Kolesnik, M. V. | Search bioRxiv

bioRxiv2026

A Unique Type V CRISPR-Cas System Encoded by a Group of Thermus Viruses

CRISPR-Cas are widespread adaptive immune systems that protect bacteria and archaea from mobile genetic elements such as bacteriophages. Metagenomic sequencing identified CRISPR-Cas systems in phage genomes; however, their functions remain largely unknown. Here, we present Cas12r-CRISPR, a novel type V CRISPR-Cas system encoded by Lalka phages infecting thermophilic Thermus bacteria. We determined Cas12r-CRISPR PAM consensus sequence and crRNA structure and showed, that when provided with appropriate spacers and expressed in Thermus thermophilus, Cas12r-CRISPR efficiently interferes with plasmid transformation as well as infection by diverse Thermus phages. In the course of Lalka phage infection, the Cas12r-CRISPR locus is expressed with middle phage genes and its transcripts are among the most abundant phage RNAs. Notably, most Cas12r-CRISPR spacers target integrative mobile elements widespread in Thermus genomes. Both Lalka phages and targeted integrative mobile elements use host tRNA genes as attachment sites. We therefore propose that Cas12r-CRISPR participates in an inter-MGEs conflict. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/719267v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@f90c35org.highwire.dtl.DTLVardef@90a063org.highwire.dtl.DTLVardef@111f936org.highwire.dtl.DTLVardef@1008132_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Structure and function of virion RNA polymerase of crAss-like phage

CrAss-like phages are a recently described family-level group of viruses that includes the most abundant virus in the human gut1,2. Genomes of all crAss-like phages encode a large virion-packaged protein2,3 that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs)4. Using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a novel DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited, likely pre-virion-packaged state. This conformation is attained with the help of a Cleft-blocking domain that interacts with the active site motif and occupies the RNA-DNA hybrid binding grove. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs involved in RNA interference5,6, although most of phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of protein folding space. Considering the structural similarity, we propose that eukaryal RNA interference polymerases take their origin in a phage, which parallels the emergence of the mitochondrial transcription apparatus7.

molecular biology↗