Intensive “Analyzing genomic variants in bacteria”
Intensive “Analyzing genomic variants in bacteria”
On February 24-26, Skoltech hosted a bioinformatics intensive for high school and college students
At the Intensive, participants explored sequencing data that our laboratory staff obtained while working on a research project. This project is devoted to the study of CRISPR-Cas systems of hyperthermophilic bacteria Thermus thermophilus. In nature, these bacteria live in hot (60-80 ℃) springs - in Russia, for example, such springs are found in Kamchatka, and our laboratory has several times sent expeditions there to collect samples, from which we then isolated Thermus bacteria and viruses that infect these bacteria.
The life of these bacteria is not easy: in addition to having to adapt to the extreme conditions of the environment in which they live, they are also attacked by a variety of genetic parasites - bacteriophages, transposons, integrative conjugative elements. To protect themselves from these parasites, Thermus bacteria encode a large arsenal of defense systems: for example, in addition to restriction-modification systems, toxin-antitoxin loci, and prokaryotic Argonaute systems, as many as four CRISPR-Cas systems are encoded in the genome of T. thermophilus strain HB27.
T. thermophilus bacteria contain type III CRISPR-Cas systems, some of the most complexly constructed defense systems in prokaryotes (Kolesnik et al., 2021). These systems specifically recognize and cleave RNA molecules, but they also protect cells from bacteriophages with DNA genomes and from DNA plasmids by engaging a complex signaling pathway leading to the activation of additional immune components. These systems are very effective - for example, if a cell acquires a spacer (a short nucleotide sequence within the CRISPR cassette) targeting the bacteriophage transcript, the ability of these cells to resist infection increases tens of thousands of times (Artamonova et al., 2020). How do bacteriophages and plasmids manage to avoid extinction?
To find out how mobile elements manage to evade the immune response of CRISPR-Cas systems, we conducted an experiment. We introduced into T. thermophilus cells a plasmid containing an antibiotic resistance gene that is attacked by the CRISPR-Cas system. In theory, such cells should not form colonies on a medium containing an antibiotic at all - in fact, they should all destroy the plasmid that has entered them. Indeed, in our experiments the presence of immune response the number of grown plasmids is reduced by thousands of times - but nevertheless, some number of colonies grow. To understand what happened in such cells, we selected several colonies, isolated DNA from the cells, and sequenced them on Illumina and Oxford Nanopore platforms.
At the intensives, we analyzed the sequencing data obtained. We found two types of mutations that avoid an immune response. The first type is short deletions (nucleotide dropouts) that lead to inactivation (reading frame failure) in one of the key components of the CRISPR-Cas system. The second type of mutation was more unusual: some cells were found to have lost huge sections of the genome encoding the entire locus with cas genes. After assembling genomes from Oxford Nanopore's long reads, it turned out that these huge deletions result from recombination between CRISPR cassettes - with a new, hybrid CRISPR cassette being formed. We assume that such a process can occur in nature and that cells can get rid of unnecessary spacers in this way, and now we analyze this phenomenon in more detail.
We were very happy to see so many interested participants at our event. We had a little bit of a mismatch between the number of people and the capacity of our server, so it hung at one stage, but otherwise it went well!