Partitioning of Chromosomal DNA during Establishment of Cellular Asymmetry in Bacillus subtilis

The switch from symmetric to asymmetric cell division is a keyfeature of development in many organisms, including Bacillussubtilis sporulation. Here we demonstrate that, prior to theonset of asymmetric cell division, the B. subtilis chromosomeis partitioned into two unequally sized domains, with the origin-proximalone-third of the future forespore chromosome condensed nearone pole of the cell. Asymmetric chromosome partitioning isindependent of polar division, as it occurs in cells depletedof FtsZ but depends on two transcription factors that governthe initiation of sporulation, ${sigma}$ ^H and Spo0A-P. It is also independentof chromosome partitioning proteins Spo0J and Soj, suggestingthe existence of a novel mechanism controlling chromosome structure.Thus, our results demonstrate that, during sporulation, twoseparable events prepare B. subtilis for asymmetric cell division:the relocation of cell division sites to the cell poles andthe asymmetric partitioning of the future forespore chromosome.

INTRODUCTION

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Introduction

Separation of daughter chromosomes occurs prior to cell divisionin nearly all organisms. In eukaryotes, the importance of properlyorchestrating chromosome segregation and cytokinesis is highlightedby the existence of checkpoints to arrest the cell cycle whenchromosomes are damaged or incompletely segregated. In bacteria,chromosome separation and cytokinesis are regulated both temporallyand spatially, so that, even during rapid exponential growth,daughter chromosomes are well separated prior to the onset ofdivision. Bacterial chromosome segregation appears to consistof two distinct steps: the rapid movement of the duplicatedorigins apart from one another (9, 11, 38, 40) and the separatecondensation of the replicated daughter chromosomes (12, 19,35). This process results in vegetative bacterial chromosomeshaving a bilobed structure during replication, with the daughterchromosomes being well separated prior to the onset of cytokinesis,so the path of the invaginating septum appears free of chromosomalDNA (4, 14). When chromosome segregation fails, as it does inmutants defective in chromosome partitioning or condensation,the chromosomes can be sheared during daughter cell separation(24, 34, 39, 47).

How, then, are bacterial chromosomes prepared for asymmetricallypositioned division events, such as that which occurs duringthe sporulation pathway of Bacillus subtilis? At the onset ofsporulation, the chromosome is reorganized into a rod-like structure(the axial filament), which is readily distinguished from thebilobed chromosome of vegetative cells (31). Next, a septumis formed close to one cell pole, trapping the origin-proximalregion of the chromosome in the smaller daughter cell (the forespore),while the remainder is translocated across the septum afterdivision (43, 44). This translocation event requires the SpoIIIEprotein (1, 43), without which the forespore receives only theorigin-proximal 30% of a chromosome, while the remainder islocated in the mother cell (44, 45). Thus, in the B. subtilissporulation pathway, the relative order of division and chromosomesegregation is reversed, with division occurring prior to chromosomesegregation, rather than after.

Little is known about the architecture of the forespore chromosomebefore the sporulation septum is synthesized. Is the regionof the invaginating septum cleared of DNA prior to asymmetricseptation, as during vegetative growth, or is the septum requiredto partition the chromosome? Previous studies have supportedthe latter proposal, largely due to the failure to observe partitionedaxial filaments in the absence of septation (45). The presenceof a partitioned axial filament with a condensed region of thechromosome near one cell pole was therefore thought to indicatethat a sporulation septum had formed (13, 21, 29, 45), implyingthat chromosome partitioning was a consequence of cell division.However, these studies preceded reliable methods to simultaneouslyobserve the asymmetrically positioned sporulation septum andthe chromosomes (27), leaving open the possibility that individualchromosomes are reorganized into two separate domains, creatinga gap to accommodate the invaginating septum. Here, we addressthis issue using time-lapse deconvolution microscopy togetherwith fluorescent membrane stains that allow visualization ofsepta and nucleic acid stains to reveal chromosome structure.We demonstrate that, during sporulation, the bacterial chromosomeis partitioned into two domains of unequal size prior to asymmetriccell division. This asymmetric chromosome condensation eventis independent of cell division protein FtsZ, providing evidencefor an FtsZ-independent reorganization of cellular architecturethat serves to prepare the bacterium for asymmetric cell division.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains and culture conditions. PY79 was the wild-type strain used in these studies (46). Theremaining strains were PY79 derivatives and contained the followingadditional mutations or genetic constructs: KP427 P_spac-spo0H(25), KP444 P_spac-ftsZ (2), KP527 ${Delta}$ spo0J-soj (33), and KP648 ${Delta}$ spo0A::erm (17). All experiments were performed at 37°C.Sporulation was induced by resuspension (37), with the timeof resuspension being defined as the onset of sporulation (t₀).Identical results were obtained when sporulation was inducedby nutrient exhaustion (not shown). The IPTG (isopropyl-ß-D-thiogalactopyranoside)-inducibleP_spac promoter was used to allow depletion of FtsZ from strainKP444 during sporulation (27) and to deplete strain KP427 cellsof ${sigma}$ ^H. In the latter case, strain KP427 was grown and sporulatedin media lacking IPTG. Luria-Bertani medium was used for exponentialgrowth of PY79, and the culture was grown to an optical densityat 600 nm of 0.300.

