Application of molecular biology techniques in the assessment of microbial community responses to environmental perturbations

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Virginia Tech

Molecular biology techniques were used in this research to assess the changes that occur in aquatic microbial communities when exposed to chemical wastes and the response of genetically engineered microorganisms (GEMs) in irradiated soils. Different molecular and microbial approaches to monitoring both types of microbial community changes can provide information about those systems at less complex and more sensitive levels than can be achieved with more traditional methods. Traditionally, aquatic microbial communities are evaluated by taxonomic identification and enumeration. Changes in species richness (diversity of taxa) and density of representative taxa were evaluated by DNA-DNA hybridization of prokaryotic communities at stations above and below points of industrial effluent release. Preliminary studies in 1990 indicated a positive correlation (0.95) between traditional identification and enumeration and DNA-DNA hybridization. This correlation was approaching significance but limited by a small sampling size. Study sites in 1991 did not have high correlation (0.04 and 0.36). Low correlation values were the result of methodological difficulties in the hybridization process rather than actual disparities between the two approaches. Graphically, the trends in protozoan species richness and densities of represented taxa were reflected in the DNA-DNA hybridization over time. Improvements in a molecular method include the use of radioisotopes for membrane bound hybridizations or liquid hybridizations (COT curves). In irradiated clay and loam microcosms, genetically-engineered Erwinia carotovora and wildtype declined significantly (p ≤ 0.05) over a 60 day period. Interestingly, in the clay soil, both the GEM and wildtype remained at decreased but detectable levels (using the MPN technique) after 60 days. In the loam soil, the GEM declined below levels of detection while the wildtype persisted and displayed a highly variable growth die-off pattern before declining in numbers. No significant (p ≥ 0.05) differences between the GEM and wildtype were observed in the clay soil. In the loam soil, wildtype survived at significantly (p ≤ 0.05) greater densities than the GEM over 60 days. Differences in soil characteristics as well as the type of GEM used resulted in novel patterns of persistence. An alternative to the MPN method for determining low densities of organisms is the polymerase chain reaction (PCR) in conjunction with DNA probes and hybridization techniques. For environmental probes to be used, the DNA sequence or a portion of the sequence must be determined to serve as a template for PCR amplification. Both ends of a probe specific for 63 serologically distinct strains of E. carotovora were sequenced in the initial phase of the PCR amplification and detection process. This research employs several molecular and microbial methods for the determination of changes in microbial community structure and function in response to chemical or irradiation perturbations.