Critical Components of the Conjugation Machinery of the Integrative and Conjugative Element ICE Bs1 of Bacillus subtilis

Abstract

Conjugation, or mating, plays a profound role in bacterial evolution by spreading genes that allow bacteria to adapt to and colonize new niches. ICEBs1, an integrative and conjugative element of Bacillus subtilis, can transfer itself and mobilize resident plasmids. DNA transfer is mediated by a type IV secretion system (T4SS). Characterized components of the ICEBs1 T4SS include the conserved VirB4-like ATPase ConE, the bifunctional cell wall hydrolase CwlT, and the presumed VirD4-like coupling protein ConQ. A fusion of ConE to green fluorescent protein (GFP) localizes to the membrane preferentially at the cell poles. One or more ICEBs1 proteins are required for ConE's localization at the membrane, as ConE lacks predicted transmembrane segments and ConE-GFP is found dispersed throughout the cytoplasm in cells lacking ICEBs1. Here, we analyzed five ICEBs1 genes to determine if they are required for DNA transfer and/or ConE-GFP localization. We found that conB, conC, conD, and conG, but not yddF, are required for both ICEBs1 transfer and plasmid mobilization. All four required genes encode predicted integral membrane proteins. conB and, to some extent, conD were required for localization of ConE-GFP to the membrane. Using an adenylate cyclase-based bacterial two-hybrid system, we found that ConE interacts with ConB. We propose a model in which the ICEBs1 conjugation machinery is composed of ConB, ConC, ConD, ConE, ConG, CwlT, ConQ, and possibly other ICEBs1 proteins, and that ConB interacts with ConE, helping to recruit and/or maintain ConE at the membrane. IMPORTANCEConjugation is a major form of horizontal gene transfer and has played a profound role in bacterial evolution by moving genes, including those involved in antibiotic resistance, metabolism, symbiosis, and infectious disease. During conjugation, DNA is transferred from cell to cell through the conjugation machinery, a type of secretion system. Relatively little is known about the conjugation machinery of Gram-positive bacteria. Here, we analyzed five genes of the integrative and conjugative element ICEBs1 of Bacillus subtilis. Our research identifies four new components of the ICEBs1 conjugation machinery (ConB, ConC, ConD, and ConG) and shows an interaction between ConB and ConE that is required for ConE to associate with the cell membrane. Conjugation is a major form of horizontal gene transfer and plays a profound role in bacterial evolution and the acquisition of new traits (1-3). Conjugation can spread antibiotic resistance and disseminate genes involved in symbiosis, degradation of pollutants, metabolism, and pathogenesis. Conjugative elements encode specialized DNA translocation channels classified as type IV secretion systems (T4SSs) (4-7). T4SSs are composed of many interacting proteins that span the envelope of the donor cell. In addition to transferring the conjugative DNA element, the conjugation machinery also can mobilize resident plasmids or other DNA elements that do not encode their own machinery.There is a rich body of mechanis...

Identification, characterization and benefits of an exclusion system in an integrative and conjugative element of Bacillus subtilis

“…We found that both conG ICEBs1 and conG ICEBat1 complemented the transfer defect caused by the loss of conG in ICEBs1. As determined previously, conG ICEBs1 fully restored transfer to wild type levels (Babic et al, 2011;Leonetti et al, 2015). conG ICEBat1 largely restored conjugation, but to levels ~10-fold lower than those with con-G ICEBs1 (Fig.…”

“…The donor strain MA980 contains the peptidase deletion of cwlT , cwlT Δ( 207‐327 ), described previously (Dewitt and Grossman, ). Donor strains containing a deletion of conG , Δ conG ( 5‐805 ) were derived from MMB1283 (Leonetti et al , ). Donor strains containing a deletion of conQ , Δ conQ848 , were derived from CAL848 (Lee et al , ).…”

“…conG was amplified by PCR from B. subtilis or B. atrophaeus genomic DNA and fused to Pspank(hy) essentially as described (Leonetti et al , ). Constructs included Pspank(hy)‐ conG ICE Bs1 , present in the strain KPD225 and Pspank(hy)‐ conG ICE Bat1 , present in strain KPD224.…”

“…Transformants were screened for the loss of lacZ, indicating the loss of the integrated plasmid and PCR was used to identify a clone that contained the ΔyddJ allele. (Leonetti et al, 2015). Constructs included Pspank(hy)-conG ICEBs1 , present in the strain KPD225 and Pspank(hy)-conG ICEBat1 , present in strain KPD224.…”

“…The conjugation machinery encoded by ICEBs1 is a type IV secretion system (T4SS) that resembles other well-characterized T4SSs encoded by conjugative elements found in Gram-positive bacteria, such as pCW3, pIP501 and pCF10 (Goessweiner-Mohr et al, 2014;Guglielmini, et al, 2014;Leonetti et al, 2015). The T4SS of ICEBs1 is classified as a member of the MPF FA class of T4SSs, which are found in Firmicutes and Actinobacteria and do not contain dedicated adhesion proteins (Guglielmini et al, 2014).…”