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


  • Editor: Kornelia Smalla

  • Present address: Eva M. Top, Department of Biological Sciences, University of Idaho, Moscow, ID, USA.

Dirk Springael, Division of Soil and Water Management, Catholic University of Leuven (KULeuven), Kasteelpark Arenberg 20, 3001 Heverlee, Belgium. Tel: +32 0 16 321604; fax: +32 0 16 321997; e-mail: dirk.springael@biw.kuleuven.be


tmoA and related genes encode the α-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.