Functional Dissection of the Conjugative Coupling Protein TrwB

Abstract

The conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We designed a conjugation assay in which the frequency of DNA transfer is directly proportional to the amount of TrwB. A collection of point mutants was constructed in the TrwB cytoplasmic domain on the basis of the crystal structure of TrwB⌬N70, targeting the nucleotide triphosphate (NTP)-binding region, the cytoplasmic surface, or the internal channel in the hexamer. An additional set of transfer-deficient mutants was obtained by random mutagenesis. Most mutants were impaired in both DNA and protein transport. We found that the integrity of the nucleotide binding domain is absolutely required for TrwB function, which is also involved in monomer-monomer interactions. Polar residues surrounding the entrance and inside the internal channel were important for TrwB function and may be involved in interactions with the relaxosomal components. Finally, the N-terminal transmembrane domain of TrwB was subjected to random mutagenesis followed by a two-hybrid screen for mutants showing enhanced protein-protein interactions with the related TrwE protein of Bartonella tribocorum. Several point mutants were obtained with mutations in the transmembranal helices: specifically, one proline from each protein may be the key residue involved in the interaction of the coupling protein with the type IV secretion apparatus.

Transfer of R388 Derivatives by a Pathogenesis-Associated Type IV Secretion System into both Bacteria and Human Cells

“…4B). The same result was obtained when we assayed the TrwB P18S mutant, lying in the transmembranal domain of TrwB, which shows stronger interactions with B. tribocorum TrwE while maintaining its conjugative functions (10). Thus, TrwB, although not absolutely essential, is involved in DNA transfer to human cells, requiring the TrwB region involved in T4SS interaction.…”

“…Next, we assayed the mobilization of plasmid pHP132, carrying the R388 Dtr region cloned into a Bartonella-replicating vector, by the chromosomally encoded T4SS of B. tribocorum, into E. coli. No transconjugants were obtained (Table 6) either by expressing the R388 T4SS from the recipient cell or by assaying the mutation in TrwB shown to increase its interaction with the B. tribocorum TrwE homologue (P18S) (10).…”

“…Recently, a mutagenesis analysis of this protein has shown that the transmembranal region is involved in interaction with TrwE. Several point mutations in this region were shown to increase this interaction with the Bartonella TrwE homologue, without affecting its functionality in conjugative DNA transfer (10).…”

“…The amount of TrwB and TrwC in the cells was estimated by Western blotting of total protein extracts, as described previously (10). E. coli D1210 cells containing the indicated plasmids were grown overnight.…”

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

“…Although a lot of information has been gathered over the years about the activity, oligomerization state and structure as well as the importance of VirD4 with respect to substrate recognition and processing, its location within the T4S system was unknown (Cascales & Christie, 2004; Mihajlovic et al , 2009; Lang et al , 2010; de Paz et al , 2010; Whitaker et al , 2016). This is the issue we set out to address in this study.…”