DNA TRANSLOCATION ACROSS PLANAR BILAYERS CONTAINING BACILLUS-SUBTILISION CHANNELS

The mechanisms by which genetic material crosses prokaryotic membranes are incompletely understood. We have developed a new methodology to s tudy the translocation of genetic material via pores in a reconstitute d system, using techniques from electrophysiology and molecular biolog y. We report here that planar bilayer membranes become permeable to do uble-stranded DNA (kilobase range) if Bacillus subtilis membrane vesic les containing high conductance channels have been fused into them. Th e translocation is an electrophoretic process, since it does not occur if a transmembrane electrical field opposing the movement of DNA, a p olyanion, is applied. It is not an aspecific permeation through the ph ospholipid bilayer, since it does not take place if no proteins have b een incorporated into the membrane. The transport is also not due simp ly to the presence of polypeptides in the membrane, since it does not occur if the latter contains gramicidin A or a eukaryotic, multiprotei n vesicle fraction exhibiting 30-picosiemens anion-selective channel a ctivity, The presence of DNA alters the behavior of the bacterial chan nels, indicating that it interacts with the pores and may travel throu gh their lumen, These results support the idea that DNA translocation may lake place through proteic pores and suggest that some of the high conductance bacterial channels observed in electrophysiological exper iments may be constituents of the DNA translocating machinery in these organisms.