1887

Abstract

A cluster (27 kbp) of genes responsible for the biosynthesis of the amphipathic, polysaccharide bioemulsifier emulsan from the oil-degrading RAG-1 was isolated and characterized. The complete sequence of this cluster, termed , consisted of 20 ORFs. One set of 17 ORFs was transcribed in one direction, while a second set of three ORFs, 607 bp upstream of the first, was transcribed in the opposite direction. Mutations in either of the two regions caused defects in emulsan production, yielding specific activities of 5–14% of parental emulsifying activity. Putative functions could be assigned to proteins involved in production of nucleotide amino sugar precursors, transglycosylation, transacetylation, polymerization and transport. However, no JUMPstart or sequences, normally found associated with some polysaccharide biosynthetic gene clusters, were identified. Evidence is presented suggesting that the bioemulsifier may be a member of the group 1 or group 4 polysaccharides.

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2001-07-01
2019-12-13
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References

  1. Alon, R. N. & Gutnick, D. L. ( 1993; ). Esterase from the oil-degrading Acinetobacter lwoffii RAG-1: sequence analysis and overexpression in Escherichia coli. FEMS Microbiol Lett 112, 275-280.[CrossRef]
    [Google Scholar]
  2. 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]
  3. Bailey, M. J., 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]
  4. Bayer, E. A., Skutelsky, E., Goldman, S., Rosenberg, E. & Gutnick, D. L. ( 1983; ). Immunochemical identification of the major cell surface agglutinogen of Acinetobacter calcoaceticus RAG-92. J Gen Microbiol 129, 1109-1119.
    [Google Scholar]
  5. Belsky, I., Gutnick, D. L. & Rosenberg, E. ( 1979; ). Emulsifier of Arthrobacter RAG-1: determination of emulsifier-bound fatty acids. FEBS Lett 101, 175-178.[CrossRef]
    [Google Scholar]
  6. Cohen-Bazire, G., Sistrom, W. R. & Stanier, R. Y. ( 1957; ). Kinetic studies of pigment synthesis by non-sulphur purple bacteria. J Cell Comp Physiol 49, 25-68.[CrossRef]
    [Google Scholar]
  7. Comstock, L. E., Johnson, J. A., Michalski, J. M., Morris, J. G. J. & Kaper, J. B. ( 1996; ). Cloning and sequence of a region encoding a surface polysaccharide of Vibrio cholerae O139 and characterization of the insertion site in the chromosome of Vibrio cholerae O1. Mol Microbiol 19, 815-826.[CrossRef]
    [Google Scholar]
  8. Crater, D. L., Dougherty, B. A. & van de Rijn, I. ( 1995; ). Molecular characterization of hasC from an operon required for hyaluronic acid synthesis in group A streptococci. Demonstration of UDP-glucose pyrophosphorylase activity. J Biol Chem 270, 28676-28680.[CrossRef]
    [Google Scholar]
  9. Dean, C. R., Franklund, C. V., Retief, J. D., Coyne, M. J., Hatano, K., Evans, D. J., Pier, G. B. & Goldberg, J. B. ( 1999; ). Characterization of the serogroup O11 O-antigen locus of Pseudomonas aeruginosa PA103. J Bacteriol 181, 4275-4284.
    [Google Scholar]
  10. Desai, J. D. & Banat, I. M. ( 1997; ). Microbial production of surfactants and their commercial potential. Microbiol Mol Biol Rev 61, 47-64.
    [Google Scholar]
  11. 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]
  12. 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]
  13. Goldman, S., Shabtai, Y., Rubinovitz, C., Rosenberg, E. & Gutnick, D. L. ( 1982; ). Emulsan in Acinetobacter calcoaceticus RAG-1: distribution of cell-free and cell-associated cross-reacting material. Appl Environ Microbiol 44, 165-170.
    [Google Scholar]
  14. Grangeasse, C., Doublet, P., Vaganay, E., Vincent, C., Deleage, 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]
  15. Gutnick, D. L. ( 1987; ). The emulsan polymer: perspectives on a microbial capsule as an industrial product. Biopolymers 26, S223-S240.[CrossRef]
    [Google Scholar]
  16. Gutnick, D. L., Allon, R., Levy, C., Petter, R. & Minas, W. ( 1991; ). Applications of Acinetobacter as an industrial organism. In The Biology of Acinetobacter , pp. 411-441. Edited by K. J. Towner, E. Bergogne-Berezin & C. A. Fewson. New York:Plenum.
