Prof. Christof Holliger, Dr. Christophe Regeard, Dr. Céline Delbès
5th Framework Programm of the EU
February 2000 – January 2003
Dr. D. Pieper, GBF, Braunschweig, Germany ; Prof. D. Janssen, RUG, Groningen, The Netherlands ; Prof. W. Reineke, BUGHW, Wuppertal, Germany ; Bioclear BV, Groningen, The Netherlands
The general objective of the MAROC project was the development and optimization of molecular detection methods for the monitoring of bioremediation processes. A combination of detection of specific microorganisms, detection of catabolic genes and transformation activity, all in situ, will allow the accurate analysis of in situ natural attenuation. In order to reach this goal, the specific objectives were 1) to characterise a representative amount of sites concerning their pollution profile and metabolic potential, 2) to set up enrichment cultures for isolation of a representative amount of microorganisms with desired metabolic potential, 3) to initiate the characterisation of the genetic information responsible for the metabolic potential of isolated organisms and mixed cultures, 4) to develop primers and probes for quantitative PCR methods, based on the previously accumulated information, and 5) to verify their usefulness using laboratory strains, new isolates, and DNA and RNA isolated from environmental samples. Our part of the project was the development of molecular tools enabling the detection of the potential to reductively dechlorinate chlorinated ethenes.
Two strategies were chosen to detect the activity of dehalorespiring bacteria using tetrachloroethene as electron acceptor in environmental samples. The first strategy was based on the fact that Dehalobacter restrictus and Dehalococcoides ethenogenes are strict dehalorespiring bacteria. The ratio between the copy numbers of 16S rRNA and 16S rDNA present in a sample could allow getting an indication of the activity state of these bacteria in the environment investigated. To be able to quantify these two nucleic acid fractions, a real-time PCR method was developed for the 16S rRNA gene of Dehalobacter restritus. First tests with a plasmid containing the 16S rRNA gene showed a linear relationship between the Ct value and the concentration in a range of 102 and 107 copies per reaction. Ten copies were also detectable. In some environmental samples Dehalobacter restrictus was detected by this approach and a clear increase in number was observed upon enrichment of PCE dechlorination activity. The next steps will be to apply the Real-time PCR method to RNA and DNA isolated simultaneously from pure cultures and from environmental samples, steps that have not been carried out in the framework of this project.
The second strategy was to detect the key enzyme, the reductive dehalogenase in environmental samples. Sequences alignment of the amino acid sequences of reductive dehalogenases available on public data bases revealed five regions that are conserved in all reductive dehalogenases, and two that are specific for chloroethene reductive dehalogenases. Degenerated PCR primers were designed and successfully applied to pure culture and environmental sample DNA. From pure culture DNA the known pceA genes were re-isolated from the PCR products by cloning and sequencing, but also new putative genes were isolated. The use of a special bioinformatics tool allowed defining specific patterns of the conserved blocks for different groups of reductive dehalogenases. Applying this approach to newly isolated putative dehalogenase genes enabled to propose which chlorinated compounds could be used as substrate by the encoded protein. Hence, although not knowing the motif of the active site, this method could be used to make a link between the structure and the function of a putative reductive dehalogenase gene.
In the 23 environmental samples used as inocula for enrichment cultures, known pceA and tceA genes were detected by a nested PCR with specific primers on the PCR products of the degenerate PCR. The same specific primers were also applied on DNA extracted from samples of the first generation enrichment cultures. The direct PCR detection was also successful in some cases but showed that not in all enrichments the dehalogenase genes detected in the environmental samples increased significantly in number to be detected by direct specific PCR. This indicated that other yet unidentified dehalogenases were responsible for the dechlorination activity observed.
In order to investigate the diversity of chloroethene reductive dehalogenase genes, a SSCP fingerprint technique was developed. The technique is based on gene sequences of already known reductive dehalogenase genes and reveals small differences of putative dehalogenase genes with the known genes. A molecular marker was developed and tests with plasmids containing different dehalogenase genes showed that a multiplex PCR with the primers for the different genes was feasible. The SSCP approach was applied on the environmental samples. A big diversity was observed in some samples, in others only 3-5 bands were observed. Gel bands with the same migration distance as one of the molecular marker were sequenced. This showed that they were 100% identical with the target gene. Whether it is possible to make a link between the diversity observed and dechlorination activity found is not yet known.
In conclusion, the work done during this project provides molecular tools to amplify putative chloroethene reductive dehalogenase genes from environmental samples and to investigate their diversity in the same. The bioinformatics approach allows assigning the isolated putative dehalogenase genes to a certain group of reductive dehalogenases. It was unfortunately not possible to make clear links between the dehalogenase genes detected and the dechlorination activity observed in enrichment cultures.