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.

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About 10% of sequenced bacteria have multiple replicons, also known as multipartite genomes. How these multipartite genomes are maintained is still poorly understood.

Due to the enhanced labeling capability of maleimide-based fluorescent probes inin vitroexperiments, lysine-cysteine-lysine (KCK) tags are frequently added to proteins for visualization. Here we show that, although no noticeable changes were detected fromin vivofluorescence imaging and chromatin immunoprecipitation (ChIP) assays, the KCK-tag substantially altered DNA compaction rates byBacillus subtilisParB protein inin vitrosingle-molecule DNA flow-stretching experiments. Furthermore, our measurements and statistical analyses demonstrate that the KCK-tags also altered the ParB protein′s response to nucleotide (cytidine triphosphate CTP or its nonhydrolyzable analog CTPγS) binding and the presence of the specific DNA binding sequence (parS). Remarkably, the appended KCK-tags are capable of even reversing the trends of DNA compaction rates upon different experimental conditions. DNA flow-stretching experiments for both fluorescently-labeled ParB proteins and ParB proteins with an N-terminal glutamic acid-cysteine-glutamic acid (ECE) tag support the notion that electrostatic interactions between charges on the tags and the DNA backbone are an underlying cause of the protein′s property changes. While it is typically assumed that the short KCK-tag minimally perturbs protein function, our results demonstrate that this assumption must be carefully tested when using tags for protein labeling.