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The Investigation and Classification of the CRISPR/cas System in the Acidothermophilic Thaumarchaeote DS1 from Dragon Spring, Yellowstone National Park

By: Bridget Hansen

Microbiology

Faculty Advisor: Dr. José R. de la Torre

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated proteins (cas) have been associated closely with the Archaea domain and viral immunity, especially in thermophiles. Recent studies have been done to classify the types of CRISPR systems found in all microbial genomes and provide a guideline for what constitutes the classification. The archaeon, DS1, sampled from Dragon Mouth Springs, Yellowstone, is an unknown species with two CRISPR arrays and two putative gene cassettes that may function in tandem with the CRISPR but are not associated. The only other archaeon with a similar CRISPR system is Thermoplasma acidophilus, lacking in cas genes. Using IMG, GLIMMER, EasyGene, GeneMarkS, BLAST and the CRISPRdb, putative genes can be annotated assisting in a classification of DS1. The extracted spacer sequences with CRISPRfinder, will aid in the understanding of what types of viruses, if identified, could be infecting these archaeal cells. By using a sequence alignment, putative proteins can be identified and their function investigated for possible viral use. Phylogenetic analysis will compare the CRISPR and 16S rRNA of DS1 to the thermophilic archaeon Thermoplasma acidophilus. These studies will provide insight into a potentially new CRISPR system.

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Cysteine desulfurase, What is it good for? (Absolutely something)

By: Chelsea Allen

Microbiology

Faculty Advisor: Dr. José R. de la Torre

Thaumarchaeota archaeon DS1, the subject of this study, is an uncultivated archaea whose genome was sequenced from metagenomic data sourced from the low-pH, high-temperature Dragon Hot Springs in Yellowstone National Park. The uncultivated status of this organism means that any and all information is elucidated only from comparative genomic analysis. Initial gene annotations of DS1 revealed a sequence that appears to translate for a cysteine desulfurase protein. The presence of this protein indicates a system for the biosynthesis of FeS cofactors that is common in Eukarya and Bacteria, but is only rarely found in Archaea. Through genomic analysis, I will examine this putative gene and locate other genes in DS1 that are involved in FeS cluster biosynthetic pathways. The results of my research will not only illuminate an aspect of the biochemistry of DS1 but will have larger implications in the evolution and phylogenetic relationships of this Thaumarchaeota.

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Oxidation of Sulfur in Thaumarchaeota Archaeon DS1 for its Thermoacidic Environment

By: Jamie Huang

Microbiology

Faculty Advisor: Dr. José R. de la Torre

Thaumarchaeota archaeon DS1 (Dragon Thaumarchaeon) was discovered in a hot spring in Yellowstone National Park, WY, USA. DS1 was discovered as a single cell and has its DNA completely sequenced known to be similar to chemolithotrophs. Lithotrophs produce sugar through chemosynthesis, a biological conversation of carbon molecules into nutrients into organic matter and the oxidation of inorganic molecules, hydrogen sulfude (H2S), as a source of energy. DS1 uses the oxidation of hydrogen sulfide to sulfates for its production of energy using a thioredoxin-disulfide reductase protein. DS1 produces a by-product of sulfates for its hydrogen sulfide rich environment like other thermoacidiophiles also using the thioredoxin-disulfide reductase protein DS1 has present in its genome. The relationship will be shown using the relationships between different organisms on a taxonomical tree. The organism found in the same environmental conditions as DS1 should have the same genes to oxidize hydrogen sulfide. The organism related to DS1 is going to be useful in determining how sulfur oxidation mutated to be able to produce energy for DS1 in the thermophilic environment or if DS1 obtained thioredoxin-disulfide reductase protein from the organism in the environment.

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Characterization Of The Crispr/Cas System In Thaumarcheota Ds1 By A Comparative Genomic Study Against Related Thermophilic Archaea

By: Jesus Rocha

Cellular & Molecular Biology

Faculty Advisor: Dr. José R. de la Torre

CRISPR’s and their associate protein Cas have been shown to function synergistically in archaea as an adaptive immune mechanism against invasive genetic elements, such as viral DNA and plasmids. This system functions by integrating spacers resembling such invasive elements into CRISPR regions of the infected organism. The spacers can then be used by the CRISPR/Cas system to provide a sequence-specific immune response during subsequent infections. / CRISPR/Cas systems tend to be highly variable, but it has been determined that most CRISPR systems used by archaea are those that fall under the description types I and III. I will use genomic analysis software such as BLAST, TCOFEE,PSORT-B, Phobius, PFAM and IMG, to determine what type of CRISPR/Cas system is expressed in the newly sequenced Thaumarcheota DS 1. I will then compare it to other thermophilic archeon CRISPR/Cas systems and determine the level of conservation. The results from this research could allow us to understand potentially different CRISPR/Cas systems DS1 may possess. /

