Single molecule tracking of ComGC^CYS with Alexa-Fluor C5-maleimide treatment, with/ and without addition of DNA with an incubation time of 10 minutes.

Single molecule tracking of ComGCCYS with Alexa-Fluor C5-maleimide treatment, with/ and without addition of DNA with an incubation time of 10 minutes.</p

Time lapse (20 second intervals) of pilus structures of AF488-C5 maleimide stained <i>B</i>. <i>subtilis</i> cells grown to competence expressing ComGC^CYS.

Time lapse (20 second intervals) of pilus structures of AF488-C5 maleimide stained B. subtilis cells grown to competence expressing ComGCCYS.</p

Response of pilus dynamics after addition of DNA.

Panels A) and B) show heat maps of confined tracks of (AF488 C5-maleimide labelled) ComGCCYS in cells projected into a standardized B. subtilis cell. Confinement radius was 108.7 nm. Colour code on the right indicates intensity of signals. “- DNA” and “+ DNA” indicates untreated cells or cells treated with DNA for 10 minutes. C) Bubble plot shows diffusion constants [μm2/s] the fraction sizes of populations by setting a simultaneous diffusion constant which is shown in correspondence. Errors correspond to the 95% confidence intervals which are given by “confint” matlab function by using its values that result from the fit. +DNA and -DNA indicates cells which were treated with DNA for 10 minutes.</p

Dynamics of AF488 C5-maleimide-stained competence pili. A+B) Images of time-laps microscopy were acquired every 20 s.

Yellow arrow indicates pilus formation, orange pilus retraction. Movies were acquired in the GFP-channel. C) Colocalization of fluorescently labelled DNA with C5 Maleimide-stained competence pili in BF-, GFP, and TxRed-channel. Scale bars 2 μm.</p

Retraction and Extension of pilus structure of AF488-C5 maleimide stained <i>B</i>. <i>subtilis</i> cell.

Time lapse (20 second intervals) of pilus structures of AF488-C5 maleimide stained B. subtilis cells grown to competence expressing ComGCCYS. (GIF)</p

Model for extension and retraction of the competence pilus in <i>B</i>. <i>subtilis</i>.

Pre-pilins are processed by the prepilin peptidase ComC (yellow) and are assembled into a competence pilus, for which ComGB (blue) might be the platform protein. The energy could be provided by assembly-ATPase ComGA (green), while a retraction factor, or retraction activity, is yet to be identified (purple). Processed ComGC (grey) diffuses within the membrane, while some of the molecules, after having assembled into a competence pilus, are positioned bound within the pilus polymer, represented the static single molecule fraction.</p

Localization of Single molecule tracks of ComGC^CYS treated with AF488-C5 maleimide.

A) Two examples of overlays of Confinement maps in representative cells, showing confined (red) or mobile (blue) tracks, or those showing transitions (green). B) Confinement map of confined tracks projected into a standardized B. subtilis cell. Confinement radius is 108.7 nm, which corresponds to 3 times the localization error. Confined tracks are indicated in red, green tracks show tracks from a confined to a more mobile movement and vice versa, while blue tracks are defined as mobile, moving out of the confinement radius. Scale bar represents 2 μm.</p

Plasmids used in this study.

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.</div