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Volume 142, Issue 12

Insertional gene inactivation in a phototrophic sulphur bacterium: APS-reductase-deficient mutants of Free

Abstract

In purple sulphur bacteria of the family sulphite oxidation via intermediary formation of adenylylsulphate is an enzymologically well characterized process. In contrast, the role of an alternative direct oxidation pathway via the enzyme sulphite: acceptor oxidoreductase has not been resolved. This paper reports the cloning of the genes encoding the adenylylsulphate-forming enzyme adenosine-5′-phosphosulphate (APS) reductase from strain D (DSM 180), a representative of the purple sulphur bacteria, and the construction of mutations in these genes by insertion of a kanamycin Ω cartridge. The mutated genes were transferred to on suicide vectors of the pSUP series by conjugation and delivered to the chromosome by double homologous recombination. Southern hybridization and PCR analyses of the recombinants obtained verified the first insertional gene inactivation in purple sulphur bacteria. Enzymological studies demonstrated the absence of APS reductase from the mutants. Further phenotypic characterization showed no significant effect of APS reductase deficiency on the sulphite-oxidizing ability of the cells under photolithoautotrophic growth conditions. In the wild-type as well as in mutant strains, tungstate, the specific antagonist of molybdate, led to the intermediary accumulation of sulphite in the medium during sulphide oxidation and strongly inhibited growth with sulphite as photosynthetic electron donor; this indicates that a molybdoenzyme, probably sulphite: acceptor oxidoreductase, is the main sulphite-oxidizing enzyme in Specific inactivation of selected genes as developed for in this study provides a powerful genetic tool for further analysis of sulphur metabolism and other metabolic pathways in phototrophic sulphur bacteria.

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Keyword(s): adenosine-5′-phosphosulphate reductase , interposon mutagenesis , Keywords:Chromatium vinosum, , molybdenum and sulphite oxidation
Society for General Microbiology 1996
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1996-12-01
2024-04-16
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References

