In order to shed light onto life in halophilic environments, the completely sequenced halophilic genomes of Halobacterium salinarum str. R1, Natronomonas pharaonis, and Haloquadratum walsbyi, were analysed and compared amongst each other and with further haloarchaea. Though gene identification, function assignment, and metabolic pathway reconstruction might be performed straightforward by automatic means, the quality of data is usually not sufficient to establish cellular models thereafter. To avoid accumulation of prediction errors such as gene overprediction and cross-species transfer of misassigned functions (Table 1.3), the focus of this work was set on improving results of consecutive prediction steps that lead from complete genome sequences to biological models of the studied haloarchaea. This is reached by implementing computational procedures that post- process prediction results (e.g. for start codon selection, Chapter 1), by combining results from available tools (e.g. for enzyme assignment, Chapter 5), and by developing new prediction tools (e.g. for secretome analysis, Chapter 3). Apart from applying bioinformatics strategies, misprediction rates can also be reduced by integration of experimental and literature data (e.g. for metabolic pathway reconstruction, Chapter 5). Generated data from genomes and pathway analysis was stored and made available for other scientists, so that it might support the design of experiments and the generation of metabolic models for halophilic archaea in future.
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Studies have shown that hides contain extensive populations of Gram-positive and Gram-negative bacteria which may be either resident or transient bacteria. As soon as the animal is slaughtered, these bacteria can grow on the raw hides and degrade the hide quality (6–8). A mixture of salt and boric acid is commonly used to prevent the growth of these microorganisms and preserve the hides. If hides are preserved with only crude solar salt, they can be contaminated by different species of extremely halophilic archaea (9–12). These microorganisms are easily detected on hides as they are revealed by red to orange pigments. Extremely halophilic archaea produce red, orange and pink coloured colonies because of the presence of C 50 carotenoids (9–12).
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Bacteria have evolved completely different strategies for lysine degradation: either via 5-amino pentanamide (Yamanishi et al. , 2007) or via cadaverine (Fothergill & Guest, 1977) (both pathways are combined in number (2) in Figure 3.21). Both sub- strates, which are degradation products of lysine, are converted to 5-amino-pentanoate and then by 5-aminovalerate transaminase (EC 220.127.116.11) and glutarate-semialdehyde dehydrogenase (EC 18.104.22.168) to glutarate, which is converted by glutarate-CoA ligase (EC 22.214.171.124) to glutaryl-CoA - the end point of three pathways. Homology searches of the amino acid sequence of the lysine decarboxylase (EC 126.96.36.199), which con- verts lysine into cadaverine, found homologies to some archaeal proteins. Among them were some "hypothetical conserved" annotated proteins from Hrd. utahensis , Har. marismortui , Nmn. pharaonis , Nmn. moolapensis , Hqr. walsbyi and others, but no proteins from Hbt. salinarum (Table S1). All sequences included the highly con- served motif "PGGXGTXXE" (alignment not shown), which is always annotated in proteins of the lysine decarboxylase super-family, an indication that lysine is degraded in halophilic archaea via cadaverine and 5-amino-pentanoate. An unusual catabolic pathway for lysine has been detected in anaerobic bacteria such as Clostridium sp. , that are able to ferment lysine to acetate, butyrate and ammonia (Stadtman, 1973; Barker et al. , 1982) and recently all genes in this pathway could be identified in Clostridium sp. (Kreimeyer et al. , 2007).