Fluorescent stains. Fluorescent membrane stains FM 4-64, MitoTracker red CMXros,and MitoTracker green and DNA stains SYTO 16 and DAPI (4',6'-diamidino-2-phenylindole)were obtained from Molecular Probes (Eugene, Oreg.) and resuspendedas recommended by the manufacturer. Final concentrations of2 µg of FM 4-64/ml, 0.2 µg of MitoTracker red CMXros/ml,10 µg of MitoTracker green/ml, 0.2 µg of SYTO 16/ml,and 0.3 µg of DAPI/ml were used.

Preparation of samples for immunofluorescence microscopy. Samples were prepared for immunofluorescence microscopy as describedpreviously (28, 33) using rabbit antibodies specific for ${sigma}$ ^F (agift from Richard Losick) and affinity-purified chicken antibodiesspecific for FtsZ (a gift from Petra Levin). The secondary antibodieswere Cy5-labeled goat anti-chicken (Jackson Immunolabs) andOregon green-labeled goat anti-rabbit (Molecular Probes). DAPIand FM 4-64 were added to Slow Fade equilibration buffer (MolecularProbes), as previously described (33).

Microscopy. Membrane and chromosome morphology was assessed using an AppliedPrecision optical sectioning microscope (27), with samples stainedand slides prepared as described previously (27, 33). Briefly,the bacteria were stained with the appropriate fluorescent membranestain and DAPI on a slide and immobilized with a poly-L-lysine(Sigma)-treated coverslip. However, MitoTracker red CMXros stainingwas more uniform if it was mixed with the cells in an Eppendorftube prior to applying cells to a slide. Between 6 and 12 opticalsections were immediately collected from each field, and theimages were deconvolved using Delta Vision software.

We noted that the chromosomes of vegetatively growing B. subtiliscells are extremely sensitive to exposure to the UV light usedto visualize DAPI-stained chromosomes. During time-lapse microscopy,DAPI-stained chromosomes undergo a cycle of decondensation andrecondensation upon exposure to UV excitatory light. For example,after focusing on the cells using FM 4-64 fluorescence, thefirst image of DAPI-stained chromosomes collected during a 2-sexposure to UV light shows the nucleoids to be condensed; however,after an additional 2 s of exposure to UV light, the chromosomesdecondense and appear to fill the entire cytoplasm. This effectis likely due to UV-induced nicking of DNA and the consequentrelaxation of supercoils that normally contribute to chromosomecondensation (15). After more-prolonged UV exposure (>90s), the nucleoids recondense and appear similar to their originalstate. In contrast, nucleoids stained with SYTO 16 appear condensedand do not change appearance even after prolonged exposure toblue excitation light; however, after a 5-s exposure to UV light,they rapidly decondense. We therefore minimized exposure ofthe cells to UV light prior to image acquisition. This phenomenonmay explain the somewhat variable structure of the chromosomesof growing B. subtilis, which can appear either well condensed(as shown here) or more diffuse and unstructured. The chromosomesof sporulating cells also decondensed, but only after prolongedexposure to UV.

