The parABS system is a widely employed mechanism for plasmid partitioning and chromosome segregation in bacteria. ParB binds to parS sites on plasmids and chromosomes and associates with broad regions of adjacent DNA, a phenomenon known as spreading. Although essential for ParB function, the mechanism of spreading remains poorly understood. Using single-molecule approaches, we discovered that Bacillus subtilis ParB (Spo0J) is able to trap DNA loops. Point mutants in Spo0J that disrupt DNA bridging are defective in spreading and recruitment of structural maintenance of chromosomes (SMC) condensin complexes in vivo. DNA bridging helps to explain how a limited number of Spo0J molecules per parS site (~20) can spread over many kilobases and suggests a mechanism by which ParB proteins could facilitate the loading of SMC complexes. We show that DNA bridging is a property of diverse ParB homologs, suggesting broad evolutionary conservation.

Structural maintenance of chromosomes (SMC) complexes play critical roles in chromosome dynamics in virtually all organisms but how they function remains poorly understood. In Bacillus subtilis, SMC condensin complexes are topologically loaded at centromeric sites adjacent to the replication origin. Here we provide evidence that these ring-shaped assemblies tether the left and right chromosome arms together while traveling from the origin to the terminus (>2 Mb) at rates >50kb/min. Condensin movement scales linearly with time arguing for an active transport mechanism. These data support a model in which SMC complexes function by processively enlarging DNA loops. Loop formation followed by processive enlargement provides a mechanism for how condensin complexes compact and resolve sister chromatids in mitosis and how cohesin generates topologically associating domains (TADs) during interphase.

SMC condensin complexes play a central role in compacting and resolving replicated chromosomes in virtually all organisms, yet how they accomplish this remains elusive. In Bacillus subtilis, condensin is loaded at centromeric parS sites, where it encircles DNA and individualizes newly replicated origins. Using chromosome conformation capture and cytological assays, we show that condensin recruitment to origin-proximal parS sites is required for the juxtaposition of the two chromosome arms. Recruitment to ectopic parS sites promotes alignment of large tracks of DNA flanking these sites. Importantly, insertion of parS sites on opposing arms indicates that these "zip-up" interactions only occur between adjacent DNA segments. Collectively, our data suggest that condensin resolves replicated origins by promoting the juxtaposition of DNA flanking parS sites, drawing sister origins in on themselves and away from each other. These results are consistent with a model in which condensin encircles the DNA flanking its loading site and then slides down, tethering the two arms together. Lengthwise condensation via loop extrusion could provide a generalizable mechanism by which condensin complexes act dynamically to individualize origins in B. subtilis and, when loaded along eukaryotic chromosomes, resolve them during mitosis.

DNA replication divides the circular Escherichia coli chromosome into equal arms (replichores). Visualization of pairwise combinations of multiple genetic loci reveals that the two replichores occupy separate nucleoid halves, with the replication origin between; positions of loci on each replichore recapitulate the genetic map. Sequential replication-segregation regenerates the structure by sequentially layering newly replicated replichore DNA to specific inner and outer edges of the developing sister nucleoids. Replication fork-dependent locus positions are imprinted, so that in most generations the chromosome orientation in a mother cell is recreated as a arrangement of sister chromosomes in daughter cells. Although it has been known for many years that bacterial circular chromosomes are replicated bidirectionally from a defined origin, precisely how this DNA is organized into the bacterial nucleoid, and how and when chromosomal DNA is segregated, has remained elusive (Sherratt 2003;Wu 2004;Gitai et al. 2005). Replication divides the chromosome into two equal arms (replichores), each being transcribed predominantly in the same direction as replication, and each containing distinctive asymmetric base-skewed DNA sequences that are important for chromosome processing (Blattner et al. 1997;Rocha 2004;Lesterlin et al. 2005). In recent years a combination of improved cytological methods, in which specific genetic loci were visualized using either fluorescent in situ hybridization (FISH), or the binding of fluorescent DNA-binding proteins to their cognate DNAbinding sites (FROS), have begun to reveal that bacterial chromosomes are highly organized yet dynamic structures (e.g., see Gordon et al. 1997;Niki et al. 2000;Li et al. 2002;Bates and Kleckner 2005;Wang et al. 2005), with DNA replication apparently being confined to specific cellular "replication factories" (Lemon and Grossman 2000;Adachi et al. 2005;Bates and Kleckner 2005).Models of chromosome organization based on these analyses for Escherichia coli, Bacillus subtilis, and Caulobacter crescentus have favored an arrangement in which the ori-ter chromosome axis runs longitudinally along the cell, with left and right chromosome arms (replichores) placed above and below this axis, or interwound (Teleman et al. 1998;Niki et al. 2000;Viollier et al. 2004;Gitai et al. 2005;Thanbichler et al. 2005). Such an organization is clear in C. crescentus, where in a resting cell, the replication origin region, ori, localizes to one cell pole, and ter, the replication termination region, to the other pole, and other loci lie between the two. Sequential replication-segregation generates two daughter cells that are mirror symmetrical with respect to chromosome orientation ( Fig. 1; Viollier et al. 2004;Gitai et al. 2005). The E. coli data have provided conflicting views of how DNA is organized in vivo and how/when it segregates. Nevertheless, the idea of chromosome organization about a longitudinal axis has been implicit (Niki a...

The bacterial chromosome must be compacted over 1000-fold to fit into its cellular compartment. How it is condensed, organized and ultimately segregated has been a puzzle for over half a century. Recent advances in live-cell imaging and genome-scale analyses have led to new insights into these problems. We argue that the key feature of compaction is orderly folding of DNA along adjacent segments, and that this organization provides easy and efficient access for protein-DNA transactions and plays a central role in driving segregation. Similar principles and common proteins are used in eukaryotes to condense and resolve sister chromatids at metaphase.