  17. 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]
  18. Hopwood, D. A., Bibb, M. J., Chater, K. F. & 7 other authors (1985). Genetic Manipulation of Streptomyces: a Laboratory Manual. Norwich: John Innes Foundation.
  19. Kereszt, A., Kiss, E., Reuhs, B. L., Carlson, R. W., Kondorosi, A. & Putnoky, P. ( 1998; ). Novel rkp gene clusters of Sinorhizobium meliloti involved in capsular polysaccharide production and invasion of the symbiotic nodule: the rkpK gene encodes a UDP-glucose dehydrogenase. J Bacteriol 180, 5426-5431.
    [Google Scholar]
  20. Leahy, J. G., Jones-Meehan, J. M., Pullias, E. L. & Colwell, R. R. ( 1993; ). Transposon mutagenesis in Acinetobacter calcoaceticus RAG-1. J Bacteriol 175, 1838-1840.
    [Google Scholar]
  21. Lee, F. K., 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]
  22. Lin, W. S., Cunneen, T. & Lee, C. Y. ( 1994; ). Sequence analysis and molecular characterization of genes required for the biosynthesis of type 1 capsular polysaccharide in Staphylococcus aureus. J Bacteriol 176, 7005-7016.
    [Google Scholar]
  23. Liu, D., Cole, R. A. & Reeves, P. R. ( 1996; ). An O-antigen processing function for Wzx (RfbX): a promising candidate for O-unit flippase. J Bacteriol 178, 2102-2107.
    [Google Scholar]
  24. Meier-Dieter, U., Starman, R., Barr, K., Mayer, H. & Rick, P. D. ( 1990; ). Biosynthesis of enterobacterial common antigen in Escherichia coli. Biochemical characterization of Tn10 insertion mutants defective in enterobacterial common antigen synthesis. J Biol Chem 265, 13490-13497.
    [Google Scholar]
  25. Nieto, J. M., Bailey, M. J., 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]
  26. Ofir, I., Bloch, Y., Frankel, G., Ullrich, H., Geider, K. & Rosenshine, I. ( 1999; ). Protein tyrosine kinases in bacterial pathogens are associated with virulence and production of exopolysaccharide. EMBO J 18, 3241-3248.[CrossRef]
    [Google Scholar]
  27. Paulsen, I. T., Beness, A. M. & Saier, M. H. J. ( 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]
  28. Pines, O. & Gutnick, D. ( 1981; ). Relationship between phage resistance and emulsan production, interaction of phages with the cell-surface of Acinetobacter calcoaceticus RAG-1. Arch Microbiol 130, 129-133.[CrossRef]
    [Google Scholar]
  29. Pines, O., Bayer, E. A. & Gutnick, D. L. ( 1983; ). Localization of emulsan-like polymers associated with the cell surface of Acinetobacter calcoaceticus. J Bacteriol 154, 893-905.
    [Google Scholar]
  30. 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]
  31. Reddy, P. G., Allon, R., Mevarech, M., Mendelovitz, S., Sato, Y. & Gutnick, D. L. ( 1989; ). Cloning and expression in Escherichia coli of an esterase-coding gene from the oil-degrading bacterium Acinetobacter calcoaceticus RAG-1. Gene 76, 145-152.[CrossRef]
    [Google Scholar]
  32. Reeves, P. ( 1993; ). Evolution of Salmonella O antigen variation by interspecific gene transfer on a large scale. Trends Genet 9, 17-22.[CrossRef]
    [Google Scholar]
  33. 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]
  34. Roberts, I. S. ( 1996; ). The biochemistry and genetics of capsular polysaccharide production in bacteria. Annu Rev Microbiol 50, 285-315.[CrossRef]
    [Google Scholar]
  35. Rosenberg, E., Zuckerberg, A., Rubinovitz, C. & Gutnick, D. L. ( 1979; ). Emulsifier of Arthrobacter RAG-1: isolation and emulsifying properties. Appl Environ Microbiol 37, 402-408.
