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Isolation of mutants of Alcaligenes eutrophus unable to derepress the fermentative alcohol dehydrogenase

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Abstract

Eight representative strains of Alcaligenes eutrophus, two strains of Alcaligenes hydrogenophilus and three strains of Paracoccus denitrificans were examined for their ability to use different alcohols and acetoin as a carbon source for growth. A. eutrophus strains N9A, H16 and derivative strains were unable to grow on ethanol or on 2,3-butanediol. Alcohol-utilizing mutants derived from these strains, isolated in this study, can be categorized into two major groups: Type I-mutants represented by strain AS1 occurred even spontaneously and were able to grow on 2,3-butanediol (t d=2.7–6.4 h) and on ethanol (t d=15–50 h). The fermentative alcohol dehydrogenase was present on all substrates tested, indicating that this enzyme in vivo is able to oxidize 2,3-butanediol to acetoin which is a good substrate for wild type strains. Type II-mutants represented by strain AS4 utilize ethanol as a carbon source for growth (t d=3–9 h) but do not grow on butanediol. In these mutants the fermentative alcohol dehydrogenase is only present in cells cultivated under conditions of restricted oxygen supply, but a different NAD-dependent alcohol dehydrogenase is present in ethanol grown cells. Cells grown on ethanol, acetoin or 2,3-butanediol synthesized in addition two proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity and acetate thiokinase. An acylating acetaldehyde dehydrogenase (EC 1.2.1.10) was not detectable. Applying the colistin- and pin point-technique for mutant selection to strain AS1, mutants, which lack the fermentative alcohol dehydrogenase even if cultivated under conditions of restricted oxygen supply, were isolated; the growth pattern served as a readily identifiable phenotypic marker for the presence or absence of this enzyme.

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References

  • Beisenherz G, Bolze HJ, Bücher T, Czok R, Garbade KH, Meyer-Arendt E (1953) Diphosphofructose-Aldolase, Phosphoglyceraldehyd-Dehydrogenase, Glycerophosphat-Dehydrogenase und Pyruvat-Kinase aus Kaninchenmuskel in einem Arbeitsgang. Z Naturforsch 8b:555–577

    Google Scholar 

  • Beudeker RF, De Boer W, Kuenen JG (1981) Heterolactic fermentation of intracellular polyglucose by the obligate chemolithotroph Thiobacillus neapolitanus under anaerobic conditions. FEMS Microbiol Lett 12:337–342

    Google Scholar 

  • Bowien B, Schlegel HG (1981) Physiology and biochemistry of aerobic hydrogen-oxidizing bacteria. Ann Rev Microbiol 35:405–452

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254

    Google Scholar 

  • Brown TDK, Jones-Mortimer MC, Kornberg HL (1977) The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J Gen Microbiol 102:327–336

    Google Scholar 

  • Clark D, Cronan JE (1980) Escherichia coli mutants with altered control of alcohol dehydrogenase and nitrate reductase. J Bacteriol 141:177–181

    Google Scholar 

  • Davis DH, Stanier RY, Doudoroff M (1970) Taxonomic studies on some Gram negative polarly flagellated “hydrogen bacteria” and related species. Arch Mikrobiol 70:1–13

    Google Scholar 

  • Don RH, Pemperton JM (1981) Properties of six pesticide degradation plasmids isolated from Alcaligenes paradoxus and Alcaligenes eutrophus. J Bacteriol 145:681–686

    Google Scholar 

  • Friedrich B, Friedrich CG, Meyer M, Schlegel HG (1984) Expression of hydrogenase in Alcaligenes spp. is altered by interspecific plasmid exchange. J Bacteriol 158:331–339

    Google Scholar 

  • Friedrich B, Kortlüke C, Hogrefe C, Eberz G, Silber B, Warrelmann J (1986) Genetics of hydrogenase from aerobic lithoautotrophic bacteria. Biochimie 68:133–145

    Google Scholar 

  • Hogrefe C, Friedrich B (1984) Isolation and characterization of megaplasmid DNA from lithoautotrophic bacteria. Plasmid 12:161–169

    Google Scholar 

  • Jendrossek D, Steinbüchel A, Schlegel HG (1987) Purification and properties of two different acetaldehyde dehydrogenases from Alcaligenes eutrophus involved in the utilization of ethanol. Eur J Biochem (in press)

  • Juni E, Heym GA (1956) A cyclic pathway for the bacterial disimilation of 2,3-butanediol, acetylmethylcarbinol and diacetyl. I. General aspects of the 2,3-butanediol cycle. J Bacteriol 71:425–432

    Google Scholar 

  • Kaudewitz F (1959) Inaktivierende und mutagene Wirkung salpetriger Säure auf Zellen von Escherichia coli. Z Naturforsch 14b:528–537

    Google Scholar 

  • King PP (1982) Biotechnology. An industrial view. J Chem Tech Biotechnol 32:2–8

    Google Scholar 

  • Kuhn M, Steinbüchel A, Schlegel HG (1984) Hydrogen evolution by strictly aerobic hydrogen bacteria under anaerobic conditions. J Bacteriol 159:633–639

    Google Scholar 

  • Lopez J, Thoms B, Fortnagel P (1973) Mutants of Bacillus subtilis blocked in acetoin reductase. Eur J Biochem 40:479–483

