Conjugative plasmid transmission leads to an accelerated propagation of bacteria with multiple antibiotic resistances. In order to understand gram-positive conjugation on a molecular level, we are working on the functional characterization of individual transfer proteins derived from the conjugative plasmid pIP501. The respective transfer region is arranged in a single operon encoding fifteen putative transfer proteins which resemble a simplified type IV secretion system (T4SS) [1, 2]. So far, we could determine the high resolution structures of TraK, TraN as well as the C-terminal domain of TraM which exhibits a VirB8-like fold [3, 4]. Low resolution SAXS data were collected for soluble truncations of TraH and TraG, for the latter peptidoglycan degrading activity has been demonstrated thereby facilitating insertion of the T4SS machinery in the bacterial cell envelope [5].
Within this work, different biophysical and biochemical methods have been employed to functionally characterize the N–terminal domain of the bitopic protein TraM (TraMΔN) and the soluble truncations of TraF. The proteins have been successfully expressed, purified and biophysically characterized. The solution structure of the N-terminal domain was determined through SAXS; further NMR experiments are being conducted. Preliminary data has shown that TraMΔN binds to DNA, suggesting a direct involvement in the conjugative transfer of the T-DNA strand. Currently, NMR experiments are carried out and DNA band-shift experiments are performed to elucidate the functional aspects of TraMΔN. Crystallization trials are carried out in parallel for TraMΔN and TraF to determine their structure.