FIGURE 4.

Incompatibility between pVG1 derivatives and pSA564. (A) Schematic linear map of the pVG1 construct. The orange section is from the vector backbone, which includes a ColE1 origin of replication for E. coli (but not for S. aureus), an ampicillin resistance cassette for E. coli and a chloramphenicol resistance cassette for S. aureus. The green section was cloned from pSA564, and includes (from left to right) a truncated rep1_N gene, the rac gene, the RNA1 gene and the repA_N gene. The location of the rac promoter is unknown. Further details about the pSA564 insert can be found in Supplementary Figure 1. (B) The pVG1 plasmid and its derivatives were transformed into PR01, plated on MHC agar plates and incubated over night at 37°C. Colonies were picked directly from the transformation MHC agar plate and resuspended in MH medium. These suspensions were used for serial dilutions that were then spotted on MH, MHC, MHP, and MHCP agar-plates and incubated over night at 37°C. Growth on MHC indicates the presence of pVG1 or its derivatives (or the pEB01 control), growth on MHP indicates the presence of pSA564, and growth on MHCP indicates the presence of both pSA565 and a pVG1 plasmid. Details of the mutations in pVG1 can be found in Table 2.