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

Abstract

Structural Maintenance of Chromosomes (SMC) proteins and their complex partners (ScpA and ScpB in many bacteria) are involved in chromosome compaction and segregation in all kinds of organisms. We employed single molecule tracking (SMT), tracking of chromosomal loci, and single molecule counting in Bacillus subtilis to show that in slow growing cells, ∼30 Smc dimers move throughout the chromosome in a constrained mode, while ∼60 ScpA and ScpB molecules travel together in a complex, but independently of the nucleoid. Even an Smc truncation that lacks the ATP binding head domains still scans the chromosome, highlighting the importance of coiled coil arm domains. When forming a complex, 10–15 Smc/ScpAB complexes become essentially immobile, moving slower than chromosomal loci. Contrarily, SMC-like protein RecN, which forms assemblies at DNA double strand breaks, moves faster than chromosome sites. In the absence of Smc, chromosome sites investigated were less mobile than in wild type cells, indicating that Smc contributes to chromosome dynamics. Thus, our data show that Smc/ScpAB clusters occur at several sites on the chromosome and contribute to chromosome movement.

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

“…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).…”

“…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.…”

“…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.…”

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

“…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.…”

Single molecule dynamics at a bacterial replication fork after nutritional downshift

“…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).…”