1887

Abstract

The moderately halophilic strain S-30 produces a high-molecular-mass acidic polymer (4·7×10 Da) composed of repeating units of mannose, galactose, glucose and glucuronic acid. This exopolysaccharide (EPS), known as mauran, has interesting functional properties that make it suitable for use in many industrial fields. Analysis of the flanking regions of a mini-Tn insertion site in an EPS-deficient mutant of , strain TK71, led to the identification of five ORFs (), which form part of a gene cluster () with the same structural organization as others involved in the biosynthesis of group 1 capsules and some EPSs. Conserved genetic features were found such as JUMPstart and elements, which are characteristically located preceding the gene clusters for bacterial polysaccharides. On the basis of their amino-acid-sequence homologies, their putative hydropathy profiles and the effect of their mutations, it is predicted that EpsA (an exporter-protein homologue belonging to the OMA family) and EpsC (a chain-length-regulator homologue belonging to the PCP family) play a role in the assembly, polymerization and translocation of mauran. The possibility that mauran might be synthesized via a Wzy-like biosynthesis system, just as it is for many other polysaccharides, is also discussed. This hypothesis is supported by the fact that EpsJ is homologous with some members of the PST-exporter-protein family, which seems to function together with each OMA–PCP pair in polysaccharide transport in Gram-negative bacteria, transferring the assembled lipid-linked repeating units from the cytoplasmic membrane to the periplasmic space. Maximum induction of the genes is reached during stationary phase in the presence of 5 % (w/v) marine salts.

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2005-09-01
2019-11-21
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References

  1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. ( 1990; ). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef]
    [Google Scholar]
  2. Arakawa, Y., Wacharotayankun, R., Nagatsuka, T., Ito, H., Kato, N. & Ohta, M. ( 1995; ). Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid. J Bacteriol 177, 1788–1796.
    [Google Scholar]
  3. Argandoña, A., Martínez-Checa, F., Llamas, I., Quesada, E. & del Moral, A. ( 2003; ). Megaplasmids in Gram-negative, moderately halophilic bacteria. FEMS Microbiol Lett 227, 81–86.[CrossRef]
    [Google Scholar]
  4. Arias, S., del Moral, A., Ferrer, M. R., Tallon, R., Quesada, E. & Béjar, V. ( 2003; ). Mauran, an exopolysaccharide produced by the halophilic bacterium Halomonas maura, with a novel composition and interesting properties for biotechnology. Extremophiles 7, 319–326.[CrossRef]
    [Google Scholar]
  5. Artsimovitch, I. & Landick, R. ( 2002; ). The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed non-template DNA strand. Cell 109, 193–203.[CrossRef]
    [Google Scholar]
  6. Bailey, M. J. A., Hughes, C. & Koronakis, V. ( 1996; ). Increased distal gene transcription by the elongation factor RfaH, a specialized homologue of NusG. Mol Microbiol 22, 729–737.[CrossRef]
    [Google Scholar]
  7. Bailey, M. J. A., Hughes, C. & Koronakis, V. ( 1997; ). RfaH and the ops element, components of a novel system controlling bacterial transcription elongation. Mol Microbiol 26, 845–851.[CrossRef]
    [Google Scholar]
  8. Béjar, V., Llamas, I., Calvo, C. & Quesada, E. ( 1998; ). Characterization of exopolysaccharides produced by 19 halophilic strains included in the species Halomonas eurihalina. J Biotechnol 61, 135–141.[CrossRef]
    [Google Scholar]
  9. Bik, E. M., Bunschoten, A. E., Willems, R. J. L., Chang, A. C. Y. & Mooi, F. R. ( 1996; ). Genetic organization and functional analysis of the otn DNA essential for cell-wall polysaccharide synthesis in Vibrio cholerae O139. Mol Microbiol 20, 799–811.[CrossRef]
    [Google Scholar]
  10. Blumenkrantz, N. & Asboe-Hansen, G. ( 1973; ). New method for quantitative determination of uronic acids. Anal Biochem 54, 484–489.[CrossRef]
    [Google Scholar]
  11. Bouchotroch, S., Quesada, E., del Moral, A., Llamas, I. & Béjar, V. ( 2001; ). Halomonas maura sp. nov., a novel moderately halophilic, exopolysaccharide-producing bacterium. Int J Syst Evol Microbiol 51, 1625–1632.[CrossRef]
    [Google Scholar]
  12. Bradford, M. M. ( 1976; ). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72, 248–254.[CrossRef]
    [Google Scholar]
  13. Bullock, W. O., Fernández, J. M. & Short, J. M. ( 1987; ). XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection. Biotechniques 5, 376–379.
