RESUMO
The sulfate-reducing highly enriched culture N47 is capable to anaerobically degrade naphthalene, 2-methylnaphthalene, and 2-naphthoic acid. A proteogenomic investigation was performed to elucidate the initial activation reaction of anaerobic naphthalene degradation. This lead to the identification of an alpha-subunit of a carboxylase protein that was two-fold up-regulated in naphthalene-grown cells compared to 2-methylnaphthalene-grown cells. The putative naphthalene carboxylase subunit showed 48% similarity to the anaerobic benzene carboxylase from an iron-reducing, benzene-degrading culture and 45% to alpha-subunit of phenylphosphate carboxylase of Aromatoleum aromaticum EbN1. A gene for the beta-subunit of putative naphthalene carboxylase was located nearby on the genome and was expressed with naphthalene. Similar to anaerobic benzene carboxylase, there were no genes for gamma- and delta-subunits of a putative carboxylase protein located on the genome which excludes participation in degradation of phenolic compounds. The genes identified for putative naphthalene carboxylase subunits showed only weak similarity to 4-hydroxybenzoate decarboxylase excluding ATP-independent carboxylation. Several ORFs were identified that possibly encode a 2-naphthoate-CoA ligase, which is obligate for activation before the subsequent ring reduction by naphthoyl-CoA reductase. One of these ligases was exclusively expressed on naphthalene and 2-naphthoic acid and might be the responsible naphthoate-CoA-ligase.
Assuntos
Carboxiliases/metabolismo , Deltaproteobacteria/enzimologia , Naftalenos/metabolismo , Proteoma/metabolismo , Anaerobiose , Deltaproteobacteria/genética , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Genoma Bacteriano , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Anaerobic polycyclic aromatic hydrocarbon (PAH) degradation is a key process for natural attenuation of oil spills and contaminated aquifers. Assessments by stable isotope fractionation, however, have largely been limited to monoaromatic hydrocarbons. Here, we report on measured hydrogen isotope fractionation during strictly anaerobic degradation of the PAH naphthalene. Remarkable large hydrogen isotopic enrichment factors contrasted with much smaller values for carbon: ε(H) = -100 ± 15, ε(C) = -5.0 ± 1.0 (enrichment culture N47); ε(H) = -73 ± 11, ε(C) = -0.7 ± 0.3 (pure culture NaphS2). This reveals a considerable potential of hydrogen isotope analysis to assess anaerobic degradation of PAHs. Furthermore, we investigated the conclusiveness of dual isotope fractionation to characterize anaerobic aromatics degradation. C and H isotope fractionation during benzene degradation (ε(C) = -2.5 ± 0.2; ε(H) = -55 ± 4 (sulfate-reducing strain BPL); ε(C) = -3.0 ± 0.5; ε(H) = -56 ± 8 (iron-reducing strain BF)) resulted in dual isotope slopes (Λ = 20 ± 2; 17 ± 1) similar to those reported for nitrate-reducers. This breaks apart the current picture that anaerobic benzene degradation by facultative anaerobes (denitrifiers) can be distinguished from that of strict anaerobes (sulfate-reducers, fermenters) based on the stable isotope enrichment factors.