Time-lapse microscopy. Sporulating PY79 cells were stained with FM 4-64 (1 µg/ml)and SYTO 16 (0.2 µg/ml) at the time of resuspension, andtime-lapse microscopy was performed as described previously(27). Briefly, 40 µl of culture was placed on a coverslipat 1.0 h after the onset of sporulation (t_1.0), covered witha chambered slide, and heated to 30°C. Three optical sectionsspaced 0.15 µm apart were collected every 15 min for 1h. Under these conditions, the frequency of polar septationwas low, likely due to fluorescence toxicity: only about 5%of the cells that were in focus throughout the experiment synthesizeda polar septum by the end of the experiment. Of these sporangia,about 50% had a clearly partitioned forespore chromosome priorto the onset of septal biogenesis. The remaining sporangia hada partial sporulation septum at the start of the experimentand a partitioned chromosome.

Quantitation of the DNA content of the polar condensed regions. The DNA content of the polar condensed regions of the foresporechromosome (or future forespore chromosome) was quantified usingDelta Vision, version 2.10, software (Applied Precision). Identicalresults were obtained when DNA content was determined both beforeand after deconvolution. Images were collected from 3 to 6 focalplanes of DAPI- and FM 4-64-stained bacteria at either t_1.5for sporangia with partial septa or at both t_1.0 and t_1.5 forsporangia lacking septa. Separate stacks of images were takenfor each fluorophore, and the DAPI images were collected beforethe FM 4-64 images. Exposure times were adjusted to be withinthe linear range of the camera yet to contain sufficient datafor accurate deconvolution and quantitation. The average maximumpixel values were $~$ 3,000 counts/pixel. Following the completionof DNA translocation into the forespore, measurements of DAPIstaining intensity underestimate the amount of DNA in the forespore,probably due to the higher concentration of DNA in the smallforespore than in the larger mother cell and consequently thegreater quenching of the DAPI fluorescence in the forespore(32). Because the measurements presented here are of incompletelytranslocated forespore chromosomes, in which the concentrationof the DNA should be lower than after translocation is complete,these measurements may be less affected by quenching. Nonetheless,our measurements may somewhat underestimate the proportion ofDNA in the polar condensed region.

The DNA content of the polar condensed region relative to thatof the remaining portion was determined by using the "edit polygon"function of the Delta Vision, version 2.10, software. We definedone polygon that enclosed the small condensed region (polarDNA) and a second that enclosed the entire chromosome (totalDNA). The "polygon statistics function" was used to determinethe integrated intensity of each of these regions, a measurethat reflects the total fluorescence intensity within each region(DNA content). At this stage of sporulation, the total DNA contentof the cell is expected to be equivalent to two complete chromosomes.Therefore, to determine the proportion of the future foresporechromosome that was within the polar condensed region, we multipliedthe ratio of polar DNA content to total DNA content by 2. Atotal of 18 wild-type sporangia (from t_1.5) were quantified,10 that lacked a partial or complete sporulation septum by FM4-64 staining and 8 that had a partial sporulation septum asshown by FM 4-64 staining that failed to completely traversethe cell.

RESULTS AND DISCUSSION

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Results and Discussion

Asymmetric chromosome condensation precedes asymmetric division. We have previously shown that vital membrane stain FM 4-64 readilydetects both complete (Fig. 1A to E, arrows 4) and incomplete(Fig. 1A to E, arrows 3) sporulation septa, which form veryclose to the cell pole (27). We therefore stained wild-typeB. subtilis at t_1.5 with FM 4-64 and the DNA-specific stainDAPI and observed that many sporangia containing an incompletesporulation septum also contained a small, condensed regionof one chromosome near the cell pole (Fig. 1A to E, arrows 3).Furthermore, many cells in which there was no evidence of apolar septum contained a small portion of the chromosome condensednear one or both poles (Fig. 1A to E, arrows 1 and 2). Thesecondensed DNA regions are in the same location as those in cellscontaining a partial sporulation septum (Fig. 1A to E, arrows3) and are readily seen before (Fig. 1E, arrows 1 and 3) orafter deconvolution (Fig. 1C and D, arrows 1 and 3), as wellas in fixed cells. We estimate that the gap between the twochromosome domains is approximately 5 to 10 times the spacenecessary to accommodate two lipid bilayers and the interveningcell wall material of the future sporulation septum.

