Effects of the chromosome partitioning protein Spo0J (ParB) on oriC positioning and replication initiation in Bacillus subtilis

Abstract

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.

FIG. 1.

(A) B. subtilis chromosome with parS sites shown. B. subtilis has a circular chromosome with the origin of replication at 0°/360° and the terminus region at 180°. Spo0J binds eight known parS sites located in the origin-proximal 20% of the chromosome (37). The parS sites are located at 330°, 354°, 355°, 356°, 359°, 4°, 15°, and 40° and are indicated by flags. (B) Strains with an array of 16 parS sites inserted in different regions of the chromosome (90°, 181°, 210°, and 270°), indicated by the shaded rectangles, were constructed. In each strain, six of the eight known endogenous parS sites were inactivated. The remaining parS sites were located at 355° and 15°, indicated by flags.

FIG. 2.

Locations of sister copies of various regions of the chromosome. Strains producing Spo0J-GFP, LacI-GFP, LacI-CFP, or Rtp-YFP were grown at 30°C in defined minimal medium. Only cells with two foci of the region of interest were analyzed. The distance from each focus to the same cell pole was measured from images of live cells in exponential growth (Materials and Methods) and is plotted on the x axis, and cell length is plotted on the y axis. Cell length and the midcell positions are indicated by solid lines. Cell quarter positions are indicated by dotted lines. The number of cells analyzed (n) is indicated in the lower right corner of each panel. One focus is indicated with red open circles and the other with blue crosses. (A) Position of Spo0J-GFP bound to endogenous parS sites (strain PSL10). (B, C) Position of sister origin regions (359°) visualized with LacI-GFP bound to lac operator arrays in spo0J⁺ (strain DCL696) and spo0J mutant (strain DCL705) cells. (D, E) Position of Spo0J-GFP bound to a parS array (D, strain PSL25) or LacI-CFP bound to a lac operator array (E, strain KPL718) inserted at 90°. (F) Position of Spo0J-GFP bound to a parS array inserted at 210° (strain PSL27). (G, H) Position of Spo0J-GFP bound to a parS array (G, strain DCL631) or LacI-CFP bound to a lac operator array (H, strain KPL686) inserted at 181°. (I) Position of Rtp-YFP bound to endogenous ter sites (strain IRN424). (J, K) Position of Spo0J-GFP bound to a parS array (J, strain PSL23) or LacI-CFP bound to a lac operator array (K, strain KPL716) inserted at 270°.

FIG. 3.

Relationship between cell length and interfocal distance. Data from Fig. 2A to C were used to determine the distance between the two foci in each cell (the interfocal distance). This is plotted as a function of cell length. (A) Spo0J-GFP from Fig. 2A. (B) LacI-GFP in spo0J⁺ cells from Fig. 2B. (C) LacI-GFP from spo0J mutant cells from Fig. 2C. The correlation coefficient, r, which measures how strongly the interfocal distance and cell length are correlated, is shown for each plot (r = 1 for two perfectly, positively correlated variables). An ellipse was drawn around the distribution of wild-type cells (B), and this ellipse was superimposed on the corresponding plots in panels A andC. The interfocal distances in most of the spo0J mutant cells were within this ellipse; a subset (≈15%) fell below and to the right of the wild-type distribution. These cells had replicated origins that were closer together than the origins in wild-type cells of similar length.

FIG. 4.

Visualization of chromosomal regions during sporulation and growth. (A to E) Cells were grown in defined minimal medium and induced to sporulate by the addition of mycophenolic acid. Samples were taken 4 h after the initiation of sporulation, and membranes were stained with FM4-64. The positions of origin regions were visualized with LacI-GFP bound to an array of lac operators inserted at 359°. Note that for cells with two or more foci, there was no detectable effect of spo0J on the frequency of positioning an origin region in the forespore (see text). Images illustrate some of the different types of sporangia observed. (A) Typical origin position in spo0J⁺ cells (strain PSL62 spo0J⁺ ΔspoIIIE::tet). (B to E) spo0J mutant cells [strain PSL73 Δ(soj-spo0J)::spc ΔspoIIIE::tet] with two origin foci located in the mother cell (B) and the chromosomal DNA visualized with DAPI (C), indicating that there is DNA in the forespore. (D) A spo0J mutant cell with a single focus of the origin region that is excluded from the forespore. (E) A spo0J mutant cell with four copies of the origin region, one of which is in the forespore. (F) Increased copies of the terminus region, visualized with LacI-CFP bound to an array of lac operators inserted at 181°, in a spo0J mutant (strain PSL110) during exponential growth.

FIG. 5.

Early and asynchronous initiation of replication in spo0J mutant cells. spo0J⁺ (A, strain CRK6000) and spo0J mutant (B, strain NIK6074) cells were grown in Difco antibiotic medium 3, and samples were taken during exponential growth and treated for analysis by flow cytometry (see Materials and Methods). Histograms show the number of cells with a given amount of DNA per cell after completion of rounds of replication, as determined by flow cytometry. The numbers on the x axis represent the number of origins per cell at the time the sample was taken. More than 20,000 cells were analyzed in each experiment.