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

Abstract

F plasmids circulate widely among the Enterobacteriaceae through encoded type IV secretion systems (T4SSFs). Assembly of T4SSFs 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 T4SSFs 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 T4SSF 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 T4SSF‐associated activities, (ii) TraU, TraN, and TrbC are nonessential but contribute significantly to distinct T4SSF 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 T4SSF assembly or activation to regulate DNA transfer and F pilus dynamics and deposition onto alternative platforms.

Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machines

“…As our previous assays, e.g., phage infection, only indirectly tested for F pilus production, we additionally deployed a sensitive fluorescent labeling procedure that enabled direct visualization of ED208 pili [25]. Strains of interest were engineered to produce Cys-derivatized TraA ED pilins (TraA.C116), shown previously to support pED208 transfer at WT levels [25]. The Cys118 residues are surface-exposed on ED208 pili and accessible to labeling with fluorescent maleimide conjugates, e.g., FM488-mal.…”

“…Deletions of TraF, TraH, or TraW rendered the pED208 system completely nonfunctional ( Fig. 5 ) [25] Swaps of F-encoded TraF, TraH, and TraW for their pED208 counterparts failed to restore functionality of the pED208 system, as shown by lack of DNA transfer, resistance to M13K07 infection, and absence of ED208 pili ( Fig. 5A,B, S3D ).…”

“…F and pED208 mutations. E. coli strain HME45 were used to generate deletions of the tra genes from pED208 or pOX38 (the F plasmid) by recombineering as previously described [25,73]. A FRT-Kan r -FRT cassette from pKD13 was amplified with primers listed in Table S2 to carry homologous sequence to the upstream and downstream regions of the region targeted for deletion.…”

“…The structures, coupled with other biochemical and genetic findings, also predict regions of conformational flexibility that might be important for structural transitions associated with machine activation. These inherently flexible regions include the T4CP -channel interface, as suggested by difficulties in isolation of stable T4CP -channel complexes and recent evidence for spatial repositioning of VirD4-like TrwB to polar regions phylogenetically distantly-related F-like plasmids, classical F and pED208 [23][24][25]. F is designated as IncF1B/MOB F12 group A and pED208 and its progenitor plasmid F 0 -lac are IncFV/MOB F12 group C plasmids [26].…”

Capsules and their traits shape phage susceptibility and plasmid conjugation efficiency

“…The three most prevalent MPF types of plasmids in Proteobacteria are MPFF (named after plasmid F), MPFT (named after the Ti plasmid) and MPFI (named after the IncI R64 plasmid) [38]. While MPFF conjugative pili are typically long (up to 20 µm) [39,40], flexible and retractable [41], MPFT and MPFI are shorter and rigid (<1 µm) [41][42][43][44]. Even though there is very little published information on the effect of capsules on conjugation, we showed recently that conjugation of one plasmid from a non-capsulated Escherichia coli strain to different Klebsiella strains was higher in ΔwcaJ mutants which do not produce capsules than in the capsulated wild type strains [12].…”