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, and 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 molecules 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 support a model in which transport of environmental DNA occurs through the entire cell surface by a dynamic pilus, mediating efficient uptake through the cell wall into the periplasm, where DNA diffuses to a cell pole containing the localized transport machinery mediating passage into the cytosol.

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.

The Bacillus subtilis dCMP deaminase ComEB acts as a dynamic polar localization factor for ComGA within the competence machinery.

Bacillus subtilis can import DNA from the environment by an uptake machinery that localizes to a single cell pole. We investigated the roles of ComEB and of the ATPase ComGA during the state of competence. We show that ComEB plays an important role during competence, possibly because it is necessary for the recruitment of GomGA to the cell pole. ComEB localizes to the cell poles even upon expression during exponential phase, indicating that it can serve as polar marker. ComEB is also a deoxycytidylate monophosphate (dCMP) deaminase, for the function of which a conserved cysteine residue is important. However, cysteine-mutant ComEB is still capable of natural transformation, while a comEB deletion strain is highly impaired in competence, indicating that ComEB confers two independent functions. Single-molecule tracking (SMT) reveals that both proteins exchange at the cell poles between bound and unbound in a time scale of a few milliseconds, but turnover of ComGA increases during DNA uptake, whereas the mobility of ComEB is not affected. Our data reveal a highly dynamic role of ComGA during DNA uptake and an unusual role for ComEB as a mediator of polar localization, localizing by diffusion-capture on an extremely rapid time scale and functioning as a moonlighting enzyme.