smSRM of bacteria in agarose pads.

<p><b> A. smSRM imaging of bacteria in agarose pads.</b> (i) A double-side adhesive o-ring was placed on a coverslip and melted agarose was added to create an adhering surface for the bacteria. (ii) Bacterial cells, previously stained with the membrane dye FM4-64 mixed with fiducial marks, were deposited on agarose and the pad was sealed with a clean coverslip. The sample was finally fixed on an Attofluor cell (Invitrogen) to avoid bacterial motion during microscopy. (iii–iv) Sequential imaging of bacterial membrane and SpoIIIE (iii) Epi-fluorescence image of the cell membrane was collected by exiting at 532 nm. (iv) smSRM images were collected by using continuous excitation with a 532 nm laser and by applying regular pulses of photo-activation with a 405 nm laser. <b>B–C. Lateral drift during smSRM acquisition in agarose pads.</b> Lateral drift over the full acquisition period was assessed by plotting the trajectories of fluorescent beads in <i>x</i> (B) and <i>y</i> (C) coordinates over time. Each colored trajectory corresponds to a single fluorescent bead. <b>D–E. Alignment correction in smSRM experiments in agarose pads.</b> Distortion arising from chromatic aberrations was quantified from the distance between the same fluorescent beads observed in two different emission channels (D) and corrected by using a linear transformation procedure (E) (see Materials and Methods). Each dot represents a different bead and the abcissa represents the <i>x</i> coordinate of each bead. Error bars represent the precision of localization before (D) and after (E) drift and alignment correction. <b>F–G. Bleed-through of the membrane staining agent FM4-64 during smSRM imaging in agarose pads. (i)</b> Image of a cell in the SpoIIIE-PA (SpoIIIE-eosFP) (F) and FM4-64 (G) channels. (ii) Line scans of the fluorescence signal across a <i>B. subtilis</i> cell (white dotted line in panels F-i and G-i) in the two observation channels (green and red lines, respectively). For comparison, the line scan of the fluorescence intensity emitted by a single SpoIIIE-PA protein was overlapped in F-ii (black dotted line). As expected, the signal-to-noise ratio and contrast in the red channel are adequate (SNR = 40/contrast = 2.3, panel G-ii). However, even at low dye concentrations the fluorescence signal from FM4-64 bleeds into the SpoIIIE-PA channel (SNR = 8/contrast = 1.3, panel F-ii), compromising single-molecule detection, lowering the localization precision, and often leading to false positive localizations. For comparison, in the single-molecule trace shown in F-ii the signal to noise ratio is 30, and the contrast is 3. <b>H. SpoIIIE localization observed by smSRM in agarose pads.</b> Pointillist representation of SpoIIIE-PA localization in <i>B. subtilis</i> at different cell stages. Each green dot represents a single fluorescent event detected in a single frame during the smSRM acquisition. False positive localizations can be observed scattered homogeneously over the cell membrane.</p

Super-Resolution Imaging of Bacteria in a Microfluidics Device

<div><p>Bacteria have evolved complex, highly-coordinated, multi-component cellular engines to achieve high degrees of efficiency, accuracy, adaptability, and redundancy. Super-resolution fluorescence microscopy methods are ideally suited to investigate the internal composition, architecture, and dynamics of molecular machines and large cellular complexes. These techniques require the long-term stability of samples, high signal-to-noise-ratios, low chromatic aberrations and surface flatness, conditions difficult to meet with traditional immobilization methods. We present a method in which cells are functionalized to a microfluidics device and fluorophores are injected and imaged sequentially. This method has several advantages, as it permits the long-term immobilization of cells and proper correction of drift, avoids chromatic aberrations caused by the use of different filter sets, and allows for the flat immobilization of cells on the surface. In addition, we show that different surface chemistries can be used to image bacteria at different time-scales, and we introduce an automated cell detection and image analysis procedure that can be used to obtain cell-to-cell, single-molecule localization and dynamic heterogeneity as well as average properties at the super-resolution level.</p></div

Cell flatness, stability and growth in microfluidics chambers.

