Assessment of the diversity, abundance, and ecological distribution of members of candidate division SR1 reveals a high level of phylogenetic diversity but limited morphotypic diversity

Abstract

We used a combination of 16S rRNA gene clone library surveys, quantitative PCR (qPCR) analysis, and fluorescent in situ hybridization to investigate the diversity, abundance, and distribution of members of candidate division SR1 in multiple habitats. Using SR1-specific 16S rRNA gene primers, we identified multiple novel SR1 lineages in four different anaerobic environments: sediments from Zodletone Spring, a sulfide- and sulfur-rich spring in southwestern Oklahoma; inner layers of microbial mats obtained from Sperm Pool, a high-temperature, low-pH pool (55 degrees C, pH 2.5) in Yellowstone National Park; fresh bovine ruminal contents; and anaerobic freshwater pond sediments (Duck Pond) in Norman, Oklahoma. qPCR analysis indicated that SR1 members constitute a small fraction (<0.01%) of the microbial communities in Duck Pond and ruminal samples but constitute a significant fraction (11.6 and 48.7%) of the total number of bacterial 16S rRNA genes in Zodletone Spring and the inner layers of Sperm Pool microbial mat samples, respectively. By using SR1-specific fluorescent probes, filamentous cells were identified as the sole SR1 morphotype in all environments examined, with the exception of Sperm Pool, where a second bacillus morphotype was also identified. Using a full-cycle 16S rRNA approach, we show that each of these two morphotypes corresponds to a specific phylogenetic lineage identified in the Sperm Pool clone library. This work greatly expands the intralineage phylogenetic diversity within candidate division SR1 and provides valuable quantification and visualization tools that could be used for investigating the ecological roles, dynamics, and genomics of this as-yet-uncultured bacterial phylum.

FIG. 1.

Phylogenetic tree based on 16S rRNA gene sequences of members of candidate division SR1 encountered in this study. Bootstrap values (expressed as percentages) are based on 1,000 replicates and are shown for branches with bootstrap values of more than 50%. Sequences generated in this study are in boldface type, with the number of clones in each OTU reported in parentheses. GenBank accession numbers are shown in parentheses for other clones. The tree was constructed with the neighbor-joining algorithm with Jukes-Cantor corrections as described in Materials and Methods.

FIG. 2.

Whole-cell hybridization of paraformaldehyde-fixed cells with Alexa Fluor 488-labeled candidate division SR1-427 probe. Panels A and B, C and D, and E and F depict DAPI-stained cells (A, C, and E) versus FISH-labeled cells (B, D, and F) of anaerobic sulfur spring (Zodletone Spring) source sediment sample (A and B), anaerobic freshwater sediment sample (Duck Pond) (C and D), and Yellowstone National Park Sperm Pool microbial mat sample (E and F). Note the scarcity of SR1 cells in Zodletone Spring (B) and Duck Pond (D) ecosystems, as opposed to the abundance of SR1 cells in Sperm Pool ecosystem (F). The solid white arrows point to the filamentous SR1 morphotypes in all three environments, while the white broken arrows point to the bacillus morphotype in Sperm Pool. The bars (10 μm) in panels A and B applies to all panels of the figure.

FIG. 3.

Whole-cell hybridization of Yellowstone microbial mat paraformaldehyde-fixed cells with Alexa Fluor 488-labeled probe SR1-232 (specific for SR1 BD2-14 subgroup I) (A) and Alexa Fluor 488-labeled probe SR1-575 (specific for SR1 BD2-14 subgroup V) (C). Panels B and D are the phase-contrast images for the same microscopic fields shown in panels A and C, respectively. Solid white arrows point to bacillus-like cells labeled with probe SR1-232 but not with probe SR1-575, while white broken arrows point to filamentous cells labeled with probe SR1-575 but not with probe SR1-232. Bars, 10 μm.