Simultaneous recovery of extracellular and intracellular DNA suitable for molecular studies from marine sediments

Abstract

The occurrence of high extracellular DNA concentrations in aquatic sediments (concentrations that are 3 to 4 orders of magnitude greater than those in the water column) might play an important role in biogeochemical cycling, as well as in horizontal gene transfer through natural transformation. Since isolation of extracellular DNA from sediments is a difficult and unsolved task, in this study we developed an efficient procedure to recover simultaneously DNA associated with microbial cells and extracellular DNA from the same sediment sample. This procedure is specifically suitable for studying extracellular DNA because it avoids any contamination with DNA released by cell lysis during handling and extraction. Applying this procedure to different sediment types, we obtained extracellular DNA concentrations that were about 10 to 70 times higher than the intracellular DNA concentrations. Using specific targeted prokaryotic primers, we obtained evidence that extracellular DNA recovered from different sediments did not contain amplifiable 16S rRNA genes. By contrast, using DNA extracted from microbial cells as the template, we always amplified 16S rRNA genes. Although 16S rRNA genes were not detected in extracellular DNA, analyses of the sizes of extracellular DNA indicated the presence of high-molecular-weight fragments that might have contained other gene sequences. This protocol allows investigation of extracellular DNA and its possible participation in natural transformation processes.

FIG. 1.

Gel electrophoresis of the 16S rRNA gene amplified with primers 530f and 1492r and with primers 27f and 1492r from samples utilized to test for DNA contamination due to cell lysis. Lane 1, DNA marker (100 bp; Promega); lanes 2 and 3, PCR products from extracellular DNA recovered from muffle furnace-treated sediment samples inoculated with V. mediterranei; lanes 4 and 5, PCR products from samples containing an internal standard of purified DNA from V. mediterranei; lanes 6 and 7, PCR products from intracellular DNA recovered from muffle furnace-treated sediment samples inoculated with V. mediterranei.

FIG. 2.

Gel electrophoresis analysis of the 16S rRNA gene amplified with primers 530f and 1492r and with primers 27f and 1492r from extracellular and intracellular DNA extracted from sediment samples collected in the southern Pacific Ocean, in the deep western Mediterranean Sea, and in the Adriatic Sea. Lanes 1 and 10, DNA marker (100 bp; Promega); lanes 2 and 3, intracellular DNA from southern Pacific Ocean sediments; lanes 4 and 5, extracellular DNA from southern Pacific Ocean sediments; lanes 6 and 7, intracellular DNA from western Mediterranean Sea sediments; lanes 8 and 9, extracellular DNA from western Mediterranean Sea sediments; lanes 11 and 12, intracellular DNA from Adriatic Sea sediments; lanes 13 and 14, extracellular DNA from Adriatic Sea sediments.

FIG. 3.

Dot blot analysis of extracellular and intracellular DNA with the eubacterial 341 oligonucleotide probe. Equal amounts of the DNA standard from E. coli or of sediment samples were blotted in each row (the amounts [in micrograms] are indicated on the left). The results are for intracellular and extracellular DNA fractions from sediments collected in the deep western Mediterranean Sea (WMS), in the Adriatic Sea (AS), and in the southern Pacific Ocean (SPO).

FIG. 4.

Gel electrophoresis of extracellular DNA extracted from different sediment samples. Lane 1, DNA marker (1 kb; Promega); lanes 2, 3, and 4, DNA from sediments collected in the southern Pacific Ocean, Adriatic Sea, and deep western Mediterranean Sea, respectively.