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Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16

II. The isolation and characterization of mutants auxotrophic for phenylalanine and tyrosine

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Abstract

  1. 1.

    Mutants derived from the hydrogen bacterium Alcaligenes eutrophus strain H16 auxotrophic for phenylalanine and tyrosine were isolated employing mutagenic agents (EMS, nitrite), the colistine counterselection technique and the “pin-point” isolation method. Three different types of mutants were found: (1) Mutants, requiring phenylalanine or phenylpyruvate for growth, were affected in chorismate mutase as well as prephenate dehydratase. Both activities were regained by reversion to prototrophy. The auxotrophic strains accumulated chorismic acid. (2) Strains with a growth response similar to that of the first group lacked only prephenate dehydratase activity which was partially regained by reversion. Chorismate mutase and prephenate dehydrogenase were derepressed up to two-fold. Mutants grown in minimal medium excreted prephenic acid. (3) The third type of mutants required phenylalanine or phenylpyruvate and grew slowly when supplemented with chorismate or prephenate. The enzymes involved in the specific pathway of phenylalanine and tyrosine were found to be present. Some of them were even more active than in the wild-type.

  2. 2.

    Mutants accumulating chorismic acid or prephenic acid were able to grow on minimal medium when incubated long enough. The chemical instability of the excretion products resulted in their nonenzymatic conversion to subsequent intermediates which were taken up by the cells, allowing growth.

  3. 3.

    A method is described for preparing barium prephenate using the auxotrophic mutant 6B-1 derived from A. eutrophus H16. Prephenic acid, excreted by this strain, was obtained from the culture filtrate with a purity of at least 70% and a yield of approximately 180 mg per 2 l of medium.

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Abbreviations

CTAB:

cetyltrimethylammoniumbromide

EMS:

ethyl methanesulfonate

References

  • Ahmed,S.I., Campbell,J.J.R.: A bifunctional enzyme in Pseudomonas aeruginosa: a new pattern in the organization of enzymes concerned with phenylalanine and tyrosine biosynthesis. J. Bact. 115, 205–212 (1973)

    Google Scholar 

  • BakerT.I.: Phenylalanine-tyrosine biosynthesis in Neurospora crassa. Genetics 58, 351–359 (1968)

    Google Scholar 

  • Calhoun,D.H., Pierson,D.L., Jensen,R.A.: Channelshuttle mechanism for the regulation of phenylalanine and tyrosine synthesis at a metabolic branch point in Pseudomonas aeruginosa. J. Bact. 113, 241–251 (1973)

    Google Scholar 

  • Champney,W.S., Jensen,R.A.: The enzymology of prephenate dehydrogenase in Bacillus subtilis. J. biol. Chem. 245, 3763–3770 (1970)

    Google Scholar 

  • Colburn,R.W., Tatum,E.L.: Studies of a phenylalaninetyrosine requiring mutant of Neurospora crassa (strain S4342). Biochim. biophys. Acta (Amst.) 97, 442–448 (1965)

    Google Scholar 

  • Cotton,R.G.H., Gibson,F.: The biosynthesis of phenylalanine and tyrosine. Enzymes converting chorismic acid into prephenic acid and their relationships to prephenate dehydratase and prephenate dehydrogenase. Biochim. biophys. Acta (Amst.) 100, 76–88 (1965)

    Google Scholar 

  • Cotton,R.G.H., Gibson,F.: The biosynthesis of phenylalanine and tyrosine in the pea (Pisum sativum): Chorismate mutase. Biochim. biophys. Acta (Amst.) 156, 187–189 (1968)

    Google Scholar 

  • Davidson,B.E., Blackburn,E.H., Dopheide,T.A.A.: Chorismate mutase—prephenate dehydratase from Escherichia coli K-12. 1. Purification, molecular weight, and amino acid composition. J. biol. Chem. 247, 4441–4446 (1972)

    Google Scholar 

  • Davis,B.D.: Aromatic biosynthesis: IV. Preferential conversion in incompletely blocked mutants of a common precursor of several metabolites. J. Bact. 64, 729–748 (1952)

    Google Scholar 

  • Davis,B.D., Mingioli,E.S.: Aromatic biosynthesis. VII. Accumulation of two derivatives of shikimic acid by bacterial mutants. J. Bact. 66, 129–136 (1953)

    Google Scholar 

  • Dayan,J., Sprinson,D.B.: Enzyme alterations in tyrosine and phenylalanine auxotrophs of Salmonella typhimurium. J. Bact. 108, 1174–1180 (1971)

    Google Scholar 

  • DeMoss,J.A.: The conversion of shikimic acid to anthranilic acid by extracts of Neurospora crassa. J. biol. Chem. 240, 1231–1235 (1965)

