Assessing the Active Bacterial Community Composition in Response to Drying and Rewetting Stresses in a California Grassland Soil.

Microorganisms experience environmental changes to which they must acclimate to survive. Understanding how microbes persist during environmental stresses has been an ongoing challenge for microbial ecologists. Evaluating overall community composition can reveal population dynamics and diversity, but a large proportion of microbial cells in a given environment may be inactive at any time. Approaches are needed, therefore, to identify the microbial populations that are active and may have important ecological roles under specific conditions.

Recently, the bromodeoxyuridine (BrdU) technique was developed to label actively growing cells. BrdU, a thymidine analog, is incorporated into newly synthesized DNA, and the BrdU-labeled DNA is then isolated from total extractable DNA by immunocapture using a BrdU-specific antibody. Analyzing the BrdU-labeled DNA allows for assessing the actively growing community, which can then be compared to the unlabeled DNA that represents the total community. However, applying the BrdU approach to study soils has been problematic due to low DNA yields and contaminants. To address these challenges, we developed a protocol, optimizing specificity and reproducibility, to amplify BrdU-labeled gene fragments encoding 16S rRNA. Our research showed that the determining factor was the Taq polymerase: among the 13 different Taq DNA polymerases we tested, only 3 provided adequate yield with minimal contamination.

We tested our protocol using dry California grassland soil that was rewetted with a BrdU solution. The composition of the dominant bacterial community within the actively growing bacteria was analyzed by Terminal-Restriction Fragment Length Polymorphism (T-RFLP) and compared to the total community. We found that optimized BrdU technique was useful for revealing the active bacterial taxonomic groups following a drying and rewetting event and for distinguishing differences between active and total communities.