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. 1997 Mar;63(3):1131–1138. doi: 10.1128/aem.63.3.1131-1138.1997

Characterization of the genes encoding the three-component membrane-bound alcohol dehydrogenase from Gluconobacter suboxydans and their expression in Acetobacter pasteurianus.

K Kondo 1, S Horinouchi 1
PMCID: PMC168402  PMID: 9055427

Abstract

The three-component membrane-bound alcohol dehydrogenase (ADH) of Gluconobacter suboxydans IFO12528 was purified, and the NH2-terminal amino acid sequence of each subunit was determined. On the basis of the amino acid sequences, the genes adhA, encoding the 72-kDa dehydrogenase, adhB, encoding the 44-kDa cytochrome c-553 (a CO-binding cytochrome c), and adhS, encoding a 15-kDa protein, were cloned and the amino acid sequences of their products were deduced from the nucleotide sequences. The dehydrogenase and cytochrome genes were clustered with the same transcription polarity, as is the case in species of Acetobacter, another genus of acetic acid bacteria. These AdhA and AdhB subunits showed similarity in amino acid sequence to those from Acetobacter spp., whereas AdhS showed no similarity to the corresponding subunit of the ADH complex of Acetobacter pasteurianus. Consistent with this, adhS of G. suboxydans could not complement a defect in the corresponding subunit of A. pasteurianus. When the adhA-adhB gene cluster of G. suboxydans was expressed in an ADH-deficient mutant of A. pasteurianus, the transformant showed distinct ADH activity. The ADH complex was purified to near homogeneity and consisted of two subunits, the dehydrogenase and the cytochrome c subunits derived from G. suboxydans, without any other subunit. These data suggested that AdhS, the smallest subunit of ADH, from G. suboxydans is not essential for ADH activity in A. pasteurianus, in contrast to the essential role of A. pasteurianus AdhS, which is required for correct assembly of the dehydrogenase and cytochrome c subunits on the membrane.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Anderson D. J., Morris C. J., Nunn D. N., Anthony C., Lidstrom M. E. Nucleotide sequence of the Methylobacterium extorquens AM1 moxF and moxJ genes involved in methanol oxidation. Gene. 1990 May 31;90(1):173–176. doi: 10.1016/0378-1119(90)90457-3. [DOI] [PubMed] [Google Scholar]
  3. Bairoch A. PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res. 1992 May 11;20 (Suppl):2013–2018. doi: 10.1093/nar/20.suppl.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Cleton-Jansen A. M., Goosen N., Fayet O., van de Putte P. Cloning, mapping, and sequencing of the gene encoding Escherichia coli quinoprotein glucose dehydrogenase. J Bacteriol. 1990 Nov;172(11):6308–6315. doi: 10.1128/jb.172.11.6308-6315.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cleton-Jansen A. M., Goosen N., Odle G., van de Putte P. Nucleotide sequence of the gene coding for quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. Nucleic Acids Res. 1988 Jul 11;16(13):6228–6228. doi: 10.1093/nar/16.13.6228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Edman P., Begg G. A protein sequenator. Eur J Biochem. 1967 Mar;1(1):80–91. doi: 10.1007/978-3-662-25813-2_14. [DOI] [PubMed] [Google Scholar]
  8. Fukaya M., Tayama K., Tamaki T., Tagami H., Okumura H., Kawamura Y., Beppu T. Cloning of the Membrane-Bound Aldehyde Dehydrogenase Gene of Acetobacter polyoxogenes and Improvement of Acetic Acid Production by Use of the Cloned Gene. Appl Environ Microbiol. 1989 Jan;55(1):171–176. doi: 10.1128/aem.55.1.171-176.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  10. Inoue T., Sunagawa M., Mori A., Imai C., Fukuda M., Takagi M., Yano K. Cloning and sequencing of the gene encoding the 72-kilodalton dehydrogenase subunit of alcohol dehydrogenase from Acetobacter aceti. J Bacteriol. 1989 Jun;171(6):3115–3122. doi: 10.1128/jb.171.6.3115-3122.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kondo K., Beppu T., Horinouchi S. Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus. J Bacteriol. 1995 Sep;177(17):5048–5055. doi: 10.1128/jb.177.17.5048-5055.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lahti R., Pitkäranta T., Valve E., Ilta I., Kukko-Kalske E., Heinonen J. Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12. J Bacteriol. 1988 Dec;170(12):5901–5907. doi: 10.1128/jb.170.12.5901-5907.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Matsushita K., Yakushi T., Takaki Y., Toyama H., Adachi O. Generation mechanism and purification of an inactive form convertible in vivo to the active form of quinoprotein alcohol dehydrogenase in Gluconobacter suboxydans. J Bacteriol. 1995 Nov;177(22):6552–6559. doi: 10.1128/jb.177.22.6552-6559.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Matsushita K., Yakushi T., Toyama H., Shinagawa E., Adachi O. Function of multiple heme c moieties in intramolecular electron transport and ubiquinone reduction in the quinohemoprotein alcohol dehydrogenase-cytochrome c complex of Gluconobacter suboxydans. J Biol Chem. 1996 Mar 1;271(9):4850–4857. doi: 10.1074/jbc.271.9.4850. [DOI] [PubMed] [Google Scholar]
  16. Megnet R. Mutants partially deficient in alcohol dehydrogenase in Schizosaccharomyces pombe. Arch Biochem Biophys. 1967 Jul;121(1):194–201. doi: 10.1016/0003-9861(67)90024-0. [DOI] [PubMed] [Google Scholar]
  17. Meyer T. E., Kamen M. D. New perspectives on c-type cytochromes. Adv Protein Chem. 1982;35:105–212. doi: 10.1016/s0065-3233(08)60469-6. [DOI] [PubMed] [Google Scholar]
  18. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  22. Takemura H., Kondo K., Horinouchi S., Beppu T. Induction by ethanol of alcohol dehydrogenase activity in Acetobacter pasteurianus. J Bacteriol. 1993 Nov;175(21):6857–6866. doi: 10.1128/jb.175.21.6857-6866.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tamaki T., Fukaya M., Takemura H., Tayama K., Okumura H., Kawamura Y., Nishiyama M., Horinouchi S., Beppu T. Cloning and sequencing of the gene cluster encoding two subunits of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes. Biochim Biophys Acta. 1991 Feb 16;1088(2):292–300. doi: 10.1016/0167-4781(91)90066-u. [DOI] [PubMed] [Google Scholar]
  24. Wong H. C., Fear A. L., Calhoon R. D., Eichinger G. H., Mayer R., Amikam D., Benziman M., Gelfand D. H., Meade J. H., Emerick A. W. Genetic organization of the cellulose synthase operon in Acetobacter xylinum. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8130–8134. doi: 10.1073/pnas.87.20.8130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. doi: 10.1016/0022-2836(85)90046-4. [DOI] [PubMed] [Google Scholar]

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