Meta-Analysis of Quantification Methods Shows that Archaea and Bacteria Have Similar Abundances in the Subseafloor

Abstract

There is no universally accepted method to quantify bacteria and archaea in seawater and marine sediments, and different methods have produced conflicting results with the same samples. To identify best practices, we compiled data from 65 studies, plus our own measurements, in which bacteria and archaea were quantified with fluorescent in situ hybridization (FISH), catalyzed reporter deposition FISH (CARD-FISH), polyribonucleotide FISH, or quantitative PCR (qPCR). To estimate efficiency, we defined "yield" to be the sum of bacteria and archaea counted by these techniques divided by the total number of cells. In seawater, the yield was high (median, 71%) and was similar for FISH, CARD-FISH, and polyribonucleotide FISH. In sediments, only measurements by CARD-FISH in which archaeal cells were permeabilized with proteinase K showed high yields (median, 84%). Therefore, the majority of cells in both environments appear to be alive, since they contain intact ribosomes. In sediments, the sum of bacterial and archaeal 16S rRNA gene qPCR counts was not closely related to cell counts, even after accounting for variations in copy numbers per genome. However, qPCR measurements were precise relative to other qPCR measurements made on the same samples. qPCR is therefore a reliable relative quantification method. Inconsistent results for the relative abundance of bacteria versus archaea in deep subsurface sediments were resolved by the removal of CARD-FISH measurements in which lysozyme was used to permeabilize archaeal cells and qPCR measurements which used ARCH516 as an archaeal primer or TaqMan probe. Data from best-practice methods showed that archaea and bacteria decreased as the depth in seawater and marine sediments increased, although archaea decreased more slowly.

A new view of the tree of life

“…We do not attribute the smaller scope of the Archaea relative to Bacteria to sampling bias because metagenomics and single-cell genomics methods detect members of both domains equally well. Consistent with this view, Archaea are less prominent and less diverse in many ecosystems (for example, seawater 27 , hydrothermal vents 28 , the terrestrial subsurface 15 and human-associated microbiomes 29 ). The lower apparent phylogenetic diversity of Eukarya is fully expected, based on their comparatively recent evolution.…”

Influence of DNA Extraction Method, 16S rRNA Targeted Hypervariable Regions, and Sample Origin on Microbial Diversity Detected by 454 Pyrosequencing in Marine Chemosynthetic Ecosystems

“…For example, real-time PCR with different primer sets appeared to be very useful for obtaining more details about microbial group quantities (40). In addition, since we do not really know the numbers of 16S rRNA gene copies within bacterial and archaeal genomes, which could drastically increase microbial diversity results (104,105), a fluorescence in situ hybridization (FISH/catalyzed reporter deposition [CARD]-FISH) approach (90,106) could allow for confirmation of previous results.…”

The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology

“…Two enzymatic lysis buffers, TE-L lysis buffer (20 mM Tris/HCL; 2 mM EDTA; pH 8, supplemented by 15 mg/mL lysozyme, Sigma, 62970) and TE-A lysis buffer (20 mM Tris/HCL; 2 mM EDTA; pH 8, supplemented by 10000 units/ml achromopeptidase, Sigma, A3547-100KU) were prepared for enzymatic lysis. We used achromopeptidase in tandem with lysozyme for DNA extraction as this enzyme has been used to breakdown the cell wall of lysozyme resistant bacteria (Barsotti et al, 1988, Ezaki and Suzuki, 1982, Goto et al, 2007, Lloyd et al, 2013, Said et al, 2014, Ueno et al, 2011. Mechanical cell lysis was performed using 0.1 mm glass beads tubes (Sigma, Z763721) and 0.1 mm zirconium beads tubes (Pathtech, BMD1032-01).…”