    [Google Scholar]
  14. Bugert, P. & Geider, K. ( 1995; ). Molecular analysis of the ams operon required for exopolysacchaide synthesis of Erwinia amylovora. Mol Microbiol 15, 917–933.[CrossRef]
    [Google Scholar]
  15. 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]
  16. Collins, F. S. & Weissman, S. M. ( 1984; ). Directional cloning of DNA fragments at a large distance from an initial probe: a circularization method. Proc Natl Acad Sci U S A 81, 6812–6816.[CrossRef]
    [Google Scholar]
  17. Davey, M. E. & O'Toole, G. A. ( 2000; ). Molecular biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 64, 847–867.[CrossRef]
    [Google Scholar]
  18. de Lorenzo, V., Herrero, M., Jakubzik, U. & Timmis, K. N. ( 1990; ). Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative eubacteria. J Bacteriol 172, 6568–6572.
    [Google Scholar]
  19. 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]
  20. 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.[CrossRef]
    [Google Scholar]
  21. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. ( 1956; ). Colorimetric method for determination of sugars and related substances. Anal Chem 28, 350–356.[CrossRef]
    [Google Scholar]
  22. Fang, C. T., Chuang, Y. P., Shun, C. T., Chang, S. C. & Wang, J. T. ( 2004; ). A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications. J Exp Med 199, 697–705.[CrossRef]
    [Google Scholar]
  23. Glöckner, F. O., Kube, M., Bauer, M. & 11 other authors ( 2003; ). Complete genome sequence of the marine planctomycete Pirellula sp. strain 1. Proc Natl Acad Sci U S A 100, 8298–8303.[CrossRef]
    [Google Scholar]
  24. Glucksmann, M. A., Reuber, T. L. & Walker, G. C. ( 1993; ). Genes needed for the modification, polymerization, export, and processing of succinoglycan by Rhizobium meliloti: a model for succinoglycan biosynthesis. J Bacteriol 175, 7045–7055.
    [Google Scholar]
  25. González, J. E., Semino, C. E., Wang, L. X., Castellano-Torres, L. E. & Walker, G. C. ( 1998; ). Biosynthetic control of molecular weight in the polymerisation of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti. Proc Natl Acad Sci U S A 95, 13477–13482.[CrossRef]
    [Google Scholar]
  26. Grangeasse, C., Doublet, P., Vaganay, E., Vicent, C., Deléage, G., Duclos, B. & Cozzone, A. J. ( 1997; ). Characterization of a bacterial gene encoding an autophosphorylating protein tyrosine kinase. Gene 204, 259–265.[CrossRef]
    [Google Scholar]
  27. Hobbs, M. & Reeves, P. R. ( 1994; ). The JUMPstart sequence: a 39 bp element common to several polysaccharide gene clusters. Mol Microbiol 12, 855–856.[CrossRef]
    [Google Scholar]
  28. Huang, J. & Schell, M. ( 1995; ). Molecular characterization of the eps gene cluster of Pseudomonas solanacearum and its transcriptional regulation at a single promoter. Mol Microbiol 16, 977–989.[CrossRef]
    [Google Scholar]
  29. Jones, B. E. ( 2004; ). Industrial enzymes: do halophilic and alkaliphiles have a role to play? In Halophilic Microorganisms, pp. 275–284. Edited by A. Ventosa. Heidelberg: Springer.