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FIG. 1. Asymmetric chromosome condensation during sporulation precedes asymmetric cell division and is morphologically distinct from the chromosome structure of symmetrically dividing cells. Samples were harvested and stained with FM 4-64 to visualize membranes (red; A, B, F, and G) and with DAPI to infer chromosome structure (blue; B, C, G, and H and D, E, I, and J). The inverted gray-scale images in panels D, E, I, and J enhance visualization of chromosome structure. Panels E and J show chromosome structure prior to deconvolution; panels D and I show the same fields after deconvolution. (A to E) Sporulating cells of wild-type B. subtilis at t_1.5. Arrows 1 and 2, cells that lack sporulation septa (A) and that contain an asymmetrically condensed forespore chromosome (C to E); arrows 3, cell with a sporulation septum that is incomplete (A), as it fails to completely traverse the cell, and with an asymmetrically condensed forespore chromosome (C to E). Arrows 4, sporangium with a sporulation septum that appears to completely traverse the cell (A), creating the larger mother cell and smaller forespore. (F to J) Exponentially growing cells of wild-type B. subtilis, which tend to remain attached following completion of division, thereby forming long chains of individual cells. Arrowheads, partial septum that is forming in a single, long cell (F). One of the two nascent daughter cells contains a bilobed nucleoid (arrows 6), while the second contains two distinct nucleoids that are already well separated (arrows 7). Arrows 5, second nascent daughter cell in which the two visible nucleoids are well separated. Bar (A), 1 µm.

This chromosome structure was distinct from that of exponentiallygrowing cells, which showed symmetrically condensed (or bilobed)chromosomes that were well separated prior to the onset of celldivision (Fig. 1F to J). A large cell in the process of medialcell division is shown in Fig. 1F to J. Within this dividingcell there is a single bilobed nucleoid on the right side ofthe nascent septum (arrows 6) and two well-separated nucleoidson the left side (arrows 7).

Biological membranes can be partitioned into domains differingin lipid composition, which might affect the ability of FM 4-64to stain certain regions of the cell, such as the sporulationseptum. We therefore used two structurally distinct membrane-permeablestains to examine septal structure: MitoTracker red CMXros (Fig.2A) and MitoTracker green (not shown). Identical results wereobtained with these two stains, as many sporangia showed a condensedregion of DNA near one pole in the absence of a sporulationseptum (Fig. 2B, arrow 1). Thus, regardless of the fluorescentmembrane stain used, forespore chromosome condensation was observedprior to the onset of asymmetric cell division.

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FIG. 2. Cells of the wild type and several mutants stained with membrane stain MitoTracker red CMXros (A) or FM 4-64 (C, E, G, and I) and DNA stain DAPI (B, D, F, H, and J). Sporulation was induced by resuspension, and samples were harvested at t_1.5. More than 300 sporangia were examined for each strain; representative examples are shown. (A and B) Wild-type sporangia (PY79) stained with MitoTracker red CMXros (A) and DAPI (B). Both cells lack a sporulation septum and contain an asymmetrically condensed forespore chromosome (arrows 1). (C and D) In the absence of ${sigma}$ ^H (strain KP427), polar septation is blocked and the chromosomes appear decondensed. More than 400 cells were examined by fluorescence microscopy; none showed polar chromosome condensation or polar septa. (E and F) In the presence of IPTG, ${sigma}$ ^H is expressed in strain KP427. Under these conditions, polar septation and chromosome condensation are identical to those in wild-type cells. (G and H) Asymmetric chromosome condensation does not occur in a strain (KP648) lacking the spo0A gene. More than 1,000 cells were examined; representative examples are shown. (I and J) In ${Delta}$ (spo0J-soj) strain KP527, some cells lacking a polar septum show clear asymmetric chromosome condensation (arrows 2 and 3). Arrows 4, cell with a medial nascent septum without separated nucleoids.

