris, R., Jarosch, M., Danhorn, T., Sleytr, U. B. & Sára, M. ( 2000; ). ISBst12, a novel type of insertion-sequence element causing loss of S-layer-gene expression in Bacillus stearothermophilus ATCC 12980. Microbiology 146, 2175–2183.
    [Google Scholar]
  12. Egelseer, E. M., Danhorn, T., Pleschberger, M., Hotzy, C., Sleytr, U. B. & Sára, M. ( 2001; ). Characterization of an S-layer glycoprotein produced in the course of S-layer variation of Bacillus stearothermophilus ATCC 12980 and sequencing and cloning of the sbsD gene encoding the protein moiety. Arch Microbiol 177, 70–80.[CrossRef]
    [Google Scholar]
  13. Fox, A., Stewart, G. C., Waller, L. N., Fox, K. F., Harley, W. M. & Price, R. L. ( 2003; ). Carbohydrates and glycoproteins of Bacillus anthracis and related bacilli: targets for biodetection. J Microbiol Methods 54, 143–152.[CrossRef]
    [Google Scholar]
  14. Giraud, M.-F. & Naismith, J. H. ( 2000; ). The rhamnose pathway. Curr Opin Struct Biol 10, 687–696.[CrossRef]
    [Google Scholar]
  15. Glaser, L. & Kornfeld, S. ( 1961; ). The enzymatic synthesis of thymidine-linked sugars. II. Thymidine diphosphate l-rhamnose. J Biol Chem 236, 1795–1799.
    [Google Scholar]
  16. Graninger, M., Kneidinger, B., Bruno, K., Scheberl, A. & Messner, P. ( 2002; ). Homologs of the Rml enzymes from Salmonella enterica are responsible for dTDP-β-l-rhamnose biosynthesis in the Gram-positive thermophile Aneurinibacillus thermoaerophilus DSM 10155. Appl Environ Microbiol 68, 3708–3715.[CrossRef]
    [Google Scholar]
  17. Guo, D., Bowden, M. G., Pershad, R. & Kaplan, H. B. ( 1996; ). The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development. J Bacteriol 178, 1631–1639.
    [Google Scholar]
  18. Heinrichs, D. E., Monteiro, M. A., Perry, M. B. & Whitfield, C. ( 1998; ). The assembly system for the lipopolysaccharide R2 core-type of Escherichia coli is a hybrid of those found in Escherichia coli K-12 and Salmonella enterica. Structure and function of the R2 WaaK and WaaL homologs. J Biol Chem 273, 8849–8859.[CrossRef]
    [Google Scholar]
  19. Jarosch, M., Egelseer, E. M., Mattanovich, D., Sleytr, U. B. & Sára, M. ( 2000; ). S-layer gene sbsC of Bacillus stearothermophilus ATCC 12980: molecular characterization and heterologous expression in Escherichia coli. Microbiology 146, 273–281.
    [Google Scholar]
  20. Jiang, S.-M., Wang, L. & Reeves, P. R. ( 2001; ). Molecular characterization of Streptococcus pneumoniae type 4, 6B, 8, and 18C capsular polysaccharide gene clusters. Infect Immun 69, 1244–1255.[CrossRef]
    [Google Scholar]
  21. Keenleyside, W. J. & Whitfield, C. ( 1999; ). Genetics and biosynthesis of lipopolysaccharide O-antigens. In Endotoxin in Health and Disease, pp. 331–358. Edited by H. Brade, S. M. Opal, S. N. Vogel & D. C. Morrison. New York & Basel: Marcel Dekker.
  22. Kneidinger, B., Graninger, M. & Messner, P. ( 2001; ). Chromosome walking by cloning of distinct PCR fragments. Biotechniques 30, 248–249.
    [Google Scholar]
  23. Kornfeld, S. & Glaser, L. ( 1961; ). The enzymatic synthesis of thymidine-linked sugars. I. Thymidine diphosphate glucose. J Biol Chem 236, 1791–1794.
    [Google Scholar]
  24. Mahillon, J. & Chandler, M. ( 1998; ). Insertion sequences. Microbiol Mol Biol Rev 62, 725–744.
    [Google Scholar]
  25. Maier, R. M. & Soberón-Chávez, G. ( 2000; ). Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications. Appl Microbiol Biotechnol 54, 625–633.[CrossRef]
    [Google Scholar]
  26. Marumo, K., Lindqvist, L., Verma, N., Weintraub, A., Reeves, P. R. & Lindberg, A. A. ( 1992; ). Enzymatic synthesis and isolation of thymidine diphosphate-6-deoxy-d-xylo-4-hexulose and thymidine diphosphate-l-rhamnose. Production using cloned gene products and separation by HPLC. Eur J Biochem 204, 539–545.[CrossRef]
    [Google Scholar]
  27. Messner, P. & Schäffer, C. ( 2000; ). Surface layer glycoproteins of Bacteria and Archaea. In Glycomicrobiology, pp. 93–125. Edited by R. J. Doyle. New York: Kluwer Academic/Plenum Publishers.
  28. Messner, P. & Schäffer, C. ( 2003; ). Prokaryotic glycoproteins. In Progress in the Chemistry of Organic Natural Products, vol. 85, pp. 51–124. Edited by W. Herz, H. Falk & G. W. Kirby. Wien: Springer.
  29. Messner, P., Hollaus, F. & Sleytr, U. B. ( 1984; ). Paracrystalline cell wall surface layers of different Bacillus stearothermophilus strains. Int J Syst Bacteriol 34, 202–210.[CrossRef]
    [Google Scholar]
  30. Nölling, J., Breton, G., Omelchenko, M. V. & 16 other authors ( 2001; ). Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol 183, 4823–4838.[CrossRef]
    [Google Scholar]
  31. Ochman, H., Lawrence, J. G. & Groisman, E. A. ( 2000; ). Lateral gene transfer and the nature of bacterial innovation. Nature 405, 299–304.[CrossRef]
    [Google Scholar]
  32. Power, P. M. & Jennings, M. P. ( 2003; ). The genetics of glycosylation in Gram-negative bacteria. FEMS Microbiol Lett 218, 211–222.[CrossRef]
    [Google Scholar]
  33. Raetz, C. R. H. & Whitfield, C. ( 2002; ). Lipopolysaccharide endotoxins. Annu Rev Biochem 71, 635–700.[CrossRef]
    [Google Scholar]
  34. Rahim, R., Ochsner, U. A., Olvera, C., Graninger, M., Messner, P., Lam, J. S. & Soberón-Chávez, G. ( 2001; ). Cloning and functional characterization of the Pseudomonas aeruginosa rhlC gene that encodes rhamnosyltransferase 2, an enzyme responsible for di-rhamnolipid biosynthesis. Mol Microbiol 40, 708–718.[CrossRef]
    [Google Scholar]
  35. Reeves, P. R., Hobbs, M., Valvano, M. A. & 8 other authors ( 1996; ). Bacterial polysaccharide synthesis and gene nomenclature. Trends Microbiol 4, 495–503.[CrossRef]
    [Google Scholar]
  36. 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]
  37. Rocchetta, H. L., Burrows, L. L. & Lam, J. S. ( 1999; ). Genetics of O-antigen biosynthesis in Pseudomonas aeruginosa. Microbiol Mol Biol Rev 63, 523–553.
    [Google Scholar]
  38. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  39. Schäffer, C. & Messner, P. ( 2001; ). Glycobiology of surface layer proteins. Biochimie 83, 591–599.[CrossRef]
    [Google Scholar]
  40. Schäffer, C., Wugeditsch, T., Kählig, H., Scheberl, A., Zayni, S. & Messner, P. ( 2002; ). The surface layer (S-layer) glycoprotein of Geobacillus stearothermophilus NRS 2004/3a. Analysis of its glycosylation. J Biol Chem 277, 6230–6239.[CrossRef]
    [Google Scholar]
  41. Schnaitman, C. A. & Klena, J. D. ( 1993; ). Genetics of lipopolysaccharide biosynthesis in enteric bacteria. Microbiol Rev 57, 655–682.
