RNAseq analysis indicated that the phage protein Dip is transcribed in the early phase of infection, and is highly expressed (Ceyssens et al, 2021). To verify the timing of the corresponding protein translation, and to assess Dip stability during phage infection, we performed a western blot on φKZ-infected P. aeruginosa samples every 3 min during infection (Figure 6A). Using anti-Dip antibodies, Dip was first detected 9 min after the start of infection, reaching a peak at 24 min. Although a decrease in Dip levels is visible at approximately 30 min post infection, the quantity of Dip proteins subsequently increases, after which its levels appear to plateau. This indicates that Dip is produced during the early phase of infection and subsequently persists during the whole infection cycle.
RNase E, a member of the RNase E/G family, is a tetrameric enzyme and can be broadly divided into two functional halves. The N-terminal half (NTH) comprises the catalytic domain, while the non-conserved C-terminal half (CTH) is natively unstructured and acts as a scaffold to assemble the complex (Aït-Bara et al, 2021; Callaghan et al, 2005). Despite the predicted lack of structure within the scaffold domain, several short segments having structural propensity were identified in the E. coli CTH that mediate the interaction between RNase E and the cell membrane, enolase and PNPase. Moreover, the CTH contains two arginine-rich regions that have been shown to bind RNA: the RNA binding domain (RBD)/Arginine-rich region 1 (AR1) and AR2 (Callaghan et al, 2004).