Characterization of growing microorganisms in soil by stable isotope probing with H218O

Abstract

A new approach to characterize growing microorganisms in environmental samples based on labeling microbial DNA with H(2)(18)O is described. To test if sufficient amounts of (18)O 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 H(2)(18)O and labeled DNA was separated from [(16)O]DNA on a cesium chloride gradient. Soil samples were incubated with H(2)(18)O 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 (18)O can be used in the stable isotope probing of soil samples. DNA extracted from soil incubated for 21 days with H(2)(18)O 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 H(2)(18)O 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.

FIG. 1.

Separation of [¹⁸O]DNA from unlabeled DNA on a cesium chloride gradient. Tube A, DNA extracted from E. coli grown in H₂¹⁶O; tube B, DNA extracted from E. coli grown in H₂¹⁶O (top band) and DNA extracted from E. coli grown in 47.5 atom% H₂¹⁸O (bottom band); tube C, DNA extracted from E. coli grown in H₂¹⁶O (top band) and DNA extracted from E. coli grown in 23.75 atom% H₂¹⁸O (bottom band).

FIG. 2.

Time course of ¹⁸O labeling of DNA in soil. DNA was extracted from replicate samples of soil incubated with H₂¹⁸O for 0 days (tubes A), 6 days (tubes B), and 21 days (tubes C) and from replicate samples of soil incubated with H₂¹⁶O for 6 days (tubes D) and 21 days (tubes E).

FIG. 3.

Impact of g force on separation of DNA extracted from soil incubated with H₂¹⁸O. Tube A, DNA extracted from soil incubated with H₂¹⁶O and centrifuged at 176,000 × g; tubes B, DNA extracted from three replicate samples of soil incubated with H₂¹⁸O and centrifuged at 176,000 × g; tube C, DNA extracted from soil incubated with H₂¹⁶O and centrifuged at 126,000 × g; tubes D, DNA extracted from two replicate samples of soil incubated with H₂¹⁸O and centrifuged at 126,000 × g.

FIG. 4.

DNA contents in fractions, expressed as the percentage of total DNA taken from an individual centrifuge tube. DNA was extracted from soils incubated with H₂¹⁸O for 0 days (○) or 21 days (•). Fraction 1 was retrieved from the bottom of the tube, while fraction 20 was taken from the top.

FIG. 5.

Cluster analysis of TRFLP patterns generated from fractions taken from tubes C described in the legend to Fig. 2. The first number in a sample's label describes the fraction, while the second number denotes the replicate. Fraction 1 was taken from the bottom of the tube, while fraction 17 originated from the top.

FIG. 6.

Nonmetric multidimensional scaling analysis of TRFLP patterns generated from subsamples taken from tubes C described in the legend to Fig. 2. □, fractions 1 to 4 from replicate 1 and fractions 2 to 5 from replicate 2; •, fractions 5 to 11 from replicate 1 and fractions 6 to 11 from replicate 2; ○, fractions 12 to 17 from both replicates.

FIG. 7.

Fraction of fluorescence contributed by an individual terminal restriction fragment to the total fluorescence of the entire TRFLP pattern. Terminal fragments had sizes of 146 bp (•), 159 bp (□), and 491 bp (○).

FIG. 8.

Soil bacterial community composition as revealed by TRFLP patterns generated from DNA extracted from soils incubated with H₂¹⁸O for 21 days (tubes C described in the legend to Fig. 2). The average peak size of duplicates of each terminal restriction fragment is expressed as a percentage of the total fluorescence in the entire TRFLP pattern. Fraction 1 was retrieved from the bottom of the tube, while fraction 17 was taken from the top.