Quantitation of the DAPI staining intensity showed that theamount of DNA in the asymmetrically condensed region in cellscompletely lacking a sporulation septum was identical to theDNA content in forespores of sporangia in which synthesis ofthe sporulation septum had started but was incomplete (30% ±4% versus 29% ± 4% of the total forespore chromosomecontent, measured as described in Materials and Methods). Thus,the proportion of the forespore chromosome present in the condensedregion prior to the formation of the polar septum is similarto the DNA content of forespores in mutants that lack a functionalcopy of spoIIIE (30% [44, 45]) and that are defective in postseptationalchromosome translocation (43). These observations suggest thatpartitioning of the origin-proximal 30% of the future foresporechromosome precedes asymmetric cell division.

Timing and frequency of asymmetric chromosome condensation. The timing of chromosome condensation relative to septationwas quantitatively assessed by comparing the frequencies ofeach event at t_1.0 and t_1.5 (Fig. 3). At t_1.0, when very few(1%) cells had formed a polar septum, 9% had asymmetricallycondensed chromosomes (Fig. 3A). Thirty minutes later, 10% ofcells had polar septa and condensed chromosomes, while an additional11% had condensed chromosomes but no polar septa. At both timepoints, we could readily detect condensation at both poles ofthe sporangium, in which cases it appeared that about 30% ofeach chromosome was condensed near each pole. Approximately42% of sporangia containing partitioned chromosomes were partitionedat both poles at t_1.0, whereas, by t_1.5, only 19% displayedbipolar condensation (Fig. 3B). The decreased occurrence ofbipolar condensed chromosomes at later times during sporulationsuggests that the mother cell chromosome may decondense as sporulationproceeds (a point to which we will return later). Furthermore,some condensed chromosomes may not be observed due to the limitedresolution of light microscopy.

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FIG. 3. Timing of polar chromosome condensation. Sporulating cultures of PY79 were stained with FM 4-64 and DAPI, and the numbers of cells containing septa and condensed chromosomes were determined for samples collected 60 (334 cells) and 90 min (285 cells) after the onset of sporulation. (A) The percentage of total cells containing a polar condensed chromosome is plotted next to the percentage of cells containing a polar septum. (B) Percentages of sporulating cells with unipolar or bipolar condensed chromosomes.

Visualization of chromosome dynamics and asymmetric septation with time-lapse microscopy. The results presented above suggested that asymmetric chromosomecondensation preceded formation of the asymmetrically positionedsporulation septum; we used time-lapse microscopy to providequalitative support for this proposal. A sporulating culturewas stained with vital DNA stain SYTO 16 (Molecular Probes)and membrane stain FM 4-64, and deconvolution time-lapse microscopywas performed as described in Materials and Methods, with imagescollected every 15 min. Figure 4 shows two sporangia from thistime-lapse experiment (of the nine that synthesized a polarseptum during the experiment). The first sporangium (Fig. 4A to C)shows a clear partitioning of the future forespore chromosomeinto two domains, with the smaller domain near the cell pole.By the next time point (Fig. 4A' to C'), a partial sporulationseptum was synthesized in the gap between the chromosome domains,and by the next time point (Fig. 4A" to C"), the septum wascomplete.

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FIG. 4. Time-lapse microscopy: asymmetric DNA condensation precedes formation of the asymmetrically positioned sporulation septum. Sporulating cultures of PY79 were stained with vital DNA stain SYTO 16 (green; B, C, E, and F) and membrane stain FM 4-64 (red; A, C, D, and F) and prepared for time-lapse deconvolution microscopy as described in Materials and Methods. Images were acquired every 15 min. Shown are images taken 0, 15 (primes), and 30 min (double primes) after the start of the time-lapse experiment. The predivisional sporangium (A to C) shows a condensed region of the future forespore chromosome near one cell pole. Arrowheads, sites of synthesis of the sporulation septum. (D to F) Sporangium that contains a sporulation septum at the beginning of the experiment (arrowheads) and an asymmetrically condensed chromosome at the forespore distal pole (arrows), where a second partial septum is synthesized (D') and later degraded (D") in an abortive division event that we have previously described (27). Bar, 1 µm.

The second sporangium demonstrates that asymmetric chromosomepartitioning also occurs at the forespore distal pole of themother cell (Fig. 4D to F), preceding the abortive cell divisionevent that often occurs at the second potential site of polarseptation (Fig. 4D' to F') (27). When this second septum regressed,the partitioned domain in the mother cell decondensed (Fig.4E, E', and E"). Thus, asymmetric chromosome condensation precedesasymmetric septation and is reversible.

