Recruitment, Assembly, and Molecular Architecture of the SpoIIIE DNA Pump Revealed by Superresolution Microscopy

<div><p></p><p>ATP-fuelled molecular motors are responsible for rapid and specific transfer of double-stranded DNA during several fundamental processes, such as cell division, sporulation, bacterial conjugation, and viral DNA transport. A dramatic example of intercompartmental DNA transfer occurs during sporulation in <i>Bacillus subtilis</i>, in which two-thirds of a chromosome is transported across a division septum by the SpoIIIE ATPase. Here, we use photo-activated localization microscopy, structured illumination microscopy, and fluorescence fluctuation microscopy to investigate the mechanism of recruitment and assembly of the SpoIIIE pump and the molecular architecture of the DNA translocation complex. We find that SpoIIIE assembles into ∼45 nm complexes that are recruited to nascent sites of septation, and are subsequently escorted by the constriction machinery to the center of sporulation and division septa. SpoIIIE complexes contain 47±20 SpoIIIE molecules, a majority of which are assembled into hexamers. Finally, we show that directional DNA translocation leads to the establishment of a compartment-specific, asymmetric complex that exports DNA. Our data are inconsistent with the notion that SpoIIIE forms paired DNA conducting channels across fused membranes. Rather, our results support a model in which DNA translocation occurs through an aqueous DNA-conducting pore that could be structurally maintained by the divisional machinery, with SpoIIIE acting as a checkpoint preventing membrane fusion until completion of chromosome segregation. Our findings and proposed mechanism, and our unique combination of innovating methodologies, are relevant to the understanding of bacterial cell division, and may illuminate the mechanisms of other complex machineries involved in DNA conjugation and protein transport across membranes.</p></div

SpoIIIE localization at superresolution.

<p>(a) SpoIIIE is observed during all stages of the cell cycle. Individual cells were recognized and classified as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001557#pbio-1001557-g001" target="_blank">Figure 1f</a>. Pixel size was 110 nm. From each 55 ms image, we automatically determined the localization of each single molecule in the image by using MTT <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001557#pbio.1001557-Serge1" target="_blank">[57]</a>. Each of these localizations is called a single-molecule event. In our pointilist representation, each single-molecule event is represented by a single green dot, whereas membrane stain is shown in white (see SM10 in Methods S1 for more details). Cells without septum were classified as stage 1 (vegetative/pre-divisional, left panel). Cells having a symmetric division septum were classified as stage 2 (division, middle panel), whereas those showing an asymmetric septum (at 1/5^th or 4/5^th of the total cell length) were classified as stage 3 (sporulating, right panel). (b) SpoIIIE clusters were automatically detected and classified depending on their size and composition. FWHM, full width at half maximum. (c) Analysis of the cluster size distribution versus the number of single-molecule events shows two distinct cluster types: PALM-limited clusters (red dots) have a size equal or smaller (∼45 nm FWHM) than the resolution of PALM in our conditions and contain a large number of events (>1,000), whilst dynamic clusters (orange dots) are large (>100 nm FWHM) and contain fewer events (<1,000). (d) The size of PALM-limited clusters is independent of cell cycle stage and the most typical size is ∼45 nm FWHM. (e) PALM-limited cluster sizes as a function of imaging time (total time used to image each single cluster) show that these clusters are extremely stable.</p

SpoIIIE clusters assemble asymmetrically in sporulation septa.

