PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites.
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.
Hendrickx
Barbara
B
Environmental and Process Technology, Flemish Institute for Technological Research (Vito), Mol, Belgium.
Dejonghe
Winnie
W
Faber
Folkert
F
Boënne
Wesley
W
Bastiaens
Leen
L
Verstraete
Willy
W
Top
Eva M
EM
Springael
Dirk
D
D001419:Bacteria / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics; D001554:Benzene / Q000378:metabolism; D001555:Benzene Derivatives / Q000378:metabolism; D044822:Biodiversity; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D044223:Pseudomonas stutzeri / Q000235:genetics; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D012988:Soil Microbiology; D012989:Soil Pollutants / Q000378:metabolism*; D014050:Toluene / Q000378:metabolism; D014992:Xylenes / Q000378:metabolism
D001426:Bacterial Proteins; D001555:Benzene Derivatives; D004269:DNA, Bacterial; D012989:Soil Pollutants; D014992:Xylenes; D014050:Toluene; D006899:Mixed Function Oxygenases; D001554:Benzene; C004912:ethylbenzene