title: Anaerobic activation of p-cymene in denitrifying betaproteobacteria: methyl group hydroxylation versus addition to fumarate.
abstract: The betaproteobacteria "Aromatoleum aromaticum" pCyN1 and "Thauera" sp. strain pCyN2 anaerobically degrade the plant-derived aromatic hydrocarbon p-cymene (4-isopropyltoluene) under nitrate-reducing conditions. Metabolite analysis of p-cymene-adapted "A. aromaticum" pCyN1 cells demonstrated the specific formation of 4-isopropylbenzyl alcohol and 4-isopropylbenzaldehyde, whereas with "Thauera" sp. pCyN2, exclusively 4-isopropylbenzylsuccinate and tentatively identified (4-isopropylphenyl)itaconate were observed. 4-Isopropylbenzoate in contrast was detected with both strains. Proteogenomic investigation of p-cymene- versus succinate-adapted cells of the two strains revealed distinct protein profiles agreeing with the different metabolites formed from p-cymene. "A. aromaticum" pCyN1 specifically produced (i) a putative p-cymene dehydrogenase (CmdABC) expected to hydroxylate the benzylic methyl group of p-cymene, (ii) two dehydrogenases putatively oxidizing 4-isopropylbenzyl alcohol (Iod) and 4-isopropylbenzaldehyde (Iad), and (iii) the putative 4-isopropylbenzoate-coenzyme A (CoA) ligase (Ibl). The p-cymene-specific protein profile of "Thauera" sp. pCyN2, on the other hand, encompassed proteins homologous to subunits of toluene-activating benzylsuccinate synthase (termed [4-isopropylbenzyl]succinate synthase IbsABCDEF; identified subunits, IbsAE) and protein homologs of the benzylsuccinate β-oxidation (Bbs) pathway (termed BisABCDEFGH; all identified except for BisEF). This study reveals that two related denitrifying bacteria employ fundamentally different peripheral degradation routes for one and the same substrate, p-cymene, with the two pathways apparently converging at the level of 4-isopropylbenzoyl-CoA.
authors.affiliation: Institute for Chemistry and Biology of the Marine Environment, Carl von Ossietzky University Oldenburg, Oldenburg, Germany Max Planck Institute for Marine Microbiology, Bremen, Germany.
authors.forename: Annemieke
authors.initials: A
authors.lastname: Strijkstra
authors.affiliation: Institute for Chemistry and Biology of the Marine Environment, Carl von Ossietzky University Oldenburg, Oldenburg, Germany Max Planck Institute for Marine Microbiology, Bremen, Germany.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Trautwein
authors.affiliation: Organische Geochemie, Helmholtz Zentrum Potsdam Deutsches GeoForschungsZentrum, Potsdam, Germany.
authors.forename: René
authors.initials: R
authors.lastname: Jarling
authors.affiliation: Institute for Chemistry and Biology of the Marine Environment, Carl von Ossietzky University Oldenburg, Oldenburg, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Wöhlbrand
authors.affiliation: Institute for Chemistry and Biology of the Marine Environment, Carl von Ossietzky University Oldenburg, Oldenburg, Germany.
authors.forename: Marvin
authors.initials: M
authors.lastname: Dörries
authors.affiliation: Max Planck Genome Centre Cologne, Cologne, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Reinhardt
authors.affiliation: School of Chemistry, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Marta
authors.initials: M
authors.lastname: Drozdowska
authors.affiliation: School of Chemistry, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Bernard T
authors.initials: BT
authors.lastname: Golding
authors.affiliation: Organische Geochemie, Helmholtz Zentrum Potsdam Deutsches GeoForschungsZentrum, Potsdam, Germany.
authors.forename: Heinz
authors.initials: H
authors.lastname: Wilkes
authors.affiliation: Institute for Chemistry and Biology of the Marine Environment, Carl von Ossietzky University Oldenburg, Oldenburg, Germany Max Planck Institute for Marine Microbiology, Bremen, Germany rabus@icbm.de.
authors.forename: Ralf
authors.initials: R
authors.lastname: Rabus
mesh_terms: D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000378:metabolism; D020562:Betaproteobacteria / Q000201:enzymology / Q000378:metabolism*; D000081007:Cymenes; D058440:Denitrification; D005650:Fumarates / Q000378:metabolism*; D006900:Hydroxylation; D039821:Monoterpenes / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D019802:Succinic Acid / Q000378:metabolism; D020628:Thauera / Q000201:enzymology / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000081007:Cymenes; D005650:Fumarates; D039821:Monoterpenes; C007210:4-cymene; D019802:Succinic Acid; D010088:Oxidoreductases