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

Abstract

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.

Fig 1. Transformation frequencies of strains encoding ComGC from the amyE locus and mutations.

The bar plot indicates the CFU (colony forming unit) defined as the number of viable cells per millilitre per microgram added chromosomal DNA. Experiments were done in technical and biological duplicates. Error bars indicate the standard deviations. AF C5-Mal: AF-488 C5-maleimide stain.

Fig 2. Epifluorescence microscopy of AF488 C5-maleimide stained cells of mutated cells (PY79 amyE::comGC^CYS) and cells (PY79 amyE::comGC) encoding ComGC at the amyE locus.

With 0.5 mM IPTG induction (panel A and B), without IPTG induction (panel C) and with low (0.01 mM) concentrations of IPTG (panel D). Left images of a panel show bright field images (BF-channel), right panels show epifluorescence pictures (GFP-channel, i.e. imaging of 488 nm fluorescence excitation). Scale bars represent 2 μm.

Fig 3. 3D-reconstructions of Structured illumination microscopy of AF488 C5-maleimide-stained cells encoding ComGC^CYS from the amyE-locus.

Left panel shows 0°C, middle panel 180° and right panel 270°. Corresponding S1 movie. Scale bars represent 2 μm.

Fig 4. 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.

Fig 5. 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.

Fig 6. Squared Displacemend analysis (SQD) of ComGC^CYS cells treated with AF488 C5-maleimide in B. subtilis cells grown to competence.

A and B (upper panel) show Rayleigh fits of the generated data of one and two populations. The plot shows the frequency of diffusion constants depending on the diffusion coefficient of the tracks in a histogram. The probability R² is annotated for each histogram, a two population fit is shown in B, which better describes the data as in A. Lower Panels of A and B show quantile–quantile plots for one (A) and two populations (B) which illustrate how well measured (dashed line) and the modelled (blue line) data fit together.

Fig 7. Response of pilus dynamics after addition of DNA.

Panels A) and B) show heat maps of confined tracks of (AF488 C5-maleimide labelled) ComGC^CYS 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.

Fig 8. Model of DNA uptake in B. subtilis as a two-tier process.

The periplasmatic uptake of double stranded (dsDNA) occurs over the whole cell surface via a true pilus structure, showing dynamic assembly and disassembly. The pilus structure is made out of minor (ComGD, ComGE, ComGG) and major pilins (ComGC) shown in grey. The anchor of the structure might be ComGB (light blue). Via pilus retraction, bound DNA will be pulled into the periplasm where it will bind to the DNA receptor ComEA (red), and diffuse throughout the periplasm. Once reaching the poles, it can be taken up by ComEC (dark blue). ComFA (violet) might provide the energy for the uptake. Direct passage through the pilus, via ComEA to ComEC, may be possible at the pole. ComEC processes double stranded (ds) DNA into single stranded (ss) DNA, which once inside the cytosol will be coated by single strand DNA binding proteins SsbA, SsbB (purple) and DprA (green). This is followed by an integration into the chromosome via dynamic RecA filaments.

Fig 9. Model for extension and retraction of the competence pilus in B. subtilis.

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.