Quantification of bias related to the extraction of DNA directly from soils.

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 lambda 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.

Isolation and characterization of a novel gamma-hexachlorocyclohexane-degrading bacterium.

The natural biotic capacity of soils to degrade gamma-hexachlorocyclohexane (gamma-HCH, lindane) was estimated using an enrichment technique based on the ability of soil bacteria to develop on synthetic media and degrade the xenobiotic compound, used as the sole source of carbon and energy. Bacterial inocula from relatively highly contaminated soils (from wood treatment factories) were found to promote efficiently the degradation of gamma-HCH, which subsequently permitted isolation of a competent gamma-HCH-degrading microorganism. The decrease of gamma-HCH concurrently with the release of chloride ions and the production of CO2 demonstrated the complete mineralization of gamma-HCH mediated by the isolate. This was confirmed by gas chromatography-mass spectrometry analyses showing that degradation subproducts of gamma-HCH included an unidentified tetrachlorinated compound and subsequently 1,2,4-trichlorobenzene and 2,5-dichlorophenol. The two linA- and linB-like genes coding, respectively, for a gamma-HCH dehydrochlorinase and a dehalogenase were characterized by using a PCR strategy based on sequence homologies with previously published sequences from Sphingomonas paucimobilis UT26. Nucleotide sequence analysis of the linA-like region revealed the presence of a 472-bp open reading frame exhibiting high homology with the linA gene from S. paucimobilis, while a preliminary study also indicated strong homology among the two linB genes. All enzymes involved in the gamma-HCH degradative pathway appear to be extracellular and encoded by genes located on the chromosome, although numerous cryptic plasmids have been detected.

Phenotypic drift inBradyrhizobium japonicum populations after introduction into soils as established by numerical analysis.

The degree of phenotypic variation of the bacterial strains USDA 125-Sp, USDA 138 and USDA 138-SmBradyrhizobium japonicum a long time after introduction was studied in three experimental fields. A total of 54 phenotypic characters were analyzed by constructing a dendrogram based on an hierarchic classification. Strong similarities (92.6, 94 and 95%) were found between the isolates introduced into soil 8, 10 and 13 years ago and between their respectiveB. japonicum parental clones. The dendrogrammic analysis detected a small amount of phenotypic drift, however, between soil isolates and parental clones belonging to the same serogroup (selective effects were found to have generated 0 to 3.9% variation for the USDA 125-Sp inoculum introduced 8 years ago, and 3.2-3.5% after 10 and 13 years, respectively, for the USDA 138 and USDA 138-Sm bacterial inocula) and within the serogroup 125 soil isolates (2.7%). We found a similar evolution of serogroup 125 isolates when compared with parental clones conserved on slant agar at 4°C. When a drift was observed, the isolates from soil presented a lower activity for several enzymes and lower diversity compared with the parental clones.