Finally, we investigated the evolutionary importance of PL spacers since they come at such a fitness cost. We showed that a triggered growth arrest provides broad-spectrum immunity and can protect cells from later infection by untargeted phages!

Without Cas10 activity, all spacers behave like PL suggesting that Cas10 normally prevents growth arrest in PE. Compared to PE, PL spacers are rarely acquired so there must be a mechanism to retain them. We wondered if Cas10 could allow eventual recovery from growth arrest.

Mutating the accessory Type III RNase, Csm6, showed that downstream PE spacers, in addition to PL, fail to provide immunity without Csm6 activity. Downstream PE regions become increasingly dependent on this RNase but do not seem to trigger the prolonged arrest seen with PL.

Growth assays and microscopy showed that targeting PE transcripts allows uninhibited growth of infected cells but targeting PL transcripts causes a growth arrest allowing only the uninfected cells to proliferate. Surprisingly, some regions of downstream PE behave like PL.

We characterized the immune response mediated by spacers across the entire phage genome. RNA-seq revealed that phage transcription is controlled by 2 promoters. Spacers targeting early transcripts (PE) are enriched, but those targeting late transcripts (PL) also provide immunity.