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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Samenvatting

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.

Originele taal-2English
Pagina's (van-tot)262-273
TijdschriftFEMS microbiology ecology
Volume55
Nummer van het tijdschrift2
DOI's
StatusPublished - feb 2006

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Keywords

  • bodemverontreiniging

Citeer dit

Hendrickx, B., Dejonghe, W., Faber, F., Boënne, W., Bastiaens, L., Verstraete, W., ... Springael, D. (2006). PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites. FEMS microbiology ecology, 55(2), 262-273. https://doi.org/10.1111/j.1574-6941.2005.00018.x