FIG. 1.

A simplified depiction of gram-negative bacterial conjugation and entry exclusion by TraG in F-like T4SSs. (a) Donor bacteria possessing F-like plasmids coding for conjugative T4SS are piliated and will repeatedly extend and retract conjugative pili to contact a neighboring bacterium.¹⁰¹ (b) Pilus retraction occurs when a neighboring recipient bacterium's cell wall has been contacted by the pilus tip of the donor cell, allowing for adhesion.¹⁰² (c) Donor and recipient bacteria are brought into close proximity, and after surface and entry exclusion steps, a mating junction forms resulting in the fusion of lipid bilayers. The synchronous transfer and replication of a single strand of nicked plasmid DNA from the donor to recipient is then mediated by the relaxasome (pink) and transferosome (purple).⁶ Rolling circle replication by DNA replication machinery (light blue) in the recipient allows for synthesis of the complementary strand of the plasmid DNA. (d) The conjugative transfer event ends after the single-stranded DNA is transferred to the recipient cell and the mating junction has closed. The recipient cell, following synthesis of the complementary strand of the transferred plasmid DNA, is now a donor cell for the conjugative plasmid. (e) In a colony of bacteria carrying plasmids with identical transfer (tra) regions, conjugation between donor cells is prevented through entry exclusion (Eex), proposedly in the event of faulty surface exclusion.^32,100 The formation of a complete mating pore and the transfer of redundant plasmid DNA is prevented through interaction of TraG in the donor cell with a cognate TraS embedded in the recipient's IM. [Figures (a)–(e) made with Biorender]. (f) Generalized domain organization of TraG. The N-terminal transmembrane region of the protein responsible for aiding in pilus assembly is colored in magenta and the C-terminal periplasmic region termed TraG* is colored in green. These regions are separated by a signal I peptidase cleavage site at residue A451.³² The 45-residue truncation mutant construct is labeled as _ΔTraG* and contains the region responsible for interaction with TraS. The non-homologous region, which differs between TraG_F and TraG_R100, is responsible for Eex. The region shaded in light green from A452 to A496 represents a flexible linker region between the two domains.