1887

Abstract

SUMMARY

The hydrogenase enzyme of Clostridium acetobutylicum plays a pivotal role in controlling electron flow, and hence carbon flow, during the complex biphasic fermentation of carbohydrates to the neutral solvents acetone and butanol. We report here the cloning and molecular characterization of the hydrogenase-encoding gene (hydA) from C. acetobutylicum P262. This gene was isolated by colony hybridization, using the Clostridium pasteurianum hydrogenase-1 gene as a probe. The DNA sequence encoding the hydA gene from C. acetobutylicum was determined, and revealed an ORF (1722 bp) encoding a 574 amino-acid protein. This C. acetobutylicum hydrogenase protein product has 82% similarity and 67% identity with the C. pasteurianum hydrogenase-1 protein. Northern blot analysis of RNA isolated from C. acetobutylicum indicates that the C. acetobutylicum hydrogenase protein product is translated from a monocistronic operon. RNA was isolated from the different morphological and physiological stages of a batch C. acetobutylicum fermentation, and further Northern blot analyses revealed no differences in the expression of the gene during acidogenesis as opposed to solventogenesis. Primer extension experiments confirmed these results and identified the 5' start of the mRNA transcript. These results correlated well with the physiological need for this organism to dispose of excess reducing equivalents.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-141-1-171
1995-01-01
2021-05-12
Loading full text...

Full text loading...

/deliver/fulltext/micro/141/1/mic-141-1-171.html?itemId=/content/journal/micro/10.1099/00221287-141-1-171&mimeType=html&fmt=ahah

