Uptake of environmental DNA in Bacillus subtilis occurs all over the cell surface through a dynamic pilus structure

At the transition to stationary phase, a subpopulation of Bacillus subtilis cells can enter the developmental state of competence, where DNA is taken up through the cell envelope, is processed to single stranded DNA, which is incorporated into the genome if sufficient homology between sequences exists. We show here that the initial step of transport across the cell wall occurs via a true pilus structure, with an average length of about 500 nm, which assembles at various places on the cell surface. Once assembled, the pilus remains at one position and can be retracted in a time frame of seconds. The major pilin, ComGC, was studied at a single molecule level in live cells. ComGC was found in two distinct populations, one that would correspond to ComGC freely diffusing throughout the cell membrane, and one that is relatively stationary, likely reflecting pilus-incorporated molecules. The ratio of 65% diffusing and 35% stationary ComGC changed towards more stationary molecules upon addition of external DNA, while the number of pili in the population did not strongly increase. These findings suggest that the pilus assembles stochastically, but engages more pilin monomers from the membrane fraction in the presence of transport substrate. Our data reveal that localized transport of DNA into the cytosol at a single cell pole is preceded by localization throughout the periplasm and efficient uptake through the entire cell surface by a dynamic pilus. Importance Horizontal gene transfer (HGT) is important for bacterial evolution and the adaption to new environments. Through HGT, genes can be transferred between bacteria, which can lead to antibiotic resistance development, especially critical with regards to pathogenic bacterial species. One mechanism of HGT is natural competence, through which bacteria can take up DNA under natural conditions and incorporate it into their chromosomes. For DNA uptake, a complex will be formed that is conserved among Gram-positive and Gram-negative bacteria, in spite of different envelope architectures. We show that in Bacillus subtilis , where the part of the uptake complex spanning the cell membrane localizes to a single cell pole, transport through the cell wall is mediated by a visible pilus structure that can extend and retract to pull DNA into the periplasm. Thus, DNA uptake from the environment is not limited to polar sites, but is channelled into the cytosol at the pole. Single molecule dynamics of ComGC revealed a static, likely pilus-bound, and a mobile fraction of ComGC within the cell membrane. Addition of environmental DNA increased the static fraction of molecules, showing that pilus dynamics respond to DNA binding. ### Competing Interest Statement The authors have declared no competing interest.