Subcellular location of the coupling protein TrwB and the role of its transmembrane domain

Conjugation is the most important mechanism for horizontal gene transfer and it is the main responsible for the successful adaptation of bacteria to the environment. Conjugative plasmids are the DNA molecules transferred and a multiprotein system encoded by the conjugative plasmid itself is necessary. The high number of proteins involved in the process suggests that they should have a defined location in the cell and therefore, they should be recruited to that specific point. One of these proteins is the coupling protein that plays an essential role in bacterial conjugation. TrwB is the coupling protein of R388 plasmid that is divided in two domains: i) The N-terminal domain referred as transmembrane domain and ii) a large cytosolic domain that contains a nucleotide-binding motif similar to other ATPases. To investigate the role of these domains in the subcellular location of TrwB, we constructed two mutant proteins that comprised the transmembrane (TrwBTM) or the cytoplasmic (TrwBΔN70) domain of TrwB. By immunofluorescence and GFP-fusion proteins we demonstrate that TrwB and TrwBTM mutant protein were localized to the cell pole independently of the remaining R388 proteins. On the contrary, a soluble mutant protein (TrwBΔN70) was localized to the cytoplasm in the absence of R388 proteins. However, in the presence of other R388-encoded proteins, TrwBΔN70 localizes uniformly to the cell membrane, suggesting that interactions between the cytosolic domain of TrwB and other membrane proteins of R388 plasmid may happen. Our results suggest that the transmembrane domain of TrwB leads the protein to the cell pole.This work was supported by funds from the Spanish Ministry of Economy (Grant No BFU2012-36241), from MICINN (Grant No. BFU2010-22103) and from EC FP6 Program (LSHM-CT-2005-019023). R.L.S. was a postdoctoral scientist supported by CSIC I3P and Basque Government postdoctoral fellowships. A.J.V was a postdoctoral scientist supported by the University of the Basque Country. S.A-A. was a pre-doctoral student supported by the Basque Government. B.U-U. was supported by a pre-doctoral fellowship from the University of the Basque Country.Peer Reviewe