# CRISPR adaptation in Streptococcus thermophilus benefits from phage environmental DNA

## TL;DR

This study shows that free phage DNA in the environment helps bacteria develop CRISPR immunity, but it is not used to create the immune memory.

## Contribution

The study reveals that phage environmental DNA enhances CRISPR immunity in bacteria in a sequence-specific and phage-specific manner.

## Key findings

- Phage environmental DNA (eDNA) is involved in CRISPR immunity, as its digestion reduces the generation of phage-immune colonies.
- Only phage eDNA increases the generation of phage-immune colonies, showing its specific role in CRISPR immunity.
- The effect of eDNA is linked to early-expressed genes in phages and is not used as a source of genetic information for spacer acquisition.

## Abstract

The CRISPR-Cas system is a bacterial adaptive immune system that protects against infection by phages: viruses that infect bacteria. To develop immunity, bacteria integrate spacers—fragments of the invading nucleic acids—into their CRISPR array to serve as the basis for sequence-targeted DNA cleavage. However, upon infection, a phage quickly takes over the metabolism of the bacterium, leaving little time for the bacterium to acquire new spacers, transcribe them, and use them to cut the invading DNA. To develop CRISPR immunity, bacteria must be safely exposed to phage DNA. Phage infection releases environmental DNA (eDNA) which could be involved in the development of CRISPR immunity. Using Streptococcus thermophilus and phages 2972 and 858 as a model for CRISPR immunity, we show that eDNA is involved in CRISPR immunity, as generation of phage-immune bacterial colonies decreases with eDNA digestion. Furthermore, it is phage eDNA specifically that impacts CRISPR immunity since only its addition increases the generation of phage-immune colonies. We also show that the effect of eDNA is phage-specific, sequence-specific, and can even be traced to a region of the genome covering the early-expressed genes, which differ between phages 2972 and 858. However, we also show that eDNA is not used as a source of genetic information for spacer acquisition. These results link eDNA to the CRISPR-Cas system, providing a better understanding of the context of the emergence of CRISPR immunity and could inform our understanding of the mechanisms through which bacteria detect phage infection.

How can a bacterial adaptive immune system (the CRISPR-Cas system) exist at all, when exposure to a virulent phage is so consistently lethal? We proposed that bacteria might actively sample their genetic environment for phage DNA through natural competence. In testing this hypothesis, we revealed that free phage DNA is important to CRISPR immunity—but not as the source of the immunological memory.