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Item Functional and Phylogenetic Diversity of Cas10 Proteins(Mary Ann Liebert Inc, 2023-04) Wiegand, Tanner; Wilkinson, Royce; Santiago-Frangos, Andrew; Lynes, Mackenzie; Hatzenpichler, Roland; Wiedenheft, BlakeCas10 proteins are large subunits of type III CRISPR RNA (crRNA)-guided surveillance complexes, many of which have nuclease and cyclase activities. Here, we use computational and phylogenetic methods to identify and analyze 2014 Cas10 sequences from genomic and metagenomic databases. Cas10 proteins cluster into five distinct clades that mirror previously established CRISPR-Cas subtypes. Most Cas10 proteins (85.0%) have conserved polymerase active-site motifs, while HD-nuclease domains are less well conserved (36.0%). We identify Cas10 variants that are split over multiple genes or genetically fused to nucleases activated by cyclic nucleotides (i.e., NucC) or components of toxin–antitoxin systems (i.e., AbiEii). To clarify the functional diversification of Cas10 proteins, we cloned, expressed, and purified five representatives from three phylogenetically distinct clades. None of the Cas10s are functional cyclases in isolation, and activity assays performed with polymerase domain active site mutants indicate that previously reported Cas10 DNA-polymerase activity may be a result of contamination. Collectively, this work helps clarify the phylogenetic and functional diversity of Cas10 proteins in type III CRISPR systems.Item Roadmap for naming uncultivated Archaea and Bacteria(2020-08) Murray, Alison E.; Freudenstein, John; Gribaldo, Simonetta; Hatzenpichler, Roland; Hugenholtz, Philip; Kampfer, Peter; Konstantinidis, Konstantinos T.; Lane, Christopher E.; Papke, R. Thane; Parks, Donovan H.; Rossello-Mora, Ramon; Stott, Matthew B.; Sutcliffe, Iain C.; Thrash, J. Cameron; Venter, Stephanus N.; Whitman, William B.; Acinas, Silvia G.; Amann, Rudolf I.; Anantharaman, Karthik; Armengaud, Jean; Baker, Brett J.; Barco, Roman A.; Bode, Helge B.; Boyd, Eric S.; Brady, Carrie L.; Carini, Paul; Chain, Patrick S. G.; Colman, Daniel R.; DeAngelis, Kristen M.; Asuncion de los Rios, Maria; Estrada-de los Santos, Paulina; Dunlap, Christopher A.; Eisen, Jonathan A.; Emerson, David; Ettema, Thisjs J. G.; Eveillard, Damien R.; Girguis, Peter R.; Hentschel, Ute; Hollibaugh, James T.; Hug, Laura A.; Inskeep, William P.; Ivanova, Elena P.; Klenk, Hans-Peter; Li, Wen-Jun; Lloyd, Karen G.; Loffler, Frank E.; Makhalanyane, Thulani P.; Moser, Duane P.; Nunoura, Takuro; Palmer, Marike; Parro, Victor; Pedros-Alio, Carlos; Probst, Alexander J.; Smits, Theo H. M.; Steen, Andrew D.; Steenkamp, Emma T.; Spang, Anja; Stewart, Frank J.; Tiedje, James M.; Vandamme, Peter; Wagner, Michael; Wang, Feng-Ping; Yarza, Pablo; Hedlund, Brian P.; Reysenbach, Anna-LouiseThe assembly of single-amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) has led to a surge in genome-based discoveries of members affiliated with Archaea and Bacteria, bringing with it a need to develop guidelines for nomenclature of uncultivated microorganisms. The International Code of Nomenclature of Prokaryotes (ICNP) only recognizes cultures as ‘type material’, thereby preventing the naming of uncultivated organisms. In this Consensus Statement, we propose two potential paths to solve this nomenclatural conundrum. One option is the adoption of previously proposed modifications to the ICNP to recognize DNA sequences as acceptable type material; the other option creates a nomenclatural code for uncultivated Archaea and Bacteria that could eventually be merged with the ICNP in the future. Regardless of the path taken, we believe that action is needed now within the scientific community to develop consistent rules for nomenclature of uncultivated taxa in order to provide clarity and stability, and to effectively communicate microbial diversity.