The Bacterial SMC Complex Displays Two Distinct Modes of Interaction with the Chromosome

Abstract

The bacterial SMC (structural maintenance of chromosomes) complex binds nonspecifically to DNA in vitro and forms two discrete subcellular centers in vivo, one in each cell half. How this distribution is maintained is unclear. We show by time-lapse imaging of single molecules that the localization is achieved through limited, yet rapid movement of the SMC subunits through the nucleoid. Accessory ScpAB subunits mediate the arrest of 20% of SMC molecules at the center of a cell half and do not move together with the 80% mobile SMC molecules. Only free SMC, but not the preformed SMC/ScpAB complex, was able to bind to DNA in vitro, revealing distinct functions of SMC fractions. Thus, whereas SMC alone dynamically interacts with many sites on the chromosome, it forms static assemblies together with ScpAB complex partners. Our findings reveal two distinct modes of interaction of SMC with the chromosome and indicate that limited diffusion within a confined space and transient arrest may be a general mechanism for positioning proteins within a chromosome and within a noncompartmentalized cell.

Condensin- and Replication-Mediated Bacterial Chromosome Folding and Origin Condensation Revealed by Hi-C and Super-resolution Imaging

“…This model is consistent with two populations of SMC-ScpAB coexisting in the cell: a static population at the origin, and a highly mobile population that appears to scan the nucleoid (Kleine Borgmann et al, 2013). The concomitant spreading of replichore pairing with replication may occur in a regular or discontinuous manner and may help in condensation of the two newly replicated chromosomes to facilitate segregation (Gruber et al, 2014;Wang et al, 2014b).…”

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

“…Full length Smc and headless Smc (containing the coiled coil up the N- and C-terminal domain, i.e. including the ScpA neck-binding region) were purified as Strep fusions via streptavidin affinity chromatography and ensuing gel filtration chromatography (Supplementary Figure S3) (23,38). Headless Smc could only be obtained up to 370 nM, which was tested for binding 500 bp DNA immobilized on an SPR chip (via biotinylation).…”

“…Headless Smc could only be obtained up to 370 nM, which was tested for binding 500 bp DNA immobilized on an SPR chip (via biotinylation). Wild type Smc bound to the DNA in a sequence-independent fashion (the more Smc is added, the more is bound), and an 8-fold molar excess of ScpAB markedly reduced binding, ∼2-fold (Supplementary Figure S4A) (23). Addition of ATP to the reaction mixture led to a mild increase in binding avidity of Smc (Supplementary Figure S4A).…”

“…We have previously shown that static Smc molecules depend on the presence of ScpA and of ScpB (23). We therefore wondered if in Smc depleted cells ScpA-YFP would become entirely mobile.…”

“…However, it has still not been demonstrated that these clusters are directly responsible for chromosome condensation and to the sites of entrapment in vivo . Only recently, single molecule microscopy studies additionally reported significant mobile fractions of Smc and the E. coli homologue MukB and its accessory proteins also outside of these clusters (22,23), which were not detectable using traditional wide-field microscopy with long exposure times due to motion blurring of these molecules. Thereby, two fractions of Smc molecules could be distinguished: static molecules mainly associated with the ‘condensation centers’ and mobile molecules that appeared to scan the chromosome.…”