Single molecule tracking reveals that the bacterial SMC complex moves slowly relative to the diffusion of the chromosome

Schibany, Sonja; Borgmann, Luise A K Kleine; Rösch, Thomas C.; Knust, Tobias; Ulbrich, Maximilian H.; Graumann, Peter L.

doi:10.1093/nar/gky581

Cited by 31 publications

(38 citation statements)

References 64 publications

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“…1C), showing further that Smc foci apparently do not always colocalize with origin foci (we found colocalization in 43.4% of foci) and revealing that Smc is also statically bound to DNA away from origin regions. This is consistent with chromatin immunoprecipitation with microarray technology (ChIP-chip) data revealing Smc enrichment at several sites away from oriC (20) and with the results of our previous SMT experiments (8).…”

Section: Resultssupporting

confidence: 92%

“…Previously, Smc had been described to be clustered around oriC regions, forming foci that colocalize with oriC and with ParB foci. However, using single-molecule tracking, we recently found that Smc forms immobile clusters at many sites on the chromosome (8). To clarify this issue, we constructed a strain in which a functional Smc-mVenus (Smc-mVenus fluorescent protein) fusion can be localized relative to origin regions.…”

Section: Resultsmentioning

confidence: 99%

“…We next asked if isolated head domains can still be recruited to condensation centers, since we observed that a Headless-mVenus fusion was no longer recruited to these structures (8). Therefore, we connected the N-and C-terminal domains of smc with a linker, fused this head-domain construct to mVenus, and expressed the fusion ectopically.…”

Section: Resultsmentioning

confidence: 99%

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The Major Chromosome Condensation Factors Smc, HBsu, and Gyrase in Bacillus subtilis Operate via Strikingly Different Patterns of Motion

Schibany

Hinrichs

Hernández-Tamayo

et al. 2020

mSphere

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Although DNA-compacting proteins have been extensively characterized in vitro, knowledge of their DNA binding dynamics in vivo is greatly lacking. We have employed single-molecule tracking to characterize the motion of the three major chromosome compaction factors in Bacillus subtilis, Smc (structural maintenance of chromosomes) proteins, topoisomerase DNA gyrase, and histone-like protein HBsu. We show that these three proteins display strikingly different patterns of interaction with DNA; while Smc displays two mobility fractions, one static and one moving through the chromosome in a constrained manner, gyrase operates as a single slow-mobility fraction, suggesting that all gyrase molecules are catalytically actively engaged in DNA binding. Conversely, bacterial histone-like protein HBsu moves through the nucleoid as a larger, slow-mobility fraction and a smaller, high-mobility fraction, with both fractions having relatively short dwell times. Turnover within the SMC complex that makes up the static fraction is shown to be important for its function in chromosome compaction. Our report reveals that chromosome compaction in bacteria can occur via fast, transient interactions in vivo, avoiding clashes with RNA and DNA polymerases. IMPORTANCE All types of cells need to compact their chromosomes containing their genomic information several-thousand-fold in order to fit into the cell. In eukaryotes, histones achieve a major degree of compaction and bind very tightly to DNA such that they need to be actively removed to allow access of polymerases to the DNA. Bacteria have evolved a basic, highly dynamic system of DNA compaction, accommodating rapid adaptability to changes in environmental conditions. We show that the Bacillus subtilis histone-like protein HBsu exchanges on DNA on a millisecond scale and moves through the entire nucleoid containing the genome as a slow-mobility fraction and a dynamic fraction, both having short dwell times. Thus, HBsu achieves compaction via short and transient DNA binding, thereby allowing rapid access of DNA replication or transcription factors to DNA. Topoisomerase gyrase and B. subtilis Smc show different interactions with DNA in vivo, displaying continuous loading or unloading from DNA, or using two fractions, one moving through the genome and one statically bound on a time scale of minutes, respectively, revealing three different modes of DNA compaction in vivo.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

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The Major Chromosome Condensation Factors Smc, HBsu, and Gyrase in Bacillus subtilis Operate via Strikingly Different Patterns of Motion

Schibany

Hinrichs

Hernández-Tamayo

et al. 2020

mSphere

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“…5E ). Interestingly, the diffusion constant for the fast subpopulation of AbrB (total mass of 156 kDa including YFP) is very similar to the diffusion constant of 0.53 µm 2 /s for the freely diffusive fraction of the Smc dimer 33 (total mass of 330 kDa including YFPs), and 0.51 µm 2 /s for the freely diffusive fraction of the DnaA dimer 9 (total mass of 156 kDa including YFPs), suggesting that this corresponds to the freely diffusive state of AbrB. In contrast, the previously reported diffusion constant for the DNA-bound state of Spo0J (0.02 µm 2 /s) 9 is about 4-fold lower than that for the static subpopulation of AbrB (0.084 µm 2 /s), suggesting that this value might be the result of a mixture between two diffusive states, for instance by AbrB frequently interacting with DNA in a non-specific manner, in search of its binding sites.…”

Section: Resultsmentioning

confidence: 62%

SMTracker: a tool for quantitative analysis, exploration and visualization of single-molecule tracking data reveals highly dynamic binding of B. subtilis global repressor AbrB throughout the genome

Rösch

Oviedo-Bocanegra

Fritz

et al. 2018

Sci Rep

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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.

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“…We next turned towards SMT to quantify changes in protein dynamics at a single molecule level, using an experimental setup that has been described before (43,45,46). We used SMTracker software 1.5 (36) to analyse tracks that were generated using u-track software (33).…”

Section: Single Molecule Tracking Reveals Poor Enrichment Of Dnag At mentioning

confidence: 99%

Single molecule dynamics at a bacterial replication fork after nutritional downshift

Hernández-Tamayo

Schmitz

Graumann

2020

Preprint

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Replication forks must respond to changes in nutrient conditions, especially in bacterial cells. By investigating the single molecule dynamics of replicative helicase DnaC, DNA primase DnaG, and of lagging strand polymerase DnaE in the model bacterium Bacillus subtilis in response to transient replication blocks due to DNA damage, to inhibition of the replicative polymerase, or to downshift of serine availability, we show that proteins react differentially to the stress conditions. DnaG appears to be recruited to the forks by a diffusion and capture mechanism, becomes more statically associated after arrest of polymerase PolC, but binds much less often after fork blocks due to DNA damage or to nutritional downshift. These results indicate that binding of the alarmone ppGpp due to the stringent response prevents DnaG from binding to forks rather than blocking bound primase. Dissimilar behaviour of DnaG and of DnaE suggest that both proteins are recruited independently to the forks, rather than jointly. Turnover of all three proteins was increased during replication block after nutritional downshift, different from the situation due to DNA damage or polymerase inhibition, showing high plasticity of forks in response to different stress conditions. Forks persisted during all stress conditions, apparently ensuring rapid return to replication extension.

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Single molecule tracking reveals that the bacterial SMC complex moves slowly relative to the diffusion of the chromosome

Cited by 31 publications

References 64 publications

The Major Chromosome Condensation Factors Smc, HBsu, and Gyrase in Bacillus subtilis Operate via Strikingly Different Patterns of Motion

The Major Chromosome Condensation Factors Smc, HBsu, and Gyrase in Bacillus subtilis Operate via Strikingly Different Patterns of Motion

SMTracker: a tool for quantitative analysis, exploration and visualization of single-molecule tracking data reveals highly dynamic binding of B. subtilis global repressor AbrB throughout the genome

Single molecule dynamics at a bacterial replication fork after nutritional downshift

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