References

  1. Adams M. W. W. 1990; The structure and mechanism of iron-hydrogenases. Biochim Biophys Acta 1020115–145
    [Google Scholar]
  2. Allcock E. R., Reid S.J., Jones D.T., Woods D. R. 1982; Clostridium acetobutylicum protoplast formation and regeneration . Appl Environ Microbiol 43:719–721
    [Google Scholar]
  3. Altschul S. F., Gish W., Miller W., Myers E. M., Lipman D. J. 1990; Basic local alignment search tool. J Mol Biol 215:403–410
    [Google Scholar]
  4. Andersch W., Bahl H., Gottschalk G. 1983; Level of enzymes involved in acetate, butyrate, acetone and butanol formation by Clostridium acetobutylicum . Eur J Appl Microbiol Biotechnol 18:327–332
    [Google Scholar]
  5. Beinert H. 1990; Recent developments in the field of iron-sulfur proteins. FASEB J 4:2483–2491
    [Google Scholar]
  6. Bertram J., Dürre P. 1989; Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum . Arch Microbiol 151:551–557
    [Google Scholar]
  7. Brendel V., Trifonov E. N. 1984; A computer algorithm for testing potential prokaryotic terminators. Nucleic Acids Res 12:4411–4427
    [Google Scholar]
  8. Burchhardt G., Dürre P. 1990; Isolation and characterization of DNase-deficient mutants of Clostridium acetobutylicum . Curr Microbiol 21:307–311
    [Google Scholar]
  9. Cunningham R. P., Asahara H., Bank J. F., Scholes C. P., Salerno J. C., Surerus K., Munck E., McCracken J., Peisach J., Emptage M. H. 1989; Endonuclease III is an iron-sulfur protein. Biochemistry 28:4450–4455
    [Google Scholar]
  10. Datta R., Zeikus J. G. 1985; Modulation of acetone-butanol-ethanol fermentation by carbon monoxide and organic acids. Appl Environ Microbiol 49:522–529
    [Google Scholar]
  11. Devereux J., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395
    [Google Scholar]
  12. Gapes J. R., Larsen V. F., Maddox I. S. 1982; Microbial production of n -butanol: some problems associated with the fermentation . In Energy from Biomass, Proceedings of the 14th Biotechnology Conference90–101 Palmerston North; New Zealand:
    [Google Scholar]
  13. Gerischer U., Dürre P. 1992; mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solvento-genesis . J Bacteriol 174:426–433
    [Google Scholar]
  14. Graves M. C., Rabinowitz J. C. 1986; In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene . J Biol Chem 261:11409–11415
    [Google Scholar]
  15. Gribskov M., Burgess R. R. 1986; Sigma factors from E. coli, B. subtilis, phage SP01 and phage T4 are homologous proteins . Nucleic Acids Res 14:6745–6763
    [Google Scholar]
  16. Hausinger R. P., Howard J. B. 1983; Thiol reactivity of the nitrogenase Fe-protein from Azotobacter vinelandii. J Biol Chem 258:13486–13492
    [Google Scholar]
  17. Henikoff S. 1984; Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 228:351–359
    [Google Scholar]
  18. Ish-Horowicz D., Burke J. F. 1981; Rapid and efficient cosmid cloning. Nucleic Acids Res 9:2989–2998
    [Google Scholar]
  19. Jones D. T., Woods D. R. 1986; Acetone-butanol fermentation revisited. Microbiol Rev 50:484–524
    [Google Scholar]
  20. Jones D. T., van der Westhuizen A., Long S., Allcock E. R., Reid S. J., Woods D. R. 1982; Solvent production and morphological changes in Clostridium acetobutylicum . Appl Environ Microbiol 43:1434–1439
    [Google Scholar]
  21. Keilty S., Rosenberg M. 1987; Constitutive function of a positively regulated promoter reveals new sequences essential for activity. J Biol Chem 2626389–6395
    [Google Scholar]
  22. Kim B. H., Zeikus J. G. 1985; Importance of hydrogen metabolism in regulation of solventogenesis by Clostridium acetobutylicum . Dev Ind Microbiol 26:1–14
    [Google Scholar]
  23. Kim B. H., Bellows P., Datta R., Zeikus J. G. 1984; Control of carbon and electron flow in Clostridium acetobutylicum fermentations: utilization of carbon monoxide to inhibit hydrogen production and to enhance butanol yields . Appl Environ Microbiol 48:764–770
    [Google Scholar]
  24. Lin Y.-L., Blaschek H. P. 1984; Transformation of heat-treated Clostridium acetobutylicum protoplasts with pUBHO plasmid DNA . Appl Environ Microbiol 48:737–742
    [Google Scholar]
  25. Manoil C., Beckwith J. 1985; TnphoA : a transposon probe for protein export signals . Proc Natl Acad Sci USA 82:8129–8133
    [Google Scholar]
  26. Marmur J. 1961; A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3:208–218
    [Google Scholar]
  27. Martin J. R., Petitdemange H., Marczak R., Gay R. 1982 A study of NAD(P) oxidoreductases of Clostridium acetobutylicum involved in the metabolism of glucose . In Industrielle et Bio technologic Production d’Intermediares Industries par Culture Anaerobie123–138 Edited by Chaude O., Durand G. Paris; Societe Francaise de Microbiologic:
    [Google Scholar]
  28. Meyer J., Gagnon J. 1991; Primary structure of hydrogenase I from Clostridium pasteurianum . Biochemistry 30:9697–9704
    [Google Scholar]
  29. Meyer C. L., McLaughlin J. K., Papoutsakis E. T. 1985; The effect of CO on growth and product formation in batch cultures of Clostridium acetobutylicum . Biotechnol Lett 7:37–42
    [Google Scholar]
  30. Meyer C. L., Roos J. W., Papoutsakis E. T. 1986; Carbon monoxide gassing leads to alcohol production and butyrate uptake without acetone formation in continuous cultures of Clostridium acetobutylicum . Appl Microbiol Biotechnol 24:159–167
    [Google Scholar]
  31. Mortenson L. E., Chen J. S. 1975 Hydrogenases. In Microbial Production and Utilisation of Gases69–74 Edited by Schlegel H. G., Gottschalk G., Pfennig N. Gottingen, Germany: Goltze;
    [Google Scholar]
  32. Needleman S. B., Wunsch C. D. 1970; A general method applicable to search for similarities in the amino acid sequence of two proteins. J Mol Biol 48:443–453
    [Google Scholar]
  33. O'Brien R. W., Morris J. G. 1971; Oxygen and the growth and metabolism of Clostridium acetobutylicum . J Gen Microbiol 68:307–318
    [Google Scholar]
  34. Oelmüller U., Krüger N., Steinbüchel A., Friedrich C. G. 1990; Isolation of prokaryotic RNA and detection of specific mRNA with biotinylated probes. J Microbiol Methods 11:73–84
    [Google Scholar]
  35. Ponnambalam S., Webster C., Bringham A., Busby S. 1986; Transcription initiation in the Escherichia coli galactose operon promoters in the absence of the normal —35 region sequences . J Biol Chem 261:16043–16048
    [Google Scholar]
  36. 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]
  37. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Set USA 74:5463–5467
    [Google Scholar]
  38. Sauer U., Dürre P. 1993; Sequence and molecular characterization of a DNA region encoding a small heat shock protein of Clostridium acetobutylicum . J Bacteriol 175:3394–3400
    [Google Scholar]
  39. Southern E. M. 1975; Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
    [Google Scholar]
  40. Spivey M. J. 1978; The acetone/butanol/ethanol fermentation. Process Biochem 13:2–5
    [Google Scholar]
  41. Stokkermans J., van Dongen W., Kaan A., van den Berg W., Veeger C. 1989; hydy, a gene from Desulfovibrio vulgaris (Hildenborough) encodes a polypeptide homologous to the peri-plasmic hydrogenase . FEMS Microbiol Lett 58:217–222
    [Google Scholar]
  42. Tabor S., Richardson C. C. 1987; DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84:4767–4771
    [Google Scholar]
  43. Thauer R. K., Kaufer B., ZShringer M., Jungermann K. 1974; The reaction of the iron-sulfur protein hydrogenase with carbon monoxide. Eur J Biochem 42:447–452
    [Google Scholar]
  44. Vasconcelos I., Girbal L., Soucaille P. 1994; Regulation of a carbon and electron flow in Clostridium acetobutylicum grown in chemostat culture at neutral pH on mixtures of glucose and glycerol . J Bacteriol 176:1443–1450
    [Google Scholar]
  45. Voordouw G., Brenner S. 1985; Nucleotide sequence of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough) . Eur J Biochem 148:515–520
    [Google Scholar]
  46. Voordouw G., Strang J. D., Wilson F. R. 1989; Organization of all genes encoding [Fe] hydrogenase in Desulfovibrio vulgaris subsp. oxamicus Monticello . J Bacteriol 171:3881–3889
    [Google Scholar]
  47. Wilkinson S. R., Young M. 1993; Wide diversity of genome size among different strains of Clostridium acetobutylicum . J Gen Microbiol 139:1069–1076
    [Google Scholar]
  48. Woods D. R., Jones D. T. 1986; Physiological responses of Bacteroides and Clostridium strains to environmental stress factors . Adv Microb Physiol 27:1–64
    [Google Scholar]
  49. Yanisch-Perron D., Vieira J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of M13mp8 and pUC19 vectors. Gene 33:103–119
    [Google Scholar]
  50. Yerushalmi L., Volesky B. 1985; Importance of agitation in acetone-butanol fermentation. Biotechnol Bioeng 27:1297–1305
    [Google Scholar]
  51. Young M., Minton N. P., Staudenbauer W. L. 1989; Recent advances in the genetics of the Clostridia. FEMS Microbiol Rev 63:301–325
    [Google Scholar]
  52. Zappe H., Jones D. T., Woods D. R. 1986; Cloning and expression of Clostridium acetobutylicum endoglucanase, cellobiase and amino acid biosynthesis genes in Escherichia coli . J Gen Microbiol 132:1367–1372
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-141-1-171
Loading
/content/journal/micro/10.1099/00221287-141-1-171
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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