Sequence-dependent catalytic regulation of the SpoIIIE motor activity ensures directionality of DNA translocation

Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane-bound DNA translocase SpoIIIE and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE-DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from non-specific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. These findings are relevant in vivo in the context of chromosome transport through an open channel, where SpoIIIE can rapidly explore DNA while directionality is modulated by the probability of translocation initiation upon interaction with SRS versus non-specific DNA.Instituto de Física de Líquidos y Sistemas Biológico

Sequence-dependent catalytic regulation of the SpoIIIE motor activity ensures directionality of DNA translocation

Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane-bound DNA translocase SpoIIIE and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE-DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from non-specific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. These findings are relevant in vivo in the context of chromosome transport through an open channel, where SpoIIIE can rapidly explore DNA while directionality is modulated by the probability of translocation initiation upon interaction with SRS versus non-specific DNA.Instituto de Física de Líquidos y Sistemas Biológico

Behavior of Tn3 Resolvase in Solution and Its Interaction with res

The solution properties of Tn3 resolvase (Tn3R) were studied by sedimentation equilibrium, sedimentation velocity analytical ultracentrifugation, and small-angle neutron scattering. Tn3R was found to be in a monomer-dimer self-association equilibrium, with a dissociation constant of [Formula: see text] Sedimentation velocity and small-angle neutron scattering data are consistent with a solution structure of dimeric Tn3R similar to that of γδ resolvase in a co-crystal structure, but with the DNA-binding domains in a more extended conformation. The solution conformations of sites I, II, and III were studied with small angle x-ray scattering and modeled using rigid-body and ab initio techniques. The structures of these sites do not show any distortion, at low resolution, from B-DNA. The equilibrium binding properties of Tn3R to the individual binding sites in res were investigated by employing fluorescence anisotropy measurements. It was found that site II and site III have the highest affinity for Tn3R, followed by site I. Finally, the affinity of Tn3R for nonspecific DNA was assayed by competition experiments

Low-Resolution Reconstruction of a Synthetic DNA Holliday Junction

We have studied the low-resolution solution conformation of a Holliday (or four-way) DNA junction by using small-angle x-ray scattering, sedimentation velocity, and computational modeling techniques. The scattering data were analyzed in two independent ways: firstly, by rigid-body modeling of the scattering data using previously suggested models for the Holliday junction (HJ), and secondly, by ab initio reconstruction methods. The models found by both methods agree with experimentally determined sedimentation coefficients and are compatible with the results of previous studies using different techniques, but provide a more direct and accurate determination of the solution conformation of the HJ. Our results show that addition of Mg(2+) alters the conformation of the HJ from an extended to a stacked arrangement

Sequence-dependent catalytic regulation of the SpoIIIE motor activity ensures directionality of DNA translocation

Abstract Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane-bound DNA translocase SpoIIIE and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE-DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from non-specific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. These findings are relevant in vivo in the context of chromosome transport through an open channel, where SpoIIIE can rapidly explore DNA while directionality is modulated by the probability of translocation initiation upon interaction with SRS versus non-specific DNA

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