<p><b>A. Fluorescence intensity profiles of flat and inclined cells.</b> Schematic fluorescence intensity profiles are drawn across a line passing through the cell poles and the center of a closing septum at different axial positions (left panels). z represents the direction of the optical axis, with z = 0 corresponding to the plane in which the center of the septum is on focus. (i) On a flat cell, the distance between the peaks corresponding to the positions of the cell poles and the center of the cell (d_R and d_L, respectively) either remain constant or diminish as the axial focal position increases or decreases. (ii) In contrast, in inclined cells the cell pole peaks move together to the left or the right as the axial position increases, and move to the opposite direction when the axial position decreases. This movement translates into an increase of d_R and a decrease of d_L on one side of the focal plane and the reverse on the opposite side. Intensity profiles are schematic and not from real data. <b>B. 3D-SIM membrane imaging of a dividing </b><b><i>B. subtilis</i></b><b> cell and profile quantification.</b> (i) Three planes of a single dividing cell are shown with the x-axis representing the axis of the cell. (ii) Three views of a constant intensity level reconstruction of the cell shown in (i). (iii) Intensity profiles drawn in the <i>x</i> direction at the three focal plane positions shown in (i). <b>C. Integrity and stability of </b><b><i>B. subtilis</i></b><b> during smSRM imaging on poly-L-lysine-treated surfaces.</b> Cells were incubated for 2 h in sporulation media, injected into a poly-L-lysine-coated microfluidics chamber, and attached to the surface as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076268#pone-0076268-g002" target="_blank">Figure 2B</a>. Bright field images of sporulating <i>B. subtilis</i> cells before (left panel) and after (right panel) smSRM imaging (∼40 min). Notice that cells do not show any movement or sign of damage due the poly-L-lysine surface coating or due to smSRM imaging. Medium was continuously renewed by flow (5 µl/min of LB20%). <b>D–E. </b><b><i>B. subtilis</i></b><b> can grow and divide after smSRM imaging on chitosan-treated surfaces.</b> Exponentially growing cells were injected into a chitosan-treated microfluidics chamber, and attached to the surface as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076268#pone-0076268-g002" target="_blank">Figure 2B</a>. Representative examples of <i>B. subtillis</i> spotted on chitosan and followed by time-lapse bright field imaging under two conditions: (D) at 18°C with no medium renewal, and (E) at 23°C while renewing the growing medium (LB20%). Time between frames was 8 min. Images were taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076268#pone.0076268.s003" target="_blank">Movie S1</a>. Frame numbers (white) are indicated in the time-lapse montage of panel D. Color-coded arrows indicate elongating (green), pre-divisional (orange) and recently divided (yellow) cells. Notice that bacteria are not affected by the smSRM imaging procedure and can grow and divide successfully when attached to the surface coated with chitosan.</p

smSRM of bacteria in a microfluidics chamber.

