Abstract
We identified a functional single strand origin of replication (sso) in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons) are DNA elements typically found integrated into a bacterial 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 circle 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) maintenance 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 similar 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.
Highlights
Horizontal gene transfer, the ability of cells to acquire DNA from exogenous sources, is a driving force in bacterial evolution, facilitating the movement of genes conferring antibiotic resistance, pathogenicity, and other traits [1]
We found that ICEBs1, from Bacillus subtilis, contains at least two regions that enable conversion of single-stranded DNA (ssDNA) to double-stranded DNA
[31] ICEBs1 for sequences that are similar to known sso's from plasmids that replicate in B. subtilis by rolling circle replication
Summary
Horizontal gene transfer, the ability of cells to acquire DNA from exogenous sources, is a driving force in bacterial evolution, facilitating the movement of genes conferring antibiotic resistance, pathogenicity, and other traits [1]. Conjugation, a form of horizontal gene transfer, is the contact-dependent transfer of DNA from a donor to a recipient, generating a transconjugant. The DNA to be transferred is processed and protein machinery in the donor mediates transfer to the recipient. The proteins involved in DNA processing and conjugation are encoded by a conjugative element. Integrative and conjugative elements (ICEs, called conjugative transposons), appear to be more prevalent than conjugative plasmids [2]. The conjugation machinery (a type IV secretion system) encoded by ICEs is homologous to that of conjugative plasmids, and much of what is known about the mechanisms of transfer have come from studies of conjugative plasmids [3,4]. The defining feature of ICEs that distinguish them from conjugative plasmids is that ICEs are typically found integrated into a host chromosome and are passively propagated during chromosomal replication and cell division
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