  1. Bazaral M., amp;Helinski D. R. 1968; Circular DNA forms of colicinogenic factors El, E2 and E3 from Escherichia coli. J Mol Biol 36:185–194
     [Google Scholar]
  2. Beck E., Ludwig G., Auerswald E. A., Reiss B., amp;Schaller H. 1982; Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19:327–336
     [Google Scholar]
  3. Bockholt R., Masepohl B., amp;Pistorius E. K. 1991; Insertional inactivation of the psbO gene encoding the manganese stabilizing protein of photosystem II in the cyanobacterium Sjnechococcus PCC7942. Effect on photosynthetic water oxidation and L-amino acid oxidase activity. FEBS Lett 294:59–63
     [Google Scholar]
  4. Brune D. C. 1989; Sulfur oxidation by phototrophic bacteria. Biochim Biophys Acta 975:189–221
     [Google Scholar]
  5. Brune D. C. 1995; Sulfur compounds as photosynthetic electron donors. In Anoxygenic Photosynthetic Bacteria, pp. 847–970 Blankenship R. E., Madigan M. T., Bauer C. E. Edited by Dordrecht: Kluwer.;
     [Google Scholar]
  6. Cooper B. P., Trüper H. G. 1979; Improved synthesis and rapid isolation of millimole quantities of adenylylsulfate. Z Naturforsch 34c:346–349
     [Google Scholar]
  7. Dahl C., Trüper H. G. 1989; Comparative enzymology of sulfite oxidation in Thiocapsa roseopersicina strains 6311, Ml and BBS under chemotrophic and phototrophic conditions. Z Naturforsch 44c:617–622
     [Google Scholar]
  8. Dahl C., Trüper H. G. 1994; Enzymes of dissimilatory sulfide oxidation in phototrophic bacteria. Methods Enzymol 243:400–421
     [Google Scholar]
  9. Dahl C., Speich N., Trüper H. G. 1994; Enzymology and molecular biology of sulfate reduction in the extremely thermophilic archaeon Archaeoglobus fulgidus. Methods Enzymol 243:331–349
     [Google Scholar]
  10. Donohue T. J., Kaplan S. 1991; Genetic techniques in Rhodospirillaceae. Methods Enzymol 204:459–485
     [Google Scholar]
  11. Fauque G., LeGall J., Barton L. L. 1991; Sulfate-reducing and sulfur-reducing bacteria. In Variations in Autotrophic Life, pp 271–337 Shively J. M., Barton L. L. Edited by New York: Academic Press.;
     [Google Scholar]
  12. Fellay R., Frey J., Krisch H. M. 1987; Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vivo insertional mutagenesis of Gram-negative bacteria. Gene 52:147–154
     [Google Scholar]
  13. Fischer U. 1989; Enzymatic steps in dissimilatory sulfur metabolism by whole cells of anoxyphotobacteria. In Biogenic Sulfur in the Environment pp. 262–279 Saltzman E., Cooper W. Edited by Washington, DC: American Chemical Society.;
     [Google Scholar]
  14. Frey J., Krisch H. M. 1985; Q mutagenesis in Gram-negative bacteria: a selectable interposon which is strongly polar in a wide range of bacterial species. Gene 36:143–150
     [Google Scholar]
  15. Fry B., Gest H., Hayes J. M. 1985; Isotope effects associated with the anaerobic oxidation of sulfite and thiosulfate by the photosynthetic bacterium, Chromatium vinosum. FEMS Microbiol Lett 27:227–232
     [Google Scholar]
  16. Giordano G., Haddock B.A.8tBoxer. 1980; Molybdenum- limited growth achieved either phenotypically or genotypically and its effect on the synthesis of formate dehydrogenase and nitrate reductase by Escherichia coli K12. FEMS Microbiol Lett 8:229–235
     [Google Scholar]
  17. Gisselmann G., Niehaus A. 1992; Homologies in the structural genes coding for sulphate reducing enzymes from higher plants and prokaryotes. Bot Acta 105:133–226
     [Google Scholar]
  18. Hanahan D. 1983; Studies on transformation of Escherichia coliwith plasmids. J Mol Biol 166:557–580
     [Google Scholar]
  19. Ivanovski R. N., Petushkova Y. P. 1976; Substrate phosphorylation during oxidation of sulfite by Thiocapsa roseopersicina depending on growth conditions. Microbiology (English translation of Mikrobiologiya). 45:941–945
     [Google Scholar]
  20. Kelly D. P., Chambers L. A., Trudinger P. A. 1969; Cyanolysis and spectrophotometric estimation of trithionate in mixture with thiosulfate and tetrathionate. Anal Chem 41:898–901
     [Google Scholar]
  21. Krone F. A., Westphal G. 1991; Characterisation of the gene cysH and of its product phospho- adenylylsulphate reductase from Escherichia coli. Mol Gen Genet 225:314–319
     [Google Scholar]
  22. Leinweber F. -J., Monty K. J. 1987; Sulfite determination: fuchsin method. Methods Emymol 143:15–17
     [Google Scholar]
  23. Neutzling O., Pfleiderer C. 1985; Dissimilatory sulphur metabolism in phototrophic ‘non-sulphur’ bacteria. J Gen Microbiol . 131:791–798
     [Google Scholar]