<p><b>A. Micro-fluidic chamber assembly.</b> A a coverslip and a 1-way inlet and single outlet ports were sealed together by a parafilm mask melted at 90 °C during 1 minute. <b>B. Sequential smSRM imaging procedure in microfluidic chamber.</b> (i) The microfluidic chamber was filled with a 0.01% (w/v) solution of poly-L-Lysine or 0.015% (w/v) chitosan and incubated for at least 5 minutes at room temperature. After washing with sporulation media, 100 μL of a concentrated solution of bacterial cells along with fiducial marks were injected and let settle onto the coated surface. (i–ii) A high flow force was applied by pumping sporulation medium to rinse the channel, wash away unattached bacteria and ensure that attached bacteria laid completely flat on the surface. (ii) DNA was imaged by epi-fluorescence microscopy, and (iii) SpoIIIE was imaged by smSRM. (iv) Finally, the FM4-64 membrane staining agent was injected allowing for bacterial membrane detection by epi-fluorescence. <b>C–E. Lateral drift during smSRM acquisition in micro-fluidics chambers. C.</b> Bright field image of <i>B. subtilis</i> in a microfluidics chamber coated with chitosan. During bright field image acquisition the 561 nm laser was turned on to simultaneously detect fiducial marks. Colored squares indicate the selected beads for drift calculation and correction. <b>D–E.</b> Lateral drift over the full acquisition period was assessed by plotting the trajectories of fluorescent beads in x (D) and y (E) coordinates over time. Each colored trajectory corresponds to a single fluorescent bead selected in C. Blue line represent the mean of all trajectories. <b>F. Quantification of lateral displacement after drift correction.</b> The lateral displacements of the different fiducial marks were recalculated after subtraction of the drift from the mean trajectory. Grey line represents the mean drift obtained for the selected group of beads (σ_x = 5.4 nm and σ_y = 6.1 nm). <b>G. Effect of drift correction on smSRM imaging.</b> Pointillist reconstruction of detected events during smSRM imaging of SpoIIIE-PA (SpoIIIE-mMaple) in sporulating <i>B. subtillis</i>. Red and green dots represent the detected events before (i) and after (ii) drift correction, respectively. The size of the dots representing single molecule detections has been artificially increased for visualization purposes. <b>H. Background in the SpoIIIE-PA channel during smSRM.</b> Line scan of the fluorescence signal across a <i>B. subtilis</i> cell in the green (SpoIIIE-PA) detection channel (white dotted line in panels H-i). The background in the green channel corresponding to cell autofluorescence is extremely low (SNR = 0.3/contrast = 1.04) as compared to that observed in the presence of low amounts of membrane dye in agarose pads (SNR = 8/contrast = 1.3) and considerably lower than the signal from single-molecules (SNR∼30/contrast ∼5–10). <b>I. Imaging bacterial membranes after smSRM acquisition.</b> Line scans of the fluorescence signal across a <i>B. subtilis</i> cell in the red channel after injection of FM4-64 (white dotted line in panels I-i). As expected, the signal-to-noise ratio and contrast in the red channel are excellent (SNR = 200/contrast = 40, panel I-ii).</p

Automatic cluster detection and characterization.

<p><b>A.</b> Scheme of the clusterization algorithm. (i) Red dots represent single-molecule localizations with black lines representing pixel boundaries of the smSRM image in the inset. (ii) Initially, the field of view is divided in virtual pixels of a size smaller than our localization precision (virtual pixel size is typically 5–10 nm) and a binary image of the localizations map is built (white pixels). (iii) The binary image is then analyzed, and independent objects are automatically detected and classified. Objects showing a minimum number of events (5 to 50) and area (>1 px) were further processed whereas the rest were discarded. (iv) Each cluster was classified depending on their size, number of events and trajectories. A dynamic (orange dots) and a PALM-limited cluster (green dots, see main text) were detected. Dots and pixel sizes were arbitrary modified for visualizing purposes. <b>B. PALM-limited clusters.</b> (i) smSRM reconstruction of the distribution of SpoIIIE-PA (SpoIIIE-mMaple), and (ii) a pointillist representation of a PALM-limited cluster in which single localizations are color-coded by time (complete time-series). <b>C–D. Analysis of single-molecule detections in a typical cluster. C.</b> Number of events detected in the PALM-limited cluster shown in B are plotted as a function of time. Single, non-overlapping localization events are detected and dark times between events are longer than average emission times, consistent with each photo-activated protein being imaged with no overlapping between events. <b>D.</b> Cumulative number of events detected as a function of time in the PALM-limited cluster shown in B. This trace shows that photo-activation rates are in average homogeneous during acquisition. <b>E–F Characterization of dynamic clusters. E.</b> A pointillist representation of a dynamic cluster (highlighted by a dotted line) in which single localizations are color-coded by time (complete time-series). Here, localization events spread over several pixels and follow a path along the cell pole. <b>F.</b> Representative trajectories generated from tracking the motion of single-localizations identified in panel E (color coded by time of detection: from latest to earliest red, green and blue).</p

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

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