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

Abstract

The detection of anaerobic hydrocarbon degrader populations via catabolic gene markers is important for the understanding of processes at contaminated sites. Fumarate-adding enzymes (FAEs; i.e., benzylsuccinate and alkylsuccinate synthases) have already been established as specific functional marker genes for anaerobic hydrocarbon degraders. Several recent studies based on pure cultures and laboratory enrichments have shown the existence of new and deeply branching FAE gene lineages, such as clostridial benzylsuccinate synthases and homologues, as well as naphthylmethylsuccinate synthases. However, established FAE gene detection assays were not designed to target these novel lineages, and consequently, their detectability in different environments remains obscure. Here, we present a new suite of parallel primer sets for detecting the comprehensive range of FAE markers known to date, including clostridial benzylsuccinate, naphthylmethylsuccinate, and alkylsuccinate synthases. It was not possible to develop one single assay spanning the complete diversity of FAE genes alone. The enhanced assays were tested with a range of hydrocarbon-degrading pure cultures, enrichments, and environmental samples of marine and terrestrial origin. They revealed the presence of several, partially unexpected FAE gene lineages not detected in these environments before: distinct deltaproteobacterial and also clostridial bssA homologues as well as environmental nmsA homologues. These findings were backed up by dual-digest terminal restriction fragment length polymorphism diagnostics to identify FAE gene populations independently of sequencing. This allows rapid insights into intrinsic degrader populations and degradation potentials established in aromatic and aliphatic hydrocarbon-impacted environmental systems.

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.…”

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

“…This difference should not be due to energetic constraints, as both strains can yield the same high energy from p-cymene degradation (⌬G 0= ϭ Ϫ5,354 kJ mol Ϫ1 p-cymene). The distinctiveness of the herein reported enzymes from other known anaerobic hydrocarbon-activating enzymes with respect to substrate type (p-cymene) and phylogenetic branching could add to (i) the compound-specific resolution power of biogeographic studies on genes encoding these enzymes (78) and (ii) the prospects of biomimetic approaches for the industrially relevant COH bond activation (79). Further degradation of 4-isopropylbenzoylCoA is currently being investigated by pursuing possibilities of degradation via a route similar to the recently described 4-methylbenzoyl-CoA pathway (80) or the classical anaerobic benzoylCoA pathway (8).…”

Anodes Stimulate Anaerobic Toluene Degradation via Sulfur Cycling in Marine Sediments

“…The ␣-subunit of the dissimilatory sulfite reductase (dsrA) and the ␣-subunit of the benzylsuccinate synthase (bssA) are genetic markers commonly used for the identification and quantification of sulfate reducers and anaerobic toluene degraders, respectively, that use the fumarate addition pathway for toluene degradation (37,47). In order to assess the extent of the enrichment of anaerobic toluene-degrading and sulfate-reducing bacteria, the quantification of 16S rRNA, dsrA, and bssA genes was carried out on DNA samples extracted from the anode and bulk anolyte of reactors polarized at 0 mV and ϩ300 mV.…”