Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Feb;178(3):871–880. doi: 10.1128/jb.178.3.871-880.1996

Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri.

B Söhling 1, G Gottschalk 1
PMCID: PMC177737  PMID: 8550525

Abstract

A region of genomic DNA from Clostridium kluyveri was cloned in Escherichia coli by a screening strategy which was based on heterologous expression of the clostridial 4-hydroxybutyrate dehydrogenase gene. The gene region (6,575 bp) contained several open reading frames which encoded the coenzyme A (CoA)- and NADP+-dependent succinate-semialdehyde dehydrogenase (sucD), the 4-hydroxybutyrate dehydrogenase (4hbD), and a succinyl-CoA;CoA transferase (cat1), as analyzed by heterologous expression in E. coli. An open reading frame encoding a putative membrane protein (orfY) and the 5' region of a gene encoding a sigma 54-homologous sigma factor (sigL) were identified as well. Transcription was investigated by Northern (RNA) blot analysis. Protein sequence comparisons of SucD and 4HbD revealed similarities to the adhE (aad) gene products from E. coli and Clostridium acetobutylicum and to enzymes of the novel class (III) of alcohol dehydrogenases. A comparison of CoA-dependent aldehyde dehydrogenases is presented.

Full Text

The Full Text of this article is available as a PDF (549.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BARTSCH R. G., BARKER H. A. A vinylacetyl isomerase from Clostridium kluyveri. Arch Biochem Biophys. 1961 Jan;92:122–132. doi: 10.1016/0003-9861(61)90226-0. [DOI] [PubMed] [Google Scholar]
  2. BERGMEYER H. U., HOLZ G., KLOTZSCH H., LANG G. PHOSPHOTRANSACETYLASE AUS CLOSTRIDIUM KLUYVERI. ZUECHTUNG DES BACTERIUMS, ISOLIERUNG, KRISTALLISATION UND EIGENSCHAFTEN DES ENZYMS. Biochem Z. 1963;338:114–121. [PubMed] [Google Scholar]
  3. Bader J., Günther H., Schleicher E., Simon H., Pohl S., Mannheim W. Utilization of (E)-2-butenoate (crotonate) by Clostridium kluyveri and some other Clostridium species. Arch Microbiol. 1980 Mar;125(1-2):159–165. doi: 10.1007/BF00403214. [DOI] [PubMed] [Google Scholar]
  4. Bairoch A. PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res. 1991 Apr 25;19 (Suppl):2241–2245. doi: 10.1093/nar/19.suppl.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barker H. A., Taha S. M. Clostridium kluyverii, an Organism Concerned in the Formation of Caproic Acid from Ethyl Alcohol. J Bacteriol. 1942 Mar;43(3):347–363. doi: 10.1128/jb.43.3.347-363.1942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bochner B. R., Savageau M. A. Generalized indicator plate for genetic, metabolic, and taxonomic studies with microorganisms. Appl Environ Microbiol. 1977 Feb;33(2):434–444. doi: 10.1128/aem.33.2.434-444.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  8. Clark D. P. The fermentation pathways of Escherichia coli. FEMS Microbiol Rev. 1989 Sep;5(3):223–234. doi: 10.1016/0168-6445(89)90033-8. [DOI] [PubMed] [Google Scholar]
  9. Conway T., Ingram L. O. Similarity of Escherichia coli propanediol oxidoreductase (fucO product) and an unusual alcohol dehydrogenase from Zymomonas mobilis and Saccharomyces cerevisiae. J Bacteriol. 1989 Jul;171(7):3754–3759. doi: 10.1128/jb.171.7.3754-3759.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Conway T., Sewell G. W., Osman Y. A., Ingram L. O. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis. J Bacteriol. 1987 Jun;169(6):2591–2597. doi: 10.1128/jb.169.6.2591-2597.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Donnelly M. I., Cooper R. A. Two succinic semialdehyde dehydrogenases are induced when Escherichia coli K-12 Is grown on gamma-aminobutyrate. J Bacteriol. 1981 Mar;145(3):1425–1427. doi: 10.1128/jb.145.3.1425-1427.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fischer R. J., Helms J., Dürre P. Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis. J Bacteriol. 1993 Nov;175(21):6959–6969. doi: 10.1128/jb.175.21.6959-6969.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Freier S. M., Kierzek R., Jaeger J. A., Sugimoto N., Caruthers M. H., Neilson T., Turner D. H. Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9373–9377. doi: 10.1073/pnas.83.24.9373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gerischer U., Dürre P. Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region from Clostridium acetobutylicum. J Bacteriol. 1990 Dec;172(12):6907–6918. doi: 10.1128/jb.172.12.6907-6918.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gerischer U., Dürre P. mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis. J Bacteriol. 1992 Jan;174(2):426–433. doi: 10.1128/jb.174.2.426-433.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Goodlove P. E., Cunningham P. R., Parker J., Clark D. P. Cloning and sequence analysis of the fermentative alcohol-dehydrogenase-encoding gene of Escherichia coli. Gene. 1989 Dec 21;85(1):209–214. doi: 10.1016/0378-1119(89)90483-6. [DOI] [PubMed] [Google Scholar]
  18. Hempel J., Harper K., Lindahl R. Inducible (class 3) aldehyde dehydrogenase from rat hepatocellular carcinoma and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated liver: distant relationship to the class 1 and 2 enzymes from mammalian liver cytosol/mitochondria. Biochemistry. 1989 Feb 7;28(3):1160–1167. doi: 10.1021/bi00429a034. [DOI] [PubMed] [Google Scholar]
  19. Hidalgo E., Chen Y. M., Lin E. C., Aguilar J. Molecular cloning and DNA sequencing of the Escherichia coli K-12 ald gene encoding aldehyde dehydrogenase. J Bacteriol. 1991 Oct;173(19):6118–6123. doi: 10.1128/jb.173.19.6118-6123.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jörnvall H., Persson B., Jeffery J. Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases. Eur J Biochem. 1987 Sep 1;167(2):195–201. doi: 10.1111/j.1432-1033.1987.tb13323.x. [DOI] [PubMed] [Google Scholar]
  21. Kessler D., Leibrecht I., Knappe J. Pyruvate-formate-lyase-deactivase and acetyl-CoA reductase activities of Escherichia coli reside on a polymeric protein particle encoded by adhE. FEBS Lett. 1991 Apr 9;281(1-2):59–63. doi: 10.1016/0014-5793(91)80358-a. [DOI] [PubMed] [Google Scholar]
  22. Kletzin A. Molecular characterization of the sor gene, which encodes the sulfur oxygenase/reductase of the thermoacidophilic Archaeum Desulfurolobus ambivalens. J Bacteriol. 1992 Sep;174(18):5854–5859. doi: 10.1128/jb.174.18.5854-5859.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  24. Lee F. J., Lin L. W., Smith J. A. A glucose-repressible gene encodes acetyl-CoA hydrolase from Saccharomyces cerevisiae. J Biol Chem. 1990 May 5;265(13):7413–7418. [PubMed] [Google Scholar]
  25. Lin T. W., Bridger W. A. Sequence of a cDNA clone encoding pig heart mitochondrial CoA transferase. J Biol Chem. 1992 Jan 15;267(2):975–978. [PubMed] [Google Scholar]
  26. Marathe S., Connerton I. F., Fincham J. R. Duplication-induced mutation of a new Neurospora gene required for acetate utilization: properties of the mutant and predicted amino acid sequence of the protein product. Mol Cell Biol. 1990 Jun;10(6):2638–2644. doi: 10.1128/mcb.10.6.2638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nair R. V., Bennett G. N., Papoutsakis E. T. Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824. J Bacteriol. 1994 Feb;176(3):871–885. doi: 10.1128/jb.176.3.871-885.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Palosaari N. R., Rogers P. Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum. J Bacteriol. 1988 Jul;170(7):2971–2976. doi: 10.1128/jb.170.7.2971-2976.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Parales R. E., Harwood C. S. Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida. J Bacteriol. 1992 Jul;174(14):4657–4666. doi: 10.1128/jb.174.14.4657-4666.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  32. Reid M. F., Fewson C. A. Molecular characterization of microbial alcohol dehydrogenases. Crit Rev Microbiol. 1994;20(1):13–56. doi: 10.3109/10408419409113545. [DOI] [PubMed] [Google Scholar]
  33. SAITO H., MIURA K. I. PREPARATION OF TRANSFORMING DEOXYRIBONUCLEIC ACID BY PHENOL TREATMENT. Biochim Biophys Acta. 1963 Aug 20;72:619–629. [PubMed] [Google Scholar]
