1887

Abstract

sp. strain 12S produces an exopolysaccharide (EPS), methanolan, composed of glucose, mannose and galactose. Twenty-four ORFs flanking a Tn insertion site in an EPS-deficient mutant were identified, and 21 genes () were predicted to participate in methanolan synthesis on the basis of the features of the primary sequence. Gene disruption analyses revealed that and are required for methanolan synthesis, whereas and are not essential. EpsFG and EpsE showed homology with Wzc (chain length regulator) and Wza (export protein) of group 1 capsule-producing , suggesting that methanolan was synthesized via a Wzy-like biosynthesis system. This possibility was supported by the fact that the putative hydropathy profiles of EpsH and EpsM were similar to those of Wzx and Wzy, which are also involved in the flipping of the repeating unit in the cytoplasmic membrane and the polymerization of the capsule in the Wzy-dependent system. EpsBJNOP and EpsR are probably glycosyltransferases involved in the synthesis of the repeating unit onto the lipid carrier. In particular, EpsB appeared to catalyse the initial transfer of the glucose moiety. On the basis of their predicted location in the cells, it is proposed that EpsI and EpsL are involved in methanolan export to the cell surface. strains expressing EpsQ, EpsS and EpsT showed enhanced activities of GDP-mannose pyrophosphorylase, UDP-galactose 4-epimerase and UDP-glucose pyrophosphorylase, respectively, revealing that they were responsible for the production of the activated compositional sugars of methanolan. EpsU contains a conserved a lytic transglycosylase motif, indicating that it could participate in the degradation of polysaccharides. EpsA and EpsK, which have conserved DNA-binding and cAMP-binding motifs, respectively, were deduced to be transcriptional regulators. In particular, EpsA seems to positively regulate the transcription of methanolan synthesis genes, since the constitutive expression of in strain 12S increased the EPS production. Interestingly, EpsD showed homology with peptidyl prolyl isomerases that catalyse the folding of proteins following translocation across the cytoplasmic membrane.

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2003-02-01
2024-03-28
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References

  1. Allen P, Hart C. A., Saunders J. R. 1987; Isolation from Klebsiella and characterization of two rcs genes that activate colanic acid capsular synthesis in Escherichia coli . J Gen Microbiol 133:331–340
    [Google Scholar]
  2. Altschul S. F, Warren G, Miller W, Myers E. W., Lipman D. J. 1990; Basic local alignment search tool. J Mol Biol 215:403–410
    [Google Scholar]
  3. Attwood M. M., Quayle J. R. 1984; Formaldehyde as a central intermediary metabolite of methylotrophic metabolism. In Microbial Growth on C-1 Compounds pp 315–323 Edited by Crawford R. L., Hanson R. S. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  4. Becker A, Niehaus K., Puhler A. 1995; Low-molecular-weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterized by a periplasmic N-terminal domain and a missing C-terminal domain. Mol Microbiol 16:191–203
    [Google Scholar]
  5. Becker A, Katzen F, Puhler A., Ielpi L. 1998; Xanthan gum synthesis and application: a biochemical/genetic perspective. Appl Microbiol Biotechnol 50:145–152
    [Google Scholar]
  6. Bugert P., Geider K. 1995; Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora . Mol Microbiol 15:917–933
    [Google Scholar]
  7. 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]
  8. Chida K, Shen G. J, Kodama T., Minoda Y. 1983; Acidic polysaccharide production from methane by a new methane-oxidizing bacterium H-2. Agric Biol Chem 47:275–280
    [Google Scholar]
