Validation of Heavy-Water Stable Isotope Probing for the Characterization of Rapidly Responding Soil Bacteria

Abstract

ABSTRACT Rapid 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 (H 2 18 O-SIP) to identify bacteria that respond to soil rewetting. First, we conducted experiments to address uncertainties regarding the H 2 18 O-SIP method. Using liquid chromatography-mass spectroscopy (LC-MS), we determined that oxygen from H 2 18 O was incorporated into all structural components of DNA. Although this incorporation was uneven, we could effectively separate 18 O-labeled and unlabeled DNAs derived from laboratory cultures and environmental samples that were incubated with H 2 18 O. We found no evidence for ex vivo exchange of oxygen atoms between DNA and extracellular H 2 O, suggesting that 18 O incorporation into DNA is relatively stable. Furthermore, the rate of 18 O incorporation into bacterial DNA was high (within 48 to 72 h), coinciding with pulses of CO 2 generated from soil rewetting. Second, we examined shifts in the bacterial composition of grassland soils following rewetting, using H 2 18 O-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 of Alphaproteobacteria , Betaproteobacteria , and Gammaproteobacteria increased, while the relative recovery of Chloroflexi and Deltaproteobacteria decreased. Together, our results suggest that H 2 18 O-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.