Quantitative discrimination of 16 S rRNA genes of Dehalococcoides species by MagSNiPer, a quantitative single-nucleotide-polymorphism genotyping method.

Previously we developed MagSNiPer, an SNP (single nucleotide polymorphism) genotyping method. In the present paper we show development of an automated system for MagSNiPer, namely MagSNiPer Station, and its application for quantitative discrimination of Dehalococcoides species, which perform anaerobic dechlorination of chloroethenes. MagSNiPer Station is equipped with a thermal cycler, a tip stand, a microtitre-plate automated stacker, an eight-channel tip dispenser, a magnetic separation unit for Magtration technology, and a chemiluminescence detector. It can automatically perform all processes required for SNP genotyping by MagSNiPer. A primer was designed for discriminating single nucleotide difference between 16 S rRNA genes of Dehalococcoides ethenogenes and Dehalococcoides BAV1. Chemiluminescence intensities for the 16 S rRNA genes obtained by MagSNiPer were proportional to their quantity. MagSNiPer analysis of 16 S rRNA genes amplified on the DNA purified from groundwater gave a ratio of these two 16 S rRNA genes similar to that obtained by cloning and sequencing. MagSNiPer is much easier, more rapid and more cost-effective than conventional sequencing. Compared with denaturing gradient-gel electrophoresis, MagSNiPer has the advantage of being quantitative. Therefore, by applying MagSNiPer at several sites where single base differences exist among Dehalococcoides species, it is possible to analyse Dehalococcoides consortia with ease, yielding useful information on anaerobic bioremediation of chloroethenes.

Detection and identification of Dehalococcoides species responsible for in situ dechlorination of trichloroethene to ethene enhanced by hydrogen-releasing compounds.

Previous studies have shown that Dehalococcoides species are responsible for the anaerobic bioremediation of chloroethene pollution. It has been thought that co-operation of several species is required for complete dechlorination to ethene. In the present study, we used quantitative PCR of 16 S rRNA and RDase (reductive dehalogenase) genes to examine species changes and the population of Dehalococcoides species in ground water in which the dechlorination of TCE (trichloroethene) to ethene was enhanced by delivery of hydrogen-releasing compounds. The results have shown that at least two different Dehalococcoides species co-operate in the dechlorination of TCE to ethene. Initially, the number of strains equipped with TCE RDase increased approx. 10(5)-fold. This was followed by a decrease to the original level, according to the exhaustion of TCE. Subsequently, another strain appeared, which had a VC (vinyl chloride) RDase gene similar to bvcA of Dehalococcoides sp. BAV1 and is probably responsible for the dechlorination of VC to ethene. Analysis of several genes has suggested that the former strain is like Dehalococcoides sp. FMC-TCE, and the latter strain is similar to the Dehalococcoides sp. strain that exists in the Dehalococcoides-containing mixed culture KB1. These results support the notion that monitoring Dehalococcoides species by the presence of RDase genes as genetic markers provides detailed information on the progress of bioremediation of chloroethenes, which will be useful to improve the efficiency of bioremediation.