The biogeography, ecophysiology, and functional potential of phototrophic Chloroflexi in alkaline hot springs: from marker genes to metagenomes
2022-07
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The biogeography, ecophysiology, and functional potential of phototrophic Chloroflexi in alkaline hot springs: from marker genes to metagenomes
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2022-07
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Alkaline hot springs in Yellowstone National Park (YNP) provide a framework to study the relationship between photoautotrophs and temperature. Previous work has focused on understanding how Cyanobacteria (oxygenic phototrophs) vary with temperature, sulfide, and pH – but many questions remain regarding the ecophysiology of anoxygenic photosynthesis due to the taxonomic and metabolic diversity of these taxa. Phototrophs within the Cyanobacteria and Chloroflexi groups are frequently observed in alkaline hot springs. Decades of research has determined that temperature constrains Cyanobacteria in alkaline hot springs, but factors that constrain the distribution of phototrophic Chloroflexi remain unresolved. In Chapter 1, I review the key findings from Chloroflexi isolate and in situ studies in the two hot springs that have been the focus of decades of work in YNP. I highlight the metabolic and ecological diversity of characterized phototrophic Chloroflexi as it relates to nitrogen fixation, carbon cycling, and sulfur cycling. Additionally, I introduce how foundational studies have informed next generation sequencing efforts in this dissertation and in other works.
In Chapter 2, I examined the distribution of genes involved in phototrophy, carbon fixation, and nitrogen fixation in eight alkaline (pH 7.3-9.4) hot spring sites near the upper temperature limit of photosynthesis (~71ºC) in YNP using metagenome sequencing. Based on genes encoding key reaction center proteins, geographic isolation plays a larger role than temperature in selecting for distinct phototrophic Chloroflexi while genes typically associated with autotrophy in anoxygenic phototrophs did not have distinct distributions with temperature. However, I recovered Calvin Cycle gene variants associated with Chloroflexi, an alternative carbon fixation pathway in anoxygenic photoautotrophs. Lastly, I recovered several abundant nifH (nitrogen fixation gene) sequences associated with Roseiflexus providing further evidence that genes involved in nitrogen fixation in Chloroflexi are more common than previously assumed. Together, these results add to the body of work focused on the distribution and functional potential of phototrophic bacteria in Yellowstone National Park hot springs and support the hypothesis that a combination of abiotic and biotic factors impact the distribution of phototrophic bacteria in hot springs.
In Chapter 3, I employed a combination of 16S rRNA gene sequencing and inorganic carbon photoassimilation microcosms, to test the hypothesis that temperature would constrain the activity and composition of phototrophic Cyanobacteria and Chloroflexi. I expected diversity and rates of photoassimilation to decrease with increasing temperature. I report 16S rRNA amplicon sequencing along with carbon isotope signatures and photoassimilation from 45-72ºC in two alkaline hot springs. I found that Roseiflexus, Chloroflexus (Chloroflexi) and Leptococcus (Cyanobacteria) operational taxonomic units (OTUs) have distinct distributions with temperature. This distribution suggests that, like phototrophic Cyanobacteria, temperature selects for specific phototrophic Chloroflexi taxa. The richness of phototrophic Cyanobacteria decreased with increasing temperature along with a decrease in oxygenic photosynthesis, whereas Chloroflexi richness and rates of anoxygenic photosynthesis did not decrease with increasing temperature, even as temperatures approaches the upper limit of photosynthesis (~72 - 73ºC). Our carbon isotopic data suggest an increasing prevalence of 3-hydroxypropionate bicycle (3-HPB) with decreasing temperature coincident with photoautotrophic Chloroflexi. Together these results indicate temperature plays a role in defining the niche space of phototrophic Chloroflexi (as has been observed for Cyanobacteria), but other factors such as morphology, geochemistry, or metabolic diversity of Chloroflexi, in addition to temperature, could determine the niche space of this highly versatile group.
Finally, in Chapter 4, I build on the work in Rabbit Creek from Chapter 3 by conducting a phylogenomic and pangenome analysis of 17 Chloroflexales metagenome assembled genomes (MAGs). I hypothesized that Chloroflexus and Roseiflexus would harbor unique core genomes and that temperature would select for certain accessory genes. I built a phylogenomic tree with 17 Rabbit Creek MAGs and 15 NCBI isolate genomes and found that the Rabbit Creek MAGs represent genera with characterized isolates as well as novel taxa. I examined the functional potential of Roseiflexus and Chloroflexus MAGs and found that Roseiflexus core genomes were depleted in both 3-HPB and sulfide oxidation functions while Chloroflexus core genomes contained sulfide:quinone oxidoreductases (SQRs) and several steps in the 3-HPB. Furthermore, I found both Roseiflexus and Chloroflexus core genomes contained genes for carbon assimilation and storage, suggesting this is an integral function for Chloroflexi in the Rabbit Creek ecosystem. Lastly, I recovered several MAGs that did not align with reference genomes but contained genes for type-II reaction centers and thiosulfate oxidation. Additionally, the unclassified MAGs were more like other Rabbit Creek MAGs. This work builds on the body of work in phototrophic Chloroflexi in alkaline hot springs and further constrains the role of Chloroflexi in carbon and sulfur cycling.
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University of Minnesota Ph.D. dissertation. July 2022. Major: Plant and Microbial Biology. Advisor: Trinity Hamilton. 1 computer file (PDF); xi, 115 pages.
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Bennett, Annastacia. (2022). The biogeography, ecophysiology, and functional potential of phototrophic Chloroflexi in alkaline hot springs: from marker genes to metagenomes. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/243079.
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