  9. Claros M. G., von-Heijne G. 1994; TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci 10:685–686
    [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
    [Google Scholar]
  11. Drummelsmith J., Whitfield C. 2000; Translocation of group 1 capsular polysaccharide to the surface of Escherichia coli requires a multimeric complex in the outer membrane. EMBO J 19:57–66
    [Google Scholar]
  12. Dubois M, Gilles K. A, Hamilton J. K, Rebers P. A., Smith F. 1956; Colorimetric method for determination of sugar and related substances. Anal Chem 28:350–356
    [Google Scholar]
  13. Erik L. L. S, Gunnar H., Anders K. 1998; A hidden Markov model for predicting transmembrane helices in protein sequences. In Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology pp 175–182 Edited by Glasgow J., Littlejohn T., Major F., Lathrop R., Sankoff D., Sensen C. Menlo Park, CA: AAAI Press;
    [Google Scholar]
  14. Gonzalez J. E, Semino C. E, Wang L. X, Castellano-Torres L. E., Walker G. C. 1998; Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti . Proc Natl Acad Sci U S A 95:13477–13482
    [Google Scholar]
  15. Gottesman S., Stout V. 1991; Regulation of capsular polysaccharide synthesis in Escherichia coli K12. Mol Microbiol 5:1599–1606
    [Google Scholar]
  16. Holtje J. V. 1996; Lytic transglycosylases. EXS 75:425–429
    [Google Scholar]
  17. Hou C. T, Laskin A. I., Patel R. N. 1978; Growth and polysaccharide production by Methylocystis parvus OBBP on methanol. Appl Environ Microbiol 37:800–804
    [Google Scholar]
  18. Ian S. R. 1996; The biochemistry and genetics of capsular polysaccharide production in bacteria. Annu Rev Microbiol 50:285–315
    [Google Scholar]
  19. Kolkman M. A, Morrison D. A, van-der-Zeijst B. A., Nuijten P. J. 1996; The capsule polysaccharide synthesis locus of Streptococcus pneumoniae serotype 14: identification of the glycosyl transferase gene cps14E . J Bacteriol 178:3736–3741
    [Google Scholar]
  20. Koo H. M, Yim S. W, Lee C. S, Pyun Y. R., Kim Y. S. 2000; Cloning, sequencing, and expression of UDP-glucose pyrophosphorylase gene from Acetobacter xylinum BRC5. Biosci Biotechnol Biochem 64:523–529
    [Google Scholar]
  21. Kovach M. E, Elzer P. H, Hill D. S, Robertson G. T, Farris M. A, Roop R. M., Peterson K. M. 1995; Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176
    [Google Scholar]
  22. Linton J. D, Watts P. D, Austin R. M, Haugh D. E., Neikus H. G. D. 1986; The energetics and kinetics of extracellular polysaccharide production from methanol by micro-organisms possessing different pathways of C1 assimilation. J Gen Microbiol 132:779–788
    [Google Scholar]
  23. Llull D, Lopez R, Garcia E., Munoz R. 1998; Molecular structure of the gene cluster responsible for the synthesis of the polysaccharide capsule of Streptococcus pneumoniae type 33F. Biochim Biophys Acta 1443217–224
    [Google Scholar]
  24. Meyer M, Dimroth P., Bott M. 2001; Catabolite repression of the citrate fermentation genes in Klebsiella pneumoniae : evidence for involvement of the cyclic AMP receptor protein. J Bacteriol 183:5248–5256
    [Google Scholar]
  25. Missiakas D, Betton J. M., Raina S. 1996; New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH. Mol Microbiol 21:871–884
    [Google Scholar]
  26. Monday S. R., Schiller N. L. 1996; Alginate synthesis in Pseudomonas aeruginosa : the role of AlgL (alginate lyase) and AlgX. J Bacteriol 178:625–632
    [Google Scholar]
  27. Moreno F, Rodicio R., Herrero P. 1981; A new colorimetric assay for UDP-glucose 4-epimerase activity. Cell Mol Biol 27:589–592
    [Google Scholar]
  28. Morona R, Mavris M, Fallarino A., Manning P. A. 1994; Characterization of the rfc region of Shigella flexneri . J Bacteriol 176:733–747
    [Google Scholar]
