Identification of a toluene-degrading bacterium from a soil sample through H ₂ ¹⁸O DNA stable isotope probing

DNA stable isotope probing (DNA-SIP) with H ₂ ¹⁸O was used to identify a toluene-degrading bacterium in soil amended with 48 ppm toluene. After quantification of toluene degradation rates in soil, DNA was extracted from soil incubated with H ₂ ¹⁸O, H ₂ ¹⁶O, H ₂ ¹⁶O and 48 ppm toluene, or H ₂ ¹⁸O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H ₂ ¹⁶O. With extracts from soils to which only H ₂ ¹⁸O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H ₂ ¹⁸O and toluene was analyzed. We suggest that this third band formed because toluene does not contain any oxygen atoms and toluene-degrading organisms had to transfer oxygen atoms from H ₂ ¹⁸O into metabolic intermediates to form nucleic acids de novo. We extracted the third DNA band and amplified a large fraction of the bacterial 16S rRNA gene. Direct sequencing of the PCR product obtained from the labeled DNA, as well as cloned 16S rRNA amplicons, identified a known toluene degrader, Rhodococcus jostii RHA1. A toluene-degrading bacterial strain was subsequently isolated from soil and shown to be Rhodococcus jostii RHA1. Finally, quantitative real-time PCR analysis showed that the abundance of the 16S rRNA gene of Rhodococcus jostii RHA1 increased in soil after toluene exposure but not in soils from which toluene was withheld. This study indicates that H ₂ ¹⁸O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.