Polar chromosome condensation depends on transcription factors ${sigma}$ ^H and Spo0A. Spo0A-P is required for the switch from medial to bipolar FtsZring assembly (21); thus mutants lacking Spo0A fail to relocalizethe FtsZ rings and continue to divide medially. They also maintainthe bilobed chromosome structure typical of vegetative growth(Fig. 2G and H) (21). In contrast, ${sigma}$ ^H is not required for thebipolar localization of FtsZ, although, for reasons that areunclear, mutants that lack ${sigma}$ ^H fail to form polar septa (21).

We tested the dependence of asymmetric chromosome condensationon ${sigma}$ ^H by utilizing a strain in which expression of the gene encoding ${sigma}$ ^H (spo0H) was under the control of an IPTG-inducible promoter(18). In the presence of IPTG, polar septa and polar condensedchromosomes appeared at the same rate and frequency as in thewild type (Fig. 2E and F). However, in the absence of ${sigma}$ ^H, neitherpolar septa nor polar chromosome domains were formed (Fig. 2C and D).Indeed, the chromosomes of ${sigma}$ ^H-deficient cells were poorlycondensed, filling the cytoplasm to a much greater extent thanthe wild type or any of the other mutants examined, even infixed cells. It is possible that this chromosome condensationdefect inhibits polar septation, which could be governed bya checkpoint monitoring asymmetric chromosome condensation.Alternatively, the septation defect may be caused by the reducedexpression of cell division proteins such as FtsZ and FtsA inthe absence of ${sigma}$ ^H (8, 10) or by the decreased Spo0A-directedgene expression in the absence of ${sigma}$ ^H (36).

Chromosome-partitioning proteins Spo0J and Soj are not required for asymmetric chromosome condensation. Two candidates for proteins that control chromosome architectureduring sporulation are Spo0J and Soj, homologues of chromosome-and plasmid-partitioning proteins (6). Spo0J and Soj are requiredfor efficient chromosome segregation (16) and together regulatethe transcription of genes required for polar septation: Sojacts as a transcriptional repressor, whereas Spo0J inhibitsSoj, allowing sporulation-specific gene expression and polarseptation (3, 30). Spo0J and Soj provide attractive candidatesfor proteins involved in modulating chromosome architecture,since Spo0J binds to the origin-proximal region of the chromosomelikely to be in the polar condensed region and assembles intofoci near the cell poles (9, 23). Furthermore, it has been suggestedthat Soj compacts Spo0J foci (26), an activity that could causedramatic changes in chromosome architecture, such as asymmetricchromosome partitioning.

We therefore investigated the structure of the chromosome inthe spo0J soj double mutant, which is able to express early-sporulation-specificgenes and thereby to synthesize the polar septum. If eitherSpo0J or Soj is required for asymmetric chromosome condensation,then, in the double mutant, polar septation should occur beforechromosome partitioning. This was not the case, as we observedchromosome partitioning before polar septation in some sporangia(Fig. 2I and J, arrows 2), although in many cases the polarchromosome domain was less clearly separated from the remainderof the chromosome than in the wild type (Fig. 2I and J, arrows3). This could suggest that Spo0J or Soj or both stabilize thepartitioned chromosome or that the multiple copies of the chromosomefound in the spo0J soj double mutant (41) preclude efficientpacking. In keeping with the importance of these proteins forchromosome partitioning, we noted that the spo0J soj doublemutant occasionally formed medial septa directly through a chromosome(Fig. 2I and J, arrows 4) and that it produced anucleate cellsand minicells (not shown). Thus neither Spo0J or Soj is essentialfor asymmetric chromosome condensation, although it remainspossible that they stabilize the partitioned chromosome domain.

Depletion of FtsZ during sporulation does not prevent asymmetric chromosome condensation. An early event in the asymmetric division pathway of B. subtilisis the relocalization of FtsZ from the midpoint to the cellpoles (21), an event likely to be necessary to localize otherproteins to polar division sites (5, 20, 22). The results describedabove demonstrate that asymmetric chromosome condensation occursprior to the onset of septal biogenesis but did not addresswhether it is independent of FtsZ ring assembly, which precedescell division. We therefore depleted cells of FtsZ during sporulationby utilizing a strain in which the ftsZ gene is under the controlof the IPTG-inducible P_spac promoter (2). When cells are grownwithout IPTG, FtsZ levels fall, ultimately preventing cell division,but not cell growth, thereby causing the production of long,multinucleate cells (filaments) lacking detectable FtsZ. Undersporulation conditions, these filaments fail to synthesize thesporulation septum, although the cells express early sporulation-specificproteins (2).

