Abstract: Type IV secretion systems (T4SS) translocate DNA and protein substrates across the cell envelope of bacteria to a widely distributed number of eukaryotic and prokaryotic target cells. Conjugative bacteria use T4SS to mediate the transfer of DNA and proteins to recipient cells. T4SS are macromolecular assemblies composed of 11 mating pair subunits (VirB1 to VirB11) and a coupling protein (VirD4) that span inner and outer bacterial membranes. Three of these subunits are ATPases that energize DNA and protein substrate transfer as well as pilus assembly. VirB4 proteins are the largest and most evolutionarily conserved proteins in T4SS and are essential for virulence and plasmid transfer.VirB4 proteins have been suggested to energize substrate translocation across the T4SS. Recently, we demonstrated that TrwK, the VirB4 homologue in the R388 conjugative system, is able to hydrolyze ATP in the absence of potential substrates. The atomic structure of VirB4 proteins is unknown. Based on computing predictions, a model of the C-terminal (residues 413 to 772) of Escherichia coli TrwK has been suggested using the atomic coordinates of the coupling protein TrwB of plasmid R388 as a template. Secondary structure predictions of TrwK and TrwB revealed the presence of three ?-helices in the C-terminus that are conserved in all VirB4 proteins but are absent in TrwB. Therefore, we decided to generate truncated variants of TrwK where these ?-helical structures were sequentially removed and we have analyzed their in vitro and in vivo properties. The results suggest that the C-terminal end of VirB4 proteins could play a key functional regulatory role in the biological activity of T4SS.

Autoría

Descargar referencia