Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment

Abstract

This study reports improvements in two of the key steps, lysis of indigenous cells and DNA purification, required for achieving a rapid nonselective protocol for extracting nucleic acids directly from sodium dodecyl sulfate (SDS)-treated sediment rich in organic matter. Incorporation of bead-mill homogenization into the DNA extraction procedure doubled the densitometrically determined DNA yield (11.8 jig of DNA* g [dry weight] of sediment-') relative to incorporation of three cycles of freezing and thawing (5.2 ,ug of DNA* g [dry weight] of sediment-'). The improved DNA extraction efficiency was attributed to increased cell lysis, measured by viable counts of sediment microorganisms which showed that 2 and 8%, respectively, survived the bead-mill homogenization and freeze-thaw procedures. Corresponding measurements of suspensions of viable Bacillus endospores demonstrated that 2 and 94% of the initial number survived. Conventional, laser scanning epifluorescence phase-contrast, and differential interference-contrast microscopy revealed that small coccoid bacterial cells (1.2 to 0.3 ,um long) were left intact after combined SDS and bead-mill homogenization of sediment samples. Estimates of the residual fraction of the fluorescently stained cell numbers indicated that 6% (2.2 X 108 cellsg [dry weight] of sediment-1) of the original population (3.8 X 109 cells * g [dry weight] of sediment-') remained after treatment with SDS and bead-mill homogenization. Thus, lysis of total cells was less efficient than that of cells which could be cultured. The extracted DNA was used to successfully amplify nahR, the regulatory gene for naphthalene catabolism in Pseudomonas putida G7, by PCR. By scaling down the mass of sediment extracted to 0.5 g and by using gel purification and SpinBind DNA purification cartridges, the time required to extract DNA from whole sediment samples was reduced to 2 h. Microbial ecologists, systematicists, and population geneticists have become increasingly interested in methods for complete, unbiased isolation of DNA (7, 9, 12, 16, 29, 30) and RNA (6, 8, 11, 19, 34, 36) from soils and sediments because such procedures promise to make the genomes of uncultured indigenous microorganisms available for molecular analysis. The ideal (2, 35, 36) is to circumvent the biases implicit in culture-based procedures by directly accessing the genes of naturally occurring microbial communities. But achieving this ideal requires overcoming a variety of interferences that diminish the quality, yield, and diversity of extracted nucleic acids. These interferences raise questions about the completeness of nucleic acid extraction, and about the representativeness of results based on the procedures. The popular direct lysis approach to DNA extraction and purification (24) may be dissected into the following conceptual steps: (i) washing the material to remove soluble components that may impair manipulation of subsequently isolated DNA; (ii) disruption of cells in the material to release DNA or RNA from the cells; (iii) sep...

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