Community ecology of an anoxygenic phototrophic mat inhabiting a sulfidic hot spring in Yellowstone National Park
1Taylor Desiree Mitchem, 1Ryan Christopher Holloway, 2Dave M. Ward, and 1Eric Daniel Becraft1University of North Alabama, Department of Biology, Florence, AL. 2Montana State University, Department of Land Resources and Environmental Sciences, Bozeman, MT
Microbial mats in sulfidic Yellowstone hot springs constructed by filamentous anoxygenic phototrophic bacteria, termed green Chloroflexus mats (GCF mats), were first described by the late Dick Castenholz in 1973. They occur in sulfidic hot springs of the Mammoth Terraces group at temperatures between 50 and 65°C and are also found in similar springs in other locations within the United States, Iceland and New Zealand. The laminated nature of GCF mats suggests that, like laminated oxygenic cyanobacterial mats in non-sulfidic hot springs, these anoxygenic mats can be considered modern analogs of stromatolite fossils. Thus, their existence challenges the notion that stromatolites necessarily reflect the presence of microbial mat communities driven by oxygenic phototrophic, a metabolism that oxygenated the Earth. Due to their low numbers on modern Earth, GCF mats have not been metagenomically analyzed using modern sequencing methods. Here were examine the first metagenomes of New Pit GCF mat in Yellowstone National Park. We assembled and binned metagenomic assembled genomes (MAGs), and the most abundant lineages were targeted for metabolic analysis. The community consists primarily of Chloroflexota, as expected, but also contains numerous Thermodesulfobacteriota lineages known for their sulfate-reducing metabolism. In addition, candidate phyla KSB1 was identified in abundance. Annotated metabolisms consisted of lesser-known carbon fixation pathways, hydrogen oxidation, sulfur oxidation and reduction, nitrogen reduction and fixation, and diverse heterotrophic pathways. This is the first known metagenomic and metabolic analysis of an anoxygenic GCF mat, which are likely modern analogs of ancient microbial mats on early Earth.
Sample collection
Samples were collected from “New Pit” Spring on 20 September 2012 and 2014 (Figure 1). The water temperature was 57.8°C, and sulfide concentration was 28.17 to 43.27 µM. The spring and mat lie at the base of a deep depression in constant shade, such that irradiance near the mat was 182 to 261 µM photons compared to 2185 to 2284 µM in full sun. Samples for molecular analysis were frozen on liquid N2 and kept frozen until extraction of DNA. Samples for microscopy were embedded in 1% agar (in 0.2 µm filtered spring water) containing 2.5% glutaraldehyde or 4% formaldehyde and kept at ambient temperature until analysis.
For scalar irradiance microsensor measurements, a cohesive piece of mat was placed in a 50 ml Falcon tube on solidifying agar made from spring water and then covered with “New Pit” Spring water. Upon arrival in the laboratory, the mat and the upper ~5mm of the agar plug were transferred to a slightly larger sample container. Loose parts at the mat periphery were carefully glued to the agar with small droplets of agar applied from the side avoiding any agar covering the mat surface. The mounted samples were then covered with “New Pit” spring water. These operations were done over ~15 min in dim light and light measurements commenced within 2 hours after arrival.
Molecular analyses
DNA was extracted from the mat core using the MP Bio soil DNA extraction kit (CAT 47014) and Illumina metagenomic sequencing was conducted at the Joint Genome Institute (JGI). Metagenomes were assembled with META SPAdes (1) and binned into Metagenome Assembled Genomes (MAGs) with Metabat2 (2) on the Kbase platform (3). GTDB (4) and CheckM (5) were used to predict taxonomic classification and estimated genome completeness and contamination. Average nucleotide identity (ANI) was calculated using FastANI (6) on Kbase and MAGs with ≥95% ANI were combined for metabolic analysis. Relative abundance was determined by metagenomic read-mapping to MAGs using Bowtie2 (7) on Kbase. MAGs >1% of the population and >70% completeness and <5% contamination were used for metabolic analyses. Roseiflexus was included because it was highly abundant, even though quality genomes were lacking due to population heterogeneity. Proteins were identified on RAST (8) and metabolisms were mapped and analyzed using Blast Koala (KEGG pathway) (9).
Figure 1. “New Pit” Spring, Mammoth Terraces, Yellowstone National Park and the green microbial mat within. (A) Landscape view of depression in which the spring occurs (B and C) closeup of microbial mat and (D) mat slice with mm scale bar on right.
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