Structural basis of cooperative DNA recognition by the plasmid conjugation factor, TraM

The conjugative transfer of F-like plasmids such as F, R1, R100 and pED208, between bacterial cells requires TraM, a plasmid-encoded DNA-binding protein. TraM tetramers bridge the origin of transfer (oriT) to a key component of the conjugative pore, the coupling protein TraD. Here we show that TraM recognizes a high-affinity DNA-binding site, sbmA, as a cooperative dimer of tetramers. The crystal structure of the TraM–sbmA complex from the plasmid pED208 shows that binding cooperativity is mediated by DNA kinking and unwinding, without any direct contact between tetramers. Sequence-specific DNA recognition is carried out by TraM’s N-terminal ribbon–helix–helix (RHH) domains, which bind DNA in a staggered arrangement. We demonstrate that both DNA-binding specificity, as well as selective interactions between TraM and the C-terminal tail of its cognate TraD mediate conjugation specificity within the F-like family of plasmids. The ability of TraM to cooperatively bind DNA without interaction between tetramers leaves the C-terminal TraM tetramerization domains free to make multiple interactions with TraD, driving recruitment of the plasmid to the conjugative pore

Diversitaet und Gentransfer in denitrifizierenden Bakteriengemeinschaften. Teilprojekt: Analyse des Gentransferpotentials fuer Konjugation und Transformation Abschlussbericht

SIGLEAvailable from TIB Hannover: F00B1241 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

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

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