RESEARCH ARTICLE Identification of a Single Strand Origin of Replication in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis

We identified a functional single strand origin of replication (sso) in the integrative and conju-gative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons) are DNA elements typically found integrated into a bac-terial chromosome where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICEs become activated and excise from the host chromosome and can transfer to neighboring cells via the element-encoded conjugation machinery. Activated ICEBs1 undergoes autonomous rolling circle replication that is needed for the maintenance of the excised element in growing and dividing cells. Rolling cir-cle replication, used by many plasmids and phages, generates single-stranded DNA (ssDNA). In many cases, the presence of an sso enhances the conversion of the ssDNA to double-stranded DNA (dsDNA) by enabling priming of synthesis of the second DNA strand. We initially identified sso1 in ICEBs1 based on sequence similarity to the sso of an RCR plasmid. Several functional assays confirmed Sso activity. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1) mainte-nance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2) stable acquisition of ICEBs1 following transfer to a new host. We identified sequences simi-lar to known plasmid sso's in several other ICEs. Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements. Author Summary Mobile genetic elements facilitate movement of genes, including those conferring antibi-otic resistance and other traits, between bacteria. Integrative and conjugative element