Culture-independent identification of microorganisms that respond to specified stimuli

A new approach that permits culture-independent identification of microorga nisms that respond to specified stimuli was developed. This approach was il lustrated by examination of microorganisms that grew in response to various nutrient supplements added to soil. A thymidine nucleotide analog, bromode oxyuridine (BrdU), and supplements were added to soil and incubated for 3 d ays. DNA was extracted from the soil, and the newly synthesized DNA was iso lated by immunocapture of the BrdU-labeled DNA. The unique perspective this approach offers was demonstrated by comparing the microbial community stru ctures obtained from total soil DNA and the BrdU-labeled fraction in an rRN A gene (rDNA) analysis. The traditional total DNA analysis revealed no nota ble differences between the treatments, whereas the BrdU-labeled DNA showed significantly different banding patterns between the nutrient supplement t reatments and compared with total DNA banding patterns. PCR primers were de veloped to specifically amplify the intergenic region of an rDNA sequence u nique to the BrdU analysis of a phosphate supplement treatment. Amplificati on of DNA from all treatments using these primers showed that it was unique to the phosphate treatment and that it was present in both the total DNA a nd BrdU-labeled DNA fractions. This result demonstrates the promise of this new strategy because it was able to permit identification of a sequence fr om a phosphate-responsive organism that was not discernable in the traditio nal total DNA community structure analysis.