This paper was presented at the International Workshop "Microorganisms - Water and Aquifers", organized by the Zuckerberg Institute For Water Research, J. Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, held on September 20-21, 2005 in the Sede Boqer Campus, Israel.
Introduction
Millions of tonnes of xenobiotic compounds are each year applied globally as pesticides in agricultural production as well as on consolidated urban areas, along railways and roads and within farmyards. As an outcome of this extensive environmental input natural waters such as rivers, lakes and groundwater have been contaminated with pesticide residues. In Denmark, where over 99% of the drinking water originates from groundwater, the detection of pesticide residues has resulted in the closure of numerous water supply wells. The economic costs associated with closing contaminated drinking water wells and finding new unaffected water resources have raised the question whether natural attenuation processes within the groundwater environment is sufficient to reduce contaminant concentrations or purification of groundwater should be introduced.
A toolbox using state-of-the-art nucleic acid techniques enabling monitoring of presence and activity of key degradative bacteria in environmental samples are currently being developed. The project involves selection and molecular characterisation of degradative bacteria in regards to phylogeny, metabolic pathways, functional genes and degradation efficiency under various environmental conditions. Genetic markers suitable for detecting abundance and specific catabolic activity will be elucidated among the degradative strains, with the aim of eventually applying microchip array technologies as well as mRNA-based approached for rapid and extensive analysis of environmental samples. Potential genetic markers will involve genes encoding catabolic enzymes and gene fragments specific for different phylogenetic groups. Additionally, quantitative PCR methods will be employed for quantifying functional gene abundance and expression. The following contains a brief run-trough of techniques undergoing development and optimisation at the Microbiology lab at Department of Geochemistry, GEUS.
Results and discussion
Elucidating key members of degradative communities by PCR and RT-PCR DGGE 16S rRNA gene fingerprinting: Simple pin-pointing of consortia members benefiting from mineralization of the herbicide linuron by targeting 16S rRNA.
The phenylurea herbicide linuron (N-(3,4-dichloro-phenyl)-N'-methoxy-N'-methylurea) is used worldwide in the conventional production of corn, cereals, vegetables and fruit. The dissipation rates determined in agricultural soils by laboratory and field experiments are highly variable, with values ranging from days to several years. Linuron is frequently detected in surface and ground waters near or below areas with intensive use, and in one extreme case linuron was detected in a drinking water well in concentrations up to 2800 g l-1.
Unfortunately, linuron and some of its metabolites are suspected of being endocrine disruptors and of having toxic effects on various aquatic and soil organisms which have stimulated research aimed at obtaining linuron-mineralizing microorganisms. Bacterial communities capable of rapid linuron mineralization was enriched from Danish agricultural soil by inoculating soil into mineral salt solution with 10-mg l-1 linuron as sole source of carbon, nitrogen and energy. One enrichment culture, designated Sp8-3, mineralized 14C-linuron with approximately 60 – 70% metabolized to 14CO2 within 10 days with high turbidity and extensive cell. No linuron or the metabolites N-(3,4-dichlorophenyl)-N-methylurea, N-(3,4-dichlorophenyl)urea were detected at the end of the experiment.
The community structure of Sp8-3 was determined during linuron mineralization by DNA-extraction and analysis of PCR-amplified 16S rRNA genes and the metabolically active Sp8-3 members was studied by RNA extraction subjected to reverse transcription (RT) after DNase treatment. Reverse transcribed cDNA and DNA extracted directly from Sp8-3 were amplified using Bacteria-specific forward primer PRBA338f with a 40 bp clamp and the reverse primer PRUN518r targeting the V3 16S rRNA gene region and separated by denaturing gradient gel electrophoresis (DGGE).
Bands of interest were excised, reamplified and the PCR products were purified and subsequently sequenced. The DGGE-based characterization of Sp8-3 indicates that the mineralization activity primarily is related to the metabolism performed by one single bacterium with similarities to Variovorax spp. However, the presence of additional DGGE-bands suggest the involvement of secondary strains in linuron mineralization. DGGE-fingerprinting enabled us to pinpoint active members benefiting from linuron mineralization facilitating easier cultivation and selection of appropriate strains for detailed analysis without prior knowledge on the catabolic genes. The study is descried in details in Sørensen et al. (2005).
Quantification of catabolic genes: Quantitative PCR targeting tfdA genes during mineralization of the herbicide MCPA.
