Contributions of F‐specific subunits to the F <scp>plasmid‐encoded</scp> type <scp>IV</scp> secretion system and F pilus

Abstract

F plasmids circulate widely among the Enterobacteriaceae through encoded type IV secretion systems (T4SS_F s). Assembly of T4SS_F s and associated F pili requires 10 VirB/VirD4-like Tra subunits and eight or more F-specific subunits. Recently, we presented evidence using in situ cryoelectron tomography (cryoET) that T4SS_F s undergo structural transitions when activated for pilus production, and that assembled pili are deposited onto alternative basal platforms at the cell surface. Here, we deleted eight conserved F-specific genes from the MOBF12C plasmid pED208 and quantitated effects on plasmid transfer, pilus production by fluorescence microscopy, and elaboration of T4SS_F structures by in situ cryoET. Mutant phenotypes supported the assignment of F-specific subunits into three functional Classes: (i) TraF, TraH, and TraW are required for all T4SS_F -associated activities, (ii) TraU, TraN, and TrbC are nonessential but contribute significantly to distinct T4SS_F functions, and (iii) TrbB is essential for F pilus production but not for plasmid transfer. Equivalent mutations in a phylogenetically distantly related MOB12A F plasmid conferred similar phenotypes and generally supported these Class assignments. We present a new structure-driven model in which F-specific subunits contribute to distinct steps of T4SS_F assembly or activation to regulate DNA transfer and F pilus dynamics and deposition onto alternative platforms.