1887

Abstract

GDP-mannose dehydrogenase (GMD) is a key regulatory enzyme and the committal step in alginate biosynthesis. In this study, a metabolic approach has been used to investigate GMD activity in non-mucoid and isogenically related mucoid strains of . Intracellular concentrations of GDP-mannose and GDP-mannuronate have been quantified using HPLC separation methods, and their concentrations have been related to GMD activity and total alginate production. In all strains of tested, GDP-mannose accumulated particularly during the exponential phase of growth in batch culture; the GDP-mannose concentrations in mucoid strains were significantly lower compared with isogenic non-mucoid strains. The product of GMD activity, GDP-mannuronate, was detectable only in mucoid strains, albeit at low but relatively constant levels irrespective of growth phase. The GDP-mannose concentrations in mucoid strains were always significantly greater than those of GDP-mannuronate, indicating that GMD is a rate-limiting enzyme in the biosynthesis of alginate. Significant GMD activity and extracellular alginate production were detected only in mucoid strains. The metabolic data reported here, together with previous genetic studies, provide strong evidence that GMD is the key regulatory enzyme controlling alginate biosynthesis in mucoid strains of .

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-139-1-119
1993-01-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/139/1/mic-139-1-119.html?itemId=/content/journal/micro/10.1099/00221287-139-1-119&mimeType=html&fmt=ahah

References

  1. Anastassiou E.D., Mintzas A.C., Konuavis C., Dimitra-COPOULIS G. 1987; Alginate production BY clinical non-mucoid Pseudomonas aeruginosa strains. Journal of Clinical Microbiology 25:656–659
    [Google Scholar]
  2. Berry A., Devault J.D., Chakrabarty A.M. 1989; High osmolarity is a signal for enhanced algD transcription in mucoid and non-mucoid Pseudomonas aeruginosa strains. Journal of Bacteriology 171:2312–2317
    [Google Scholar]
  3. Blumenkrantz N., Asboe-hansen G. 1973; New method for the quantitative determination of uronic acids. Analytical Biochemistry 54:484–489
    [Google Scholar]
  4. Bradford M.M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
    [Google Scholar]
  5. Darzins A., Wang S.K., Vanags R.I., Chakrabarty A.M. 1985; Clustering of mutations affecting alginic acid biosynthesis in mucoid Pseudomonas aeruginosa. Journal of Bacteriologv 164:516–524
    [Google Scholar]
  6. Deretic V., Konyecsni W.M. 1989; Control of mucoidy in Pseudomonas aeruginosa; transcriptional regulation of algR and identification of the second regulatory gene algQ. Journal of Bacteriology 171:3680–3688
    [Google Scholar]
  7. Deretic V., Gill J.F., Chakrabarty A.M. 1987a; Pseudomonas aeruginosa infection in cystic fibrosis; nucleotide sequence and transcriptional regulation of the algD gene. Nucleic Acids Research 15:4567–4581
    [Google Scholar]
  8. Deretic V., Gill J.F., Chakrabarty A.M. 1987 b; Gene algD encoding GDP-mannose dehydrogenase is transcriptionally activated in mucoid Pseudomonas aeruginosa. Journal of Bacteriology 169:351–358
    [Google Scholar]
  9. Deretic V., Dikshit R., Konyecsni M., Chakrabarty A.M., Misra T.K. 1989; The algR gene which regulates mucoidy in Pseudomonas aeruginosa belongs to a class of environmentally responsive genes. Journal of Bacteriology 171:1278–1283
    [Google Scholar]
