Persistence of bacterial DNA in soil

It is a well established fact that only a very small portion of micro-organisms are cultivable. Fortunately during the last decades a couple of molecular methods have been established which allow a culture- independent investigation of micro-organisms and enable new insights into the microbial world. Especially several fingerprinting methods like DGGE are frequently used to characterize the structure and dynamics of microbial populations. However, the first step and thus the basis and of all these methods is an exact and correct DNA extraction but it is this extraction which was shown to cause several, partially severe problems especially in connection with complex matrices like sludge or soil. Both, own results and investigations of other colleagues point to the possibility of false positive results, thus to the possibility of overestimation of organismic DNA and to an erroneous discrimination between living and dead microbial cells. Selective extraction procedures using EMA or PMA, as recently introduced and also applied at our lab, were also not able to completely solve the problem but were shown to cause additional, new problems in connection with DNA quantification. However, irrespective of the progress in this field of research most investigations do not even care with the possibility of false DNA extraction and thus overestimation of living cells. The extend of the possible bias and the impact of matrix-effects are also not known yet. Within the proposed project we therefore want to investigate if there is a discrepancy between vitality and activity of microbial cells on the one hand and extractability of microbial DNA on the other hand. For the proposed investigation a standard soil will be used because humic substances and clays are known to retain nucleic acids and will therefore strengthen the investigated effects. Besides that the use of a standard soil opens the possibility to use a complex but common available substrate enabling replication and reproduction of the proposed experiments. The experimental setup will be divided into three main tasks: i) extraction of total and free DNA from soil, ii) the dynamics of DNA-persistence in soil and iii) the protective role of soil on DNA-persistence. Special emphasis will be laid on basic soil properties (pH, contents of humic substances and clays etc.) and their impact on DNA- persistence and extractability. Besides investigating total DNA-contents of the autochthonous microflora we will also focus on species specific DNA which will first be added to the soil and is afterwards again extracted and quantified via quantitative-PCR. Although it is not the basic goal of the proposed project to develop a new or at least better DNA-extraction method, the results of the present investigation might contribute to do so. If our working hypothesis (that conventional DNA-extraction out of complex environmental samples distinctly overestimates DNA-content of "living" cells) turns out to be true, several results of investigations ignoring this problem will have to be reconsidered critically.

Molecular biological methods are essential for the culture independent investigation of the microflora of different habitats. Different fingerprinting methods such as DGGE etc., but also highly modern sequencing techniques are currently used to investigate and characterize microbial populations. The fundament of all these methods is based on a successful, complete but also not wrong-positive extraction of nucleic acids. In matrices like soil this turned out to be quite challenging and especially the differentiation between DNA derived from alive vs dead cells is extremely difficult or hardly possible with many molecular biological methods. In the present project the influence of different DNA extraction methods and protocols was evaluated in standard soil using different disintegration strategies in order to evaluate the total amount of extractable DNA and the effect on microbial fingerprints. Moreover, a method for the selective separation of DNA derived from dead cells was tested and optimized extensively. However, a comprehensive and reproducible separation could not be obtained. Nevertheless, weaknesses and flaws of different standard extraction method could be demonstrated. Variation of different abiotic soil parameters demonstrated their impact on total extractable DNA, but also on the quality of the extracted DNA.