Specificity and selective advantage of an exclusion system in the integrative and conjugative element ICEBs1ofBacillus subtilis

Abstract

Integrative and conjugative elements (ICEs) are mobile genetic elements capable of transferring their own and other DNA. They contribute to the spread of antibiotic resistances and other important traits for bacterial evolution. Exclusion is a mechanism used by many conjugative plasmids and a few ICEs to prevent their host cell from acquiring a second copy of the cognate element. ICEBs1 of Bacillus subtilis has an exclusion mechanism whereby the exclusion protein YddJ in a potential recipient inhibits the activity of the ICEBs1-encoded conjugation machinery in a potential donor. The target of YddJ-mediated exclusion is the conjugation protein ConG (a VirB6 homolog). Here we defined the regions of YddJ and ConG that confer exclusion specificity and determined the importance of exclusion to host cells. Using chimeras that had parts of ConG from ICEBs1 and the closely related ICEBat1 we identified a putative extracellular loop of ConG that conferred specificity for exclusion by the cognate YddJ. Using chimeras of YddJ from ICEBs1 and ICEBat1 we identified two regions in YddJ needed for exclusion specificity. We also found that YddJ-mediated exclusion reduced death of donor cells following conjugation into recipients. Donor death was dependent on the ability of transconjugants to themselves become donors and was reduced under osmo-protective conditions, indicating that death was likely due to alterations in the donor cell envelope caused by excessive conjugation. We postulate that elements that can have high frequencies of transfer likely evolved exclusion mechanisms to protect the host cells from excessive death.ImportanceHorizontal gene transfer is a driving force in bacterial evolution, responsible for the spread of many traits, including antibiotic and heavy metal resistances. Conjugation, one type of horizontal gene transfer, involves DNA transfer from donor to recipient cells through conjugation machinery and direct cell-cell contact. Exclusion mechanisms allow conjugative elements to prevent their host from acquiring additional copies of the element, and are highly specific enabling hosts to acquire heterologous elements. We defined regions of the exclusion protein and its target in the conjugation machinery that convey high specificity of exclusion. We found that exclusion protects donors from cell death during periods of high transfer. This is likely important for the element to enter new populations of cells.

Biology and engineering of integrative and conjugative elements: Construction and analyses of hybrid ICEs reveal element functions that affect species-specific efficiencies

“…ICE Bs1 (∼20.5 kb) of B. subtilis encodes conjugation and replication machinery homologous to that of Tn 916 . ICE Bs1 has only naturally been observed in B. subtilis , although related sequences are found in several other Bacillus species (Avello et al, 2019; Davis and Grossman, 2021). ICE Bs1 transfers into a variety of other Bacillus species (including B. anthracis, B. cereus, B. thuringiensis ) and also a variety of Enterococcus, Streptococcus , and Listeria species (Auchtung et al, 2005; Brophy et al, 2018; DeWitt and Grossman, 2014).…”