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 ␣-, ␤-, and ␥-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 acetylCoA 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.Polycyclic aromatic hydrocarbons (PAHs) are constantly released into the environment by anthropogenic activities such as industrial use or by accidental contamination. Due to the low chemical reactivity caused by the resonance energy of the aromatic ring structure and the low bioavailability of PAHs, they are persistent in the environment (15). The understanding of microbial metabolic capabilities in terms of anaerobic PAH degradation is in its infancy. However, natural amelioration of contaminated sites relies on the degradation capacities of microorganisms, and therefore, it is an essential prerequisite to broaden knowledge about the microorganisms involved and their potentials concerning PAH breakdown.Numerous microorganisms that can degrade PAHs under aerobic conditions have alread...

Comparative analysis of metagenomes from three methanogenic hydrocarbon-degrading enrichment cultures with 41 environmental samples

“…Analogous genes (bns) have also been reported in the degradation of 2-methylnaphthalene (Figure 2, pathway 5, Selesi et al, 2010). We found matches to all HMMs for bbs genes in TOLDC and for the majority of bbs genes in NAPDC and SCADC (Figure 2), showing that these genes are present in all cultures regardless of the hydrocarbon substrate used for enrichment.…”

“…Recent evidence suggests that some fumarate addition genes that cluster with nmsA may function in the activation of toluene or other monoaromatics rather than polycyclic aromatic hydrocarbons (Acosta-González et al, 2013;von Netzer et al, 2013); thus, the physiological role of nms-like genes in SCADC and NAPDC is unclear as polycyclic aromatic hydrocarbon degradation has not been tested with these cultures. Previous studies noted that bssA and nmsA exhibit a high degree of homology (for example, Selesi et al, 2010), thus enzymes involved in fumarate addition might potentially co-activate several structurally diverse substrates. For example, Azoarcus sp.…”

Genome sequencing of a single cell of the widely distributed marine subsurface Dehalococcoidia, phylum Chloroflexi

“…Two subunits (gamma and beta) were present on one contig, whereas genes for possible alpha and delta subunits were present on a separate contig. The alpha and delta subunits contain ATP-binding sites of the acetate kinase/sugar kinase/Hsp70 actin family domains and therefore an ATP-dependent reduction of an aromatic ring typical of facultative anaerobes could be hypothesised (Selesi et al, 2010). The only other gene predicted to encode an enzyme involved in the oxidation of aromatic compounds to acetylCoA was a gene annotated as a subunit of succinylCoA:benzylsuccinate CoA-transferase.…”

Anaerobic Activation of p -Cymene in Denitrifying Betaproteobacteria: Methyl Group Hydroxylation versus Addition to Fumarate

“…strain T (12). This type of reaction proved to be archetypical among diverse facultatively and obligately anaerobic bacteria for the initial activation of the benzylic methyl group in toluene, all three xylene isomers (13)(14)(15), or 2-methylnaphthalene (16,17), as well as the subterminal methylene group in n-alkanes (18,19) and the benzylic methylene group in ethylbenzene (20). In the case of the latter, addition to fumarate is only known for energy-limited sulfate-reducing bacteria, while denitrifying "Aromatoleum aromaticum" strains EbN1 and EB1 activate ethylbenzene via hydroxylation to (S)-1-phenylethanol (21)(22)(23).…”