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

  • Barbara H
  • Winnie D
  • Folkert F
  • et al.
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Abstract

tmoA and related genes encode the 03B1-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.

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Barbara, H., Winnie, D., Folkert, F., Wesley, B., Leen, B., Willy, V., … Dirk, S. (2006). PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites. FEMS Microbiology Ecology, 55(2), 262–273. Retrieved from http://dx.doi.org/10.1111/j.1574-6941.2005.00018.x

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