The tfdA gene is known to be involved in the first step of the degradation of phenoxy acid herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) in several soil bacteria – but bacteria containing other tfdA-like genes have been isolated as well. A quantitative real-time PCR method was used to follow the increase in the concentration of tfdA genes during degradation of MCPA in a sandy top- and subsoil over a period of 115 days. Quantitative PCR revealed growth in the tfdA containing bacterial community from 500 genes g-1 soil to app. 3×104 genes g-1 soil and from 500 genes g-1 soil to 7×105 genes g-1 soil for topsoils initially added 2.3 mg MCPA kg-1 soil (dw) and 20 mg MCPA kg-1 soil (dw), respectively. We analysed the diversity of the tfdA gene during the degradation experiment. Analyses of melting curves of real-time PCR amplification products showed that a shift in the dominant tfdA population structure occurred during the degradation period. The shift was observed as a decrease in the optimum for the melting curve from 90.5°C to 87.5°C. Further DGGE revealed that at least 5 distinct bands were visible at the end of the degradation period. DNA from the distinct bands were sequenced and a phylogenetic analysis revealed that the tfdA genes responsible for the degradation of the MCPA belonged to the class III tfdA genes, while the tfdA genes present in the soil before the occurrence of degradation belonged to the class I tfdA genes. This work is presented in Baelum et al. (in-revision).
Fishing for mRNA from specific strains: Magnetic capture hybridization RT-PCR.
Quantitative detection of Salmonella sp. in soil and chicken manure is accomplished using the magnetic capture hybridization technique as a purification technique. The detection’s were targeted the invA gene present in all hitherto investigated Salmonella sp. using conserved primers. After cell lysis, phenol/chloroform purification and isopropanol precipitation the RNA extract was combined with the hybridization probe conjugated to paramagnetic beads. After hybridization the beads are washed with dH20, and the captured nucleic acids were released by denaturation and purified for contaminating DNA using DNase. The purified nucleic acid had a high purity and there was no need for dilution of the samples prior to RT-PCR. The developed procedure was reproducibly used to quantify Salmonella sp in high organic agricultural soil. The detection limit using ordinary quantitative PCR (cybergreen, melting curves and gelphoto) showed detection of 5x104 cells per gram of soil. Chicken manure combined into soil (1:4) did not result in reduced ability to quantify Salmonella sp. mRNA. Pasteurizing (65°C, 30 min) of Salmonella sp. containing chicken manure resulted in a five log units decrease in RT-PCR signal as compared to DNA based quantitative PCR. The mRNA based detection of Salmonella sp. is thus superior to DNA based detection, in terms of quantifying Salmonella sp. that might be viable and thus mRNA based detection would not generate false positives as DNA based PCR can do. This work is compiled in Jacobsen and Holben (in-prep).
Future directions
Gene expression profiling with single degradative strains and environmental samples targeting inducible catabolic genes: Differentially expressed genes involved in degradation of xenobiotic compounds may be elucidated by differential display methods (Liang and Pardee 1992)) such as restriction fragment differential display-PCR (RFDD-PCR). RFDD-PCR displays fragments from the coding region of mRNA and allows detection and identification of differentially expressed genes in prokaryotes (Gravesen et al. 2000; Brzostowicz et al. 2003). RFDD-PCR can be used to compare mRNA patterns from cells grown either with or without various xenobtics of interest aiming at detecting inducible catabolic genes. The involvement of detected differentially expressed genes in the biodegradation may subsequently be demonstrated by cloning, sequencing and expression in host organisms. Differential display potentially offers a fast and high-throughput approach for elucidating new catabolic genes, and have proved well-suited for elucidating inducible genes in slow-growing degradative strains with minor growth yield (Brzostowicz et al. 2005), which is a characteristic of oligotrophic microorganisms from subsurface environments.
Optimising mRNA extraction from environmental samples: There is a well-known challenge associated with extracting nucleic acids from environmental samples (Martin-Laurent et al. 2001; Hurt et al. 2001). Especially extraction of intact mRNA is troublesome as mRNA is highly unstable (Hurt et al. 2001), which will affect the use of mRNA-based monitoring systems for detecting in situ metabolic activities in environmental samples. Several different methods have been shown to be able to extract RNA from soil including the bead-beading method by Yu and Mohn, (1999) and the frozen-grinding method by Hurt et al. (2001). However, neither of these methods are particularly robust, especially in terms of how fast the mRNA is degraded. In a current project we used high quality artificial radio-labelled mRNA to pinpoint critical steps in the current procedure with the aim of optimising the procedure.
Conclusion
In conclusion the current activities are directed towards the development and application of further advanced molecular methods for the detection of presence and activity of soil and groundwater inhabiting microorganisms. The overall challenge is to validate that the ever increasing sequence information is representative for the microbial communities being present and active in the environment.
Acknowledgement
This work was primarily supported by the Danish Technical Research Council, talent grant 26-04-0051 (funding for SRS) and the Danish Agricultural and Veterinary Research Council through the SOUND project.
References:
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