PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites

Abstract

tmoA and related genes encode the alpha-subunit of the hydroxylase component of the major group (subgroup 1 of subfamily 2) of bacterial multicomponent mono-oxygenase enzyme complexes involved in aerobic benzene, toluene, ethylbenzene and xylene (BTEX) degradation. A PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of tmoA-like gene sequences in environmental samples using a newly designed moderately degenerate primer set suitable for that purpose. In 35 BTEX-degrading bacterial strains isolated from a hydrocarbon polluted aquifer, tmoA-like genes were only detected in two o-xylene degraders and were identical to the touA gene of Pseudomonas stutzeri OX1. The diversity of tmoA-like genes was examined in DNA extracts from contaminated and non-contaminated subsurface samples at a site containing a BTEX-contaminated groundwater plume. Differences in DGGE patterns were observed between strongly contaminated, less contaminated and non-contaminated samples and between different depths, suggesting that the diversity of tmoA-like genes was determined by environmental conditions including the contamination level. Phylogenetic analysis of the protein sequences deduced from the amplified amplicons showed that the diversity of TmoA-analogues in the environment is larger than suggested from described TmoA-analogues from cultured isolates, which was translated in the DGGE patterns. Although different positions on the DGGE gel can correspond to closely related TmoA-proteins, relationships could be noticed between the position of tmoA-like amplicons in the DGGE profile and the phylogenetic position of the deduced protein sequence.

Use of 16S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies

“…Lateral gene transfer has uncoupled function from phylogeny for prokaryotes, so the identity of a bacterium responsible for a function is unlikely to be uncovered through diversity studies. This has already led researchers to use functional genes, such as the [NiFe] hydrogenase gene, amoA, pmoA, nirS, nirK, nosZ, and pufM, in conjunction with DGGE (1,19,22,39,41) and to detect pmoA and dysB mRNAs with CARD-FISH (14,26), leading the way for in situ identification of protein-encoding genes. The potential for alternative housekeeping genes to study diversity or the use of operational genes in molecular microbial ecology is an exciting prospect.…”

Removal of dissolved organics from produced water by forward osmosis

“…Several studies have been conducted to develop suitable technologies for the treatment of produced water [5][6][7][8][9][10][11][12]. Treatment processes such as oxidation, extraction, stripping, sorption, biological treatment, and membrane technologies have been widely investigated.…”

“…Water initially makes up a small percentage of fluids when a well is new, whereas over time the volume of water tends to *Corresponding author. (2013) [1][2][3][4][5][6][7][8][9][10] increases as the product yield declines. The estimated average global production of produced water is 210 million barrels (bbl) per day, resulting in an annual estimation of 77 billion bbl per year [4].…”

Biological monitoring for carbon capture and storage – A review and potential future developments

“…As with the applications for biosensors, PCR has primarily been used in other fields for monitoring chemical pollutants. Examples include the use of PCR to characterise the microbial community in groundwater and/or soil contaminated with tetracycline (Aminov et al, 2002), BTEX (Hendrickx et al, 2006), phenol and tar acids (Pickup et al, 2001). Some of these quantitative PCR methods have also been developed for the detection of BTEX breakdown products by isolating genes from hydrocarbon consuming anaerobic bacteria (Baldwin et al, 2010;Beller et al, 2002;Junca and Pieper, 2004).…”