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P23-77 virus elements are present in plasmids and viruses of halophilic archaea. P23-77 has a predicted packaging ATPase and two major capsid proteins (see above). Interestingly, we dis- covered similar gene products in Haloarcula hispanica virus SH1 (GenBank no. AY950802), in the genome of Haloarcula maris- mortui ATCC 43049 (AY596297), and in Halobacterium salina- rium plasmid pHH205 (AY048850 ). Despite its name, Halobacterium is an archaeal organism. The putative integrated plasmid or virus in Haloarcula marismortui (ATCC 43049) is sit- uated within ca. 540 to 560 kb of the 3.13-Mb genome. This element is designated here as IHP (for integrated Haloarcula provirus). Most other putative proteins encoded by IHP are re- lated to halophilic archaea (data not shown). However, the puta- tive IHP protein rrnAC0597 (AAV45608) is 35% identical to Halorubrum phage HF2 putative protein CAOIfh, and one FIG. 4. Comparison of putative ATPase sequences from membrane-containing dsDNA viruses, plasmids, or genome integrated genetic elements of bacterial, archaeal, and eukaryotic origin. Walker A and B regions, as well as the phage PRD1 packaging ATPase P9 region that is conserved in all known PRD1-like viruses. Putative or experimentally demonstrated ATPase sequences from bacterial (P23-77, IN93, PRD1, AP50, Bam35, Gil16c, and PM2), archaeal (SH1, pHH205, IHP, and STIV), and eukaryotic viruses (mimivirus), plasmids, or genome integrated sequences are aligned (the acronyms IHP and STIV are defined in the text). Sequences were chosen on the basis of previously proposed evolutionary relationships of the viruses, structural and genetic comparisons, or sequence similarity to P23-77, as detected by BLAST. Amino acid residues identical in all sequences are depicted in red, conservative sequences are depicted in blue, and blocks of similar amino acids are depicted with a green background. The GenBank or RefSeq numbers of putative ATPase sequences are indicated in parentheses as follows: IN93 (BAC55291), SH1 (AY950802), pHH205 (YP_01687808), IHP (AAV45617), PRD1 (P27381), AP50 (ACB54903), Bam35 (NP_943760), STIV (AAS89100), PM2 (AF155037), and mimivirus (AAV50705).
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Our understanding of the third domain of life, Archaea, has greatly increased since its establishment some 20 years ago. The in- creasing information on archaea has also brought their viruses into the limelight. Today, about 100 archaeal viruses are known, which is a low number compared to the numbers of characterized bacterial or eukaryotic viruses. Here, we have performed a comparative biological and structural study of seven pleomorphic viruses infecting extremely halophilic archaea. The pleomor- phic nature of this novel virion type was established by sedimentation analysis and cryo-electron microscopy. These nonlytic viruses form virions characterized by a lipid vesicle enclosing the genome, without any nucleoproteins. The viral lipids are unse- lectively acquired from host cell membranes. The virions contain two to three major structural proteins, which either are em- bedded in the membrane or form spikes distributed randomly on the external membrane surface. Thus, the most important step during virion assembly is most likely the interaction of the membrane proteins with the genome. The interaction can be driven by single-stranded or double-stranded DNA, resulting in the virions having similar architectures but different genome types. Based on our comparative study, these viruses probably form a novel group, which we define as pleolipoviruses.
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Pfeiffer et al. 2008b; Teufel et al. 2008]. These rearrangements may have been caused by evolution in the laboratory [Pfeiffer et al. 2008b]. Examples for the genetic flexibility of halophilic archaea attributed to diverged evolution have been discussed previously [Konstantinidis et al. 2007]. In contrast to Hbt. NRC-1, Hbt. salinarum R1 codes for eight tfbs (tfbA-H), three located on the plasmid pHS2 and five on the chromosome . Two of the tbps in the strain R1 (tbpB and tbpF) carry an ISH2 insertion and are not functional. From the four functional TBPs (tbpACDE), three are encoded by plasmids (pHS1 or pHS4) and only one (tbpE) by the chromosome (www.halolex.mpg.de) [Pfeiffer et al. 2008a; Pfeiffer et al. 2008b]. Since formation of the preinitiation complex requires binding of a TBP with a TFB to recruit RNAP to the promoter, it seems possible that the four functional TBPs and eight TFBs interact in up to 32 different combinations, driving transcription from a diverse set of promoters. This number of possible combinations constitutes the second highest after that of Hbt. NRC-1, when comparing with to date fully archaeal sequenced genomes [Facciotti et al. 2007]. The quantity of different combinations and the possibility of interactions with different regulators may explain the diversity of halophilic promoters [Baliga and DasSarma 1999; Soppa 1999]. In archaea, upregulation of a tfb gene in response to heat shock has been noted on the transcriptional level in both Haloferax volcanii (Hfx. volcanii) [Thompson et al. 1999] and Pyrococcus furiosus [Shockley et al. 2003]. In Hfx. volcanii, Western analysis also proved TFB-protein level to be elevated, but to a much lesser extent (≈ twofold increase). In addition, exposure to heat shock induced transcription of the transcription regulator phr in Pyrococcus furiosus, with a concomitant weak stimulation of the Phr protein, also detected by Western blotting [Vierke et al. 2003]. Similarly, mRNA levels of a homologous transcriptional regulators (AF1298) in Archaeoglobus fulgidus [Rohlin et al. 2005] have also been found significant increased in response to heat shock.
