Polyploidy, regular patterning of genome copies, and unusual control of DNA partitioning in the Lyme disease spirochete.

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.

HBsu Is Required for the Initiation of DNA Replication in Bacillus subtilis.

Nucleoid-associated proteins (NAPs) help structure bacterial genomes and function in an array of DNA transactions, including transcription, recombination, and repair. In most bacteria, NAPs are nonessential in part due to functional redundancy. In contrast, in Bacillus subtilis the HU homolog HBsu is essential for cell viability. HBsu helps compact the B. subtilis chromosome and participates in homologous recombination and DNA repair. However, none of these activities explain HBsu's essentiality. Here, using two complementary conditional HBsu alleles, we investigated the terminal phenotype of the mutants. Our analysis revealed that cells without functional HBsu fail to initiate DNA replication. Importantly, when the chromosomal replication origin (oriC) was replaced with a plasmid origin (oriN) whose replication does not require the initiator DnaA, cells without HBsu initiated DNA replication normally. However, HBsu was still essential in this oriN-containing strain. We conclude that HBsu plays an essential role in the initiation of DNA replication, likely acting to promote origin melting by DnaA, but also has a second essential function that remains to be discovered. IMPORTANCE 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.

Conformation and dynamic interactions of the multipartite genome in Agrobacterium tumefaciens.

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.

DNA-loop-extruding SMC complexes can traverse one another in vivo.

Chromosome organization mediated by structural maintenance of chromosomes (SMC) complexes is vital in many organisms. SMC complexes act as motors that extrude DNA loops, but it remains unclear what happens when multiple complexes encounter one another on the same DNA in living cells and how these interactions may help to organize an active genome. We therefore created a crash-course track system to study SMC complex encounters in vivo by engineering defined SMC loading sites in the Bacillus subtilis chromosome. Chromosome conformation capture (Hi-C) analyses of over 20 engineered strains show an amazing variety of chromosome folding patterns. Through three-dimensional polymer simulations and theory, we determine that these patterns require SMC complexes to bypass each other in vivo, as recently seen in an in vitro study. We posit that the bypassing activity enables SMC complexes to avoid traffic jams while spatially organizing the genome.

XerD unloads bacterial SMC complexes at the replication terminus.

Bacillus subtilis structural maintenance of chromosomes (SMC) complexes are topologically loaded at centromeric sites adjacent to the replication origin by the partitioning protein ParB. These ring-shaped ATPases then translocate down the left and right chromosome arms while tethering them together. Here, we show that the site-specific recombinase XerD, which resolves chromosome dimers, is required to unload SMC tethers when they reach the terminus. We identify XerD-specific binding sites in the terminus region and show that they dictate the site of unloading in a manner that depends on XerD but not its catalytic residue, its partner protein XerC, or the recombination site dif. Finally, we provide evidence that ParB and XerD homologs perform similar functions in Staphylococcus aureus. Thus, two broadly conserved factors that act at the origin and terminus have second functions in loading and unloading SMC complexes that travel between them.