Extended Data Fig. 8: I ΦKZ155 involvement in the phage replication cycle.

a. PAO1 cells were pretreated with 6 µM ASO against chmA for 30 min. Cells were infected with ΦKZ at an MOI = 5 and incubated for the indicated times followed by RNA extraction and sequencing of the transcriptomes. Relative quantification of protein-coding transcript (CDS) reads and normalisation to the maximum for ΦKZ155 over all conditions (analysis based on data described in Fig. 3a,b). b. Sequences of the targeted ΦKZ155 (ΦKZ) or gp176 (ΦPA3) transcripts and ASOs. c. PAO1 cells were transformed with a complementation plasmid encoding ΦKZ155 or the catalytically dead mutant ΦKZ155(D102N). PAO1 cells were pretreated with 6 µM ASO against ΦKZ155 for 30 min. Cells were infected with ΦKZ at an MOI = 0.0001, plasmid-encoded ΦKZ155 was induced with 0.2% arabinose, and cells were incubated for 180 min followed by CFU/PFU determination. Representative result of two independent experiments. d. RNA was 5′-[³²P]phosphorylated and left single-stranded (top) or duplexed with complementary DNA (bottom). ΦKZ155 and the catalytically dead D102N mutant were produced by in vitro translation and added to the oligomer for the cleavage reaction. Subsequently, the oligomers were analysed on an Urea-PAGE gel and autoradiographed. Commercially available E. coli RNase H protein was used as control. Representative result of two independent experiments. e. PAO1 cells were transformed as in (c). PAO1 cells were pretreated with 8 µM ASO against ΦKZ155 for 30 min. Cells were infected with ΦKZ at an MOI = 10, plasmid-encoded ΦKZ155 was induced with 0.2% arabinose, and the cells were incubated for 30 min followed by chemical crosslinking and staining with DAPI to visualise DNA and FM4-64 to visualize membranes. Data for pempty and pΦKZ155 are based on the same images as shown in Fig. 4d. TIR, translation initiation region. f. C-terminally GFP-tagged ΦKZ155 (ΦKZ155_GFP) was ectopically expressed from a plasmid in PAO1. PAO1 cells were infected with ΦKZ at an MOI = 10, the expression of ΦKZ155_GFP was induced with 0.2% arabinose, and incubated for 30 min followed by chemical crosslinking and staining with DAPI to visualise DNA and FM4-64 to visualize membranes. Representative result of two independent experiments. g. PAO1 cells were pretreated with 8 µM ASO against gp176 (ΦPA3). Cells were infected with ΦPA3 at an MOI = 5 and incubated for the indicated times followed by DNA extraction and dot-blotting. RNA was eliminated by alkaline treatment. The phage genomic DNA was detected with the radiolabelled oligo probe JVO−23279 followed by autoradiography. No host, no phage, and the non-targeting control ASO served as controls. Representative result of two independent experiments. h. PAO1 cells were pretreated with 6 µM ASO against ΦKZ155 for 30 min. Cells were infected with ΦKZ at an MOI = 5 and incubated for the indicated times post infection followed by DNA extraction and dot blotting, RNA was eliminated by alkaline treatment. Phage genomic DNA was detected with the radiolabelled oligo probe JVO-23213 (complementary to the chmA ORF) and JVO-15848 (complementary to the 5S rRNA gene of PAO1) followed by autoradiography. A representative result of two independent experiments is shown. Uncropped images of blots are available in Supplementary Fig. 1.