ORCID Profile
0000-0002-8033-5171
Current Organisation
Philipps-Universität Marburg
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Publisher: American Society for Microbiology
Date: 15-04-2018
DOI: 10.1128/AEM.02610-17
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 ision 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 ision 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 ision 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 ision septum, in agreement with the idea that it binds membrane-bound DNA, which can occur through cell ision across nonsegregated chromosomes. In contrast, SftA behaves like a soluble protein and is recruited to the ision septum as a component of the ision machinery. We show that FtsA contributes to the recruitment of SftA, revealing a dual role of FtsA at the ision 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.
Publisher: Oxford University Press (OUP)
Date: 28-06-2019
DOI: 10.1093/NAR/GKZ554
Abstract: DNA replication forks are intrinsically asymmetric and may arrest during the cell cycle upon encountering modifications in the DNA. We have studied real time dynamics of three DNA polymerases and an exonuclease at a single molecule level in the bacterium Bacillus subtilis. PolC and DnaE work in a symmetric manner and show similar dwell times. After addition of DNA damage, their static fractions and dwell times decreased, in agreement with increased re-establishment of replication forks. Only a minor fraction of replication forks showed a loss of active polymerases, indicating relatively robust activity during DNA repair. Conversely, PolA, homolog of polymerase I and exonuclease ExoR were rarely present at forks during unperturbed replication but were recruited to replications forks after induction of DNA damage. Protein dynamics of PolA or ExoR were altered in the absence of each other during exponential growth and during DNA repair, indicating overlapping functions. Purified ExoR displayed exonuclease activity and preferentially bound to DNA having 5′ overhangs in vitro. Our analyses support the idea that two replicative DNA polymerases work together at the lagging strand whilst only PolC acts at the leading strand, and that PolA and ExoR perform inducible functions at replication forks during DNA repair.
Publisher: Springer Science and Business Media LLC
Date: 06-11-2018
DOI: 10.1038/S41598-018-34572-8
Abstract: Single-particle (molecule) tracking (SPT/SMT) is a powerful method to study dynamic processes in living bacterial cells at high spatial and temporal resolution. We have performed single-molecule imaging of early DNA double-strand break (DSB) repair events during homologous recombination in the model bacterium Bacillus subtilis . Our findings reveal that DNA repair centres arise at all sites on the chromosome and that RecN, RecO and RecJ perform fast, enzyme-like functions during detection and procession of DNA double strand breaks, respectively. Interestingly, RecN changes its diffusion behavior upon induction of DNA damage, from a largely diffusive to a DNA-scanning mode, which increases efficiency of finding all sites of DNA breaks within a frame of few seconds. RecJ continues being bound to replication forks, but also assembles at many sites on the nucleoid upon DNA damage induction. RecO shows a similar change in its mobility as RecN, and also remains bound to sites of damage for few hundred milliseconds. Like RecN, it enters the nucleoid in damaged cells. Our data show that presynaptic preparation of DSBs including loading of RecA onto ssDNA is highly rapid and dynamic, and occurs throughout the chromosome, and not only at replication forks or only at distinct sites where many breaks are processes in analogy to eukaryotic DNA repair centres.
Publisher: Springer Science and Business Media LLC
Date: 24-10-2018
DOI: 10.1038/S41598-018-33842-9
Abstract: Single-particle (molecule) tracking (SPT/SMT) is a powerful method to study dynamic processes in living cells at high spatial and temporal resolution. Even though SMT is becoming a widely used method in bacterial cell biology, there is no program employing different analytical tools for the quantitative evaluation of tracking data. We developed SMTracker, a MATLAB-based graphical user interface (GUI) for automatically quantifying, visualizing and managing SMT data via five interactive panels, allowing the user to interactively explore tracking data from several conditions, movies and cells on a track-by-track basis. Diffusion constants are calculated a) by a Gaussian mixture model (GMM) panel, analyzing the distribution of positional displacements in x- and y- direction using a multi-state diffusion model (e.g. DNA-bound vs. freely diffusing molecules), and inferring the diffusion constants and relative fraction of molecules in each state, or b) by square displacement analysis (SQD), using the cumulative probability distribution of square displacements to estimate the diffusion constants and relative fractions of up to three diffusive states, or c) through mean-squared displacement (MSD) analyses, allowing the discrimination between Brownian, sub- or superdiffusive behavior. A spatial distribution analysis (SDA) panel analyzes the subcellular localization of molecules, summarizing the localization of trajectories in 2D- heat maps. Using SMTracker, we show that the global transcriptional repressor AbrB performs highly dynamic binding throughout the Bacillus subtilis genome, with short dwell times that indicate high on/off rates in vivo . While about a third of AbrB molecules are in a DNA-bound state, 40% diffuse through the chromosome, and the remaining molecules freely diffuse through the cells. AbrB also forms one or two regions of high intensity binding on the nucleoids, similar to the global gene silencer H-NS in Escherichia coli , indicating that AbrB may also confer a structural function in genome organization.
No related grants have been discovered for Peter Graumann.