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DS1 and the Mevalonate Pathway

By: Jordan Lapeyri

Microbiology

Faculty Advisor: Dr. José R. de la Torre

Thaumarchaeota archaeon DS1, an uncultured organism sequenced from a high temperature, low pH environment found in the Dragon Springs of Yellowstone National Park, is currently classified as a Thaumarchaeota, despite lacking the signature characteristic of Thaumarchaeota—the ability to oxidize ammonia. As an archaeon, DS1 utilizes the mevalonate pathway for isoprenoid synthesis (Arian Smit, 2000). Conserved genes encoding enzymes for isoprenoid synthesis, essential for archaeal life, are useful for determining phylogentic relationships. I hypothesize that the key genes in DS1’s mevalonate pathway are more closely related to the mevalonate pathway in other hyperthermophilic Thaumarchaeota, which will further support the classification of DS1 as a Thaumarchaeota. Genomic analysis performed using IMG Blast and KEGG pathways showed homology of the mevalonte pathway between DS1 and closely related archaea. With further analysis, I plan to show that the mevalonate pathway in DS1 is more closely related to that found in Thaumarchaeota than that found in Korarchaeota or Crenarchaeota.

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Vitamin B12 Production in Thaumarchaeota Archeon DS1

By: Kathy Moy

Microbiology

Faculty Advisor: Dr. José R. de la Torre

B12 also known as colbalamin is an essential vitamin and cofactor produced by organisms living in marine or freshwater environments. They are known to occur in bacteria and archaea. B12 originally evolved from anaerobic organisms to help in the fermentation of small molecules. Now studies shown that B12 is synthesized in bacteria both anaerobically and aerobically but in archaea only aerobically. The synthesis of B12 requires the intermediates of heme and chlorophyll at uroporphyrinogen III. After this, different enzymes follow the aerobic or anaerobic pathway to make B12. The anaerobic pathway requires the early cobalt insertion while the aerobic pathway requires the late cobalt insertion. B12 synthesis is poorly understood. All Thaumarchaeota uses the aerobic pathway to synthesize B12. This may not be true for Thaumarchaeota Archeon DS1. Thaumarchaeota are among the most abundant archaea on Earth.They are also chemolithoautotrophs, and capable of oxidizing ammonia as a means of obtaining energy. Because of this, we hypothesis Thaumarchaeota Archeon DS1 may not use both pathways to synthesize this crucial vitamin. To test this we used the Integrated Microbial Genome System to detect matches in KEGG’s pathway to compare the genes in the Thaumarchaeota phylum against DS1 containing the anaerobic and aerobic pathway. We then compared the sequences using BLAST and other sequencing software. Our analysis demonstrates the Thaumarchaeota genomes uses the anaerobic pathway to synthesize cobalamin but the Thaumarchaeota Archeon DS1 does not use either pathway. It uses the last steps of the cobalamin pathway. These results showed Thaumarchaeota Archeon DS1 genes are varied and this influences of the roles and environmental factors marine organism distributed by Thaumarchaeota Archeon DS1. /

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Thaumarchaeota Archaeon DS1 MOTILITY

By: Luciana Cardenas

Cellular & Molecular Biology

Faculty Advisor: Dr. José R. de la Torre

Thaumarchaeota Archaeon DS1 is a virtually unknown thermophile found in the Dragon Spring at Yellowstone National Park. This understudied organism grows in a unique environment where the temperature ranges between 68 and 72 degrees Celsius and the spring maintains an acidic pH level of 3.1. Thaumarchaeota Archaeon DS1 has been classified under the phylum of Thaumarchaeota, which range among most of the known archaea. So far, scientists have been able to isolate the genome sequence of this organism through metagenome sequencing. I hypothesize that that DS1 has the genetic potential for motility. Using various programs, I will find the genes known to be related to the motility of a similar organism, such as another Archaeon, another thermophile or acidophile. I will compare those genes to the sequenced genome of Thaumarchaeota Archaeon and find the differences or similarities in its motility related genes. The significance of isolating the genes responsible for the motility of the Thaumarchaeota Archaeon DS1 will provide insight into possible evolutionary adaptations of this microorganism and others similar to it. By comparing the flagellum differences and similarities of this organism with other marine archaea that exist in different temperature environments or different pH levels, we should be able to obtain a better understanding of how these environmental factors might have affected evolutionary changes.

