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

Abstract

The highly enriched deltaproteobacterial culture N47 anaerobically oxidizes the polycyclic aromatic hydrocarbons naphthalene and 2-methylnaphthalene, with sulfate as the electron acceptor. Combined genome sequencing and liquid chromatography-tandem mass spectrometry-based shotgun proteome analyses were performed to identify genes and proteins involved in anaerobic aromatic catabolism. Proteome analysis of 2-methylnaphthalene-grown N47 cells resulted in the identification of putative enzymes catalyzing the anaerobic conversion of 2-methylnaphthalene to 2-naphthoyl coenzyme A (2-naphthoyl-CoA), as well as the reductive ring cleavage of 2-naphthoyl-CoA, leading to the formation of acetyl-CoA and CO(2). The glycyl radical-catalyzed fumarate addition to the methyl group of 2-methylnaphthalene is catalyzed by naphthyl-2-methyl-succinate synthase (Nms), composed of alpha-, beta-, and gamma-subunits that are encoded by the genes nmsABC. Located upstream of nmsABC is nmsD, encoding the Nms-activating enzyme, which harbors the characteristic [Fe(4)S(4)] cluster sequence motifs of S-adenosylmethionine radical enzymes. The bns gene cluster, coding for enzymes involved in beta-oxidation reactions converting naphthyl-2-methyl-succinate to 2-naphthoyl-CoA, was found four intervening open reading frames further downstream. This cluster consists of eight genes (bnsABCDEFGH) corresponding to 8.1 kb, which are closely related to genes for enzymes involved in anaerobic toluene degradation within the denitrifiers "Aromatoleum aromaticum" EbN1, Azoarcus sp. strain 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 supposed to catalyze the dearomatization and ring cleavage reactions converting 2-naphthoyl-CoA to acetyl-CoA and CO(2). Comparative sequence analysis of the four encoding subunits (ncrABCD) showed the gene product to have the closest similarity to the Azoarcus type of benzoyl-CoA reductase. The present work provides the first insight into the genetic basis of anaerobic 2-methylnaphthalene metabolism and delivers implications for understanding contaminant degradation.

FIG. 1.

Proposed pathway for anaerobic 2-methylnaphthalene degradation and reductive dearomatization of 2-naphthoyl-CoA (3, 4, 53). Genes found in the N47 genome encode the following enzymes (shown in gray boxes): NmsABC, naphthyl-2-methyl-succinate synthase; BnsEF, naphthyl-2-methyl-succinate CoA transferase; BnsG, naphthyl-2-methyl-succinyl-CoA dehydrogenase; BnsH, naphthyl-2-methylene-succinyl-CoA hydratase; BnsCD, naphthyl-2-hydroxymethyl-succinyl-CoA dehydrogenase; BnsAB, naphthyl-2-oxomethyl-succinyl-CoA thiolase; and NcrABCD, 2-naphthoyl-CoA reductase. The position of the double bond is not known for octahydro-2-naphthoyl-CoA. COSCoA, thioester of CoA and the respective carboxyl group.

FIG. 2.

T-RFLP analysis of bacterial 16S rRNA gene sequences amplified from the enrichment culture N47 grown on naphthalene (A) and 2-methylnaphthalene (B). The lengths of major T-RFs are indicated.

FIG. 3.

Phylogenetic tree reflecting the relationships of 16S rRNA gene sequences identified in the naphthalene-grown enrichment culture N47 to selected sequences of Deltaproteobacteria and Spirochaetes. Sequences obtained from culture N47 are listed in bold. n indicates the number of identified sequences in the clone library. A selection of 16S rRNA genes representing selected lineages of Bacteria and Archaea was used as the out-group. The bar indicates 10% estimated sequence divergence. TCE, trichloroethene; BTEX, benzene, toluene, ethylbenzene, and xylene; TCB, trichlorobenzene.

FIG. 4.

Phylogenetic tree showing the relationships of the N47 NmsA enzyme to available pure-culture BssA enzymes and homologous fumarate-adding enzymes based on amino acid sequences. The tree was constructed by quartet puzzling. Numbers at nodes show branching confidence values deduced from 10,000 intermediate trees. GenBank accession numbers of available reference sequences are indicated. The scale bar represents 10% sequence divergence.

FIG. 5.

Partial alignment of the amino acid sequence of NmsA from the enrichment culture N47 with those of other NmsA, MasG/AssA, and BssA enzymes. The depicted alignment site contains the catalytic active cysteine and glycine residues with the characteristic sequence motif for glycine radical enzymes (shown in bold). Numbers refer to amino acid positions in the respective proteins. A. sp. T, Azoarcus sp. strain T; Magnetosp., Magnetospirillum.

FIG. 6.

Organization of the nms and bns gene clusters of anaerobic 2-methylnaphthalene catabolism in the sulfate-reducing culture N47 in comparison with those of the bss and bbs genes for anaerobic toluene degradation in Magnetospirillum sp. strain TS-6 (57), Azoarcus sp. strain T (1), T. aromatica strain K172 (28, 39), T. aromatica strain T1 (19), A. aromaticum strain EbN1 (35), and G. metallireducens strain GS15 (11). Genes are depicted as arrowheads. Annotation data for nms and bns genes are provided in Table 1. Genes for anaerobic toluene degradation are as follows: bssABCD, encoding benzylsuccinate synthase and the activating enzyme; bssE, encoding a putative chaperone; bssH, encoding a putative transporter; bbsEF, encoding succinyl-CoA:(R)-benzylsuccinate CoA-transferase; bbsG, encoding (R)-benzylsuccinyl-CoA dehydrogenase; bbsH, encoding phenylitaconyl-CoA hydratase; bbsCD, encoding 2-[hydoxy(phenyl)methyl]-succinyl-CoA dehydrogenase; bbsAB, encoding benzoylsuccinyl-CoA thiolase; and bssF, bssG, bbsI, and bbsJ, encoding hypothetical proteins.

FIG. 7.

Scale model of the organization of the ncr genes coding for the 2-naphthoyl-CoA reductase and additional genes coding for putative enzymes catalyzing ring cleavage and beta-oxidation, leading to the formation of acetyl-CoA and CO₂. Enzymes were detected in protein extracts from 2-methylnaphthalene-grown N47 cells. Symbols for the genes encoding the four subunits of 2-naphthoyl-CoA reductase are highlighted in gray. The scale indicates the nucleotide position on the DNA contig.