Figure 2.

Excision of SPβ_ICEBs1 and ICEBs1_SPβ

(A) Excision mechanisms for SPβ_kan and SPβ_ICEBs1. SPβ_kan and SPβ_ICEBs1 phage excision was induced by MMC treatment. 5′-spsM and spsM-3′, as well as 5′-trnS-leu2 and trnS-leu2-3′, were combined to generate attB_SPβ and attB_ICEBs1 in host genomes, respectively. Horizontal black arrowheads indicate the positions of primers for PCR amplification.

(B) Analysis of SPβ_kan and SPβ_ICEBs1 genome excision. Total genomic DNA was extracted from MMC-treated cells. The presence of attB was confirmed by PCR (top panel) and quantitative PCR (qPCR) analysis (bottom panel). SPβ_kan and SPβ_ICEBs1 lysogens were grown in Luria-Bertani (LB) medium. Vegetative cells in the early log phase (OD₆₀₀ ~ 0.2) were treated with 0.5 μg/mL MMC, and the cells were harvested at indicated times. The X axis represents time after MMC treatment in minutes.

(C) Excision mechanisms for ICEBs1_cat and ICEBs1_SPβ. ICEBs1_cat and ICEBs1_SPβ excisions were induced by MMC treatment. 5′-trnS-leu2 and trnS-leu2-3′, as well as 5′-spsM and spsM-3′, were recombined to generate attB_ICEBs1 and attB_SPβ in host genomes, respectively. Horizontal black arrowheads indicate the positions of primers for PCR amplification.

(D) Analysis of ICEBs1_cat and ICEBs1_SPβ genome excision. Total genomic DNAs were extracted from MMC-treated cells, and the presence of attB was confirmed using PCR amplification (top panel) and qPCR analysis (bottom panel).

In (B) and (D), amplification of attB_SPβ (PCR and qPCR, 305 bp) and attB_ICEBs1 (PCR and qPCR, 497 bp) by PCR and qPCR analysis. Data are mean ± SD; n = 3 independent experiments.

See also Table S4.