  30. Kalogeraki, V. S. & Winans, S. C. ( 1997; ). Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria. Gene 188, 69–75.[CrossRef]
    [Google Scholar]
  31. Katzen, F., Ferreiro, D. U., Oddo, C. G., Ielmini, M. V., Becker, A., Pühler, A. & Ielpi, L. ( 1998; ). Xanthomonas campestris pv. campestris gum mutants: effects on xanthan biosynthesis and plant virulence. J Bacteriol 180, 1607–1617.
    [Google Scholar]
  32. Kim, Y. R., Lee, S. E., Kim, C. M. & 9 other authors ( 2003; ). Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients. Infect Immun 71, 5461–5471.[CrossRef]
    [Google Scholar]
  33. Llamas, I., del Moral, A., Béjar, V., Girón, M. D., Salto, R. & Quesada, E. ( 1997; ). Plasmids from Halomonas eurihalina, a microorganism which produces an exopolysaccharide of biotechnological interest. FEMS Microbiol Lett 156, 251–257.[CrossRef]
    [Google Scholar]
  34. Llamas, I., Argandoña, M., Quesada, E. & del Moral, A. ( 2000; ). Transposon mutagenesis in Halomonas eurihalina. Res Microbiol 151, 1–7.
    [Google Scholar]
  35. Llamas, I., Suárez, A., Quesada, E., Béjar, V. & del Moral, A. ( 2003; ). Identification and characterization of the carAB genes responsible for encoding carbamoylphosphate synthetase in Halomonas eurihalina. Extremophiles 7, 205–211.
    [Google Scholar]
  36. Llamas, I., Keshavan, N. & González, J. E. ( 2004; ). Use of Sinorhizobium meliloti as an indicator for specific detection of long-chain N-acyl homoserine lactones. Appl Environ Microbiol 70, 3715–3723.[CrossRef]
    [Google Scholar]
  37. Margesin, R. & Schinner, F. ( 2001; ). Potential of halotolerant and halophilic microorganisms for biotechnology. Extremophiles 5, 73–83.[CrossRef]
    [Google Scholar]
  38. Markovich, D. & Murer, H. ( 2004; ). The SLC13 gene family of sodium sulphate/carboxylate cotransporters. Pflugers Arch 447, 594–602.[CrossRef]
    [Google Scholar]
  39. Marolda, C. L. & Valvano, M. A. ( 1998; ). The promoter region of the Escherichia coli O7-specific lipopolysaccharide gene cluster: structural and functional characterization of an upstream untranslated mRNA sequence. J Bacteriol 180, 3070–3079.
    [Google Scholar]
  40. Martínez-Cánovas, M. J., Béjar, V., Martínez-Checa, F. & Quesada, E. ( 2004a; ). Halomonas anticariensis sp. nov., from Fuente de Piedra, a saline-wetland, wildfowl reserve in Málaga, Southern Spain. Int J Syst Evol Microbiol 54, 1329–1332.[CrossRef]
    [Google Scholar]
  41. Martínez-Cánovas, M. J., Quesada, E., Llamas, I. & Béjar, V. ( 2004b; ). Halomonas ventosae sp. nov., a moderately halophilic, denitrifying, exopolysaccharide-producing bacterium. Int J Syst Evol Microbiol 54, 733–737.[CrossRef]
    [Google Scholar]
  42. Martínez-Checa, F., Toledo, F. L., Vilchez, R., Quesada, E. & Calvo, C. ( 2002; ). Yield production, chemical composition and functional properties of emulsifier H28 synthesized by Halomonas eurihalina strain H28 in media containing various hydrocarbons. Appl Microbiol Biotechnol 58, 358–363.[CrossRef]
    [Google Scholar]
  43. McComb, E. A. & McCready, R. M. ( 1957; ). Determination of acetyl in pectin and in acetylated carbohydrate polymers. Anal Chem 29, 819–821.[CrossRef]
    [Google Scholar]
  44. Miller, J. H. ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  45. Miller, V. L. & Mekalanos, J. J. ( 1988; ). A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170, 2575–2583.
