Requirements for TrwC Recruitment And Translocation by Its Type IV Secretion System

Abstract

Bacterial Type IV secretion systems (T4SS) are involved in conjugative DNA transfer and effector translocation. Current knowledge about substrates recruitment pathways by the T4SS remains poorly understood. Evidence has been generated for some relaxases that harbor internal translocation signals, independently mediating efficient recognition by their cognate T4SS, among which it is found TrwC, the R388 conjugative relaxase.. Beyond translocation signals, additional elements might be required to drive recruitment. An in vivo validation of the presumptive interaction between the translocation signal fragment of TrwC, and its coupling protein, conceived to be the substrate receptor, was not achieved by means of a two-hybrid assay. Surprising is the observed interaction of this fragment with the full-length protein, and its self-interaction, albeit correlates with a previously manifested oligomerization ability of TrwC under the assayed conditions. Also, a two-hybrid approach to reveal in vivo interactions with the T4SS conduit was set up, and preliminary results against the translocation signal of TrwC have been obtained. Here, further proof that DNA substrate is not imperative for TrwC translocation is shown, underscoring T4SS as ancestral machines devoted for protein translocation. Forthcoming research will characterize and validate such interactions. In addition, a higher resolution library will be constructed to narrowly determine interaction domains. Similarly, a prey library derived from the coupling protein could be assayed against different sized fragments of TrwC as the bait. Finally, biochemical assays would certainly add value to this research.