Figure 6.

The absence of RNase H1 inhibits retron-based phage defense. (A) TBE–urea gel showing production of RT-DNA in the absence of the accessory protein. (B) Schematic of full operon and ncRNA/RT constructs used in plaque assays and quantification of plaque forming units (PFU). Phage lysates were titrated onto plates of each strain/construct pair to find a concentration that yields distinct plaques. That concentration was then tested over a full plate where plaques were counted. The count was converted into PFU per mL of phage lysate. (C) Titration of phage T2 on strains containing Eco1 constructs with and without RNase H1. (D) PFU/mL of T2 on strains containing Eco1 constructs with and without RNase H1. Biological replicates of the full operon (black) and ncRNA/RT (magenta) constructs that were performed in parallel are connected with a gray line. (E) The sensitivity of each strain to T2, as expressed by the ratio of plaque-forming units on strains with the full operon versus ncRNA/RT constructs, in the presence or absence of RNase H1. A sensitivity value of 1 indicates no retron-based defense, while a value of 0 indicates total retron-based defense. Generally, as retron-based defense strengthens (approaches 0), the strain's sensitivity to phage decreases. For Eco1, there is effectively no defense against T2 and therefore no difference between strains. (F) Titration of T5 on strains containing Eco1 constructs with and without RNase H1. (G) PFU/mL of T5 on strains containing Eco1 constructs with and without RNase H1. (H) Sensitivity values indicate a significant difference in Eco1 defense against T5 with and without RNase H1. (I) Titration of T2 on strains containing Eco4 constructs with and without RNase H1. (J) PFU/mL of T2 on strains containing Eco4 constructs with and without RNase H1. (K) Sensitivity values indicate that there is no significant difference in defense between strains. (L) Titration of phage T5 on strains containing Eco4 constructs with and without RNase H1. (M) PFU/mL of T5 on strains containing Eco4 constructs with and without RNase H1. (N) Sensitivity values indicate a significant difference in Eco4 defense against T5 with and without RNase H1. (O) Titration of T2 on strains containing Eco9 constructs with and without RNase H1. (P) PFU/mL of T2 on strains containing Eco9 constructs with and without RNase H1. (Q) Sensitivity values indicate a significant difference in Eco9 defense against T2 with and without RNase H1. (R) Titration of T5 on strains containing Eco9 constructs with and without RNase H1. (S) PFU/mL of T5 on strains containing Eco9 constructs with and without RNase H1. (T) For Eco9, there is effectively no defense against T5 and therefore no difference between strains.