A new approach to characterize growing microorganisms in environmental samples based on labeling microbial DNA with H218O is described. To test if sufficient amounts of 18O could be incorporated into DNA to use water as a labeling substrate for stable isotope probing, Escherichia coli DNA was labeled by cultivating bacteria in Luria broth with H218O and labeled DNA was separated from [ 16O]DNA on a cesium chloride gradient. Soil samples were incubated with H218O for 6,14, or 21 days, and isopycnic centrifugation of the soil DNA showed the formation of two bands after 6 days and three bands after 14 or 21 days, indicating that 18O can be used in the stable isotope probing of soil samples. DNA extracted from soil incubated for 21 days with H218O was fractionated after isopycnic centrifugation and DNA from 17 subsamples was used in terminal restriction fragment length polymorphism (TRFLP) analysis of bacterial 16S rRNA genes. The TRFLP patterns clustered into three groups that corresponded to the three DNA bands. The fraction of total fluorescence contributed by individual terminal restriction fragments (TRF) to a TRFLP pattern varied across the 17 subsamples so that a TRF was more prominent in only one of the three bands. Labeling soil DNA with H218O allows the identification of newly grown cells. In addition, cells that survive but do not divide during an incubation period can also be characterized with this new technique because their DNA remains without the label.
ASJC Scopus subject areas
- Food Science
- Applied Microbiology and Biotechnology