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Abstract Structural maintenance of chromosomes (SMC) complexes organize genomes by extruding DNA loops, while replisomes duplicate entire chromosomes. These essential molecular machines must collide frequently in every cell cycle, yet how such collisions are resolved in vivo remains poorly understood. Taking advantage of the ability to load SMC complexes at defined sites in theBacillus subtilisgenome, we engineered head-on and head-to-tail collisions between SMC complexes and the replisome. Replisome progression was monitored by genome-wide marker frequency analysis, and SMC translocation was monitored by time-resolved ChIP-seq and Hi-C. We found that SMC complexes do not impede replisome progression. By contrast, replisomes restrict SMC translocation regardless of collision orientations. Combining experimental data with simulations, we determined that SMC complexes are blocked by the replisome and then released from the chromosome. Occasionally, SMC complexes can bypass the replisome and continue translocating. Our findings establish that the replisome is a barrier to SMC-mediated DNA-loop extrusion in vivo, with implications for processes such as chromosome segregation, DNA repair, and gene regulation that require dynamic chromosome organization in all organisms. 

Abstract Research on chromosome organization and cell cycle progression in spherical bacteria, particularlyStaphylococcus aureus, remains limited and fragmented. In this study, we established a working model to investigate chromosome dynamics inS. aureususing a Fluorescent Repressor-Operator System (FROS), which enabled precise localization of specific chromosomal loci. This approach revealed that theS. aureuscell cycle and chromosome replication cycle are not coupled, with cells exhibiting two segregated origins of replication at the start of the cell cycle. The chromosome has a specific origin-terminus-origin conformation, with origins localizing near the membrane, towards the tip of each hemisphere, or the “cell poles”. We further used this system to assess the role of various proteins with a role inS. aureuschromosome biology, focusing on the ParB-parSand SMC-ScpAB systems. Our results demonstrate that ParB binds fiveparSchromosomal sequences and the resulting complexes influence chromosome conformation, but play a minor role in chromosome compaction and segregation. In contrast, the SMC-ScpAB complex plays a key role inS. aureuschromosome biology, contributing to chromosome compaction, segregation and spatial organization. Additionally, we systematically assessed and compared the impact of proteins linking chromosome segregation to cell division—Noc, FtsK, SpoIIIE and XerC—on origin and terminus number and positioning. This work provides a comprehensive study of the factors governing chromosome dynamics and organization inS. aureus, contributing to our knowledge on chromosome biology of spherical bacteria.