    [Google Scholar]
  46. Moraine, R. A. & Rogovin, P. ( 1966; ). Kinetics of polysaccharide B-1459 fermentation. Biotechnol Bioeng 8, 511–524.[CrossRef]
    [Google Scholar]
  47. 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]
  48. Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. ( 1997; ). A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Int J Neural Syst 8, 581–599.[CrossRef]
    [Google Scholar]
  49. Nieto, J. J., Fernández-Castillo, R., Márquez, M. C., Ventosa, A., Quesada, E. & Ruíz-Berraquero, F. ( 1989; ). A survey of metal tolerance in moderately halophilic eubacteria. Appl Environ Microbiol 55, 2385–2390.
    [Google Scholar]
  50. Nieto, J. M., Bailey, M. J. A., Hughes, C. & Koronakis, V. ( 1996; ). Suppression of transcription polarity in the Escherichia coli haemolysin operon by a short upstream element shared by polysaccharide and DNA transfer determinants. Mol Microbiol 19, 705–713.[CrossRef]
    [Google Scholar]
  51. Ochman, H., Gerber, A. S. & Hartl, D. L. ( 1988; ). Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–623.
    [Google Scholar]
  52. Pao, S. S., Paulsen, I. T. & Saier, M. H., Jr ( 1998; ). Major facilitator superfamily. Microbiol Mol Biol Rev 62, 1–34.
    [Google Scholar]
  53. Paulsen, I. T., Beness, A. M. & Saier, M. H., Jr ( 1997; ). Computer-based analyses of the protein constituents of transport systems catalysing export of complex carbohydrates in bacteria. Microbiology 143, 2685–2699.[CrossRef]
    [Google Scholar]
  54. Quesada, E., Valderrama, M. J., Béjar, V., Ventosa, A., Gutiérrez, M. C., Ruíz-Berraquero, F. & Ramos-Cormenzana, A. ( 1990; ). Volcaniella eurihalina gen. nov., sp. nov., a moderately halophilic nonmotile Gram-negative rod. Int J Syst Bacteriol 40, 261–267.[CrossRef]
    [Google Scholar]
  55. Quesada, E., Béjar, V. & Calvo, C. ( 1993; ). Exopolysaccaharide production by Volcaniella eurihalina. Experientia 49, 1037–1041.[CrossRef]
    [Google Scholar]
  56. Quesada, E., Béjar, V., Ferrer, M. R. & 8 other authors ( 2004; ). Moderately halophilic, exopolysaccharide-producing bacteria. In Halophilic Microorganisms, pp. 297–314. Edited by A. Ventosa. Heildeberg: Springer.
  57. Rahn, A. & Whitfield, C. ( 2003; ). Transcriptional organization and regulation of the Escherichia coli K30 group 1 capsule biosynthesis (cps) gene cluster. Mol Microbiol 47, 1045–1060.[CrossRef]
    [Google Scholar]
  58. Rahn, A., Drummelsmith, J. & Whitfield, C. ( 1999; ). Conserved organization in the cps gene clusters for expression of Escherichia coli group 1 K antigens: relationship to the colanic acid biosynthesis locus and the cps genes from Klebsiella pneumoniae. J Bacteriol 181, 2307–2313.
    [Google Scholar]
  59. Reese, M. G. & Eeckman, F. H. ( 1995; ). Novel neural network algorithms for improved eukaryotic promoter site recognition. In Genome Science and Technology, vol. 1, pp. 45. Edited by J. C. Venter and D. Doyle. Hilton Head Island, SC.