    Google Scholar 

  • Lopez JM, Thoms B, Rehbein H (1975) Acetoin degradation in Bacillus subtilis by direct oxidative cleavage. Eur J Biochem 57:425–430

    Google Scholar 

  • Lorowitz W, Clark D (1982) Escherichia coli mutants with a temperature-sensitive alcohol dehydrogenase. J Bacteriol 152: 935–938

    Google Scholar 

  • Loveless A, Howarth S (1959) Mutation of bacteria at high levels of survival by ethyl methane sulfonate. Nature 184:1780–1782

    Google Scholar 

  • Mandell JD, Greenberg J (1960) A new chemical mutagen for bacteria: 1-methyl-3-nitro-1-nitrosoguanidine. Biochem Biophys Res Commun 3:575–577

    Google Scholar 

  • Mergeay M, Houba C, Gerits J (1978) Extrachromosomal inheritance controlling resistance to cadmium, cobalt, and zinc ions: evidence for curing in a Pseudomonas. Arch Int Physiol Biochim 86:440–441

    Google Scholar 

  • Oberlies G, Fuchs G, Thauer RK (1980) Acetate thiokinase and the assimilation of acetate in Methanobacterium thermoautotrophicum. Arch Microbiol 128:248–252

    Google Scholar 

  • Ohi K, Takada N, Komemushi S, Okazaki M, Miura Y (1979) A new species of hydrogen-utilizing bacterium. J Gen Appl Microbiol 25:53–58

    Google Scholar 

  • Reh M, Schlegel HG (1969) Anreicherung und Isolierung auxotropher Mutanten von Hydrogenomonas H16. Arch Mikrobiol 67:99–109

    Google Scholar 

  • Schelgel HG, Vollbrecht D (1980) Formation of the dehydrogenases for lactate, ethanol and butanediol in the strict aerobic bacterium Alcaligenes eutrophus. J Gen Microbiol 117:475–481

    Google Scholar 

  • Schlegel HG, Kaltwasser H, Gottschalk G (1961) Ein Submersverfahren zur Kultur wasserstoffoxidierender Bacterien: Wachstumsphysiologische Untersuchungen. Arch Mikrobiol 38:209–222

    Google Scholar 

  • Senior PJ, Dawes EA (1973) The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter beijerinckii. Biochem J 134:225–238

    Google Scholar 

  • Stegemann H, Francksen H, Macko V (1973) Potato proteins: genetic and physiological changes, evaluated by one or two dimensional PAA-gel-techniques. Z Naturforsch 28c:722–732

    Google Scholar 

  • Steinbüchel A, Schlegel HG (1983a) NAD-linked l(+)-lactate dehydrogenase from the strict aerobe Alcaligenes eutrophus. 1. Purification and properties. Eur J Biochem 130:321–328

    Google Scholar 

  • Steinbüchel A, Schlegel HG (1983b) NAD-linked l(+)-lactate dehydrogenase from the strict aerobe Alcaligenes eutrophus. 2. Kinetic properties and inhibition by oxaloacetate. Eur J Biochem 130:329–334

    Google Scholar 

  • Steinbüchel A, Schlegel HG (1983c) The rapid purification of lactate dehydrogenase from Alcaligenes eutrophus in a two-step procedure. Eur J Appl Microbiol Biotechnol 17:163–167

    Google Scholar 

  • Steinbüchel A, Schlegel HG (1984) A multifunctional fermentative alcohol dehydrogenase from the strict aerobe Alcaligenes eutrophus: purification and properties. Eur J Biochem 141: 555–564

    Google Scholar 

  • Steinbüchel A, Kuhn M, Niedrig M, Schlegel HG (1983) Fermentation enzymes in strictly aerobic bacteria: comparative studies on strains of the genus Alcaligenes and on Nocardia opaca and Xanthobacter autotrophicus. J Gen Microbiol 129:2825–2835

    Google Scholar 

  • Trüper HG (1965) Tricarboxylic acid cycle and related enzymes in Hydrogenomonas H16G+ grown on various carbon sources. Biochim Biophys Acta 111:565–568

    Google Scholar 

  • Weber K, Osborn M (1969) The reliability of molecular weight determination by dodecyl sulfate polyacrylamide electrophoresis. J Biol Chem 244:4406–4412

    Google Scholar 

  • Wilde E (1962) Untersuchungen über Wachstum und Speicherstoffsynthese von Hydrogenomonas. Arch Microbiol 43:109–137

    Google Scholar 

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  1. Institut für Mikrobiologie der Universität Göttingen, Grisebachstrasse 8, D-3400, Göttingen, Federal Republic of Germany

    A. Steinbüchel, C. Fründ, D. Jendrossek & H. G. Schlegel

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  1. A. Steinbüchel
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  2. C. Fründ
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  3. D. Jendrossek
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  4. H. G. Schlegel
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Steinbüchel, A., Fründ, C., Jendrossek, D. et al. Isolation of mutants of Alcaligenes eutrophus unable to derepress the fermentative alcohol dehydrogenase. Arch. Microbiol. 148, 178–186 (1987). https://doi.org/10.1007/BF00414809

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  • DOI: https://doi.org/10.1007/BF00414809

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