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Title: The dark side of the mushroom spring microbial mat: Life in the shadow of chlorophototrophs. I. Microbial diversity based on 16S rRNA gene amplicons and metagenomics

Author
item THIEL, VERA - Pennsylvania State University
item WOOD, JASON - Montana State University
item OLSEN, MILLIE - Montana State University
item TANK, MARCUS - Pennsylvania State University
item Klatt, Christian
item WARD, DAVID - Montana State University
item BRYANT, DONALD - Pennsylvania State University

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/27/2016
Publication Date: 6/17/2016
Citation: Thiel, V., Wood, J.M., Olsen, M.T., Tank, M., Klatt, C.G., Ward, D.M., Bryant, D.A. 2016. The dark side of the mushroom spring microbial mat: Life in the shadow of chlorophototrophs. I. Microbial diversity based on 16S rRNA gene amplicons and metagenomics. Frontiers in Microbiology. 7:919. doi:10.3389/fmicb.2016.00919.

Interpretive Summary: Microbial communities that live in the thermal Octopus and Mushroom Springs in Yellowstone National Park are well-studied models for understanding how contemporary microorganisms interact and utilize organic and inorganic energy sources in a densely populated system. Additionally, they serve as links to the past as analogs for the Archaean microbial communities that produced stromatolites (some of the oldest fossils known, produced between ~3.7 to 1.5 billion years ago), giving insight to early Earth processes. Whereas past work in these systems has concentrated on the roles of phototrophic microorganisms that reside near the surfaces of these microbial mats, such as cyanobacteria related to Synechoccus spp. and anoxygenic phototrophs related to Roseiflexus spp., this study examines the composition and the metabolic potential of organisms that reside in deeper layers of these mats which receive lower levels of light energy. Both DNA and RNA were extracted from samples taken from the region 3-5 mm below the surface of these mats, and were subsequently sequenced in two distinct ways. Firstly, a ‘deep’ sequencing profile was created from the highly conserved 16S rRNA gene, allowing for over 30,000 unique sequences to be assigned to species-like units. This first analysis explored the structure and the composition of these communities. Secondly, a metagenomic analysis was done that consisted of randomly sequencing portions of the genomes of these community members, which then allowed for the detection of various functional genes present in these communities. These metagenomic sequences enabled interpretations as to how these microorganisms acquire energy by determining what they produce and consume. Despite the low levels of light that reach these communities compared to those at the surface, phototrophic organisms of the genus Roseiflexus were still dominant and exhibited diversity in their 16S rRNA sequences, indicating the existence of multiple populations that may be ecologically distinct. Other dominant groups included the hydrogen (H2)-producing fermenting organisms related to Thermocrinus spp., and other disparate microbial phyla at lower levels of abundance that utilize a wide range of metabolisms for their energetic needs. IMPACT STATEMENT: This study contributes to the understanding of the composition and function of an overlooked group of organisms in a model microbial ecosystem. These metagenomic analyses allow us to attribute ecosystem services and biogeochemical processes to particular microbial populations, thus allowing us to predict how these functions would change with the prospect of fluctuations in community composition.

Technical Abstract: Microbial-mat communities in the effluent channels of Octopus and Mushroom Springs within the Lower Geyser Basin at Yellowstone National Park have been studied for nearly 50 years. The emphasis has mostly focused on the chlorophototrophic bacterial organisms of the phyla Cyanobacteria and Chloroflexi. In contrast, the diversity and metabolic functions of the heterotrophic community in the microoxic/anoxic region of the mat are not well understood. In this study we analyzed the orange-colored undermat of the microbial community of Mushroom Spring using metagenomic and rRNA-amplicon (iTag) analyses. Our analyses disclosed a highly diverse community exhibiting a high degree of unevenness, strongly dominated by a single taxon, the filamentous anoxygenic phototroph, Roseiflexus spp. The second most abundant organisms belonged to the Thermotogae, which have been hypothesized to be a major source of H2 from fermentation that could enable photomixotrophic metabolism by Chloroflexus and Roseiflexus spp. Other abundant organisms include two members of the Armatimonadetes (OP10); Thermocrinis sp.; and phototrophic and heterotrophic members of the Chloroflexi. Further, an Atribacteria (OP9/JS1) member; a sulfate-reducing Thermodesulfovibrio sp.; a Planctomycetes member; a member of the EM3 group tentatively affiliated with the Thermotogae, as well as a putative member of the Arminicenantes (OP8) represented =1% of the reads. Archaea were not abundant in the iTag analysis, and no metagenomic bin representing an archaeon was identified. A high microdiversity of 16S rRNA gene sequences was identified for the dominant taxon, Roseiflexus spp. Previous studies demonstrated that highly similar Synechococcus variants in the upper layer of the mats represent ecological species populations with specific ecological adaptations. This study suggests that similar putative ecotypes specifically adapted to different niches occur within the undermat community, particularly for Roseiflexus spp.