NicK and HelP are the only ICE–encoded factors required for autonomous replication.

1Plasmids containing spcE and ICEBs1 oriT also contained the indicated genes, helP and/or nicK, under control of ICEBs1 Pxis. nicK or helP was integrated in the chromosome as indicated.2Plasmids were isolated from E. coli and transformed into B. subtilis cells that contained no ICEBs1 (ICEBs10; strain JMA222 or derivatives). In strains with transformants, the relative copy number of the plasmid was determined by measuring the relative amount of spcE DNA. Transformants were obtained if the plasmids were capable of replication, only when both nicK and helP were present. To ensure that the plasmids were transformable, constructs were also transformed into IRN342 (containing ICEBs1) where gene expression is repressed by ImmR and the plasmids are able to integrate into the host chromosome. All plasmids were capable of transforming this recipient, and all had a relative copy number of one. Data presented are averages from at least 3 independent experiments ± standard deviation.