Single-Molecule Dynamics of DNA Receptor ComEA, Membrane Permease ComEC, and Taken-Up DNA in Competent Bacillus subtilis Cells.

In competent Gram-negative and Gram-positive bacteria, double-stranded DNA is taken up through the outer cell membrane and/or the cell wall and is bound by ComEA, which in Bacillus subtilis is a membrane protein. DNA is converted to single-stranded DNA and transported through the cell membrane via ComEC. We show that in Bacillus subtilis, the C terminus of ComEC, thought to act as a nuclease, is important not only for DNA uptake, as judged from a loss of transformability, but also for the localization of ComEC to the cell pole and its mobility within the cell membrane. Using single-molecule tracking, we show that only 13% of ComEC molecules are statically localized at the pole, while 87% move throughout the cell membrane. These experiments suggest that recruitment of ComEC to the cell pole is mediated by a diffusion/capture mechanism. Mutation of a conserved aspartate residue in the C terminus, likely affecting metal binding, strongly impairs transformation efficiency, suggesting that this periplasmic domain of ComEC could indeed serve a catalytic function as a nuclease. By tracking fluorescently labeled DNA, we show that taken-up DNA has a similar mobility as a protein, in spite of being a large polymer. DNA dynamics are similar within the periplasm as those of ComEA, suggesting that most taken-up molecules are bound to ComEA. We show that DNA can be highly mobile within the periplasm, indicating that this subcellular space can act as reservoir for taken-up DNA, before its entry into the cytosol. IMPORTANCE Bacteria can take up DNA from the environment and incorporate it into their chromosome, which is termed "natural competence" that can result in the uptake of novel genetic information. We show that fluorescently labeled DNA moves within the periplasm of competent Bacillus subtilis cells, with similar dynamics as DNA receptor ComEA. This finding indicates that DNA can accumulate in the periplasm, likely bound by ComEA, and thus can be stored before uptake at the cell pole, via integral membrane DNA permease ComEC. Assembly of ComEC at the cell pole likely occurs by a diffusion-capture mechanism. DNA uptake into cells thus takes a detour through the entire periplasm and involves a high degree of free diffusion along and within the cell membrane.