To further explore possible avenues for accessing microbial biodiversity for drug discovery from natural products, we constructed and screened a 5,000-clone "shotgun" environmental DNA library by using an Escherichia coli-Streptomyces lividans shuttle cosmid vector and DNA inserts from microbes derived directly (without cultivation) from soil. The library was analyzed by several means to assess diversity, genetic content, and expression of heterologous genes in both expression hosts. We found that the phylogenetic content of the DNA library was extremely diverse, representing mostly microorganisms that have not been described previously. The library was screened by PCR for sequences similar to parts of type I polyketide synthase genes and tested for the expression of new molecules by screening of live colonies and cell extracts. The results revealed new polyketide synthase genes in at least eight clones. In addition, at least five additional clones were confirmed by high-pressure liquid chromatography analysis and/or biological activity to produce heterologous molecules. These data reinforce the idea that exploiting previously unknown or uncultivated microorganisms for the discovery of novel natural products has potential value and, most importantly, suggest a strategy for developing this technology into a realistic and effective drug discovery tool.

In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed for the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an organism inoculated into the soil. The aim of the present investigation was to improve the extraction, purification, and quantification of DNA derived from as large a portion of the soil microbial community as possible, with special emphasis placed on obtaining DNA from gram-positive bacteria, which form structures that are difficult to disrupt. Furthermore, we wanted to identify and minimize the biases related to each step in the procedure. Six soils, covering a range of pHs, clay contents, and organic matter contents, were studied. Lysis was carried out by soil grinding, sonication, thermal shocks, and chemical treatments. DNA was extracted from the indigenous microflora as well as from inoculated bacterial cells, spores, and hyphae, and the quality and quantity of the DNA were determined by gel electrophoresis and dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield compared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage λ DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay content strongly influenced the recovery of DNA. The adsorption of DNA to clay particles decreased when the soil was pretreated with RNA in order to saturate the adsorption sites. We also investigated different purification techniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging.

All molecular analyses of soil bacterial diversity are based on the extraction of a representative fraction of cellular DNA. Methods of DNA extraction for this purpose are divided into two categories: those in which cells are lysed within the soil (direct extraction) and those in which cells are first removed from soil (cell extraction) and then lysed. The purpose of this study was to compare a method of direct extraction with a method in which cells were first separated from the soil matrix by Nycodenz gradient centrifugation in order to evaluate the effect of these different approaches on the analysis of the spectrum of diversity in a microbial community. We used a method based on polymerase chain reaction (PCR) amplification of a 16S rRNA gene fragment, followed by hybridization of the amplified fragments to a set of specific probes to assess the phylogenetic diversity of our samples. Control parameters, such as the relationship between amount of DNA template and amount of PCR product and the influence of competing DNA on PCR amplification, were first examined. Comparison between extraction methods showed that less DNA was extracted when cells were first separated from the soil matrix (0.4 microg g(-1) dry weight soil versus 38-93 microg g(-1) obtained by in situ lysis methods). However, with the exception of the gamma-subclass of Proteobacteria, there was no significant difference in the spectrum of diversity resulting from the two extraction strategies.