Conformation and dynamic interactions of the multipartite genome in Agrobacterium tumefaciens

Abstract

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.

Centromere Interactions Promote the Maintenance of the Multipartite Genome in Agrobacterium tumefaciens

“…To test how the centromeric proteins contribute to the replication and segregation of the replicons, we visualized the replication origins in the absence of ParB1 or RepB Ch2 . To label the origins in live cells, we inserted a visualization cassette, mcherry-parB P1 -parS P1 , at a position 50 kb away from ori1 , or ygfp-parB pMT1 -parS pMT1 , 57 kb away from ori2 or 11 kb away from oAt ( 11 , 27 ). Each cassette contains yGFP or mCherry fused to ParB from the pMT1 or P1 plasmid and the parS site of pMT1 or P1, respectively ( 28 ).…”

“…Like many other species, the primary chromosome (Ch1) replicates first, followed by the replication of secondary replicons (Ch2 and plasmids) ( 5 – 10 ). Our recent study ( 11 ) characterized the organization of this multipartite genome and revealed that the origins of the four replicons are clustered together and localized at the two cell poles ( Fig. 1A ).…”

“…1A ). It was found that the centromeric proteins, ParB1 for Ch1 and RepB Ch2 for Ch2, are required for origin clustering and Ch1-Ch2 alignment ( 11 ). Despite these known interactions, it is unclear how origin clustering contributes to multipartite genome maintenance and whether origin clustering is through direct interactions among the centromeric proteins or mediated by other factors.…”

“…The levels of ParB1 and RepB Ch2 are shown. ParB1 depletion strain (AtWX192) ( 11 ) contains PtraI-riboswitch-parB1 at the tetRA locus and parB1 deleted from the endogenous locus. The presence of inducers 1 μM acylhomoserine lactone (AHL) and 2 mM theophylline allows expression of ParB1.…”

“…In another example, the Vibrio cholerae polar protein HubP has also been suggested to interact with the centromeric ParB protein and localize its origin to the pole ( 26 ). In A. tumefaciens , since the replication origins of the four replicons are all localized to the cell poles ( 5 , 10 , 11 ) ( Fig. 1A ), it is possible that certain polar factors interact with each replication origin individually and serve as a platform to cluster the origins together.…”