    [Google Scholar]
  42. Sleytr, U. B. & Messner, P. ( 2003; ). Crystalline bacterial cell surface layers (S layers). In Desk Encyclopedia of Microbiology, pp. 284–291. Edited by M. Schaechter. San Diego: Elsevier Science.
  43. Sleytr, U. B., Sára, M., Pum, D., Schuster, B., Messner, P. & Schäffer, C. ( 2002; ). Self-assembly protein systems: microbial S-layers. In Biopolymers, vol. 7, Polyamides and Complex Proteinaceous Matrices I, pp. 285–338. Edited by A. Steinbüchel & S. R. Fahnestock. Weinheim: Wiley-VCH.
  44. Strauss, J., Horvath, H. K., Abdallah, B. M., Kindermann, J., Mach, R. L. & Kubicek, C. P. ( 1999; ). The function of CreA, the carbon catabolite repressor of Aspergillus nidulans, is regulated at the transcriptional and post-transcriptional level. Mol Microbiol 32, 169–178.[CrossRef]
    [Google Scholar]
  45. Tsukioka, Y., Yamashita, Y., Oho, T., Nakano, Y. & Koga, T. ( 1997a; ). Biological function of the dTDP-rhamnose synthesis pathway in Streptococcus mutans. J Bacteriol 179, 1126–1134.
    [Google Scholar]
  46. Tsukioka, Y., Yamashita, Y., Nakano, Y., Oho, T. & Koga, T. ( 1997b; ). Identification of a fourth gene involved in dTDP-rhamnose synthesis in Streptococcus mutans. J Bacteriol 179, 4411–4414.
    [Google Scholar]
  47. Wang, L., Liu, D. & Reeves, P. R. ( 1996; ). C-terminal half of Salmonella enterica WbaP (RfbP) is the galactosyl-1-phosphate transferase domain catalyzing the first step of O-antigen synthesis. J Bacteriol 178, 2598–2604.
    [Google Scholar]
  48. Whitfield, C. ( 1995; ). Biosynthesis of lipopolysaccharide O-antigens. Trends Microbiol 3, 178–185.[CrossRef]
    [Google Scholar]
  49. Whitfield, C. & Valvano, M. A. ( 1993; ). Biosynthesis and expression of cell-surface polysaccharides in Gram-negative bacteria. Adv Microb Physiol 35, 135–246.
    [Google Scholar]
  50. Winzler, R. J. ( 1955; ). Determination of serum glycoproteins. Methods Biochem Anal 11, 279–311.
    [Google Scholar]
  51. Xu, K., He, Z.-Q., Mao, Y.-M., Sheng, R.-Q. & Sheng, Z.-J. ( 1993; ). On two transposable elements from Bacillus stearothermophilus. Plasmid 29, 1–9.[CrossRef]
    [Google Scholar]
  52. Xu, Y., Murray, B. E. & Weinstock, G. M. ( 1998; ). A cluster of genes involved in polysaccharide biosynthesis from Enterococcus faecalis OG1RF. Infect Immun 66, 4313–4323.
    [Google Scholar]
  53. Yamashita, Y., Tsukioka, Y., Tomihisa, K., Nakano, Y. & Koga, T. ( 1998; ). Genes involved in cell wall localization and side chain formation of rhamnose-glucose polysaccharide in Streptococcus mutans. J Bacteriol 180, 5803–5807.
    [Google Scholar]
  54. Zhang, L., al-Hendy, A., Toivanen, P. & Skurnik, M. ( 1993; ). Genetic organization and sequence of the rfb gene cluster of Yersinia enterocolitica serotype O : 3: similarities to the dTDP-l-rhamnose biosynthesis pathway of Salmonella and to the bacterial polysaccharide transport systems. Mol Microbiol 9, 309–321.[CrossRef]
    [Google Scholar]
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