Bipolar localization of a chromosome partition protein in Bacillus subtilis

Abstract

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.

Figure 1

Localization of Spo0J during growth and sporulation. (Bars ≈1 μm.) (A–J). Growth in minimal medium. A–D and I represent one field of cells, and E–H and J a different field of cells. (A and E) DAPI staining to visualize nucleoids (blue). (B and F) Immunostaining of Spo0J with affinity-purified rabbit antibodies and secondary antibodies coupled to the fluorophore Cy-3 (orange). (C and G) Immunostaining of FtsZ with affinity-purified chicken antibodies and secondary antibodies coupled to the fluorophore fluorescein isothiocyanate (green). (D) Overlay of exposures of Spo0J (B) and FtsZ (C). Cartoons of six cells are indicated. The three cells at the top of each panel have an FtsZ ring with a single site of Spo0J staining per nucleoid (class 3 of Fig. 2). Of the three cells at the bottom, the large cell to the left has an FtsZ ring with a nucleoid on each side and two sites of Spo0J per nucleoid (class 4 of Fig. 2). The other two cells have no FtsZ ring and a single nucleoid with two sites of Spo0J (class 2 of Fig. 2). (H) Overlay of exposures of Spo0J (F) and FtsZ (G). Cartoons of five cells are indicated; all have FtsZ rings. The large cell at the top has two sites of Spo0J per nucleoid (class 4 of Fig. 2), while all of the cells at the bottom have one site of Spo0J per nucleoid (class 3 of Fig. 2). (I) Overlay of exposures of DAPI (A) and Spo0J (B). (J) Overlay of exposures of DAPI (D) and Spo0J (E). (K) Spo0J–GFP localizes to the nucleoids in a bipolar manner. Living cells were stained with 7-amino-actinomycin D to visualize the DNA (red). Endogenous fluorescence from Spo0J–GFP appears yellow when it completely overlaps with the red from the DNA. (L–O) Localization of Spo0J during early stages of sporulation in wild-type cells. Cells were stained with DAPI (blue) to visualize DNA (L and N), and immuno-stained (red) to visualize Spo0J (M and O). (L and M) Three cells are at stage I, indicated by “I,” and a sketch of one of these is shown in M. (N and O) Arrows point to the condensed forespore nucleoids from two different stage II sporangia (II). A sketch is shown of one of these at the top of O. (P–S) Spo0J colocalizes with the origin-proximal 30% of the chromosome in the forespore of a spoIIIE mutant. Two sporangia of the spoIIIE mutant stained with DAPI (blue) to visualize DNA (P), and immunostained for Spo0J (orange) (Q), and β-galactosidase (green) (R). β-Galactosidase is produced from the sspE(2G)–lacZ fusion that is expressed only in the forespore. The fusion is in the origin-proximal part of the chromosome that gets trapped in the forespore in the spoIIIE mutant. (S) Overlay of the Spo0J and β-galactosidase staining. Arrows indicate the forespore. Note that in contrast to wild-type sporangia (N) that have highly condensed forespore nucleoids, the spoIIIE mutant forespore (P) has much less staining, reflecting the absence of a complete chromosome.

Figure 2

Analysis of Spo0J localization. Shaded blobs represent the nucleoids and black balls indicate Spo0J on the nucleoid. (A) Localization of Spo0J during vegetative growth (as in Fig. 1 A–J). In several different experiments, 75–90% of the cells had discrete sites of Spo0J staining and ≈60% of all cells had visible, well-defined FtsZ rings. The proportion of cells with discrete sites of Spo0J staining was similar in cells with and without FtsZ rings, indicating that the appearance of sites of Spo0J staining does not correlate with the presence or absence of FtsZ rings. (Classes 1 and 2) Cells with no FtsZ ring and a single nucleoid body with either a single site (class 1) or two sites (class 2) of Spo0J staining. (Classes 3 and 4) Cells with an FtsZ ring (indicated by the oval at midcell) and two nucleoid bodies, one on each side of the FtsZ ring and a single site (class 3) or two sites (class 4) of Spo0J staining per nucleoid. Of the 117 cells with an FtsZ ring and two sites of Spo0J per nucleoid, 7 cells actually had two sites on one nucleoid and only one site on the other nucleoid. We infer that in a small fraction of cells, reinitiation of replication or subsequent elongation did not occur synchronously on each chromosome. Alternatively, there were two Spo0J sites, but we could not resolve them. If these cells divide before the appearance of the second site of Spo0J staining, they would produce a few cells with a single nucleoid with a single site of Spo0J staining (class 1). For simplicity, cells in all four classes are drawn the same length. In fact, there is a rough correlation with the indicated class and cell length, with class 4 cells the longest and class 1 and 2 cells the shortest (see Fig. 1). (B) Localization of Spo0J during stage I of sporulation. (C) Localization of Spo0J during stage II of sporulation.

Figure 3

Model for chromosome segregation and Spo0J localization during the cell cycle. (1) Immediately after cell division, there is no FtsZ ring and a partly replicated chromosome with two discrete sites of Spo0J staining oriented toward the cell poles. (2) As replication and cell growth continue, the FtsZ ring forms and the chromosomes continue to separate. Spo0J localization becomes markedly polar, with one site on each nucleoid oriented toward the cell pole and away from the FtsZ ring at midcell. (3) DNA replication reinitiates, most of the time on both chromosomes, and progresses far enough so that the Spo0J binding region is replicated. This is visualized as two discrete sites of Spo0J staining on each of the two nucleoids. One site on each nucleoid is toward a cell pole while the other site is closer to the FtsZ ring at midcell. (4) The FtsZ ring contracts and disassembles during septation and division, generating two cells with partly replicated chromosomes and two distinct sites of Spo0J staining.