    Google Scholar 

  • Friedrich,B., Schlegel,H.G.: Die Hydroxylierung von Phenylalanin durch Hydrogenomonas eutropha H16. Arch. Mikrobiol. 83, 17–31 (1972)

    Google Scholar 

  • Gibson,F.: Chorismic acid: Purification and some chemical and physical studies. Biochem. J. 90, 256–261 (1964)

    Google Scholar 

  • Gibson,F.: Chorismic acid. Biochem. Preparations 12, 94–97 (1968)

    Google Scholar 

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

    Google Scholar 

  • Lingens,F., Goebel,W., Uesseler,H.: Regulation der Biosynthese der aromatischen Aminosäuren in Saccharomyces cerevisiae. 2. Repression, Induktion und Aktivierung. Europ. J. Biochem. 1, 363–374 (1967)

    Google Scholar 

  • Lingens,F., Müller,G.: Über die Akkumulation von Chorisminsäure bei Mutanten und Wildstämmen von Escherichia coli und Saccharomyces cerevisiae. Z. Naturforsch. 22B, 991 (1967)

    Google Scholar 

  • Loveless,A., Howarth,S.: Mutation of bacteria at high levels of survival with mutagenic alkyl methane sulfofonates. Nature (Lond.) 184, 1780–1782 (1959)

    Google Scholar 

  • Lowry,O.H., Rosebrough,N.J., Farr,A.L., Randall,R.J.: Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265–275 (1951)

    Google Scholar 

  • Mandelstam,J., Jacoby,G.A.: Induction and multi-sensitive end-product repression in the enzymic pathway degrading mandelate in Pseudomonas fluorescens. Biochem. J. 94, 569–577 (1965)

    Google Scholar 

  • Metzenberg,R.L., Mitchell,H.K.: Isolation of prephenic acid from Neurospora. Arch. Biochem. Biophys. 64, 51–56 (1956)

    Google Scholar 

  • Nishioka,Y., Demerec,M., Eisenstark,A.: Genetic analysis of aromatic mutants of Salmonella typhimurium. Genetics 56, 341–351 (1967)

    Google Scholar 

  • Pittard,J., Wallace,B.J.: Distribution and function of genes concerned with aromatic biosynthesis in Escherichia coli. J. Bact. 91, 1494–1508 (1966)

    Google Scholar 

  • Plieninger,H.: Prephensäure, Konfiguration and Stand der Synthese. Angew. Chem. 74, 423–428 (1962)

    Google Scholar 

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

    Google Scholar 

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

    Google Scholar 

  • Schmidt,K., Liaaen-Jensen,S., Schlegel,H.G.: Die Carotinoide der Thiorhodaceae. I. Okenon als Hauptcarotinoid von Chromatium okenii Perty. Arch. Mikrobiol. 46, 117–126 (1963)

    Google Scholar 

  • Schmit,J.C., Zalkin,H.: Chorismate mutase-prephenate dehydratase. Partial purification and properties of the enzyme from Salmonella typhimurium. Biochemistry 8, 174–181 (1969)

    Google Scholar 

  • Simmonds,S.: The metabolism of phenylalanine and tyrosine in mutant strains of Escherichia coli. J. biol. Chem. 185, 755–762 (1950)

    Google Scholar 

  • Sprössler,B., Lensen,U., Lingens,F.: Eigenschaften der Chorismat-Mutase aus Saccharomyces cerevisiae S286. Hoppe-Seylers Z. physiol. Chem. 351, 1178–1182 (1970)

    Google Scholar 

  • Waltho,J.A.: Genetic analysis of phenylalanine responding mutant of Pseudomonas aeruginosa. J. Bact. 112, 1070–1075 (1972)

    Google Scholar 

  • Weber,H.L., Böck,A.: Chorismate mutase from Euglena gracilis: Purification and regulatory properties. Europ. J. Biochem. 16, 244–251 (1970)

    Google Scholar 

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Authors and Affiliations

  1. Institut für Mikrobiologie der Gesellschaft für Strahlen- und Umweltforschung mbH, München, inGöttingen

    Bärbel Friedrich & H. G. Schlegel

  2. Institut für Mikrobiologie der Universität Göttingen, Göttingen, Germany

    Bärbel Friedrich & H. G. Schlegel

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  1. Bärbel Friedrich
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  2. H. G. Schlegel
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Friedrich, B., Schlegel, H.G. Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16 . Arch. Microbiol. 103, 141–149 (1975). https://doi.org/10.1007/BF00436341

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  • DOI: https://doi.org/10.1007/BF00436341

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