Adaptation By T. Thermophilus Type III CRISPR-Cas System in the Presence of Different Phages and Appearance of Viral Escapes

In our work, we study adaptation by Type III CRISPR-Cas system using Thermus thermophilus Hb27 strain as a model organism. This hyperthermophilic bacterium encodes CRISPR–Cas systems of III–A, III–B, I–B and I–C subtypes. It has ten CRISPR arrays of three different types. Six of these arrays belong to type III (they can be shared between both III–A and III–B subtypes).

We found that infection of T. thermophilus Hb27 cells with a siphovirus phiFa resulted in a very biased adaptation. The majority of acquired spacers originated from the region of the phage genome that encodes its early genes. In contrast, adaptation in conditions of infecting T. thermophilus Hb27 with a tectivirus phiKo led to a uniform distribution of the acquired spacers over the entire genome. In the case of phiKo, spacers against each of the temporal classes of genes enabled an efficient interference. In the case of phiFa, we have not detected any middle or late gene targeting spacers that would protect cells, suggesting that there may be a phage-encoded anti-CRISPR mechanism. After re-infecting the resistant strains bearing spacers against phiFa, we detected some escape phages with lesions in protospacers, revealing the requirements for productive spacer-protospacer interactions for the Type III CRISPR-Cas system.