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Prokaryotics taxonomy is consider as a dynamic science. A handful of soil may contain many prokaryotic species. Due to molecular systematic, which led to the splitting of prokaryotes into bacteria and archaea. As molecular systematists continue to work on the phylogeny of prokaryotes. The use of polymerase chain reaction (PCR) has allowed for more rapid sequencing of prokaryote genomes. Archaea are highly diverse with respect to morphology, physiology, reproduction and ecology. As they are best known for growth in anaerobic, hypersaline, pH extremes, and high temperature habitats. Also found in marine arctic temperature and tropical waters. The research work in this is divided in two stages 1) Isolation and characterization of halophilic archaea 2) Phylogenetic identification of the haloarchaeal isolates. Section I is the introduction part Section II describes materials and methods for isolation, morphological and cultural characterization, cultivation and phylogenetic analysis of isolates. In Section III the experimental results showing cultural characteristics, growth requirements, phylogenetic trees and discussion with conclusion is included.
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extend further knowledge of thiolase, we describe with purification of β-ketothiolase from extremely halophilic Archaea which can be useful for its characterization and also in increasing the bioplastics (Poly Hydroxybutyrate) production (Senior and Dawes, 1973; Kyriakidis et al., 2005).
In archaea, the mechanism(s) by which archaella and chemosensory arrays are positioned at speciﬁc locations of the cell has not yet been studied in detail. A conical frustum that was observed in cells of the rod-shaped euryarchaeon T. kodakaraensis has been suggested to function as a polar organizing center (49). In bacteria, the mecha- nisms by which the chemosensory arrays and ﬂagella are positioned in the cell are being mapped in increasingly greater detail. The distribution of the chemosensory arrays in E. coli has been studied in detail, and various explanatory theories, such as stochastic self-assembly (41, 64), membrane curvature sorting (65, 66), and polar preferences of the clusters due to reduced clustering efﬁciency in the lateral region (35), have been devised. In contrast to E. coli, distinct proteins that are responsible for the placement of the chemosensory arrays at the cell pole have been identiﬁed in several bacterial species. In Caulobacter crescentus, the TipN and TipF proteins direct the assembly of the chemosensory arrays to the new pole at a predivisional stage (42). In other species, such as Rhodobacter sphaeroides or Vibrio sp., ParA/MinD homologs mediate the interaction with the polar organizing proteins, such as HubP, to position the chemosensory arrays (36–40). In addition to the chemosensory arrays, ParA/MinD homologs are responsible for the correct placement of many macromolecular assem- blies in bacteria. They organize cell polarity either by using an oscillation mechanism or by interacting with the polar organizing proteins (36). MinD/ParA homologs are also important to mark the cellular position of the ﬂagella and to control their numbers (36, 58, 67). Several archaea, including H. volcanii, encode one core multiple-MinD/ParA homolog. The organized positioning of the motility machinery observed in H. volcanii reveals the exciting possibility that archaea also use active mechanisms to organize the cellular placement of the macromolecular assemblies.
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Of the 174 putative protein-encoding genes that we annotate in HVTV-1, only 43 (25%) make a significant match to sequences in the GenBank database in a BLASTP search (see Table S1 in the supplemental material). Beyond the putative head and tail genes described above, many of the genes for which a possible function can be inferred appear to be involved in nucleotide metabolism or DNA replication. These are predominantly but not exclusively found in the leftward-transcribed cluster of genes bounded by genes 35 and 104, while the head and tail genes that we have iden- tified are in the rightward-transcribed cluster bounded by genes 108 and 140. Of the genes predicted to encode protein functions other than virion structure and assembly functions, we note that there are three, genes 37, 87, and 96, predicted to encode subunits of replication factor C (RFC). RFC is a two-subunit protein that serves as the DNA replication clamp loader in archaea (67). The products of genes 87 and 96 make strong matches to the large and small subunits, respectively, of the cellular RFC, and it seems likely that they constitute a virus-encoded clamp loader. The product of gene 37 makes a strong match (BLASTP E value of 3 ⫻ 10 ⫺20 ) to the cellular RFC small subunit, but this match extends over only ⬃ 20% of its length, suggesting that it may have a different func- tion. Another intriguing match is that of gp115 to Zeta-toxin, which is a component of a toxin/antitoxin system found in bacte- ria and archaea (68). There is no evidence for its cognate antitoxin from sequence searches of the HVTV-1 genome. The role of this Zeta-toxin homologue in the viral life cycle is unclear. There is one tRNA predicted in the HVTV-1 genome, an apparent tRNA Gln
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duplication, have been identified to be the most functionally divergent of all clades (Figure 1A), namely the halophilic archaea have the greatest number of amino acid sites (32 sites for TatCo, TatCt has 24 sites) under FD compared with other lineages. This is interesting as haloarchaea had to adapt to an extreme environment and FD of TatC after duplication may have enabled such adaptations through the acquisition of novel functions or the modification of its original function. In contrast to TatC, thermophilic archaea Sulfolobus presented most of its FD signal in TatA (Figure 1B). Finally, Corynebacteria accumulated most of the functionally divergent changes in TatB (Figure 1C). Importantly, 8 out of the 11 clades with the strongest signal of FD included pathogenic bacterial strains (Figure 1), which suggests that Tat translocation pathway may play a role in bacterial pathogenesis. In summary, clades showing strong FD in Tat translocation system are unique as they include microorganisms living in extreme environments always challenged by ionic concentrations, temperature or host immune response (We discuss these results with detail in the following sections).
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Halophilic archaea are characterized by the presence of multiple copies of the chromosome (Figure 1). To some extent this feature has hindered a detailed dissection of their cell cycle. Halobacterium salinarum contains approximately 30 chromosome copies during exponential phase which are reduced to around 10 in stationary phase . Interestingly, this archaeon does not have a temporally demarcated S stage, as DNA replication occurs throughout the cell cycle . However, lack of a tight replication control does not result in a deregulated cell cycle, as shown by the complete block of cell division upon inhibition of DNA polymerase . Further- more, genome segregation mechanisms that deliver equal number of chromosomes to the two daughter cells appear to be in place in H. salinarum . A different halophile, Haloferax volcanii, is also highly polyploid, with cells harbouring approximately 20 chromosome copies in expo- nential phase and approximately 12 during stationary phase . The chromosome copy number of the euryarchaeon Thermococcus kodakarensis has also been recently investigated. Analogously to the situation in other members of this phylum, T. kodakarensis cells show polyploidy with a chromosome copy number ﬂ uctuating between 19 and 7 from exponential to stationary phase .
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However, Haloferax volcanii is a notable exception in this regard: the deletion of two or more origins does not result in growth defects and the deletion of all origins leads to 7.5% faster growth than wild type; however, unlike Haloferax mediterranei, there is no activation of dormant origins . This indicates that an alternative, highly efficient mechanism for replication initiation exists in Haloferax volcanii. Given the common evolutionary history of Halobacteriales, it is likely that the core machinery for origin-independent replication exists in all species, but that Haloferax volcanii has lost an inhibitory component that prevents this mode of replication. Alternatively, it might have acquired an activating component that promotes origin-independent replication. Indeed, horizontal gene transfer is highly prevalent in Halobacteriales, as evident by a large number of gene duplications in the genome. Low species barriers exist in halophilic archaea for gene transfer and the exchange of large chromosomal fragments between Haloferax volcanii and Haloferax mediterranei has been detected in vivo . Interestingly, the dormant origin that becomes activated upon deletion of three chromosomal origins in Haloferax mediterranei is “foreign” to its genome—its chromosomal context indicates that it was acquired during a recent lateral gene transfer event . Furthermore, it is not found in Haloferax volcanii, which explains why it is not activated in an origin-less Haloferax volcanii mutant.
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E nveloped viruses of eukaryotes, including important human pathogens, such as human immunodeficiency virus, influenza virus, or Ebola virus, typically escape their host cells via budding through cellular membranes, whereby they acquire the lipid- containing envelope. Similar to many eukaryotic viruses (and un- like bacteriophages), viruses infecting archaea often contain enve- lopes and host-derived lipids (1). However, the ways of their morphogenesis and egress remain largely unexplored. As a model for studies on the release of lipid-containing viruses of Archaea, we chose Sulfolobus spindle-shaped virus 1 (SSV1), the prototypic member of the family Fuselloviridae, which represents one of the most abundant and widely distributed archaea-specific groups of viruses (2). The SSV1 virions are composed of four virus-encoded proteins (VP1 to -4) and one host-encoded chromatin protein
We could not identify a GAR formyltransferase (purN/ purT) gene in seven species of Archaea that appear to have otherwise intact purine biosynthesis pathways. The two Archaeoglobi, four Methanomicrobiales, and Methanopyrus kandleri have no genes with high similar- ity to known purT or purN genes despite otherwise complete or near-complete purine biosynthesis path- ways. Archaeoglobus fulgidus and Methanothermobacter thermoautotrophicus are known to contain methanop- terin-related folate analogues rather than tetrahydrofo- lates, so the unidentified enzymes might use formate as a carbon source (as in PurT), or may use an alternate non-folate carbon source other than methanopterin, as discussed above. Although Methanosphaera stadtmanae, Methanobrevibacter smithii, and Methanobrevibacter ruminantium are intestinal commensals and thus might rely on an environmental source of purines (despite the presence of an otherwise complete pathway), M. thermo- autotrophicus is an established purine prototroph  and so must have some means to catalyze the GAR for- myltransferase reaction. Labeling studies are inconsistent as to the substrate of the missing GAR formyltransferase in the Methanomicrobiales. In the presence of formate, M. stadtmanae has been shown to derive C8 from C2 of acetate (most consistent with the use of a tetrahydro- folate C1 carrier), while closely related M. smithii does not derive C8 from acetate .
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multiple-sequence alignment (see Text S1 in the supplemental material). All investi- gated archaea, with the exception of Thermoproteales and Nanoarchaeum equitans, contained an SD sequence ~7 to 10 bp upstream of the l7ae coding sequence, indicating that almost all archaeal l7ae mRNAs comprise a 5= UTR. This observation is striking in light of the general overabundance of leaderless transcripts in Archaea (26, 27). Potential transcriptional start sites could be assigned for most of the sequences due to the presence of conserved TATA boxes which revealed 5= UTR sizes from 10 nt to 200 nt. High sequence conservation of the leader sequences was identiﬁed only for Sulfolobales. Bioinformatic prediction of k-turn formation within the 5= UTRs is prob- lematic due to the high sequence variability of k-turn/k-loop structures. However, we were able to identify possible conserved Kt-b and Kt-n strands within the 5= UTRs by careful manual inspection. In order to investigate binding of the S. acidocaldarius L7Ae to other archaeal 5= UTRs that were identiﬁed by this approach, the gfpKt of pMD- autol7ae-gfp was exchanged with 50 bp of l7ae upstream regions of eight archaeal model organisms spread across the archaeal domain (Fig. 5a). The l7ae leader se- quences of Staphylothermus marinus, Archaeoglobus fulgidus, Haloferax volcanii, Metha- nosarcina acetivorans, Thermococcus kodakaraensis, and Methanococcus maripaludis comprised Kt-b and Kt-n strands, while only a Kt-n sequence could be identiﬁed for Aeropyrum pernix. Pyrobaculum aerophilum belongs to the order Thermoproteales, which did not comprise an l7ae 5= UTR. Control UTR strains without gfpKt showed around 80% GFP ﬂuorescence compared to strains with nonfunctional L7Ae (Fig. 5b), which might account for the residual toxicity of L7Ae. For comparison, strains with the gfpKt (S. acidocaldarius l7ae 5= UTR) showed around 40% GFP ﬂuorescence. The A. pernix UTR showed no L7Ae downregulation, probably due to the absence of the Kt-b strand. However, downregulation was observed for all other tested archaeal 5= UTRs, particu- larly for the A. fulgidus, M. acetivorans, and M. maripaludis UTRs. Strains with the P. aerophilum 5= UTR of l7ae displayed GFP signals with levels close to that measured for the background ﬂuorescence and were therefore excluded. The results highlight that S. acidocaldarius L7Ae can bind to the l7ae 5= UTR regions of various archaea, suggesting that the negative-feedback loop of L7Ae is a conserved feature and operative in most archaeal organisms.