  10. Deretic V., Govan J.R.W., Konyecsni W.M., Martin D.W. 1990; Mucoid Pseudomonas aeruginosa in cystic fibrosis: mutations in the muc loci affect transcription of the algR and algD genes in response to environmental stimuli. Molecular Microbiology 4:189–196
    [Google Scholar]
  11. Deretic V., Mohr C.D., Martin D.W. 1991; Mucoid Pseudomonas aeruginosa in cystic fibrosis; signal transduction and histone-like elements in the regulation of bacterial virulence. Molecular Microbiology 5:1577–1583
    [Google Scholar]
  12. Devault J.D., Berry A., Misra T.K., Darzins A., Chakrabarty A.M. 1989; Environmental sensory signals and microbial pathogenesis: Pseudomonas aeruginosa infection in cystic fibrosis. Biotechnology 7:352–357
    [Google Scholar]
  13. Dubois M., Giles K.A., Hamilton J.K., Rebers P.A., Smith F. 1956; Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28:350–356
    [Google Scholar]
  14. Dutta D.K., O‘donovan G. A. 1987; Separation and quantitation of bacterial ribonucleoside-triphosphates extracted with trifluoroacetic acid, by anion-exchange high performance chromatography. Journal of Chromatography 385:119–124
    [Google Scholar]
  15. Evans L.R., Linker A. 1973; Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. Journal of Bacteriology 116:915–924
    [Google Scholar]
  16. Feingold D.S., Franzen J.S. 1981; Pyridine nucleotide-linked four electron transfer dehydrogenases. Trends in Biochemical Sciences 6:103–105
    [Google Scholar]
  17. Fersht A. 1985 Enzyme Structure and Function pp. 263–290 Oxford:: Freeman.;
    [Google Scholar]
  18. Fiahlo A.M., Zielinski N.A., Fett W.A., Chakrabarty A.M., Berry A. 1990; Distribution of alginate gene sequences in the Pseudomonas rRNA homology group I-Azomonas-Azotohactcr lineage of superfamily B prokaryotes. Applied and Environmental Microbiology 56:436–443
    [Google Scholar]
  19. Flynn J.L., Ohman D.E. 1988; Use of gene replacement cosmid vector for cloning alginate conversion genes from mucoid and nonmucoid Pseudomonas aeruginosa strains: algS controls expression ofalgT. Journal of Bacteriology 170:3228–3236
    [Google Scholar]
  20. Fyfe J.A.M., Govan J.R.W. 1980; Alginate synthesis in mucoid Pseudomonas aeruginosa' chromosomal locus involved in control. Journal of General Microbiology 119:413–450
    [Google Scholar]
  21. Goldberg J.B., Ohman D.E. 1987; Construction and characterization of Pseudomonas aeruginosa algB mutant; role of algB in high level production of alginate. Journal of Bacteriology 169:1593–1602
    [Google Scholar]
  22. Govan J.R.W. 1988; Alginate biosynthesis and other unusual characteristics associated with the pathogenesis of Pseudomonas aeruginosa in cystic fibrosis. In Bacterial Infections of Respiratory and Gastrointestinal Mucosae (Special Publication of the Society of General Microbiology, pp. 2467–96, Edited by. W. Donachie,E. Griffiths,J. Stephen.; Oxford:: IRL Press.;
    [Google Scholar]
  23. Govan J.R.W. 1990; Characteristics of mucoid Pseudomonas aeruginosa in vitro and in vivo. In Pseudomonas Infection and Alginates: Biochemistry, Genetics and Pathology pp. 50–75, Edited by. Gacesa P., Russell N.J. London:: Chapman & Hall.;
    [Google Scholar]
  24. Govan J.R.W., Harris G.S. 1986; Pseudomonas aeruginosa and cystic fibrosis: unusual bacterial adaptation and pathogenesis. Microbiological Sciences 3:302–308
    [Google Scholar]
  25. Hobbie J.E., Daley R.I., Jasper S. 1977; Use of nucleopore filters for counting bacteria by fluorescence microscopy. Applied and Environmental Microbiology 33:1225–1228
