DNA Probe Method for the Detection of Specific Microorganisms in the Soil Bacterial Community

Abstract

We developed a protocol which yields purified bacterial DNA from the soil bacterial community. The bacteria were first dispersed and separated from soil particles in the presence of polyvinylpolypyrrolidone, which removes humic acid contaminants by adsorption to this insoluble polymer. The soil bacteria were then collected by centrifugation and lysed by using a comprehensive protocol designed to maximize disruption of the various types of bacteria present. Total bacterial DNA was purified from the cell lysate and remaining soil contaminants by using equilibrium density gradients. The isolated DNA was essentially pure as determined by UV spectral analysis, was at least 48 kilobases long, and was not subject to degradation, which indicated that there was no contaminating nuclease activity. The isolated DNA was readily digested by exogenously added restriction endonucleases and successfully analyzed by slot blot and Southern blot hybridizations. Using single-stranded, 32P-labeled DNA probes, we could detect and quantitate the presence of a specific microbial population in the natural soil community on the basis of the presence of a DNA sequence unique to that organism. The sensitivity of our methodology was sufficient to detect Bradyrhizobium japonicum at densities as low as 4.3 x 10 cells per g (dry weight) of soil, which corresponds to about 0.2 pg of hybridizable DNA in a l-,g DNA sample.

Selective isolation of bacteria for metagenomic analysis: Impact of membrane characteristics on bacterial filterability

“…[28][29][30] Laboratory scale applications include processes such as enzyme concentration, analysis of bacteriophages and viruses, preparation of cell-and protein-free ultrafiltrates from biological solutions, sterile filtration, enumeration of bacteria and more recently indirect DNA extraction from environmental samples. [36][37][38] Furthermore, crude filtration has been applied in several studies prior to additional treatments such as enzymatic treatment and centrifugation. [39][40][41] Two strategies are generally employed to isolate microbial populations from various environmental samples, which are dictated by the nature of the environmental samples.…”

Applications of Nucleic Acid Hybridization in Microbial Ecology

“…Thus, the ability to distinguish taxon-specific sequences in a complex mixture ofDNA holds great promise for microbial ecology. One can readily expand from detecting a specific gene in the genome of an organism to detecting the same gene in a mixture of genomes from several organisms, e.g., in the DNA obtained from the entire bacterial population in an environmental sample (Holben et al 1988).…”

“…Also, it is currently difficult to process many samples, as is often necessary for the statistical analyses needed to resolve the heterogeneity of most ecological communities. We can currently extract and purify DNA from only 6-12 soil samples simultaneously, but can analyze up to 72 DNA samples concurrently (Holben et al 1988). Also, methods for precise determination ofthe number of gene copies in a sample have not yet been developed, but there are several possible avenues to be explored.…”

“…The second approach for the recovery of DNA from soils and sediments calls for first recovering the bacterial fraction (Holben et al 1988;R. Atlas, personal communication).…”

“…Disadvantages of this approach are that the protocols are somewhat lengthy and that all of the bacteria in a sample are not recovered; however, the bacterial fraction isolated by differential centrifugation (Bone and Balkwill 1986), and thus the isolated DNA, appears to be representative ofthe total bacterial population. Currently, the quantities of bacterial DNA isolated from soils and sediments account for larger numbers of bacteria than can be detected by viable-count enumerations, and the nucleic acid methods are thus more likely to represent natural bacterial populations than are nonselective cul-turing methods (Ogram et al 1987, Holben et al 1988R. Atlas, personal communication).…”