Identification of a Single Strand Origin of Replication in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis
Fig 6
sso1 contributes to stable acquisition of ICEBs1 by recipients.
Mini-ICEBs1 with (A; strain LDW129) or without (B; strain LDW179) sso1 was crossed into recipients (strain CAL85) by conjugation. Transconjugants were selected on solid medium containing streptomycin and kanamycin. The conjugation frequency (~0.2%) is about one tenth that for wild type ICEBs1. The non-excisable element at thrC is a substrate for nicking and RCR [30,43] and produces relaxosome complexes that we suspect compete with and reduce the efficiency of transfer of the mini-ICEBs1. In addition, nicking and replication from non-excisable elements kills donor cells [43]. (A) Transconjugants that acquired mini-ICEBs1 sso1+ grew as relatively uniform, normal looking colonies. Transconjugants (including those that initially grew smaller than normal) were resistant to kanamycin after propagation under non-selective conditions, indicating that ICEBs1 was stably acquired (as described in the text). (B) Transconjugants that acquired mini-ICEBs1 Δsso1 produced at least two types of colonies, large and small. An arrow marks one large (normal) colony, and an arrowhead indicates one small colony. Most small colonies were unstable. That is, after propagation without selection, cells derived from small colonies were no longer resistant to kanamycin, indicating that ICEBs1 was not stably acquired (as described in the text).