    [Google Scholar]
  26. Holloway B.W., Krishnapillai V., Morgan A.F. 1979; Chromosomal genetics of Pseudomonas. Microbiological Reviews 43:73–102
    [Google Scholar]
  27. Kato J., Chakrabarty A.M. 1991; Purification of the regulatory protein algRl and its binding in the far upstream region of the algD promoter in Pseudomonas aeruginosa. Proceedings of the National Academy of Sciences of the United States of America 88:1760–1764
    [Google Scholar]
  28. Kelly N.M., Macdonald M.H., Martin N., Nicas T., Hancock R.E.W. 1990; Comparison of the outer membrane protein and lipopolysaccharide profiles of mucoid and nonmucoid Pseudomonas aeruginosa. Journal of Clinical Microbiology 28:2017–2021
    [Google Scholar]
  29. Konyecsni W.M., Deretic V. 1990a; DNA sequence and expression analysis of algP and algQ component of the multigene system transcriptionally regulating mucoidy in Pseudomonas aeruginosa :algP contains multiple direct repeats. Journal of Bacteriology 172:2511–2520
    [Google Scholar]
  30. Konyecsni W.M., Deretic V. 1990b; A prokaryote regulatory factor with a histone HI-like carboxy terminal domain: clonal variation of repeats within algP a gene involved in regulation of mucoidy in Pseudomonas aeruginosa. Journal of Bacteriology 172:5544–5554
    [Google Scholar]
  31. Lin T.Y., Hassid Z. 1966; Pathway of alginic acid synthesis in the marine brown alga Fucus gardneri (Silva). Journal of Biological Chemistry 241:5284–5297
    [Google Scholar]
  32. Miyamoto I., Nagase S. 1981; A simple and rapid electrophoretic method for the separation of glucuronic acid and iduronic acid. Analytical Biochemistry 115:308–310
    [Google Scholar]
  33. Mohr C.D., Hibler N.S., Deretic V. 1991; algR, a response regulator controlling mucoidy in Pseudomonas aeruginosa binds to the FUS site of the algD promoter located unusually far upstream from the mRNA start site. Journal of Bacteriology 173:5136–5143
    [Google Scholar]
  34. Narbad A., Russell N.J., Gacesa P. 1988; Radiolabelling patterns in alginate of Pseudomonas aeruginosa synthesised from specifically-labelled 14C monosaccharide precursors. Microbios 54:171–179
    [Google Scholar]
  35. Narbad A., Gacesa P., Russell N.J. 1990; Biosynthesis of alginate. In Pseudomonas Infection and Alginates. Biochemistry, Genetics and Pathology pp. 181–205, Edited by. Gacesa P., Russell N.J. London:: Chapman & Hall.;
    [Google Scholar]
  36. Ohman D.E., Chakrabarty A.M. 1981; Genetic mapping of chromosomal determinants for the production of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate. Infection and Immunity 33:142–148
    [Google Scholar]
  37. Ohman D.E., Chakrabarty A.M. 1982; Utilization of human respiratory secretions by mucoid Pseudomonas aeruginosa of cystic fibrosis origin. Infection and Immunity 37:662–669
    [Google Scholar]
  38. Ohman D.E., Goldberg J.B., Flynn J.L. 1990; Molecular analysis of the genetic switch activating alginate production. In Pseudomonas ; Biotransformation, Pathogenesis and Evolving Biotechnology pp. 28–35, Edited by. Silver S., Chakrabarty A. M., Iglewski B., Kaplan S. Washington DC:: American Society for Microbiology.;
    [Google Scholar]
  39. Phillips J. 1969; Identification of Pseudomonas aeruginosa in the clinical laboratory. Journal of Medical Microbiology 2:9–16
    [Google Scholar]
  40. Piggott N.H., Sutherland I.W., Jarman T.R. 1981; Enzymes involved in the biosynthesis of alginate in Pseudomonas aeruginosa. European Journal of Applied Microbiology and Biotechnology 13:179–183