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Defense Islands in Thaumarchaeon DS1: Identifying Putative Antiviral Genes

By: Manuela Ovalles

Microbiology

Faculty Advisor: Dr. José R. de la Torre

Organisms have developed numerous antiviral defense systems in order to survive constant viral infections. Despite how common these systems are, only a few have been identified and only some of those have been extensively studied. In this project, I will use comparative genomic analyses to identify the genes responsible for the Restriction-Modification and Toxin-Antitoxin systems in Thaumarchaeota archaeon DS1 and determine their location in the genome. Using this information, I can determine if there is any clustering of the genes in these systems in what is known as “defense islands” and note any genes of unknown function that may form a currently unknown defense system.

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Metagenomic Analyses of Missing Genes Vital to DNA Replication Pathways in Thaumarchaeota Archaeon DS1

By: Michelle S. Chu

Microbiology

Faculty Advisor: Dr. José R. de la Torre

The first generation of genome sequencing has revolutionized our understanding of biology by providing information regarding the biology of humans and phylogenetic relationships between species. However, analyzing the genomes of newly discovered organisms can be tasking since information may be inaccessible or unavailable. The Thaumarchaeota archaeon DS1 genome, published in 2012, sparked curiosity in the scientific community since this hyperthermophilic organism was initially classified as a part of the newly created phylum, Thaumarchaeota, even though it lacks the characteristic trait of being able to oxidize ammonia. In all organisms, DNA replication, a self-complementary process that does not require extraneous third parties, is responsible for synthesizing DNA daughter strands from parental DNA in the processes of mitosis and meiosis. Without DNA replication, information would be lost to successive generations in addition to inhibition of cell growth. In the DNA replication pathways associated with Thaumarchaeota archaeon DS1, several key components were missing such as DNA polymerase, an enzyme responsible for assembling nucleotides onto new synthesized strands of DNA. I hypothesize that the missing components from DNA replication pathways in Thaumarchaeota archaeon DS1 are due to incomplete genome sequencing of the un-cultured organism rather than mutations suggesting evolutionary divergences. Genomic analyses through BLAST sequencing comparing the organism of interest to similar hypthermophilic Corarchaeum and Crenarchaeota revealed the existence of missing vital DNA replication components. Additionally, the data provided has identified significant missing proportions of genes. Overall, the aims of this study were to discover missing components to provide important implications for future sequencing projects. Furthermore, improvements in the genome will serve as an important resource for researchers of Thaumarchaeota archaeon DS1 in determining phylogenetic relationships among hyperthermophilic archaea.

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Methane Metabolism in Thaumarchaeota DS1

By: Michelle Wong

Microbiology

Faculty Advisor: Dr. José R. de la Torre

Methanogens, anaerobic organisms that produce methane, play a crucial role in anaerobic environments, as they degrade complex organic compounds. In the absence of oxygen, methanogens can carry out a method known as methanogenesis, in which they oxidize hydrogen or whatever matter is available to produce methane. Closely related to methanogens are anaerobic methanotrophs, which oxidize methane as a source of energy. The exact mechanism of methane oxidation is unknown, but it has been theorized that archaea use a reversed methanogenesis pathway. Methanogenesis has both positive and negative environmental impacts, as they effectively decrease pollutants in water sources, but they can also dramatically increase methane emissions, which contribute to greenhouse gas emissions. Therefore, knowing an organism’s methane metabolic properties is vital in elucidating its role in the environment. Many methanogens are found in extreme environments in which they flourish at high temperatures, such as hot springs. Thaumarchaeota DS1, hereon referred to as DS1, is a recently sequenced strain found in Dragon Spring in Yellowstone National Park. DS1 is a chemolithoautotroph, though not much else is known about its metabolic properties. Whether DS1 carries out methanogenesis or its reverse reaction is not yet known, but it does contain many of the genes associated with methanogenic archaea and methane metabolism. In this study, I will analyze the genome sequence of DS1 and the genes it contains related to methanogenesis, and determine its role in methane metabolism.