    [Google Scholar]
  36. Sala, R. F., Morgan, P. M. & Tanner, M. E. ( 1996; ). Enzymatic formation and release of a stable glycal intermediate: the mechanism of the reaction catalyzed by UDP-N-acetylglucosamine 2-epimerase. J Am Chem Soc 118, 3033-3034.[CrossRef]
    [Google Scholar]
  37. Scupham, A. J. & Triplett, E. W. ( 1997; ). Isolation and characterization of the UDP-glucose 4′-epimerase-encoding gene, galE, from Brucella abortus 2308. Gene 202, 53-59.[CrossRef]
    [Google Scholar]
  38. Shabtai, Y. & Gutnick, D. L. ( 1985; ). Exocellular esterase and emulsan release from the cell surface of Acinetobacter calcoaceticus. J Bacteriol 161, 1176-1181.
    [Google Scholar]
  39. Shabtai, Y., Pines, O. & Gutnick, D. (1985). Emulsan: a case study of microbial capsules as industrial products. In Developments in Industrial Microbiology, pp. 291–307. Amsterdam: Elsevier.
  40. Singh, S., Singh, U., Hogan, S. E. & Feingold, D. S. ( 1990; ). Formation of UDP-2-acetamido-2-deoxy-L-galactose and UDP-2-acetamido-2-deoxy-L-galacturonic acid by Pseudomonas aeruginosa. J Bacteriol 172, 299-304.
    [Google Scholar]
  41. Skurnik, M., Venho, R., Toivanen, P. & al-Hendy, A. ( 1995; ). A novel locus of Yersinia enterocolitica serotype O:3 involved in lipopolysaccharide outer core biosynthesis. Mol Microbiol 17, 575-594.[CrossRef]
    [Google Scholar]
  42. Sonnhammer, E. L., von Heijne, G. & Krogh, A. ( 1998; ). A hidden Markov model for predicting transmembrane helices in protein sequences. In Proceedings of the International Conference on Intelligent Systems in Molecular Biology , pp. 175-182. Edited by J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff & C. Sensen. Menlo Park, CA:AAAI Press.
  43. Southern, E. M. ( 1975; ). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98, 503-517.[CrossRef]
    [Google Scholar]
  44. Stroeher, U. H., Karageorgos, L. E., Brown, M. H., Morona, R. & Manning, P. A. ( 1995; ). A putative pathway for perosamine biosynthesis is the first function encoded within the rfb region of Vibrio cholerae O1. Gene 166, 33-42.[CrossRef]
    [Google Scholar]
  45. Trefzer, A., Salas, J. A. & Bechthold, A. ( 1999; ). Genes and enzymes involved in deoxysugar biosynthesis in bacteria. Nat Prod Rep 16, 283-299.[CrossRef]
    [Google Scholar]
  46. 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]
  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. Weissborn, A. C., Liu, Q., Rumley, M. K. & Kennedy, E. P. ( 1994; ). UTP: alpha-D-glucose-1-phosphate uridylyltransferase of Escherichia coli: isolation and DNA sequence of the galU gene and purification of the enzyme. J Bacteriol 176, 2611-2618.
    [Google Scholar]
  49. Whitfield, C. ( 1995; ). Biosynthesis of lipopolysaccharide O antigens. Trends Microbiol 3, 178-185.[CrossRef]
    [Google Scholar]
  50. Whitfield, C. & Roberts, I. S. ( 1999; ). Structure, assembly and regulation of expression of capsules in Escherichia coli. Mol Microbiol 31, 1307-1319.[CrossRef]
    [Google Scholar]
  51. Ye, R. W., Zielinski, N. A. & Chakrabarty, A. M. ( 1994; ). Purification and characterization of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa involved in biosynthesis of both alginate and lipopolysaccharide. J Bacteriol 176, 4851-4857.
    [Google Scholar]
  52. Zosim, Z., Rosenberg, E. & Gutnick, D. L. ( 1986; ). Changes in hydrocarbon emulsification specificity of the polymeric bioemulsifier emulsan: effects of alkanols. Colloid Polym Sci 264, 218-223.[CrossRef]
    [Google Scholar]
  53. Zuckerberg, A., Diver, A., Peeri, Z., Gutnick, D. L. & Rosenberg, E. ( 1979; ). Emulsifier of Arthrobacter RAG-1: chemical and physical properties. Appl Environ Microbiol 37, 414-420.
    [Google Scholar]
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