Fig. 3

Genetic modification of the pac site, within the pacA coding sequence of the wildtype P1 bacteriophage genome to reduce wildtype P1 phage DNA packaging. (a) Schematic diagram showing the minimal pac sequence of the P1 phage genome, situated within the pacA coding sequence. Methylation sites, consisting of hexameric repeats, HEX4 and HEX3, are shown in black, the IHF binding site is shown in green, and the pac cleavage site is shown in orange. Synonymous mutations were introduced onto the hexameric repeats to disrupt the binding of PacA and PacB. A Comparison with the wildtype pac DNA sequence is shown, with mutated DNA bases indicated in red. (b) Schematic diagram showing the processing of the pac site, involving the binding of PacA and PacB to the hexameric repeats, as well as IHF and HU binding, which was proposed to give a bent structure of the pac site. Pacase (both PacA and PacB) enzymatic activity then leads to cleavage of the pac site.^34,35 Synonymous mutations introduced onto the hexameric repeats of the pac site lead to a reduction in pacase binding, reducing the processing and packaging of the P1 mutant pacA*::npt P1 DNA. (c) Quantification of wildtype P1 phage (in blue) and P1 transducing units (in orange) titers of lysates prepared from wildtype (WT) and pacA*::npt EMG16 cells harboring J72114 cas9-NT phagemid without spacer sequence(s) targeting E. coli or S. flexneri chromosomal sequences. (d) Ratio of wildtype P1 phage to P1 transducing units was calculated and plotted. Each data point represents a biological replicate and is the average of four technical repeats. Horizontal bars represent the group mean. The p-values (between wildtype and pacA*::npt lysates) were determined using a two-tailed unpaired t-test with significance defined by p < 0.05. p < 0.0005 between the P1 phage titer of lysates prepared from wildtype and pacA* EMG16 lysogen, as well as the PFU/TU ratio between lysates prepared from wildtype and pacA* EMG16 lysogen are shown as ***.