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Nitrogen Assimilation and Amino Acid Synthesis of DS1

By: Nicholas Kuykendall

Microbiology

Faculty Advisor: Dr. José R. de la Torre

Using IMG to find the existence of the gene pathways for nitrate reductase, nitrite reductase, ammonia transporter, glutamine synthase, glutamate dehydrogenase, will show how DS1 performs nitrogen assimilation and the use of nitrogen for the building blocks of the cell. A guess can be made that DS1 has genes for a nitrate reductase to bring nitrogen into the cell, and a glutamine synthetase and glutamate dehydrogenase. IMG will be used along with BLAST and KEGG to compare the sequences to genes and pathways in other organisms. By finding similarities using these programs will validate if one of these proteins required for one of these processes is what IMG has labeled it as. The importance to finding out the nitrogen assimilation and use of DS1 is that it is possible that DS1 has a different pathway for either of these processes that could further help us understand the environment and other organisms that it resides with.

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Determining the Origin and Role of Xenobiotic Biodegradation Genes in Thaumarchaeota archaeon DS1

By: Paul Ahn

Microbiology

Faculty Advisor: Dr. José R. de la Torre

Thaumarchaeota archaeon DS1 (henceforth DS1) is an uncultivated archeon recently sequenced from Dragon Spring in Yellowstone National Park. Dragon Spring is a hot spring which makes most of the microbes found in it thermoacidophiles, which means it lives in a high temperature and low pH environment. Despite not being able to cultivate DS1 in a laboratory setting, the genome of DS1 is already completely sequenced using metagenomic techniques. Interestingly, several genes highly associated with xenobiotic degradation were found within the genetic sequence of DS1. However, complete xenobiotic biodegradation pathways were not represented in DS1. This is most likely because different microbes within the microbiome conduct different steps in xenobiotic biodegradation pathways. / This project explores the possibility of horizontal or vertical genesis of the xenobiotic biodegradation pathway genes in Thaumarchaeota archaeon DS1 (henceforth DS1) and its place within xenobiotic biodegradation pathways. This will be done through a phylogenetic tree comparisons and analysis of the Dragon Spring metagenome. Various phylogenetic trees will be built using genes involved in xenobiotic biodegradation pathways. Then, they will be compared to the accepted standardized phylogenetic tree (built using the 16s rna). Comparing these trees will help us understand whether the genes were horizontally or vertically transferred. Likewise, a syntenic dot plot could be used for further elucidation. Then, a conceptual map of a xenobiotic biodegradation pathway will be built using the metagenomic data to understand DS1’s role in the metagenomic xenobiotic biodegradation pathway. /

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Predicting DS1's Tastes

By: Reid Griggs

Biology

Faculty Advisor: Dr. José R. de la Torre

Dragon Springs 1 is an uncultured thermoacidophillic thaumarcheon identified by metagenomic sampling from Yellowstone National Park. Part of a recently proposed clade, the thaumarcheota, DS1 belongs to a group of some of the most deeply branching archaea known. Archaea play a key role in global nutrient cycles, and all known thaumarcheotes oxidize ammonia. However, the DS1 genome does not contain amoA, the necessary enzyme for surviving by metabolizing ammonia. Based on correlation of available geochemical and genomic data, I plan to predict how DS1 survives in its geothermal hotspring environment. Genome annotation of deeply branching organsims such as DS1 represent key opportunities in understanding the evolution of metabolism on earth as well as archaeal roles in geochemical cycles.

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Metagenomic Analysis of Thaumarchaeota archaeon DS1 (Dragon Thaumarchaeon) and the potential function of the fixC gene and its role in the Nitrogen Cycle

By: Roberto Gonzalez

Microbiology

Faculty Advisor: Dr. José R. de la Torre

This project is investigating the fixC gene in the organism DS1 (Dragon thamarchaeon) which belongs to the phylum Thaumarchaeota. This organism was discovered in Dragon Spring which is a geothermal spring with a low pH and high temperature within Yellowstone National Park. The fixC gene encodes for an electron transferring dehydrogenase that has unknown function within this archaeon but the function is well studied in the model organism E.coli. In E.coli and ammonia oxidizing bacteria this gene is coexpressed with the nif gene cluster which encodes for the nitrogenase complex and is directly involved with nitrogen fixation. This gene is also related to the Carnitine biosynthesis pathway within E.coli. I will be presenting my analysis and findings of the potential carnitine biosynthesis function of the fixC gene, its relatedness to the nif gene cluster and its role in the fixation of nitrogen in the environment. The role of the fixC gene in nitrogen fixation is important because it provides additional evidence to support the theory that ammonia oxidizing archaea are the primary contributors to this process rather than the previous long held belief that ammonia oxidizing bacteria were in fact the major contributors.

