Summary The spatial organization of chromosomes by structural maintenance of chromosomes (SMC) complexes is vital to organisms from bacteria to humans 1,2 . SMC complexes were recently found to be motors that extrude DNA loops 3–11 . It remains unclear, however, what happens when multiple SMC complexes encounter one another in vivo on the same DNA, how encounters are resolved, or how interactions help organize an active genome 12 . Here, we set up a “crash-course track” system to study what happens when SMC complexes encounter one another. Using the parS /ParB system, which loads SMC complexes in a targeted manner 13–17 , we engineered the Bacillus subtilis chromosome to have multiple SMC loading sites. Chromosome conformation capture (Hi-C) analyses of over 20 engineered strains show an amazing variety of never-before-seen chromosome folding patterns. Polymer simulations indicate these patterns require SMC complexes to traverse past each other in vivo , contrary to the common assumption that SMC complexes mutually block each other’s extrusion activity 18 . Our quantitative model of bypassing predicted that increasing the numbers of SMCs on the chromosome could overwhelm the bypassing mechanism, create SMC traffic jams, and lead to major chromosome reorganization. We validated these predictions experimentally. We posit that SMC complexes traversing one another is part of a larger phenomenon of bypassing large steric barriers which enables these loop extruders to spatially organize a functional and busy genome.

DNA-loop extruding SMC complexes can traverse one anotherin vivo