Abstract
Toluene and related aromatic compounds can be mineralized to CO2 under anoxic conditions. Oxidation requires new dehydrogenase-type enzymes and water as oxygen source, as opposed to the aerobic enzymatic attack by oxygenases, which depends on molecular oxygen. We studied the anaerobic process in the denitrifying bacterium Thauera sp. strain K172. Toluene and a number of its fluoro-, chloro- and methyl-analogues were transformed to benzoate and the respective analogues by whole cells and by cell extracts. The transformation of xylene isomers to methylbenzoate isomers suggests that xylene degradation is similarly initiated by oxidation of one of the methyl groups. Toluene oxidation was strongly, but reversibly inhibited by benzyl alcohol. The in vitro oxidation of the methyl group was coupled to the reduction of nitrate, required glycerol for activity, and was inhibited by oxygen. Cells also contained benzyl alcohol dehydrogenase (NAD+), benzaldehyde dehydrogenase (NADP+), benzoate-CoA ligase (AMP-forming), and benzoyl-CoA reductase (dearomatizing). The toluene-oxidizing activity was induced when cells were grown anaerobically with toluene and also with benzyl alcohol or benzaldehyde, suggesting that benzyl alcohol or benzaldehyde acts as inducer. The other enzymes were similarly active in cells grown with toluene, benzyl alcohol, benzaldehyde, or benzoate. This is the first in vitro study of anaerobic oxidation of an aromatic hydrocarbon and of the whole-cell regulation of the toluene-oxidizing enzyme.
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References
Altenschmidt U, Fuchs G (1991) Anaerobic degradation of toluene in denitrifying Pseudomonas sp: indication for toluene methylhydroxylation and benzoyl-CoA as central aromatic intermediate. Arch Microbiol 156:152–158
Altenschmidt U, Fuchs G (1992) Anaerobic toluene oxidation to benzyl alcohol and benzaldehyde in a denitrifying Pseudomonas strain. J Bacteriol 174:4860–4862
Altenschmidt U, Oswald B, Fuchs G (1991) Purification of benzoate-coenzyme A ligase and 2-aminobenzoate-coenzyme A ligases from a denitrifying Pseudomonas sp. J Bacteriol 173: 5494–5501
Anders H-J, Kaetzke A, Kämpfer P, Ludwig W, Fuchs G (1995) Taxonomic position of aromatic degrading denitrifying pseudomonad strains K172 and KB740. Description of a new member of the genus Thauera, Thauera aromatica sp. nov. and of the genus Azoarcus, Azoarcus evansii sp. nov., members of the beta subclass of proteobacteria. Int J Sytem Bacteriol (in press)
Beller HR, Grbić-Galić D, Reinhard M (1992) Microbial degradation of toluene under sulfate-reducing conditions and the influence of iron on the process. Appl Environ Microbiol 58:786–793
Biegert T, Altenschmidt U, Eckerskorn C, Fuchs G (1993) Enzymes of anaerobic metabolism of phenolic compounds: 4-hydroxybenzoate-CoA ligase from a denitrifying Pseudomonas species. Eur J Biochem 213:555–561
Biegert T, Altenschmidt U, Eckerskorn C, Fuchs G (1995) Purification and properties of benzyl alcohol dehydrogenase from a denitrifying Thauera sp. Arch Microbiol accompanying paper
Bossert ID, Whited G, Gibson DT, Young LY (1989) Anaerobic oxidation of p-cresol mediated by a partially purified methylhydroxylase from a denitrifying bacterium. J Bacteriol 171: 2956–2962
Brackmann R, Fuchs G (1993) Enzymes of anaerobic metabolism of phenolic compounds: 4-hydroxybenzoyl-CoA reductase (dehydroxylating) from a denitrifying Pseudomonas species. Eur J Biochem 213:563–571
Bradford M M (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
Chalmers RM, Scott AJ, Fewson CA (1990) Purification of the benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by the TOL plasmid pWW53 of Pseudomonas putida MT53 and their preliminary comparison with benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase I and II from Acinetobacter calcoaceticus. J Gen Microbiol 136:637–643
Dagley S (1984) Microbial degradation of aromatic compounds. Dev Ind Microbiol 25:53–65
Dangel W, Brackmann R, Lack A, Mohammed M, Koch J, Oswald B, Seyfried B, Tschech A, Fuchs G (1991) Differential expression of enzyme activities initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA. Arch Microbiol 155:256–262
Dolfing J, Zeyer J, Binder-Eicher P, Schwarzenbach RP (1990) Isolation and characterization of a bacterium that mineralizes toluene in the absence of molecular oxygen. Arch Microbiol 154:336–341
Edwards EA, Grbić-Galić D (1994) Anaerobic degradation of toluene and o-xylene by a methanogenic consortium. Appl Environ Microbiol 60:313–322
Edwards EA, Wills LE, Reinhard M, Grbić-Galić D (1992) Anaerobic transformation of toluene and xylene by aquifer microorganisms under sulfate-reducing conditions. Appl Environ Microbiol 58:794–800
Evans PJ, Mang DT, Kim KS, Young LY (1991) Anaerobic degradation of toluene by a denitrifying bacterium. Appl Environ Microbiol 57:1139–1145