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Investigation of anaerobic behavior in Thaumarchaeota DS1

By: Stephanie Sapien

Microbiology

Faculty Advisor: Dr. José R. de la Torre

To date many archaea are known to be anaerobic as they are found in extreme environments. Thaumarchaeota DS1 is no exception as its environment encases that of a hot spring, which contains many different materials coupled with high acidity. This experiment will observe anaerobic potential in DS1 by evaluation of energy production and comparisons to other anaerobic organisms. DS1 has been recently un-cultivated and sequenced from Yellowstone hot springs. DS1 Thaumarchaeaon metabolism and behaviors remain unknown. DS1 does carry some metabolic genes for energy productions that do point to the organisms potential to possibly be an anaerobic Achaea in its natural environment. DS1 method of metabolism will be evaluated for evidence of anaerobic function by the use of organic/inorganic materials primarily oxygen. To date DS1 has an absence of cytochrome c, the terminal acceptor in the Electron Transport Chain that utilizes oxygen for the final electron acceptor. DS1 also contains an enzyme of type III Ribulose-bisphosphate carboxylase or RuBisCO. RuBisCo is an enzyme suggested to be involved in carbon fixation, which is known to be present in some anaerobic archaea that contain this enzyme in their genome. A phylogenetic analysis will be done with DS1 and those archaea known to contain type III RuBisCO using BLAST. Common behaviors of anaerobic activity will be compared to these organisms suggesting similar behavior. Identification of known methods of metabolism in DS1 and comparative archaea will be done using IMG. Energy production and key molecules used for carbon fixation will also be investigated from DS1 and other anaerobic archaea. Similar methods of energy production using pathways indicative of anaerobic status will be identified in DS1 and comparative archaea. Classification of anaerobic potential will be based on the types of pathways DS1 can utilize to generate energy.

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The synthesis of cobalamin (vitamin B12) by Dragon Spring Thaumarchaeota (DS1) and its benefits to it's surrounding environment

By: Stephany Marie Phomkhai

Microbiology

Faculty Advisor: Dr. José R. de la Torre

Cobalamin synthesis is essential for humans, as well as various Bacteria and Achaea. Many enzymes are dependent upon cobalamin and use it for different pathways. There has been research that Thaumarchaeota in marine environment synthesize cobalamin, and could possibly be the main source of cobalamin for other organisms that live in that same environment. What this project seeks, is that whether Dragon Spring Thaumarchaeota (DS1) can also synthesize cobalamin, and if so, is it the main source of cobalamin to the hot spring that it is found in. In order to proceed with this project, various genome, pathway, and alignment programs will be used to analyze DS1, and it’s ability to synthesize cobalamin.

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FabG-like Enzyme Facilitates Biosynthesis of Lipids for Use in Membrane Synthesis and Quorum Sensing in Thaumarchaeota archaeon DS1

By: Steven Chong

Microbiology

Faculty Advisor: Dr. José R. de la Torre

Thaumarchaota archaeon DS1 is a novel archaeon found the in the hot springs of Yellowstone National Park. Its place of discovery infers that this archaeon is a thermoacidophile and its cell membrane must be structured to accommodate this harsh environment. Although DS1 has never been cultured in a laboratory, its genome has been sequenced. A gene of DS1 was found to code for a putative enzyme similar to FabG; an enzyme involved in the biosynthetic pathway of fatty acid chains. This putative FabG-like protein may be critical for DS1 in the synthesis of its membrane that allows it to live in environments with high temperature and low pH. BLAST will be utilized to compare its protein sequence to that of bacteria and archaea known to contain such a protein. The protein’s evolutionary phylogenetic tree will be constructed to reveal ancestral roots. KEGG will be utilized to find the possible location of the putative FabG protein in the biosynthesis pathway. It is known that FabG performs the NADPH-dependent reduction of beta-ketoacyl-ACP substrates to beta-hydroxyacyl-ACP products but its use by DS1 to produce an end product for quorum sensing and membrane synthesis is yet to be fully explored. Study of this enzyme can provide an insight into biofilm formations and quorum sensing in DS1. My hypothesis is that DS1 contains the gene to produce an enzyme that is a putative FabG-like protein.