Autonomous replication of integrative and conjugative elements

Abstract

Mobile genetic elements facilitate movement of genes, including those conferring antibiotic resistance and other traits, between bacteria. Integrative and conjugative elements (ICEs), also known as conjugative transposons, are a large family of mobile genetic elements that can transfer between neighboring cells. ICEs are found integrated in the chromosome of their host bacterium, where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICE DNA will excise and form a circular plasmid-like intermediate. It was previously thought that ICEs were incapable of autonomous replication. However, my research, along with the work of others, shows that ICEs can replicate autonomously, and that many ICEs utilize a rolling circle replication mechanism. Plasmids and phages that use rolling circle replication encode a single strand origin (sso) that enhances priming of DNA synthesis. We identified a functional single strand origin, sso1, in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. Sso activity was important for autonomous, rolling circle replication of ICEBs1 in host cells, and for stable acquisition of the element in new host cells. I also showed that the broad-host range ICE Tn916 replicates autonomously by a rolling circle mechanism. Replication of Tn916 was dependent on the relaxase encoded by Tn916 orf20. The origin of transfer of Tn916, oriT(916), also functioned as an origin of replication. I found that the relaxase (Orf20) and the two putative helicase processivity factors (Orf22 and Orf23) encoded by Tn916 likely interact in a complex to facilitate replication. Lastly, I identified a functional single strand origin of replication (sso) in Tn916 that I predict primes second strand synthesis during rolling circle replication. The importance of autonomous replication by rolling circle in the ICE lifecycle and horizontal gene transfer processes is discussed.