High abundance of ammonia-oxidizing Archaea in coastal waters, determined using a modified DNA extraction method

Abstract

Molecular characterizations of environmental microbial populations based on recovery and analysis of DNA generally assume efficient or unbiased extraction of DNA from different sample matrices and microbial groups. Appropriate controls to verify this basic assumption are rarely included. Here three different DNA extractions, performed with two commercial kits (FastDNA and UltraClean) and a standard phenol-chloroform method, and two alternative filtration methods (Sterivex and 25-mm-diameter polycarbonate filters) were evaluated, using the addition of Nitrosopumilus maritimus cells to track the recovery of DNA from marine Archaea. After the comparison, a simplified phenol-chloroform extraction method was developed and shown to be significantly superior, in terms of both the recovery and the purity of DNA, to other protocols now generally applied to environmental studies. The simplified and optimized method was used to quantify ammonia-oxidizing Archaea at different depth intervals in a fjord (Hood Canal) by quantitative PCR. The numbers of Archaea increased with depth, often constituting as much as 20% of the total bacterial community.

FIG. 1.

Effects of filtration and DNA extraction method on DNA yield. Data are means ± SDs for 6 to 12 replicates.

FIG. 2.

Relationship between filtration volume and DNA recovery of Nitrosopumilus maritimus cells. Data are means ± SDs for four measurements each.

FIG. 3.

Vertical profile of ammonia-oxidizing Archaea determined by the modified DNA extraction method. (A) Temperature and salinity; (B) chlorophyll a; (C) dissolved oxygen; (D) ammonium and nitrite concentrations; (E) nitrate concentration; (F) total bacterial and Synechococcus cell numbers; (G) archaeal amoA and bacterial 16S rRNA gene copy numbers based on volume estimation (Nvol); (H) archaeal amoA and bacterial 16S rRNA gene copy numbers based on weight-volume estimation (Nwt vol); (I) copy number ratio between archaeal amoA and bacterial 16S rRNA genes. Quantitative PCR data are means ± SEs for four (archaeal amoA gene) and eight (bacterial 16S rRNA gene) replicates. Coccoid cells autofluorescing yellow under green-light excitation were counted as Synechococcus (38).