  60. Reuber, T. L. & Walker, G. C. ( 1993; ). Biosynthesis of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti. Cell 74, 269–280.[CrossRef]
    [Google Scholar]
  61. Rodríguez-Valera, F., Ruíz-Berraquero, F. & Ramos-Cormenzana, A. ( 1981; ). Characteristics of the heterotrophic bacterial populations in hypersaline environments of different salt concentrations. Microb Ecol 7, 235–243.[CrossRef]
    [Google Scholar]
  62. Salanoubat, M., Genin, S., Artiguenave, F. & 25 other authors ( 2002; ). Genome sequence of the plant pathogen Ralstonia solanacearum. Nature 415, 497–502.[CrossRef]
    [Google Scholar]
  63. Sambrook, J. & Russell, D. W. ( 2001; ). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  64. Sanger, F., Nicklen, S. & Coulson, A. R. ( 1977; ). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74, 5473–5477.
    [Google Scholar]
  65. Stevenson, G., Andrianopoulos, K., Hobbs, M. & Reeves, P. R. ( 1996; ). Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid. J Bacteriol 178, 4885–4893.
    [Google Scholar]
  66. Stevenson, G., Lan, R. & Reeves, P. R. ( 2000; ). The colonic acid gene cluster of Salmonella enterica has a complex history. FEMS Microbiol Lett 191, 11–16.[CrossRef]
    [Google Scholar]
  67. Stover, C. K., Pham, X. Q., Erwin, A. L. & 28 other authors ( 2000; ). Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 31, 959–964.
    [Google Scholar]
  68. Sutherland, I. W. ( 1998; ). Novel and established applications of microbial polysaccharides. Trends Biotechnol 16, 41–46.[CrossRef]
    [Google Scholar]
  69. Sutherland, I. W. ( 1999; ). Microbial polysaccharide products. Biotechnol Genet Eng Rev 16, 217–229.[CrossRef]
    [Google Scholar]
  70. Sutherland, I. W. ( 2001; ). Microbial polysaccharide from Gram-negative bacteria. Int Dairy J 11, 663–674.[CrossRef]
    [Google Scholar]
  71. Takami, H., Nakasone, K., Takaki, Y. & 9 other authors ( 2000; ). Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res 28, 4317–4331.[CrossRef]
    [Google Scholar]
  72. 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.[CrossRef]
    [Google Scholar]
  73. Ventosa, A., Nieto, J. J. & Oren, A. ( 1998; ). Biology of moderately halophilic aerobic bacteria. Microbiol Mol Biol Rev 62, 504–544.
    [Google Scholar]
  74. Vicent, 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]
  75. Vieira, J. & Messing, J. ( 1982; ). The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259–268.[CrossRef]
    [Google Scholar]
  76. Waibel, A. H., Hanazawa, T., Hinton, G., Shikano, K. & Lang, K. J. ( 1989; ). Phoneme recognition using time-delay neural networks. In IEEE transactions on acoustics, speech, and signal processing, 37, 328–339.
  77. Wang, L., Briggs, C. E., Rothemund, D., Fratamico, P., Luchansky, J. B. & Reeves, P. R. ( 2001; ). Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes. Gene 270, 231–236.[CrossRef]
    [Google Scholar]
  78. Whitfield, C., Amor, P. A. & Köplin, R. ( 1997; ). Modulation of surface architecture of Gram-negative bacteria by the action of surface polymer: lipid A-core ligase and by determinants of polymer chain length. Mol Microbiol 23, 629–638.[CrossRef]
    [Google Scholar]
  79. Yoshida, T., Ayabe, T., Yasunaga, M., Usami, Y., Habe, H., Nojiri, H. & Omori, T. ( 2003; ). Genes involved in the synthesis of the exopolysaccharide methanolan by the obligate methylotroph Methylobacillus sp. strain 12S. Microbiology 149, 431–444.[CrossRef]
    [Google Scholar]
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