<p>(a) PALM imaging of SpoIIIE (green dots) in sporulating cells overlaid with an epi-fluorescence image of the membrane (white, top panel). Intensity profiles across the direction perpendicular to the septum were used to determine the precise localization of the septal plane, which was used to calculate the distance of each single-molecule detection to the center of the septum and to automatically partition the cell into forespore (yellow) and mother cell (red). Using this partition, individual PALM-limited clusters and single-molecule events were classified as belonging to the mother cell or the forespore compartments. (b) Histogram of PALM-limited cluster localizations with respect to the center of the septum (red columns) in sporulating cells with flat septa and undergoing DNA translocation (<i>N</i> = 43). Black dotted line indicates the position of the septum. A Gaussian distribution was fitted to the data (blue solid line). SpoIIIE PALM-limited clusters preferentially localize on the mother cell side of the sporulation septum. (c) Histogram of single-molecule localizations in PALM-limited clusters (<i>N</i> = 43) in sporulating cells undergoing DNA translocation, and Gaussian fit (blue dotted line). In sporulating cells, the distribution of SpoIIIE with respect to the septum is asymmetric and biased towards the direction of the mother cell. (d) Histogram of localizations of single molecules in PALM-limited clusters of dividing cells (<i>N</i> = 71) and Gaussian fit (blue dashed line). During division, the distribution of SpoIIIE with respect to the septum is symmetric and unbiased. (e) Distance of SpoIIIE PALM-limited clusters to the center of asymmetric septa versus the amount of translocated DNA. Open blue circles represent individual distance values for individual clusters, and black squares the average distance for all clusters detected at each particular percentage of DNA translocated. The relative distance increases linearly with the amount of DNA in the forespore until ∼45% of DNA translocated, and thereon remains constant at ∼50 nm. Solid black line is a guide to the eye. (f) The size (FWHM) of individual PALM-limited clusters in the directions parallel or perpendicular to the asymmetric septa were calculated and plotted against each other to evaluate cluster symmetry (squares). The overwhelming majority of clusters are symmetric (green squares) with only a small minority being longer in the direction parallel to the septum (black squares). The dotted line is a guide to the eye.</p

SpoIIIE is recruited to future sites of septation and localizes to the leading edge of closing septa.

<p>(a–c) Statistics of SpoIIIE clusters in vegetative/pre-divisional (<i>N</i> = 705), dividing (<i>N</i> = 174), and sporulating (<i>N</i> = 2 13) cells. Clusters were automatically classified as dynamic, PALM-limited, or mixed (cells containing both cluster types). Cells with less than 50 detected events were classified as “empty.” Cells with more than one PALM-limited cluster were classified independently from those containing a single one. The proportion of single PALM-limited clusters increases from vegetative/pre-divisional to dividing cells, and is maximal in sporulating cells. (d–e) The normalized coordinates of each localized event (axial and longitudinal coordinates) were used to calculate the localization probability distribution (heat maps) of SpoIIIE for each cluster type in vegetative/pre-divisional cells. Normalized localization probability distributions were calculated for the first quartile of the cell and then reflected into the other three quartiles to impose mirror symmetry in the axis perpendicular and parallel to the cell axis. The relative average number of clusters detected in each pixel of the grid is color-coded according to the color bar (right). White lines represent cells outlines. (d) Dynamic clusters distribute homogeneously over the cell membrane. (e) In contrast, in cells in which sporulation was induced, PALM-limited clusters specifically localize to future sites of asymmetric septation. (f–h) 3D-SIM imaging of <i>B. subtilis</i> cells in the early phases of sporulation at different stages of septal constriction. Axial projections showing the distribution of FM4-64-stained membranes (red, i), fluorescently labeled SpoIIIE (ii), and a merged image (iii). A 3D reconstruction is obtained by calculating a constant fluorescence intensity profile (iv). (f) SpoIIIE localizes to future sites of asymmetric division before the onset of septal constriction as a single cluster with a size <100 nm (resolution limit in 3D-SIM). (g) In cells early in septal constriction, SpoIIIE often shows a ring-like distribution probably due to the intrinsic dynamics of the closing septal ring. (h) In cells advanced in septum constriction, SpoIIIE specifically localizes to the leading edge of the invaginating septum. A line scan (v) of the fluorescence signal across the closing septum (white dotted line in panel iii) shows that SpoIIIE fluorescence (green) is always internal to the fluorescence of the membrane (red). Scale bar, 400 nm.</p

Model for the establishment and architecture of the DNA-translocating SpoIIIE complex.

<p>(a) SpoIIIE motors are recruited to the center of constricting sporulation septa and bind nonspecifically to DNA. Early divisional proteins interact with SpoIIIE membrane domains and contribute to the formation and regulation of the aqueous channel. (b) Interactions between SpoIIIE-γ and SRS lead to the establishment of directional motion towards the mother cell compartment. (c–d) The movement of SpoIIIE motors on DNA continues until the linker domains are fully stretched, at which point further translocation by SpoIIIE causes directional chromosomal segregation. Red gradient represents the size of a PALM-limited cluster. (e) Upon completion of DNA translocation, SpoIIIE motors disengage from DNA and the last segment of circular DNA is pulled into the forespore. (f) After completion of DNA translocation, SpoIIIE or a protein interacting with it leads to membrane fission.</p

Role of SpoIIIE during chromosome segregation during sporulation in <i>B. subtilis</i> and experimental setup.

