JB Accepts, published online ahead of print on 23 October 2009

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J. Bacteriol. doi:10.1128/JB.00874-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Combined genomic and proteomic approaches identify gene clusters involved in anaerobic 2-methylnaphthalene degradation in the sulfate-reducing enrichment culture N47

Draenka Selesi, Nico Jehmlich, Martin von Bergen, Frank Schmidt, Thomas Rattei, Patrick Tischler, Tillmann Lueders, and Rainer U. Meckenstock^*

Institute of Groundwater Ecology, Helmholtz Zentrum München – German Research Center for Environmental Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany; UFZ - Helmholtz Center for Environmental Research, Department of Proteomics, Permoserstrasse 15, D-04318 Leipzig, Germany; UFZ - Helmholtz Center for Environmental Research, Department of Metabolomics, Permoserstrasse 15, D-04318 Leipzig, Germany; Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Friedrich-Ludwig-Jahn-Strasse 15a, D-17487 Greifswald, Germany; Chair f. Genome-oriented Bioinformatics, Technische Universität München, Life and Food Science Center Weihenstephan, Am Forum 1, D-85354 Freising-Weihenstephan, Germany

^* To whom correspondence should be addressed. Email: rainer.meckenstock{at}helmholtz-muenchen.de.

The highly enriched deltaproteobacterial culture N47 anaerobically oxidizes the polycyclic aromatic hydrocarbons naphthalene and 2-methylnaphthalene with sulfate as electron acceptor. Combined genome sequencing and LC-MS/MS-based shotgun proteome analysis were performed to identify genes and proteins involved in anaerobic aromatic catabolism. Proteome analysis from 2-methylnaphthalene-grown N47 cells resulted in the identification of putative enzymes catalyzing the anaerobic conversion of 2-methylnaphthalene to 2-naphthoyl-CoA as well as the reductive ring cleavage of 2-naphthoyl-CoA leading to the formation of acetyl-CoA and CO₂. The glycyl radical-catalyzed fumarate addition to the methyl group of 2-methylnaphthalene is catalyzed by naphthyl-2-methyl-succinate synthase (Nms) composed of -subunits that are encoded by the genes nmsABC. Upstream of nmsABC, nmsD is located encoding the Nms-activating enzyme which harbors the characteristic [Fe₄S₄] cluster sequence motifs of S-adenosylmethionine (SAM)-radical enzymes. The bns gene cluster, coding for enzymes involved in beta-oxidation reactions of naphthyl-2-methyl-succinate to 2-naphthoyl-CoA, was found four intervening open reading frames further downstream. It consists of eight genes (bnsABCDEFGH) corresponding to 8.1 kb, which are closely related to enzymes involved in anaerobic toluene degradation within the denitrifiers Aromatoleum aromaticum EbN1, Azoarcus sp. T, and Thauera aromatica. Another contiguous DNA sequence harbors the gene for 2-naphthoyl-CoA reductase (ncr) and 16 additional genes that were found to be expressed in 2-methylnaphthalene-grown cells. These genes code for enzymes that were correlated to catalyze the dearomatization and ring cleavage reactions of 2-naphthoyl-CoA to acetyl-CoA and CO₂. Comparative sequence analysis of the four encoding subunits (ncrABCD) showed the closest similarity to Azoarcus type of benzoyl-CoA reductase. The present work provides first insight into the genetic basis of anaerobic 2-methylnaphthalene metabolism and delivers implications for understanding contaminant degradation.