  29. Morona J. K, Paton J. C, Miller D. C., Morona R. 2000; Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in Streptococcus pneumoniae . Mol Microbiol 35:1431–1442
    [Google Scholar]
  30. Nakai K., Horton P. 1999; PSORT: a program for detecting the sorting signal of proteins and predicting their subcellular localization. Trends Biochem Sci 24:34–35
    [Google Scholar]
  31. Ohta A, Chibana H, Arisawa M., Sudoh M. 2000; The VIG9 gene products from the human pathogenic fungi Candida albicans and Candida glabrata encode GDP-mannose pyrophosphorylase. Biochim Biophys Acta 1475265–272
    [Google Scholar]
  32. Ojinnaka C, Jay A. J, Colquhoun I. J, Brownsey G. J, Morris E. R., Morris V. J. 1996; Structure and conformation of acetan polysaccharide. Int J Biol Macromol 19:149–156
    [Google Scholar]
  33. Sambrook J, Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  34. Short J. M, Fernandez J. M, Sorge J. A., Huse W. D. 1988; λ ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res 16:7583–7600
    [Google Scholar]
  35. Simon R. 1984; High frequency mobilization of Gram-negative bacterial replicons by the in vitro constructed Tn 5 - mob transposon. Mol Gen Genet 196:413–420
    [Google Scholar]
  36. Southgate G., Goodwin P. M. 1989; The regulation of exopolysaccharide production and of enzymes involved in C1 assimilation in Methylophilus methylotrophus . J Gen Microbiol 135:2859–2867
    [Google Scholar]
  37. Sutherland I. W. 1985; Synthesis and composition of gram-negative bacterial extracellular and wall polysaccharide. Annu Rev Microbiol 39:243–270
    [Google Scholar]
  38. Thompson J. D, Higgins D. G., Gibson T. J. 1994; Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
    [Google Scholar]
  39. Tolmasky M. E, Staneloni R. J., Leloir L. F. 1982; Lipid-bound saccharides in Rhizobium meliloti . J Biol Chem 257:6751–6757
    [Google Scholar]
  40. Tusnady G. E., Simon I. 1998; Principles governing amino acid composition of integral membrane proteins: application to topology prediction. J Mol Biol 283:489–506
    [Google Scholar]
  41. Velasco A, Alonso S, Garcia J. L, Perera J., Diaz E. 1998; Genetic and functional analysis of the styrene catabolic cluster of Pseudomonas sp. strain Y2. J Bacteriol 180:1063–1071
    [Google Scholar]
  42. Vincent C, Doublet P, Grangeasse C, Vaganay E, Cozzone A. J., Duclos B. 1999; Cells of Escherichia coli contain a protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb. J Bacteriol 181:3472–3477
    [Google Scholar]
  43. Wang L., Liu D. 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]
  44. Wehland M., Bernhard F. 2000; The RcsAB box: characterization of a new operator essential for the regulation of exopolysaccharide synthesis in enteric bacteria. J Biol Chem 275:7013–7020
    [Google Scholar]
  45. Whitfield C. 1995; Biosynthesis of lipopolysaccharide O antigens. Trends Microbiol 3:178–185
    [Google Scholar]
  46. Wyss O., Moreland E. J. 1968; Composition of the capsule of obligate hydrocarbon-utilizing bacteria. Appl Environ Microbiol 16:185
    [Google Scholar]
  47. Yanisch-Perron C, Vieira J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119
    [Google Scholar]
  48. Yoshida T, Ayabe T, Horinouchi M, Habe H, Nojiri H., Omori T. 2000; Saccharide production from methanol by Tn 5 -mutants derived from the extracellular polysaccharide-producing bacterium Methylobacillus sp. strain 12S. Appl Microbiol Biotechnol 54:341–347
    [Google Scholar]
  49. Yoshida T, Ayabe T, Horinouchi M, Habe H, Nojiri H., Omori T. 2001; Improvement of transformation efficiency of extracellular polysaccharide-producing methylotroph Methylobacillus sp. strain 12S by electroporation. Biotechnol Lett 23:787–791
    [Google Scholar]
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