High Abundance of Ammonia-OxidizingArchaeain Coastal Waters, Determined Using a Modified DNA Extraction Method

Abstract

. 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.Efficient DNA extraction from environmental samples is fundamental to many culture-independent characterizations (10). Thus, there was an early and concerted effort to establish appropriate methods of DNA extraction from different types of environmental samples (14,19,25,30,34,43,47). DNA extraction efficiency is particularly important for quantitative PCR (qPCR), because poor DNA extraction efficiency results in the underestimation of gene copy numbers in the samples examined (6, 42).Most methodological developments addressed DNA extraction from soil and sediment samples, with fewer comparative studies of the efficiency of collection and extraction from water samples (4, 13, 40). In part, a methodological focus on soils reflected the simplicity of filtration to collect aquatic populations and the generally good recovery of DNA from the Gramnegative bacteria making up a significant fraction of aquatic communities. However, small Archaea are now known to constitute a substantial fraction of the prokaryotic populations in marine and terrestrial systems (2,7,9,20,26,31,33,45). Since the archaeal cell wall and membrane structures are distinct from those of bacteria, there is no assurance that commonly used extraction methods are adequate. With increasing reliance on commercially available bead-beating-type DNA extraction kits, these methods are now often used for different water samples (1, 5-7, 14, 19, 36). Although most protocols incorporate mechanical disruption to ensure more-uniform extraction than is possible by using methods that rely entirely on enzymatic digestion and/or chemical disruption (4,13,40), the suitability of these protocols for the concerted analysis of archaeal and bacterial populations has not been fully evaluated.In the studies reported here, the recently isolated marine archaeon Nitrosopumilus maritimus strain SCM1 (22) was therefore used as a reference standard for evaluation of the commonly employed DNA extraction methods by using qPCR. This archaeon was then used as a reference for the development of a simple, rapid, and efficient method of extracting DNA from both ar...

Marine ammonia-oxidizing archaeal isolates display obligate mixotrophy and wide ecotypic variation

“…These distinctive differences are directly relevant to their possible contribution to nitrification in different marine environments-including lower salinity coastal regions, the photic zone of the upper water column, and the increasing acidification of ocean waters associated with climate change. (8,38,39). Predominance of AOA was consistent with generally submicromolar concentrations of ammonia at these stations, sufficient to support AOA such as SCM1 but not known AOB (24).…”

Two distinct pools of B12analogs reveal community interdependencies in the ocean

“…We quantified Thaumarchaeota via qPCR and performed direct cell counts to quantify total prokaryotes as previously described (43,44,54). DNA for 16S rRNA sequencing was extracted, amplified, and sequenced as described in SI Materials and Methods.…”

“…We combined bacterial 16S rRNA gene sequence-derived phylogenetic data with current knowledge of corrin biosynthesis genes (including our analysis) across different phylogenetic groups to infer the cobalamin biosynthetic capacity of organisms in the microbial communities at our study site as likely, unknown, or unlikely (Table S3). We quantified Thaumarchaeota by quantitative PCR (qPCR) (43,44) and calculated their contribution to the microbial population by comparing this value to direct counts of prokaryotic cells determined for each sample. Our analysis at five targeted locations in the North Pacific suggested that Thaumarchaeota represent 30-80% of prokaryotes having likely or unknown genetic capacity to synthesize cobalamin in the lower euphotic zone and deeper ( Fig.…”

Triclocarban Influences Antibiotic Resistance and Alters Anaerobic Digester Microbial Community Structure

“…(21) This extraction method may have an inherent bias toward the extraction of Bacterial DNA over Archaeal DNA. (22) …”