Conjugative coupling proteins interact with cognate and heterologous VirB10-like proteins while exhibiting specificity for cognate relaxosomes

Abstract

Conjugative coupling proteins (CPs) are proposed to play a role in connecting the relaxosome to a type IV secretion system (T4SS) during bacterial conjugation. Here we present biochemical and genetic evidence indicating that the prototype CP, TrwB, interacts with both relaxosome and type IV secretion components of plasmid R388. The cytoplasmic domain of TrwB immobilized in an affinity resin retained TrwC and TrwA proteins, the components of R388 relaxosome. By using the bacterial two-hybrid system, a strong interaction was detected between TrwB and TrwE, a core component of the conjugative T4SS. This interaction was lost when the transmembrane domains of either TrwB or TrwE were deleted, thus suggesting that it takes place within the membrane or periplasmic portions of both proteins. We have also analyzed the interactions with components of the related IncN plasmid pKM101. Its CP, TraJ, did not interact with TrwA, suggesting a highly specific interaction with the relaxosome. On the other side, CPs from three different conjugation systems were shown to interact with both their cognate TrwE-like component and the heterologous ones, suggesting that this interaction is less specific. Mating experiments among the three systems confirmed that relaxosome components need their cognate CP for transfer, whereas T4SSs are interchangeable. As a general rule, there is a correlation between the strength of the interaction seen by two-hybrid analysis and the efficiency of transfer.

Fig. 1.

Protein-protein interactions detected by affinity chromatography. Soluble lysates containing GST fusion proteins were bound to glutathione-Sepharose resin, incubated with 20 μg of the indicated added proteins, and either eluted with glutathione (a and d) or cleaved with factor Xa protease (b and c). The figure shows 10% (a) or 12% (b-d) SDS/PAGE Coomassie-stained gels of eluted proteins. Molecular weight markers shown are 97.4 (not seen in b and c), 66, 45, 31, 21.5 (not seen in a), and 14.4 kDa (not seen in a). Lanes in a: 1, markers; 2, 2 μg of purified TrwC; 3-6, resin eluates; 3 and 5, lysates from pMTX501 (containing GST-TrwBΔN75); 4 and 6, lysates from pGEX-3X (containing GST); 3 and 4, bound proteins incubated with TrwC; 5 and 6, bound proteins incubated with BSA; 7, 1 μg of purified BSA. Lanes in b:1-4, Xa cleavage products; 1, lysates from pGEX-3X; 2-4, lysates from pMTX501; 1 and 2, incubation with TrwAh; 3, incubation with TrwAhΔN35; 4, incubation with TrwAhN73; 5, markers; 6, 2 μg of purified TrwAh. Arrowheads point to the two TrwB fragments obtained by factor Xa digestion. (c) TrwB-TrwA reciprocal interaction by affinity chromatography. Eluates from either glutathione-Sepharose resin by factor Xa digestion (lanes 1 and 2) or from Ni-NTA columns (lanes 3-5). Lanes: 1, TrwBΔN75 + TrwAh; 2, TrwBΔN75 + BSA; 3, eluate from lane 1 loaded on a Ni-NTA column; 4 and 5, Ni-NTA columns preloaded with TrwAh and then eluates from Xa digestions added (lane 4, from GST + TrwAh; lane 5, from TrwBΔN75 + BSA); 6, markers. Lanes in d: 1, markers; 2-4, eluates from lysates incubated with TrwAh; 2, pMTX609 (containing GST-TraJΔN76); 3, pMTX501; 4, pGEX-3X; 5, 1 μg of purified TrwAh.

Fig. 2.

In vivo protein-protein interactions measured by using the bacterial two-hybrid assay. The indicated plasmid pairs were introduced in strain DHM1 in the absence of other conjugation functions, and β-galactosidase units were measured. Only the most representative results are shown. All other combinations between R388 proteins were tested, and they gave background levels (<30 β-galactosidase units). Tested pairs are cited by the proteins fused to T18-T25. C+, positive control [pT18zip + pT25zip (36)]; C-, negative control (pT18zip + pT25).