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. 2021 Jul 9;118(28):e2018297118. doi: 10.1073/pnas.2018297118

Fig. 1.

Fig. 1.

SPP1 DNA replication and encapsidation. (A) Schematics of the main steps of tailed bacteriophage infection exemplified by SPP1. (B) Quantification of SPP1 DNA synthesis (empty circles) and encapsidation (filled circles) determined by qPCR of total and DNase-protected DNA, respectively, in B. subtilis GSY10004 infected with SPP1lacO64. The amount of SPP1 gene 6 DNA was divided by the amount of B. subtilis gyrA reporter DNA considering that there are 2.7 nucleoids on average per cell (SI Appendix, Fig. S1 C and D) to calculate the number of SPP1 genomes per bacterium. The total number of infectious particles (filled squares; ordinate on the right) was quantified by phage titration of bacteria lysed with 10% chloroform. Note that the DNA molecule encapsidated in SPP1lacO64 particles (∼45.1 kbp) is 1.07-fold longer than the phage genome size (42.3 kbp) (15, 66), a factor that was used to convert phage particle counts into genome equivalents and reciprocally. (Right) A snapshot of phage DNA and particle yields at 30 min p.i., when experiments were stopped because cell lysis initiated. The values in bold were obtained experimentally, and those in gray were calculated based on the figure data. Note that the calculation of ∼46 capsids corresponds to particles that protect viral DNA but are noninfectious (i.e., tailless DNA-filled capsids and eventually defective tailed phage particles). The data are the average from three independent experiments. The biosynthetic effort accounting for phage DNA and virion structural proteins synthesis is depicted underneath the infection time points (Dataset S1). (C) Percentage of encapsidated DNA calculated from the data in B. (D) Phage DNA synthesis at 10 and 25 min p.i. in B. subtilis GSY10004 infected with SPP1lacO64 (15) (red), SPP1lacO64gp40− (defective in initiation of DNA synthesis) (yellow), and SPP1lacO64gp2− (defective in DNA packaging) (green). Total DNA (bars total height) and encapsidated, DNase-protected DNA (overlapping full bars) were quantified by qPCR. The data are the average from three independent experiments.