Primers: Functional Genes for Anaerobic Hydrocarbon Degrading Microbes

Abstract

The detection of anaerobic hydrocarbon degrader populations via catabolic gene markers is important for the understanding of processes at contaminated sites. The genes of fumarate-adding enzymes (FAEs; i.e., benzylsuccinate and alkylsuccinate synthases) are widely used as specific functional markers for anaerobic degraders of aliphatic and aromatic hydrocarbons. Several recent studies have shown the existence of new and deeply branching FAE gene lineages in the environment, and respective FAE gene-targeted primer systems have been advanced. Here, state-of-the art protocols for the PCR detection, T-RFLP fingerprinting as well as sequencing of FAE gene amplicons are described. These protocols can also readily be applied to other established functional markers for anaerobic degraders of petroleum hydrocarbons, such as benzoylCoA reductases as well as the ring-cleaving hydrolases involved in the central catabolism of aromatic hydrocarbons. In summary, these assays allow for rapid and directed insights into the diversity and identity of intrinsic degrader populations and degradation potentials in hydrocarbon-impacted systems.

Metagenomic Profiling and Microbial Metabolic Potential of Perdido Fold Belt (NW) and Campeche Knolls (SE) in the Gulf of Mexico

“…Hydrocarbon degraders are active and blooming whenever an oil-spill occurs, then hydrocarbon degradation genes must be present within indigenous bacterial communities. The first step, in both aerobic and anaerobic hydrocarbon degradation, involves chemical activation, and subsequently, the compound is channeled into central metabolism ( Fuchs et al, 2011 ; Lueders and Von Netzer, 2014 ). To explore hydrocarbon degradation gene families associated to the activation in aerobic degradation of alkanes have been used as are the alkane hydroxylases alkB and CYP153 ( Nie et al, 2014 ), and for aerobic degradation of aromatic compounds the Rieske non-heme iron dependent oxygenases ( Duarte et al, 2014 ).…”

An LC-MS/MS Method for a Comprehensive Determination of Metabolites of BTEX Anaerobic Degradation in Bacterial Cultures and Groundwater

“…Indeed, no other metabolism than anaerobic toluene biodegradation can produce benzylsuccinic acid [48]. The addition of fumarate to the toluene is due to a particular fumarate-adding enzyme: benzylsuccinate synthase [2,[49][50][51]. It is reasonable to assume that other enzymes close to benzylsuccinate synthase may catalyse the degradation of other BTEX of which the metabolites were found: (2-(1-phenylethyl)-succinic acid (ethylbenzene degradation), 2-(2-methylbenzyl-succinic acid (o-xylene degradation), 2-(3 -methylbenzyl)-succinic acid (p-xylene degradation), and 2-(4-methylbenzyl)-succinic acid (m-xylene degradation).…”

Microfluidic qPCR Enables High Throughput Quantification of Microbial Functional Genes but Requires Strict Curation of Primers

“…Instead, degenerate primers are often used to target a single gene in one or multiple clades, with qPCR conditions optimized to ensure acceptable reaction efficiencies (Iwai et al, 2011;Wei et al, 2015;Gaby and Buckley, 2017). Other commonly used deviations from qPCR 'best-practice' include longer amplicons to straddle an active site combined with extended elongation times (Baldwin et al, 2003;Lueders and Von Netzer, 2017), the combination of mismatched bases and lower annealing temperatures (Ishii and Fukui, 2001;Frank et al, 2008;Edwards et al, 2011), and the use of additives to combat inhibitors such as humic acids present in soil DNA extracts (Dandie et al, 2007). Given the need for flexibility in primer design and the inability to customize individual assay conditions when using MFQPCR, it is necessary to establish what the boundaries of primer variability are for MFQPCR before it can be considered a high-throughput alternative for the quantification of microbial functional genes in environmental samples.…”