<p>(a) Formation of the asymmetric sporulation septum (brown disc) divides the cell into mother cell and forespore compartments, and traps one fourth of the chromosome (black ribbon) inside the forespore (upper panel). Packaging of the remainder of the chromosome into the nascent spore (lower panel) is achieved by SpoIIIE (green disc), a septal-bound double-stranded DNA motor of the FtsK family. (b) SpoIIIE is composed of a membrane-spanning domain (orange), a 134-residue unstructured linker (brown), and a motor domain responsible for directional DNA translocation (green). A single membrane bilayer is represented by yellow circles and black sticks. (c–d) Models for the architecture of the DNA translocating complex. (c) The DNA channel model suggested that SpoIIIE hexamers on either side of fused septa assemble to form a DNA-conducting channel. (d) The sequence-directed DNA exporter model proposed that specific interactions between SpoIIIE-γ and SRS sequences (blue arrows) lead to the establishment of active SpoIIIE hexamers (green) present exclusively on the mother cell side of the septum. Inactive SpoIIIE subunits are shown in red and are located in this model on the forespore side. (e) Left panel shows a conventional epi-fluorescence image of sporulating <i>B. subtilis</i> cells, in which SpoIIIE (green) assembles in diffraction-limited foci. Membranes are shown in red. Right panel shows a PALM image of a sporulating cell undergoing sporulation, in which the green spot represents a probability density reconstruction of the localization of SpoIIIE. Scale bar, 1 µm. (f) PALM acquisition procedure. A culture of <i>B. subtilis</i> pre-incubated with the DNA intercalator sytox-green and fiducial marks are introduced into a microfluidics device (left panel). Cells are flattened by using flow force and chromosomes are imaged by epi-fluorescence microscopy. SpoIIIE is imaged by PALM (middle panel), and finally a membrane dye is introduced and an epi-fluorescence image is obtained (right panel). (g) Cells were automatically detected and their contour (dotted lines) calculated from DNA images (left image). Individual SpoIIIE proteins were detected by PALM microscopy (middle panel), and membrane images used to classify bacteria according to their stage in the cell cycle (red, dividing; blue, vegetative/pre-divisional; green, sporulating). Middle panel shows the raw image of a single frame displaying two individual single-molecules. Scale bar, 1 µm.</p

SpoIIIE clusters localize to the FtsZ ring in invaginating cells and to the septal midplane in dividing and sporulating cells.

<p>(a–b) (i) 3D-SIM imaging of SpoIIIE (green) and FtsZ (red) at two stages of septal constriction show that SpoIIIE clearly localizes to the FtsZ ring throughout the process of invagination (55% of cells, <i>N</i> = 78). Solid white line represents cell contour. (ii) A line scan of the fluorescence signal across the FtsZ ring (white arrows in panel i) shows that SpoIIIE fluorescence signal (green) overlaps to that of FtsZ (red). (iii) A 3D reconstruction of the FtsZ ring (red) and SpoIIIE (blue) is obtained by calculating a constant fluorescence intensity profile. Front (right) and side (left) views are shown. The size (FWHM) of the ring is 650 nm in (a-iii) and 280 nm in (b-iii). (c–d) Heat maps of PALM-limited clusters in (c) dividing (<i>N</i> = 87) and (d) sporulating (<i>N</i> = 180) cells show a clear SpoIIIE localization to the center of the septal plane. Normalized localization probability distributions were calculated for the first quartile of the cell and then reflected into the other three quartiles to impose mirror symmetry in the axis perpendicular and parallel to the cell axis. The relative average number of clusters detected in each pixel of the grid is color-coded according to the color bar (right). White lines represent cells outlines. (e) Probability density representation of SpoIIIE localization in dividing (top panels) and sporulating (bottom panel) cells. (f) Reconstructions of 3D-SIM images of three sporulating cells during DNA translocation show that SpoIIIE clusters localize to the center of the asymmetric septal plane.</p