The candidate division Korarchaeota comprises a group of uncultivated microorganisms that, by their small subunit rRNA phylogeny, may have diverged early from the major archaeal phyla Crenarchaeota and Euryarchaeota. Here, we report the initial characterization of a member of the Korarchaeota with the proposed name, ''Candidatus Korarchaeum cryptofilum,'' which exhibits an ultrathin filamentous morphology. To investigate possible ancestral relationships between deep-branching Korarchaeota and other phyla, we used whole-genome shotgun sequencing to construct a complete composite korarchaeal genome from enriched cells. The genome was assembled into a single contig 1.59 Mb in length with a G ؉ C content of 49%. Of the 1,617 predicted protein-coding genes, 1,382 (85%) could be assigned to a revised set of archaeal Clusters of Orthologous Groups (COGs). The predicted gene functions suggest that the organism relies on a simple mode of peptide fermentation for carbon and energy and lacks the ability to synthesize de novo purines, CoA, and several other cofactors. Phylogenetic analyses based on conserved single genes and concatenated protein sequences positioned the korarchaeote as a deep archaeal lineage with an apparent affinity to the Crenarchaeota. However, the predicted gene content revealed that several conserved cellular systems, such as cell division, DNA replication, and tRNA maturation, resemble the counterparts in the Euryarchaeota. In light of the known composition of archaeal genomes, the Korarchaeota might have retained a set of cellular features that represents the ancestral archaeal form. microbial cultivation ͉ genomics ͉ hyperthermophiles ͉ Korarchaeota ͉ phylogeny

Analysis of the genome sequence of the small hyperthermophilic archaeal parasite Nanoarchaeum equitans has not revealed genes encoding the glutamate, histidine, tryptophan and initiator methionine transfer RNA species. Here we develop a computational approach to genome analysis that searches for widely separated genes encoding tRNA halves that, on the basis of structural prediction, could form intact tRNA molecules. A search of the N. equitans genome reveals nine genes that encode tRNA halves; together they account for the missing tRNA genes. The tRNA sequences are split after the anticodon-adjacent position 37, the normal location of tRNA introns. The terminal sequences can be accommodated in an intervening sequence that includes a 12-14-nucleotide GC-rich RNA duplex between the end of the 5' tRNA half and the beginning of the 3' tRNA half. Reverse transcriptase polymerase chain reaction and aminoacylation experiments of N. equitans tRNA demonstrated maturation to full-size tRNA and acceptor activity of the tRNA(His) and tRNA(Glu) species predicted in silico. As the joining mechanism possibly involves tRNA trans-splicing, the presence of an intron might have been required for early tRNA synthesis.

The CRISPR arrays found in many bacteria and most archaea are transcribed into a long precursor RNA that is processed into small clustered regularly interspaced short palindromic repeats (CRISPR) RNAs (crRNAs). These RNA molecules can contain fragments of viral genomes and mediate, together with a set of CRISPR-associated (Cas) proteins, the prokaryotic immunity against viral attacks. CRISPR/Cas systems are diverse and the Cas6 enzymes that process crRNAs vary between different subtypes. We analysed CRISPR/Cas subtype I-B and present the identification of novel Cas6 enzymes from the bacterial and archaeal model organisms Clostridium thermocellum and Methanococcus maripaludis C5. Methanococcus maripaludis Cas6b in vitro activity and specificity was determined. Two complementary catalytic histidine residues were identified. RNA-Seq analyses revealed in vivo crRNA processing sites, crRNA abundance and orientation of CRISPR transcription within these two organisms. Individual spacer sequences were identified with strong effects on transcription and processing patterns of a CRISPR cluster. These effects will need to be considered for the application of CRISPR clusters that are designed to produce synthetic crRNAs.

CRISPR–Cas systems provide prokaryotes with adaptive immune functions against viruses and other genetic parasites. In contrast to all other types of CRISPR–Cas systems, type IV has remained largely overlooked. Here, we describe a previously uncharted diversity of type IV gene cassettes, primarily encoded by plasmid-like elements from diverse prokaryotic taxa. Remarkably, via a comprehensive analysis of their CRISPR spacer content, these systems were found to exhibit a strong bias towards the targeting of other plasmids. Our data indicate that the functions of type IV systems have diverged from those of other host-related CRISPR–Cas immune systems to adopt a role in mediating conflicts between plasmids. Furthermore, we find evidence for cross-talk between certain type IV and type I CRISPR–Cas systems that co-exist intracellularly, thus providing a simple answer to the enigmatic absence of type IV adaptation modules. Collectively, our results lead to the expansion and reclassification of type IV systems and provide novel insights into the biological function and evolution of these elusive systems.