Lalka phages and their mysterious competitors
In 2023 we visited Kunashir Island and isolated a group of odd, closely similar bacteriophages from its hot springs. Once we had sequenced and assembled their genomes, it turned out that every one of them encodes a CRISPR array with a variable set of spacers.
Bacteriophages often carry CRISPR arrays in their genomes, but usually have no cas genes of their own — they use the host’s Cas proteins instead. The CRISPR arrays of the Lalka phages, however, differed far too much from every known CRISPR-cas system of Thermus. A detailed analysis of the genomic context, followed by a series of experiments, showed that Lalka phages encode a CRISPR-Cas system of their own — a type V system belonging to a previously unknown subtype.
The system turned out to be functional and active: in our experiments it protects cells from infection by bacteriophages carrying a protospacer, and it also suppresses plasmid transformation when the plasmid is targeted. But what do bacteriophages need these CRISPR-Cas systems for? The spacers found in the arrays of the isolated phages and in metagenomic contigs target an unusual group of Thermus mobile elements.
All of these elements encode tyrosine integrases and use tRNA genes as their sites of integration into the host genome. Beyond that, the gene content of the targeted elements varies a great deal — about all that can be said is that most of them carry genes with predicted primase/polymerase and helicase functions, which are probably replication modules.
The analysis of where the spacers come from suggests that Lalka phages are competing with the mobile elements they target. What lies behind this competition we have not managed to work out yet, but we suspect that these integrative elements may behave as satellites.
We have also profiled the transcriptome of Thermus thermophilus cells infected with phage Lalka27a and found that the two most actively expressed genes at the very onset of infection encode small, negatively charged proteins (Gp5 and Gp6). We hypothesize that these proteins may mimic DNA and inhibit cellular defense systems such as restriction-modification systems, although we have not tested this yet.
Lalka phage virions also contain few proteins that are likely injected into the cell during infection. One of them is DarB, which we suspect suppresses restriction-modification systems, much like its homolog from phage P1. The second protein, which we named Lvh (large virionic helicase), is very large — around 1,700 amino acid residues — yet it contains just a single confidently identifiable domain, one with helicase function. There is one more virion protein, Gp4, but not even AF3 can predict its structure properly. In short, there is plenty that remains mysterious about these bacteriophages.