  34. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Scherf U., Buckel W. Purification and properties of 4-hydroxybutyrate coenzyme A transferase from Clostridium aminobutyricum. Appl Environ Microbiol. 1991 Sep;57(9):2699–2702. doi: 10.1128/aem.57.9.2699-2702.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Scherf U., Söhling B., Gottschalk G., Linder D., Buckel W. Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta 3-delta 2-isomerase. Arch Microbiol. 1994;161(3):239–245. doi: 10.1007/BF00248699. [DOI] [PubMed] [Google Scholar]
  37. Schoberth S., Gottschalk G. Considerations on the energy metabolism of Clostridium kluyveri. Arch Mikrobiol. 1969;65(4):318–328. doi: 10.1007/BF00412211. [DOI] [PubMed] [Google Scholar]
  38. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smith L. T., Kaplan N. O. Purification, properties, and kinetic mechanism of coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri. Arch Biochem Biophys. 1980 Sep;203(2):663–675. doi: 10.1016/0003-9861(80)90224-6. [DOI] [PubMed] [Google Scholar]
  40. Söhling B., Gottschalk G. Purification and characterization of a coenzyme-A-dependent succinate-semialdehyde dehydrogenase from Clostridium kluyveri. Eur J Biochem. 1993 Feb 15;212(1):121–127. doi: 10.1111/j.1432-1033.1993.tb17641.x. [DOI] [PubMed] [Google Scholar]
  41. Thauer R. K., Jungermann K., Henninger H., Wenning J., Decker K. The energy metabolism of Clostridium kluyveri. Eur J Biochem. 1968 Apr 3;4(2):173–180. doi: 10.1111/j.1432-1033.1968.tb00189.x. [DOI] [PubMed] [Google Scholar]
  42. Walter K. A., Bennett G. N., Papoutsakis E. T. Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes. J Bacteriol. 1992 Nov;174(22):7149–7158. doi: 10.1128/jb.174.22.7149-7158.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Weiser J. N., Love J. M., Moxon E. R. The molecular mechanism of phase variation of H. influenzae lipopolysaccharide. Cell. 1989 Nov 17;59(4):657–665. doi: 10.1016/0092-8674(89)90011-1. [DOI] [PubMed] [Google Scholar]
  44. Wierenga R. K., Terpstra P., Hol W. G. Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol. 1986 Jan 5;187(1):101–107. doi: 10.1016/0022-2836(86)90409-2. [DOI] [PubMed] [Google Scholar]
  45. Williamson V. M., Paquin C. E. Homology of Saccharomyces cerevisiae ADH4 to an iron-activated alcohol dehydrogenase from Zymomonas mobilis. Mol Gen Genet. 1987 Sep;209(2):374–381. doi: 10.1007/BF00329668. [DOI] [PubMed] [Google Scholar]
  46. Wolff R. A., Urben G. W., O'Herrin S. M., Kenealy W. R. Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri. Appl Environ Microbiol. 1993 Jun;59(6):1876–1882. doi: 10.1128/aem.59.6.1876-1882.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yan R. T., Chen J. S. Coenzyme A-acylating aldehyde dehydrogenase from Clostridium beijerinckii NRRL B592. Appl Environ Microbiol. 1990 Sep;56(9):2591–2599. doi: 10.1128/aem.56.9.2591-2599.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yang W., Li E., Kairong T., Stanley S. L., Jr Entamoeba histolytica has an alcohol dehydrogenase homologous to the multifunctional adhE gene product of Escherichia coli. Mol Biochem Parasitol. 1994 Apr;64(2):253–260. doi: 10.1016/0166-6851(93)00020-a. [DOI] [PubMed] [Google Scholar]
  49. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  50. Yoon K. H., Pack M. Y. Nucleotide sequence of the Zymomonas mobilis alcohol dehydrogenase II gene. Nucleic Acids Res. 1990 Jan 11;18(1):187–187. doi: 10.1093/nar/18.1.187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Young M., Minton N. P., Staudenbauer W. L. Recent advances in the genetics of the clostridia. FEMS Microbiol Rev. 1989 Dec;5(4):301–325. doi: 10.1111/j.1574-6968.1989.tb03402.x. [DOI] [PubMed] [Google Scholar]
  52. Youngleson J. S., Jones W. A., Jones D. T., Woods D. R. Molecular analysis and nucleotide sequence of the adh1 gene encoding an NADPH-dependent butanol dehydrogenase in the Gram-positive anaerobe Clostridium acetobutylicum. Gene. 1989 May 30;78(2):355–364. doi: 10.1016/0378-1119(89)90238-2. [DOI] [PubMed] [Google Scholar]
  53. de Vries G. E., Arfman N., Terpstra P., Dijkhuizen L. Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene. J Bacteriol. 1992 Aug;174(16):5346–5353. doi: 10.1128/jb.174.16.5346-5353.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES