Comparative Analysis Of Completely Sequenced Genomes

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The rapidly growing database of completely and nearly completely sequenced genomes of bacteria, archaea, eukaryotes and viruses (millions of genomes already available and many more in progress) creates both extensive new opportunities and major new challenges for genome research. During the year in review, we performed a variety of studies that took advantage of the genomic information to establish fundamental principles of genome evolution and to investigate the evolution of biologically and medically important groups of organisms. All extant eukaryotes descend from the last eukaryotic common ancestor (LECA), which is thought to have featured complex cellular organization. To gain insight into LECA biology and eukaryogenesis-the origin of the eukaryotic cell, which remains poorly understood- in collaboration with Drs. Valerian Dolja and Mart Krupovic, we reconstructed the LECA virus repertoire. We compiled an inventory of eukaryotic hosts of all major virus taxa and reconstructed the LECA virome by inferring the origins of these groups of viruses. The origin of the LECA virome can be traced back to a small set of bacterial-not archaeal-viruses. This provenance of the LECA virome is probably due to the bacterial origin of eukaryotic membranes, which is most compatible with two endosymbiosis events in a syntrophic model of eukaryogenesis. In the first endosymbiosis, a bacterial host engulfed an Asgard archaeon, preventing archaeal viruses from entry owing to a lack of archaeal virus receptors on the external membranes. In another major project, we mined metatranscriptomes for viroid-like, circular RNA replicons. Viroids and viroid-like covalently closed circular (ccc) RNAs are minimal replicators that typically encode no proteins and hijack cellular enzymes for replication. The extent and diversity of viroid-like agents are poorly understood. We developed a computational pipeline to identify viroid-like cccRNAs and applied it to 5,131 metatranscriptomes and 1,344 plant transcriptomes. The search yielded 11,378 viroid-like cccRNAs spanning 4,409 species-level clusters, a 5-fold increase compared to the previously identified viroid-like elements. Within this diverse collection, we discovered numerous putative viroids, satellite RNAs, retrozymes, and ribozy-like viruses. Diverse ribozyme combinations and unusual ribozymes within the cccRNAs were identified. Self-cleaving ribozymes were identified in ambiviruses, some mito-like viruses and capsid-encoding satellite virus-like cccRNAs. The broad presence of viroid-like cccRNAs in diverse transcriptomes and ecosystems implies that their host range is far broader than currently known, and matches to CRISPR spacers suggest that some cccRNAs replicate in prokaryotes. We also continued our long term investigation in comparative genomics of CRISPR loci in bacteria and archaea. CRISPR-cas loci typically contain CRISPR arrays with unique spacers separating direct repeats. Spacers along with portions of adjacent repeats are transcribed and processed into CRISPR(cr) RNAs that target complementary sequences (protospacers) in mobile genetic elements, resulting in cleavage of the target DNA or RNA. Additional, standalone repeats in some CRISPR-cas loci produce distinct cr-like RNAs implicated in regulatory or other functions. We developed a computational pipeline to systematically predict crRNA-like elements by scanning for standalone repeat sequences that are conserved in closely related CRISPR-cas loci. Numerous crRNA-like elements were detected in diverse CRISPR-Cas systems, mostly, of type I, but also subtype V-A. Standalone repeats often form mini-arrays containing two repeat-like sequence separated by a spacer that is partially complementary to promoter regions of cas genes, in particular cas8, or cargo genes located within CRISPR-Cas loci, such as toxins-antitoxins. We show experimentally that a mini-array from a type I-F1 CRISPR-Cas system functions as a regulatory guide. We also identified mini-arrays in bacteriophages that could abrogate CRISPR immunity by inhibiting effector expression. Thus, recruitment of CRISPR effectors for regulatory functions via spacers with partial complementarity to the target is a common feature of diverse CRISPR-Cas systems. In collaboration with the laboratory of Dr. Ping Xu of Gunazhou University, we explored the evolution of the genomes of spore-forming archaea. Several groups of bacteria have complex life cycles involving cellular differentiation and multicellular structures. For example, actinobacteria of the genus Streptomyces form multicellular vegetative hyphae, aerial hyphae, and spores. However, similar life cycles have not yet been described for archaea. Here, we show that several haloarchaea of the family Halobacteriaceae display a life cycle resembling that of Streptomyces bacteria. Strain YIM 93972 (isolated from a salt marsh) undergoes cellular differentiation into mycelia and spores. Other closely related strains are also able to form mycelia, and comparative genomic analyses point to gene signatures (apparent gain or loss of certain genes) that are shared by members of this clade within the Halobacteriaceae. Genomic, transcriptomic and proteomic analyses of non-differentiating mutants suggest that a Cdc48-family ATPase might be involved in cellular differentiation in strain YIM 93972. Additionally, a gene encoding a putative oligopeptide transporter from YIM 93972 can restore the ability to form hyphae in a Streptomyces coelicolor mutant that carries a deletion in a homologous gene cluster (bldKA-bldKE), suggesting functional equivalence. We propose strain YIM 93972 as representative of a new species in a new genus within the family Halobacteriaceae, for which the name Actinoarchaeum halophilum gen. nov., sp. nov. is herewith proposed. Our demonstration of a complex life cycle in a group of haloarchaea adds a new dimension to our understanding of the biological diversity and environmental adaptation of archaea. In collaboration with Drs. Allahverdyan and Khachatryan of the Yerevan Physics Institute and Dr. Lopez-Garcia of Universite Paris-Saclay, we performed a theoretical study of the coevolution of reproducers and replicators. There are two fundamentally distinct but inextricably linked types of biological evolutionary units, reproducers and replicators. Reproducers are cells and organelles that reproduce via various forms of division and maintain the physical continuity of compartments and their content. Replicators are genetic elements (GE), including genomes of cellular organisms and various autonomous elements, that both cooperate with reproducers and rely on the latter for replication. All known cells and organisms comprise a union between replicators and reproducers. We explored a model in which cells emerged via symbiosis between primordial "metabolic" reproducers (protocells) which evolved, on short time scales, via a primitive form of selection and random drift, and mutualist replicators. Mathematical modeling identifies the conditions, under which GE-carrying protocells can outcompete GE-less ones, taking into account that, from the earliest stages of evolution, replicators split into mutualists and parasites. Analysis of the model shows that, for the GE-containing protocells to win the competition and to be fixed in evolution, it is essential that the birth-death process of the GE is coordinated with the rate of protocell division. At the early stages of evolution, random, high-variance cell division is advantageous compared with symmetrical division because the former provides for the emergence of protocells containing only mutualists, preventing takeover by parasites. These findings illuminate the likely order of key events on the evolutionary route from protocells to cells.