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

Barbara Hendrickx, Winnie Dejonghe, Folkert Faber, Wesley Boënne, Leen Bastiaens, Willy Verstraete, Eva M Top, Dirk Springael

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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.

Original languageEnglish
Pages (from-to)262-273
JournalFEMS Microbiology Ecology
Issue number2
Publication statusPublished - 1 Feb 2006


  • bacteria/classification
  • bacterial proteins/genetics
  • benzene/metabolism
  • benzene derivatives/metabolism
  • biodiversity
  • dna
  • electrophoresis, polyacrylamide gel/methods
  • molecular sequence data
  • phylogeny
  • polymerase chain reaction
  • pseudomonas stutzeri/genetics
  • sequence analysis, dna
  • sequence homology
  • soil microbiology
  • soil pollutants/metabolism
  • toluene/metabolism
  • xylenes/metabolism


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