Diversity of Benzyl- and Alkylsuccinate Synthase Genes in Hydrocarbon-Impacted Environments and Enrichment Cultures

Abstract

Hydrocarbon-degrading microorganisms play an important role in the natural attenuation of spilled petroleum in a variety of anoxic environments. The role of benzylsuccinate synthase (BSS) in aromatic hydrocarbon degradation and its use as a biomarker for field investigations are well documented. The recent discovery of alkylsuccinate synthase (ASS) allows the opportunity to test whether its encoding gene, assA, can serve as a comparable biomarker of anaerobic alkane degradation. Degenerate assA- and bssA-targeted PCR primers were designed in order to survey the diversity of genes associated with aromatic and aliphatic hydrocarbon biodegradation in petroleum-impacted environments and enrichment cultures. DNA was extracted from an anaerobic alkane-degrading isolate (Desulfoglaeba alkenexedens ALDC), hydrocarbon-contaminated river and aquifer sediments, a paraffin-degrading enrichment, and a propane-utilizing mixed culture. Partial assA and bssA genes were PCR amplified, cloned, and sequenced, yielding several novel clades of assA genes. These data expand the range of alkane-degrading conditions for which relevant gene sequences are available and indicate that considerable diversity of assA genes can be found in hydrocarbon-impacted environments. The detection of genes associated with anaerobic alkane degradation in conjunction with the in situ detection of alkylsuccinate metabolites was also demonstrated. Comparable molecular signals of assA/bssA were not found when environmental metagenome databases of uncontaminated sites were searched. These data confirm that the assA gene is a useful biomarker for anaerobic alkane metabolism.

Evidence for Benzylsuccinate Synthase Subtypes Obtained by Using Stable Isotope Tools

“…To our best knowledge, this is the first report correlating phylogenetic information to enzymatic reactivity by use of cell-free assays and twodimensional isotope fractionation analysis. Our observations are supported by the results of Callaghan and colleagues (14) and von Netzer and colleagues (45), which indicate that the diversity of BssA-like sequences in environmental samples may reflect the presence of several Bss isoenzymes in nature. However, only protein exchanges modifying the active center are expected to affect stable isotope fractionation patterns.…”

“…S1 in the supplemental material). Several other aromatic and aliphatic compounds can be activated anaerobically by an analogous reaction (12), and it was recently shown by sequence analysis that numerous clades of fumarate-adding isoenzymes exist that activate different substrates (13)(14)(15). Anaerobic toluene activation is catalyzed by benzylsuccinate synthase (Bss), a member of the glycyl radical enzyme family, which also includes pyruvate formate lyase and anaerobic ribonucleotide reductase (16).…”

Reactivity and fate of secondary alkane sulfonates (SAS) in marine sediments

“…6AeF shows extracted UPLC-q-TOF-MS chromatograms for C 14 SAS and some selected metabolites, as well as the proposed anaerobic degradation pathway for SAS in marine sediments. This pathway is similar to that previously described for LAS in a recent paper by our research group (Lara-Martín et al, 2010), and confirms that the following mechanism, also proposed to explain the degradation of several types hydrocarbons (Callaghan et al, 2010;Mbadinga et al, 2011), is predominant in the marine environment. The first stage consists in the formation of dicarboxylic acids (Me-SdC) by the addition of fumarate to the subterminal carbon atom (C-2) of the alkyl chain of SAS ( Fig.…”

Enhanced Gene Detection Assays for Fumarate-Adding Enzymes Allow Uncovering of Anaerobic Hydrocarbon Degraders in Terrestrial and Marine Systems

“…F1 bssA was later identified to belong to the dominating Desulfobulbaceae degrader population (14) on-site, while F2 was identified as a novel bssA sequence type of the Peptococcaceae (15). Other studies on hydrocarbon-contaminated aquifers by Callaghan et al (5) and Yagi et al (16) and on xylene-degrading enrichments by Herrmann et al (17) also corroborated the existence of new and deeply branching FAE lineages, in addition to the described BSS, NMS, and ASS lineages. Furthermore, some new anaerobic hydrocarbon degraders belonging to the Clostridia were recently discovered: Desulfitobacterium aromaticivorans UKTL, using fumarate addition for toluene activation (18), and strain BF, possessing a bss-homologous gene (19), despite oxidizing benzene and not toluene.…”