  24. Niehaus A., Gisselmann G. 1992; Primary structure of the Synechococcus PCC 7942 PAPS reductase gene. Plant Mol Biol 20:1179–1183
     [Google Scholar]
  25. Pattaragulwanit K., Dahl C. 1995; Development of a genetic system for a purple sulfur bacterium: conjugative plasmid transfer in Chromatium vinosum. Arch Microbiol 164:217–222
     [Google Scholar]
  26. Petushkova Y. P., Ivanovski R. N. 1976; Oxidation of sulfite by Thiocapsa roseopersicina. Microbiology (English translation of Mikrobiologiya). 45:592–597
     [Google Scholar]
  27. Pfennig N. 1992; The family Chromatiaceae. In The Prokaryotes, 2nd edn. pp. 3200–3221 Edited by Balows A., Triiper H. G., Dworkin M., Harder W., Schleifer K. -H. New York:: Springer-Verlag.;
     [Google Scholar]
  28. Sambrook J., Fritsch E. F. 1989 Molecular Cloning-, a Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory.;
     [Google Scholar]
  29. Schedel M. 1980; Anaerobic oxidation of thiosulfate and elemental sulfur in Thiobacillus denitrificans. Arch Microbiol 124:205–210
     [Google Scholar]
  30. Schwenn J. D., Biere M. 1979; APS-reductase activity in the chromatophores of Chromatium vinosum strain D. FEMS Microbiol Lett 6:19–22
     [Google Scholar]
  31. Simon R., Priefer U., Pühler A. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1:784–791
     [Google Scholar]
  32. Simon R., O’Connell M., Labes M., Pühler A. 1986; Plasmid vectors for the genetic analysis and manipulation of rhizobia and other Gram-negative bacteria. Methods Enaymol 118:640–659
     [Google Scholar]
  33. Smith A. J. 1966; The role of tetrathionate in the oxidation of thiosulphate by Chromatium sp. strain D. J Gen Microbiol 42:371–380
     [Google Scholar]
  34. Smith A. J., Lascelles J. 1966; Thiosulphate metabolism and rhodanese in Chromatium sp. strain D. J Gen Microbiol 42:357–370
     [Google Scholar]
  35. Sbrbo B. 1987; Sulfate: turbidometric and nephelometric methods. Methods Emymol 143:3–6
     [Google Scholar]
  36. Speich N., Dahl C., Heisig P., Klein A., Lottspeich F., Stetter K. O., Trüper H. G. 1994; Adenylylsulphate reductase from the sulphate-reducing archaeon Archaeoglobus fulgidus: cloning and characterization of the genes and comparison of the enzyme with other iron-sulphur flavoproteins. Microbiology 140:1273–1284
     [Google Scholar]
  37. Takakuwa S. 1992; Biochemical aspects of microbial oxidation of inorganic sulfur compounds. In Organic Sulfur Chemistry : Biochemical Aspects pp. 1–43 Oae S., Okuyama T. Edited by Boca Raton, FL: CRC Press.;
     [Google Scholar]
  38. Toghrol F., Southerland W. M. 1983; Purification of Thiobacillus novellus sulfite oxidase. J Biol Chem 258:6762–6766
     [Google Scholar]
  39. Trüper H. G. 1984; Phototrophic bacteria and their sulfur metabolism. In Sulfur, Its Significance for Chemistry, for the Geo-, Bio-, and Cosmosphere and Technology pp. 367–382 Muller A., Krebs B. Edited by Amsterdam:: Elsevier.;
     [Google Scholar]
  40. Trüper H. G., Fischer U. 1982; Anaerobic oxidation of sulphur compounds as electron donors for bacterial photosynthesis. Philos Trans R Soc Lond B 298:529–542
     [Google Scholar]
  41. Trüper H. G., Rogers L. A. 1971; Purification and properties of adenylyl sulfate reductase from the phototrophic sulfur bacterium Thiocapsa roseopersicina. J Bacteriol 108:1112–1121
     [Google Scholar]
  42. Trüper H.G.8.Schlegel. 1964; Sulphur metabolism in Thiorhodaceae. I. Quantitative measurements on growing cells of Chromatium okenii. Antonie Leeuwenhoek 30:225–238
     [Google Scholar]
  43. Ulbricht H. 1984 Aspekte des Energiegewinns durch Sub- stratphosphorylierung im Zuge der Sulfitoxidation bei Chromatiaceae und Thiobacillus denitrificans. Doctoral thesis, University of Bonn.;
     [Google Scholar]
  44. Urban P. J. 1961; Colorimetry of sulfur anions. I. An improved colorimetric method for the determination of thiosulfate. Z Anal Chem 179:415–422
     [Google Scholar]
  45. Viale A. M., Kobayashi H., Akazawa T. 1989; Expressed genes for plant-type ribulose 1,5-bisphosphate carboxylase/oxygenase in the photosynthetic bacterium Chromatium vinosum which possesses two complete sets of genes. J Bacteriol 171:2391–2400
     [Google Scholar]
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Insertional gene inactivation in a phototrophic sulphur bacterium: APS-reductase-deficient mutants of Chromatium vinosum
Microbiology 142, 3363 (1996); https://doi.org/10.1099/13500872-142-12-3363
/content/journal/micro/10.1099/13500872-142-12-3363
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