The Escherichia coli SMC complex, MukBEF, forms clusters of molecules that interact with the decatenase topisomerase IV and which are normally associated with the chromosome replication origin region (ori). Here we demonstrate an additional ATP-hydrolysis-dependent association of MukBEF with the replication termination region (ter). Consistent with this, MukBEF interacts with MatP, which binds matS sites in ter. MatP displaces wild-type MukBEF complexes from ter, thereby facilitating their association with ori, and limiting the availability of topoisomerase IV (TopoIV) at ter. Displacement of MukBEF is impaired when MukB ATP hydrolysis is compromised and when MatP is absent, leading to a stable association of ter and MukBEF. Impairing the TopoIV-MukBEF interaction delays sister ter segregation in cells lacking MatP. We propose that the interplay between MukBEF and MatP directs chromosome organization in relation to MukBEF clusters and associated topisomerase IV, thereby ensuring timely chromosome unlinking and segregation.

Homologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs)1. Initially, the RecBCD complex2 resects the ends of the DSB into 3′ single-stranded DNA on which a RecA filament assembles3. Next, the filament locates the homologous repair template on the sister chromosome4. Here we directly visualize the repair of DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, in Escherichia coli, repair of DSBs between segregated sister loci is completed in 15 ± 5 min (mean ± s.d.) with minimal fitness loss. We further show that the search takes less than 9 ± 3 min (mean ± s.d) and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose that the architecture of the RecA filament effectively reduces search dimensionality. This model predicts a search time that is consistent with our measurement and is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA. Given the abundance of RecA homologues5, we believe this model to be widely conserved across living organisms.

Although the physical properties of chromosomes, including their morphology, mechanics, and dynamics are crucial for their biological function, many basic questions remain unresolved. Here we directly image the circular chromosome in live E. coli with a broadened cell shape. We find that it exhibits a torus topology with, on average, a lower-density origin of replication and an ultrathin flexible string of DNA at the terminus of replication. At the single-cell level, the torus is strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. Our data show a domain organization underlying the chromosome structure of E. coli , where MatP proteins induce site-specific persistent domain boundaries at Ori/Ter, while transcription regulators HU and Fis induce weaker transient domain boundaries throughout the genome. These findings provide an architectural basis for the understanding of the dynamic spatial organization of bacterial genomes in live cells.

Programmable control of the cell cycle has been shown to be a powerful tool in cell-biology studies. Here, we develop a novel system for controlling the bacterial cell cycle, based on binding of CRISPR/dCas9 to the origin-of-replication locus. Initiation of replication of bacterial chromosomes is accurately regulated by the DnaA protein, which promotes the unwinding of DNA at oriC. We demonstrate that the binding of CRISPR/dCas9 to any position within origin or replication blocks the initiation of replication. Serial-dilution plating, single-cell fluorescence microscopy, and flow-cytometry experiments show that ongoing rounds of chromosome replication are finished upon CRISPR/dCas9 binding, but no new rounds are initiated. Upon arrest, cells stay metabolically active and accumulate cell mass. We find that elevating the temperature from 37 to 42°C releases the CRISR/dCas9 replication inhibition, and we use this feature to recover cells from the arrest. Our simple and robust method of controlling the bacterial cell cycle is a useful asset for synthetic biology and DNA-replication studies in particular. The inactivation of CRISPR/dCas9 binding at elevated temperatures may furthermore be of wide interest for CRISPR/Cas9 applications in genomic engineering.