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Microbial and viral ecology of hot spring environments with emphasis on 454 pyrosequencing and microbial and viral interactions

Final Report Summary - MICVIRECOLHOTSPRINGS (Microbial and viral ecology of hot spring environments with emphasis on 454 pyrosequencing and microbial and viral interactions)

This project focuses on microbial and viral interaction within extreme environments in Yellowstone National Park. Indigenous lithotrophic organisms use inorganic substrate for biosynthesis, and such “rock eating” microorganisms comprise a large proportion of the biomass found on and inside our earth. Microorganisms isolated from hot springs are known to use hydrogen as an energy source. In this project hydrogen was added and growth of microbial cells monitored to study the succession of microbial populations throughout the experiment. Changes in biomass and cell numbers were monitored, and DNA was extracted and sequenced using next generation sequencing technology. Viruses are known to inhabit hot springs, and they were studied using transmission electron microscopy in combination with metagenomics analyses of potentially change of genes in their host communities.
The project aimed to study bacterial and viral interactions in batch cultures to gain knowledge of evolution over time in environments known to inhabit deeply branching microorganisms close to the root of the phylogenetic tree of life. Viruses are the only known predators in hot spring environments, and therefore we studied the effect of top down predation by viruses on the regulation of microbial and viral diversity.
Experiments were set up at hot springs in Yellowstone National Park. Obsidian Pool with its temperature ranging from 58-66°C and pH from 5.5-6.0 is known for its diverse microbial population, and has been the main sampling site throughout this project. Microcosm slurry experiments with spring water and sediments demonstrated active indigenous microbial populations. We have been able to produce repeatable results with slurry experiments amended with hydrogen and bicarbonate in batch experiments, and these were followed by larger setups in the laboratory at Colorado School of Mines. Microbial populations in sediments were shown to consume hydrogen and produce methane. We have applied methods based on 454 pyrotag amplicon sequencing of the phylogenetic marker gene 16S rRNA as well as a gene that targets the methane producing communities (methanogens), followed by bioinformatics. Analysis of 454 pyrotag amplicons and qPCR data describing microbial populations at Obsidian Pool allowed for a better understanding of the ecology of methanogens. We have seen how the genes originating from the methanogens increase in number together with other groups. Using this data we have modeled how the microbial groups interact. We have further isolated methanogenic strains from the slurry experiments to study this group in more detail. Using Illumina sequencing techniques the genomes revealed to include CRISPR sequences. These are genes known to be involved in microbial cell defense against viral infections. Hence, the isolation of methanogens strains and sequencing their genome allows us to look closer at the methanogen diversity in the hot spring environment, as well as lets us look at the potential impact that viruses have on the variation of the methanogen gene sequences over time.

Studying microbial populations in slurry experiments and isolation of interesting organisms in combination with modern sequencing techniques we can reach a better understanding of the microbial ecology of hydrogenotrophic organisms such as methanogens in Yellowstone national park. Increased knowledge on viral populations effect on microbial communities can be used in biotechnical applications, such as when microorganisms are utilized to produce methane, degrade pollutants or applied for food production such as yoghurt, beer and bread. The organisms in focus in our study, Methanothermobacter, can be found in thermal oil fields and sewage treatment plants. In these environments, viral infections have been shown to sometimes limit microbial processes and methane production. Slurry experiments turned out to be a good approach to apply in order to better understand the processes involved in microbial diversity and viral defense in mixed communities like the accessible hot springs in Yellowstone National Park.
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