    [Google Scholar]
  41. Pindar D.F., Bucke C. 1975; The biosynthesis of alginic acid by Azotobacter vinelandii. Biochemical Journal 152:617–622
    [Google Scholar]
  42. Priess J. 1964; Sugar nucleotide reactions in Arthrobacter. Part II. Biosynthesis of guanosine-diphosphomannuronate. Journal of Biological Chemistry 239:3127–3132
    [Google Scholar]
  43. Priess J. 1966; GDP-mannose dehydrogenase from Arthrobacter. Methods in Enzymology 8:285–287
    [Google Scholar]
  44. Preiss J., Wood E. 1964; Sugar nucleotide reactions in Arthrobacter. Part I. Guanosine diphosphate mannose pyrophosphorylase purification and properties. Journal of Biological Chemistry 239:3119–3126
    [Google Scholar]
  45. Pugashetti B.K., Vadas L., Pritar H.S., Feingold D.S. 1983; GDP-mannose dehydrogenase and biosynthesis of alginate-like polysaccharide in a mucoid strain of Pseudomonas aeruginosa. Journal of Bacteriology 153:1107–1110
    [Google Scholar]
  46. Randerath E., Randerath K. 1964; Resolution of complex nucleotide mixtures by two dimensionl anion-exchange thin-layer chromatography. Journal of Chromatography 16:126–129
    [Google Scholar]
  47. Roychoudhury S., May T.B., Gill J.F., Singh S.K., Feingold D.S., Chakrabarty A.M. 1989; Purification and characterization of GDP-mannose dehydrogenase, a key enzyme in the biosynthesis of alginate by Pseudomonas aeruginosa. Journal of Biological Chemistry 264:9380–9385
    [Google Scholar]
  48. Russell N.J., Gacesa P. 1988; Chemistry and biology of the alginate of mucoid strains of Pseudomonas aeruginosa in cystic fibrosis. Molecular Aspects of Medicine 10:1–91
    [Google Scholar]
  49. Russell N.J., Tatnell P.J., Gacesa P. 1992; The regulation of alginate biosynthesis by mucoid Pseudomonas aeruginosa. In Cystic Fibrosis, Basic and Clinical Research pp. 81–92, Edited by. Hoiby N., Pedersen S.S. Amsterdam:: Excerpta Medica.;
    [Google Scholar]
  50. Sherbrock-cox V., Russell N.J., Gacesa P. 1984; The purification and chemical characterization of the alginate present in the extracellular material produced by mucoid strains of Pseudomonas aeruginosa. Carbohydrate Research 135:147–154
    [Google Scholar]
  51. Sutherland I.W. 1985; Biosynthesis and composition of Gramnegative bacterial extracellular and wall polysaccharides. Annual Review of Microbiology 39:243–270
    [Google Scholar]
  52. Trevelyan W.E., Procter D.P., Harrison J.S. 1950; Detection of sugars on paper chromatograms. Nature; London: 166444–445
    [Google Scholar]
  53. Woodland H.R., Pestell R.Q.W. 1972; Determination of the nucleoside triphosphate contents of eggs and oocytes of Xenopus laevis. Biochemical Journal 127:597–605
    [Google Scholar]
  54. Wozniak D.J., Ohman D.E. 1991; Pseudomonas aeruginosa algB, a two component response regulator of the ntrC family, is required for algD transcription. Journal of Bacteriology 173:1406–1413
    [Google Scholar]
  55. Zielinski N.A., Devault J.D., Roychoudhury S., May T.B., Kimbara K., Kato J., Shinabarger D., Kitano K., Berry A., Misra T.K., Chakrabarty A.M. 1990; Molecular genetics of alginate biosynthesis. In Pseudomonas', Biotransformation. Pathogenesis and Evolving Biotechnology pp. 15–27, Edited by. Silver S., Chakrabarty A.M., Iglewski B., Kaplan S. Washington, DC:: American Society for Microbiology.;
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-139-1-119
Loading
/content/journal/micro/10.1099/00221287-139-1-119
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error