Borrelia burgdorferi, the tick-transmitted spirochete agent of Lyme disease, has a highly segmented genome with a linear chromosome and various linear or circular plasmids. Here, by imaging several chromosomal loci and 16 distinct plasmids, we show that B. burgdorferi is polyploid during growth in culture and that the number of genome copies decreases during stationary phase. B. burgdorferi is also polyploid inside fed ticks and chromosome copies are regularly spaced along the spirochete’s length in both growing cultures and ticks. This patterning involves the conserved DNA partitioning protein ParA whose localization is controlled by a potentially phage-derived protein, ParZ, instead of its usual partner ParB. ParZ binds its own coding region and acts as a centromere-binding protein. While ParA works with ParZ, ParB controls the localization of the condensin, SMC. Together, the ParA/ParZ and ParB/SMC pairs ensure faithful chromosome inheritance. Our findings underscore the plasticity of cellular functions, even those as fundamental as chromosome segregation.

Bacterial species from diverse phyla contain multiple replicons, yet how these multipartite genomes are organized and segregated during the cell cycle remains poorly understood. Agrobacterium tumefaciens has a 2.8-Mb circular chromosome (Ch1), a 2.1-Mb linear chromosome (Ch2), and two large plasmids (pAt and pTi). We used this alpha proteobacterium as a model to investigate the global organization and temporal segregation of a multipartite genome. Using chromosome conformation capture assays, we demonstrate that both the circular and the linear chromosomes, but neither of the plasmids, have their left and right arms juxtaposed from their origins to their termini, generating interarm interactions that require the broadly conserved structural maintenance of chromosomes complex. Moreover, our study revealed two types of interreplicon interactions: “ori-ori clustering” in which the replication origins of all four replicons interact, and “Ch1-Ch2 alignment” in which the arms of Ch1 and Ch2 interact linearly along their lengths. We show that the centromeric proteins (ParB1 for Ch1 and RepBCh2 for Ch2) are required for both types of interreplicon contacts. Finally, using fluorescence microscopy, we validated the clustering of the origins and observed their frequent colocalization during segregation. Altogether, our findings provide a high-resolution view of the conformation of a multipartite genome. We hypothesize that intercentromeric contacts promote the organization and maintenance of diverse replicons.

Chromosome organization by structural maintenance of chromosomes (SMC) complexes is vital to living organisms. SMC complexes were recently found to be motors that extrude DNA loops. However, it remains unclear what happens when multiple complexes encounter one another in vivo on the same DNA and how interactions help organize an active genome. We created a crash-course track system to study SMC complex encounters in vivo by engineering the Bacillus subtilis chromosome to have defined 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. Via 3D polymer simulations and theory, we find that these patterns require SMC complexes to bypass each other in vivo, contrary to the common assumption that SMC complexes mutually block each other's loop extrusion activity. We posit that the bypassing activity enables SMC complexes to spatially organize a functional and busy genome.

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While it is common for a bacterial species to express multiple nucleoid-associated proteins (NAPs), NAPs are seldomly essential for cell survival. In B. subtilis , HBsu is a NAP essential for cell viability. Here, using conditional alleles to rapidly remove or inactivate HBsu, we show that the absence of HBsu abolishes the initiation of DNA replication in vivo . Understanding HBsu’s function can provide new insights into the regulation of DNA replication initiation in bacteria.