Spo0J (ParB) of Bacillus subtilis is a DNA-binding protein that belongs to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species. We found that Spo0J contributes to the positioning of the chromosomal oriC region, but probably not by recruiting the origin regions to specific subcellular locations. In wild-type cells during exponential growth, duplicated origin regions were generally positioned around the cell quarters. In a spo0J null mutant, sister origin regions were often closer together, nearer to midcell. We found, by using a Spo0J-green fluorescent protein [GFP] fusion, that the subcellular location of Spo0J was a consequence of the chromosomal positions of the Spo0J binding sites. When an array of binding sites (parS sites) were inserted at various chromosomal locations in the absence of six of the eight known parS sites, Spo0J-GFP was no longer found predominantly at the cell quarters, indicating that Spo0J is not sufficient to recruit chromosomal parS sites to the cell quarters. spo0J also affected chromosome positioning during sporulation. A spo0J null mutant showed an increase in the number of cells with some origin-distal regions located in the forespore. In addition, a spo0J null mutation caused an increase in the number of foci per cell of LacI-GFP bound to arrays of lac operators inserted in various positions in the chromosome, including the origin region, an increase in the DNA-protein ratio, and an increase in origins per cell, as determined by flow cytometry. These results indicate that the spo0J mutant produced a significant proportion of cells with increased chromosome content, probably due to increased and asynchronous initiation of DNA replication.

In B. subtilis, the chromosome partitioning proteins Soj (ParA) and Spo0J (ParB) regulate the initiation of sporulation. Soj is a negative regulator of sporulation gene expression, and Spo0J antagonizes Soj function. Using fusions of Soj to green fluorescent protein, we found that Soj localized near the cell poles and upon entry into stationary phase oscillated from pole to pole. In the absence of Spo0J, Soj was associated predominantly with DNA. By in vivo cross-linking and immunoprecipitation, we found that Soj physically associates with developmentally regulated promoters, and this association increased in the absence of Spo0J. These results show that Soj switches localization and function depending on the chromosome partitioning protein Spo0J. We further show that mutations in the Soj ATPase domain disrupt localization and function and render Soj insensitive to regulation by Spo0J.

Background: The contour length of the circular chromosome of bacteria is greater than a millimeter but must be accommodated within a cell that is only a few micrometers in length. Bacteria do not have nucleosomes and little is known about the arrangement of the chromosome inside a prokaryotic cell. Results: We have investigated the arrangement of chromosomal DNA within the bacterium Bacillus subtilis by using fluorescence microscopy to visualize two sites on the chromosome simultaneously in the same cell. Indirect immunofluorescence with antibodies against the chromosome partition protein Spo0J were used to visualize the replication origin region of the chromosome. Green fluorescent protein fused to the lactose operon repressor Lacl was used to decorate tandem copies of the lactose operon operator lacO . A cassette of tandem operators was separately inserted into the chromosome near the origin (359°), near the replication terminus (181°), or at two points in between (90° and 270°). The results show that the layout of the chromosome is dynamic but is principally arranged with the origin and terminus maximally apart and the quarter points of the chromosome in between. Conclusions: The use of cytological methods to visualize two chromosomal sites in the same cell has provided a glimpse of the arrangement of a bacterial chromosome. We conclude that, to a first approximation, the folding of the bacterial chromosome is consistent with, and may preserve, the linear order of genes on the DNA.

smc of Bacillus subtilis encodes a homolog of eukaryotic SMC proteins involved in chromosome condensation, pairing, and partitioning. A null mutation in B. subtilis smc caused a temperature-sensitive-lethal phenotype in rich medium. Under permissive conditions, the mutant had abnormal nucleoids, approximately 10% of the cells were anucleate, and assembly of foci of the chromosome partitioning protein Spo0J was altered. In combination with a null mutation in spo0J, the smc mutation caused a synthetic phenotype; cell growth was slower and approximately 25% of the cells were anucleate. Our results demonstrate that the B. subtilis Smc protein, like its eukaryotic counterpart, plays an important role in chromosome structure and partitioning.

We have identified a DNA site involved in chromosome partitioning in B. subtilis. This site was identified in vivo as the binding site for the chromosome partitioning protein Spo0J, a member of the ParB family of partitioning proteins. Spo0J is a site-specific DNA-binding protein that recognizes a 16 bp sequence found in spo0J. Allowing two mismatches, this sequence occurs ten times in the entire B. subtilis chromosome, all in the origin-proximal approximately 20%. Eight of the ten sequences are bound to Spo0J in vivo. The presence of a site on an otherwise unstable plasmid stabilized the plasmid in a Spo0J-dependent manner, demonstrating that this site, called parS, can function as a partitioning site. This site and Spo0J are conserved in a wide range of bacterial species.

We have determined the subcellular localization of the chromosome partition protein Spo0J of Bacillus subtilis by immunofluorescence microscopy and visualizing fluorescence of a Spo0J-GFP fusion protein. Spo0J was associated with a region of the nucleoid proximal to the cell pole, both in growing cells dividing symmetrically and in sporulating cells dividing asymmetrically. Additional experiments indicated that Spo0J was bound to sites in the origin-proximal third of the chromosome. These results show that the replicating chromosomes are oriented in a specific manner during the division cycle, with the Spo0J binding region positioned toward the cell poles. Experiments characterizing cells at different stages of the cell cycle showed that chromosome orientation is established prior to the initiation of cell division. Our results indicate that there is a mechanism for orienting the chromosomes and that the chromosome partition protein Spo0J might be part of a bacterial mitotic-like apparatus.

To investigate chromosome segregation in B. subtilis, we introduced tandem copies of the lactose operon operator into the chromosome near the replication origin or terminus. We then visualized the position of the operator cassettes with green fluorescent protein fused to the LacI repressor. In sporulating bacteria, which undergo asymmetric cell division, origins localized near each pole of the cell whereas termini were restricted to the middle. In growing cells, which undergo binary fission, origins were observed at various positions but preferentially toward the poles early in the cell cycle. In contrast, termini showed little preference for the poles. These results indicate the existence of a mitotic-like apparatus that is responsible for moving the origin regions of newly formed chromosomes toward opposite ends of the cell.