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Taken together, the studies in archaea and bacteria make a compelling case that EndoMS participates in a MMR pathway. However, many important aspects of this pathway remain to be elucidated. The generation of double strand breaks by P. furiosus EndoMS is suggestive of an MMR process that functions via homologous recombination / DSBR (Ishino et al., 2016). This has the advantage that there is no need to identify nascent DNA strands to pinpoint the mismatched base, as both will be resected during DSBR. The observation that EndoMS is sometimes found in an operon with the RadA recombinase lends further support to this hypothesis (Ren et al., 2009). However, generation of a double strand break each time a mismatch is detected seems a risky strategy, unless homologous recombination is very efficient. This is probably the case in many of the euryarchaea, which are highly polyploid. It is much less obvious for the crenarchaea, which have a eukaryal-like cell cycle with monoploid and diploid stages (Lundgren & Bernander, 2007). Clearly, dissection and reconstitution of the pathway using genetic and biochemical techniques is a pressing priority. The interaction of archaeal EndoMS with the sliding clamp PCNA may provide a means to locate EndoMS at the replication fork to interrogate newly synthesised DNA, and could give the opportunity for co-location of a variety of DNA manipulation enzymes on the PCNA toolbelt (Beattie & Bell, 2011). In this regard, it will be interesting to see whether the bacterial EndoMS protein requires an interaction with the bacterial sliding clamp for activity.
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homologues in many archaea (Table 1). Also, some DNA repair mechanisms of HA resemble those of well-studied organisms, whereas others clearly deviate from them at fundamental levels. In this context, simplistic claims for the biological signifi cance of an archaeal gene based upon its most famous E. coli or yeast homologue lose their validity and deserve appropriate skepticism. At the same time, opportunities to confi rm DNA repair functions in vivo remain scarce and diffi cult, refl ecting the technical limitations of manipulating HA. As a result, we have no fi rm measure of the complexity and functional overlap of DNA stabilization and repair systems in HA, nor the extent to which they participate in “normal” or essential DNA transactions. These cautions must be borne in mind as individual components of DNA stabilization and repair become identifi ed. What we do know nevertheless suggests at least three efforts as being especially signifi cant and timely.
In contrast, asRNAs, which are by far the largest group of sRNAs found in the Archaea, are encoded in the opposite strand of their putative target. In the hyperthermophile Pyrobaculum, 3 antisense sRNAs were found opposite a ferric uptake regulator, a triose-phosphate isomerase, and transcription factor B, supporting a potential role in the regulation of iron, transcription, and core metabolism . Target enrichment of asRNAs differentially regulated by oxidative stress in H. volcanii included mRNAs involved in transposon mobility, chemotaxis signaling, peptidase activity, and transcription factors . The functional enrichment of transposon targeted by asRNAs suggests that during oxidative stress transposon activity is tightly regulated in H. volcanii, potentially explaining its increased resistance to oxidative stress conditions . Indeed, transposons are genetic elements that hop around in the genome causing double strand breaks. This added stress would likely be detrimental to a cell under oxidative stress, hence a need to be silenced [38-40]. sRNAs antisense to transposons were also reported for Thermococcus kodakarensis , S. solfataricus , and M. mazei  suggesting that, similarly to bacteria, regulation of transposition is mediated by asRNAs in archaea .
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The gut microbiome plays a central role in human health. However, at the start of this thesis project there existed methods mainly focused on characterizing the bacterial gut flora. It is known that the bacteria in the gut interact biochemically with archaea and microeukaryotes; therefore, studying bacteria in isolation provides an incomplete view of the gut community. One of the primary goals of this work was to pioneer methods to characterize these important communities. In Chapter 2, I present a sample-to-analysis pipeline to sequence fungal and microeukaryotic communities from human stool. This work analyzes the effectiveness of established primers and novel primers, developed by Greg Peterfreund in the Bushman Laboratory, and describes a classifier that I developed for classifying reads generated from those primers. In Chapter 3, I introduce a method for successfully amplifying the archaeal 16S gene from stool samples, while avoiding non-target DNA. Finally, in Chapter 4, I study the longitudinal effects of antibiotic treatment in the gut microbiome using a mouse model. This study analyzes both the bacterial and microeukaryotic communities during and after antibiotic treatment. Finally, Chapter 5 describes the impact of this work and the new possibilities that it enables.
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