Evans PJ, Ling W, Goldschmidt B, Ritter ER, Young LY (1992) Metabolites formed during anaerobic transformations of toluene and o-xylene and their proposed relationship to the initial steps of toluene mineralization. Appl Environ Microbiol 58:496–501
Fries MR, Zhou J, Chee-Sanford J, Tiedje JM (1994) Isolation characterization and distribution of denitrifying toluene-degraders from a variety of habitats. Appl Environ Microbiol 60:2801–2810
Fuchs G, Mohamed M, Altenschmidt U, Koch J, Lack A, Brackmann R, Lochmeyer C, Oswald B (1994) Biochemistry of anaerobic biodegradation of aromatic compounds. In: Ratledge C (ed) Biochemistry of microbial degradation, Kluwer, Dordrecht, pp 513–553
Gibson DT, Subramanian V (1984) Microbial degradation of organic compounds. In: Gibson DT (ed) Microbial degradation of organic compounds. Dekker, New York, pp 181–252
Gibson J, Dispensa M, Fogg GC, Evans DT, Harwood CS (1994) 4-Hydroxybenzoate-Coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation. J Bacteriol 176:634–641
Grbić-Galić D, Vogel TM (1987) Transformation of toluene and benzene by mixed methanogenic cultures. Appl Environ Microbiol 3:254–260
Hopper DJ, Bossert ID, Rhodes-Roberts ME (1991) p-Cresol methylhydroxylase from a denitrifying bacterium involved in anaerobic degradation of p-cresol. J Bacteriol 173:1298–1301
Jüttner F, Henatsch JJ (1986) Anoxic hypolimnion is a significant source of biogenic toluene. Nature 323:797–798
Koch J, Fuchs G (1992) Enzymatic reduction of benzoyl-CoA to alicyclic compounds: a key reaction in anaerobic aromatic reduction. Eur J Biochem 205:195–202
Koch J, Eisenreich W, Bacher A, Fuchs G (1993) Products of enzymatic reduction of benzoyl-CoA: a key reaction in anaerobic aromatic metabolism. Eur J Biochem 211:649–661
Kuhn EP, Colberg PJ, Schnoor JL, Wanner O, Zehnder AJB, Schwarzenbach RP (1985) Microbial transformation of substituted benzenes during infiltration of river water to groundwater: laboratory column studies. Environ Sci Technol 19:961–968
Kuhn EP, Zeyer J, Eicher P, Schwarzenbach RP (1988) Anaerobic degradation of alkylated benzenes in denitrifying laboratory aquifer columns. Appl Environ Microbiol 54:490–496
Lovley DR, Lonergan DJ (1990) Anaerobic oxidation of toluene, phenol, and p-cresol by the dissimilatory iron-reducing organism, GA-15. Appl Environ Microbiol 56:1858–1864
Lovley DR, Baedecker MJ, Lonergan DJ, Cozzarelli IM, Philips EJP, Siegel DI (1989) Oxidation of aromatic contaminants coupled to microbial iron reduction. Nature 339:297–300
MacKintosh RW, Fewson CA (1988a) Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus: purification and preliminary characterization. Biochem J 250:743–751
MacKintosh RW, Fewson CA (1988b) Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus: substrate specificities and inhibition studies. Biochem J 255:653–661
Macy JM, Rech S, Auling G, Dorsch M, Stackebrandt E, Sly LJ (1993) Thauera selenatis gen. nov., sp. nov. a member of the beta subclass of proteo bacteria with a novel type of anaerobic respiration. Int J Syst Bacteriol 213:135–142
Rabus R, Nordhaus R, Ludwig W, Widdel F (1993) Complete oxidation of toluene under strictly anoxic conditions by a new sulfate-reducing bacterium. Appl Environ Microbiol 59: 1444–1451
Rudolphi A, Tschech A, Fuchs G (1991) Anaerobic degradation of cresols by denitrifying bacteria. Arch Microbiol 155:238–248
Schocher RJ, Seyfried B, Vazquez F, Zeyer J (1991) Anaerobic degradation of toluence by pure cultures of denitrifying bacteria. Arch Microbiol 157:7–12
Seyfried B, Glod G, Schocher R, Tschech A, Zeyer J (1994) Initial reactions in the anaerobic oxidation of toluene and m-xylene by denitrifying bacteria. Appl Environ Microbiol 60:4047–4052
Shaw JP, Harayama S (1990) Purification and characterisation of TOL plasmid-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase of Pseudomonas putida. Eur J Biochem 191:705–714
Tschech A, Fuchs G (1987) Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying Pseudomonas. Arch Microbiol 148:213–217
Vogel TM, Grbić-Galić D (1986) Incorporation of oxygen from water into toluene and benzene during anaerobic fermentative transformation. Appl Environ Microbiol 52:200–202
Wilson BH, Smith GB, Rees JH (1986) Biotransformations of selected alkylbenzenes and halogenated aliphatic hydrocarbons in methanogenic aquifier material: a microcosm study. Environ Sci Technol 20:997–1002
Zeyer J, Kuhn EP, Schwarzenbach RP (1986) Rapid microbial mineralization of toluene and 1,3-dimethylbenzene in the absence of molecular oxygen. Appl Environ Microbiol 52:944–947
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Dedicated to Prof. Achim Trebst
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Biegert, T., Fuchs, G. Anaerobic oxidation of toluene (analogues) to benzoate (analogues) by whole cells and by cell extracts of a denitrifying Thauera sp.. Arch. Microbiol. 163, 407–417 (1995). https://doi.org/10.1007/BF00272129
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DOI: https://doi.org/10.1007/BF00272129