Skip to main content
. 2019 Dec 26;23(1):100805. doi: 10.1016/j.isci.2019.100805

Figure 3.

Figure 3

Excision of SPβskin Using Site-Specific Recombination (SSR) Units from a Defective Prophage

(A) Excision mechanisms for SPβ, skin, and SPβskin (from left to right). SPβ and SPβskin excisions were induced either after MMC treatment or during sporulation, whereas skin excision was observed only during sporulation, as previously reported (Kimura et al., 2010); Pv, σA-dependent promoter; PSt, stress-inducible promoter; PE/K, mother cell-specific σE/K-dependent sporulation promoter; PE+SpoIIID, mother cell-specific σE and SpoIIID-dependent sporulation promoter. Horizontal black arrowheads indicate the positions of primers for PCR amplification.

(B) Excision in the presence of MMC. SPβ and SPβskin genomes were excised from host genomes after MMC treatment, whereas the defective prophage skin (no stress-inducible promoter) was not. B subtilis 168 cells containing SPβ, skin, or SPβskin lysogens were grown in LB medium. Vegetative cells in the early log phase (OD600 ~ 0.2) were treated with 0.5 μg/mL MMC and were harvested at indicated times and analyzed by PCR amplification (top panel) and qPCR (bottom panel). N.D., not detected.

(C) Excision during sporulation. Schematics of SPβ and skin excision during sporulation are shown above figure. B. subtilis 168 sporulating cells divided asymmetrically to produce mother cells and forespores at 1–2 h after the initiation of sporulation (T1–T2). Subsequently, skin and SPβ excision was specifically induced in mother cells at approximately 3 h after the initiation of sporulation (T3) (Abe et al., 2014, Sato et al., 1990). B. subtilis 168 cells and SPβskin lysogens were grown in DSM, and samples of vegetative cells were collected (OD600 ~ 0.2; T−1) at indicated times at 1-h intervals (T1–T5). Middle and bottom panels represent PCR amplification and qPCR analysis, respectively.

In (B) and (C), Amplification of attBSPβ (PCR and qPCR, 305 bp) and attBskin (PCR and qPCR, 221 bp) by PCR and qPCR analysis. Data are mean ± SD; n = 3 independent experiments. See also Figure S3 and Table S4.