ABSTRACTRapid responses of bacteria to sudden changes in their environment can have important implications for the structure and function of microbial communities. In this study, we used heavy-water stable isotope probing (H218O-SIP) to identify bacteria that respond to soil rewetting. First, we conducted experiments to address uncertainties regarding the H218O-SIP method. Using liquid chromatography-mass spectroscopy (LC-MS), we determined that oxygen from H218O was incorporated into all structural components of DNA. Although this incorporation was uneven, we could effectively separate18O-labeled and unlabeled DNAs derived from laboratory cultures and environmental samples that were incubated with H218O. We found no evidence forex vivoexchange of oxygen atoms between DNA and extracellular H2O, suggesting that18O incorporation into DNA is relatively stable. Furthermore, the rate of18O incorporation into bacterial DNA was high (within 48 to 72 h), coinciding with pulses of CO2generated from soil rewetting. Second, we examined shifts in the bacterial composition of grassland soils following rewetting, using H218O-SIP and bar-coded pyrosequencing of 16S rRNA genes. For some groups of soil bacteria, we observed coherent responses at a relatively course taxonomic resolution. Following rewetting, the relative recovery ofAlphaproteobacteria,Betaproteobacteria, andGammaproteobacteriaincreased, while the relative recovery ofChloroflexiandDeltaproteobacteriadecreased. Together, our results suggest that H218O-SIP is effective at identifying metabolically active bacteria that influence soil carbon dynamics. Our results contribute to the ecological classification of soil bacteria while providing insight into some of the functional traits that influence the structure and function of microbial communities under dynamic soil moisture regimes.
Do It Yourself! – Experiences with Self-Synthesized CsTFA for RNA-SIP Analyses
