Single-Molecule Tracking of DNA Translocases in Bacillus subtilis Reveals Strikingly Different Dynamics of SftA, SpoIIIE, and FtsA

Abstract

Like many bacteria, Bacillus subtilis possesses two DNA translocases that affect chromosome segregation at different steps. Prior to septum closure, nonsegregated DNA is moved into opposite cell halves by SftA, while septum-entrapped DNA is rescued by SpoIIIE. We have used single-molecule fluorescence microscopy and tracking (SMT) experiments to describe the dynamics of the two different DNA translocases, the cell division protein FtsA and the glycolytic enzyme phosphofructokinase (PfkA), in real time. SMT revealed that about 30% of SftA molecules move through the cytosol, while a fraction of 70% is septum bound and static. In contrast, only 35% of FtsA molecules are static at midcell, while SpoIIIE molecules diffuse within the membrane and show no enrichment at the septum. Several lines of evidence suggest that FtsA plays a role in septal recruitment of SftA: an ftsA deletion results in a significant reduction in septal SftA recruitment and a decrease in the average dwell time of SftA molecules. FtsA can recruit SftA to the membrane in a heterologous eukaryotic system, suggesting that SftA may be partially recruited via FtsA. Therefore, SftA is a component of the division machinery, while SpoIIIE is not, and it is otherwise a freely diffusive cytosolic enzyme in vivo Our developed SMT script is a powerful technique to determine if low-abundance proteins are membrane bound or cytosolic, to detect differences in populations of complex-bound and unbound/diffusive proteins, and to visualize the subcellular localization of slow- and fast-moving molecules in live cells.IMPORTANCE DNA translocases couple the late events of chromosome segregation to cell division and thereby play an important role in the bacterial cell cycle. The proteins fall into one of two categories, integral membrane translocases or nonintegral translocases. We show that the membrane-bound translocase SpoIIIE moves slowly throughout the cell membrane in B. subtilis and does not show a clear association with the division septum, in agreement with the idea that it binds membrane-bound DNA, which can occur through cell division across nonsegregated chromosomes. In contrast, SftA behaves like a soluble protein and is recruited to the division septum as a component of the division machinery. We show that FtsA contributes to the recruitment of SftA, revealing a dual role of FtsA at the division machinery, but it is not the only factor that binds SftA. Our work represents a detailed in vivo study of DNA translocases at the single-molecule level.

Keywords: Bacillus subtilis; DNA translocase; FtsK; SpoIIIE; cell division; chromosome segregation; single-molecule tracking.

FIG 1

Localization of SftA-YFP in strains that carry mutations of cell division proteins. White arrowheads show the septal localization of SftA. (A) SftA-YFP in ΔezrA mutant; white lines show double-division septa. (B) SftA-YFP in ΔdivIVA mutant; white line shows aberrantly formed septum close to the cell pole. (C) SftA-YFP in ΔzapA mutant. (D) SftA-YFP in ΔsepF; white line points at a septum formed near the pole. (E) SftA-YFP in ΔezrA ΔftsA mutant; white lines show abnormal septa. (F) SftA-YFP in ΔftsH mutant. Images are overlays of SftA-YFP signals (green) and membranes stained with FM 4-64 (red). White bars = 2 μm.

FIG 2

(A to F) S2 Schneider cell cotransfection experiments. Cell were transfected with plasmids expressing the corresponding proteins as shown in the panels. White arrowheads point to membrane assemblies of SftA. Images were taken through the middle of the cells in panels A to C, and circles with bars indicate the focal plane in panels D to F. (G) Western blot analysis with anti-GFP antibodies of the expressed recombinant proteins in the S2 Schneider cell experiment. The letters in parentheses correspond to cells harvested from the respective experiments in images. White bars = 5 μm.

FIG 3

(A) Z-projection of all frames from a selected movie of SftA-YFP, SpoIIIE-YFP, and PfkA-YFP. (B) Heat map of single-molecule localizations of SftA-YFP, SpoIIIE-YFP, and PfkA-YFP plotted into a standardized cell. High abundance is indicated in yellow and low abundance in dark blue.

FIG 4

Single-molecule microscopy of SftA-YFP and SpoIIIE-YFP. (A) Probability density function (PDF) of displacements obtained from SftA-YFP tracked in wild-type and ftsA ezrA double-mutant cells. Histograms were simultaneously fitted to a Gaussian mixture model assuming 2 different types of diffusive behavior (green line). Indeed, the multivariate fit matched the data better than assuming a single Gaussian distribution (outer dotted line). The dashed line and the inner dotted line correspond to the distributions of the larger and smaller frame-to-frame displacements representing fast and slowly diffusing molecules, respectively. (B) PDF of SpoIIIE-YFP in untreated cells and cells treated with 50 ng/ml mitomycin C (MMC). (C) PDF of PfkA-YFP. (D) Bubble plot showing diffusion coefficients of the diffusive subfractions seen for SftA-YFP, SpoIIIE-YFP, FtsA-YFP, and PfkA-YFP.

FIG 5

Dwell time density distributions of SftA-YFP and SpoIIIE-YFP. The dwell time represents the time that a molecule stays within a radius of defined size (here, 120 nm). (A) PDF and cumulative density function (CDF) of dwell times determined for SftA in wild-type and in FtsA-depleted cells. (B) PDF and CDF of dwell times calculated for SpoIIIE-YFP in presence and absence of MMC.

FIG 6

(A to E) Determination of the subcellular localization of slow- and of fast-moving molecules. Tracks were randomly sampled from PfkA-YFP (A), FtsA-YFP (B), and SftA-YFP (C) in wild-type cells or in cells deleted for ezrA and ftsA (D), and from cells expressing SpoIIIE-YFP (E). Tracks were projected into a standardized cell of 3 by 1 μm and were sorted into slow-moving (not leaving an area of 3 by 3 pixels) molecules, indicated by red lines, and fast-moving molecules, indicated by blue lines. Note that stream rate was 30 ms, except for 15 ms for FtsA-YFP. (F to J) Superimposition of slow-moving (red line) and fast-moving (blue line) molecules sorted in x- and y-orientation (long and short axis of the cell). Shown are the mean and the standard deviation of a bootstrap analysis sampling 50 times tracks with replacement from PfkA-YFP (F), FtsA-YFP (G), or SftA-YFP (H) in wild-type cells or in cells deleted for ezrA and ftsA (I), as well as from cells expressing SpoIIIE-YFP (J). Except for SftA-YFP and SpoIIIE-YFP, for which only 750 tracks were sampled, bootstrapping was performed with 1,000 tracks.