Following FtsZ depletion, we readily observed small condensedregions of the chromosomes during sporulation, both near thecell poles (Fig. 5A and B, arrows 1) and, less frequently, withinthe filaments (Fig. 5F to H, arrows 6). Nearly 50% of the filamentsin the population contained at least one asymmetrically condensedchromosome by t₂. We confirmed that the filaments with asymmetricallycondensed chromosomes had entered the sporulation pathway byobserving that they contained the early-sporulation-specificprotein ${sigma}$ ^F by immunofluorescence microscopy (Fig. 5F to H, arrows5 and 6). In contrast, filaments without asymmetrically condensedchromosomes showed no ${sigma}$ ^F immunostaining (Fig. 5F to H, arrows4). Filaments with asymmetrically condensed chromosomes lackedFtsZ rings, as assessed by immunofluorescence microscopy (notshown); indeed, under these depletion conditions, we were unableto detect the FtsZ protein in Western blots (not shown). Thus,the polar chromosome condensation machinery may function independentlyof FtsZ ring formation. If so, then condensation would be independentof the entire cell division apparatus, whose assembly at futuredivision sites is abolished in the absence of FtsZ.

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FIG. 5. Chromosome condensation and expression of sporulation-specific protein ${sigma}$ ^F in wild-type and FtsZ-depleted cells. (A and B) Following depletion of FtsZ (strain KP444; see Materials and Methods), cell division is inhibited, producing long cells (A; FM 4-64 staining) that contain asymmetrically condensed chromosomes (B; DAPI staining) near the cell pole (arrows 1). (C to H) Cells were harvested at t₂, prepared for immunofluorescence microscopy, and stained with anti- ${sigma}$ ^F antibodies (green; D, E, G, and H) and DAPI (blue; C, E, F, and H). (C to E) Wild-type cells that show no ${sigma}$ ^F immunostaining lack polar condensed regions of their chromosomes (arrows 2), whereas those with bright ${sigma}$ ^F immunostaining show condensed chromosomes in various stages of translocation into the forespore (arrows 3). (F to H) Cells of strain KP444 (P_spac-ftsZ) were depleted of FtsZ by growth without IPTG (as described Materials and Methods), blocking cell division. Cells that show no ${sigma}$ ^F immunostaining have symmetrically condensed chromosomes (arrows 4), whereas those with bright ${sigma}$ ^F immunostaining show polar condensed regions (arrows 5 and 6). Scale bar, (A), 1 µm.

A novel determinant of cellular asymmetry. Our results demonstrate that clearing DNA from the path of aninvaginating septum normally occurs during both symmetric andasymmetric cell division (Fig. 6). However, during the asymmetriccell division of sporulation, a single chromosome is partitionedinto two separate domains of unequal sizes, whereas, duringvegetative growth, the individual daughter chromosomes are separatelyand symmetrically condensed following their replication. Whilethe mechanism by which bacteria partition a single chromosomeinto two distinct domains is presently unclear, our resultsdramatically illustrate that bacteria share with eukaryotesthe ability to precisely control their chromosomal architecture.

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FIG. 6. A model for steps in the establishment of asymmetry during sporulation in B. subtilis. (A) The switch in the assembly of FtsZ (ring) from the cell midpoint to positions near both poles requires transcription factor Spo0A-P, and the onset of polar septation requires ${sigma}$ ^H. (B) The reorganization of the chromosome condensation machinery from the symmetrically condensed bilobed structure of growing cells to form polar condensed chromosomes requires transcription factors Spo0A-P and ${sigma}$ ^H. Asymmetric division then commences at one of the two sites of FtsZ assembly, and the remaining forespore chromosome is translocated across the sporulation septum by SpoIIIE. In cells lacking Spo0A-P, symmetric chromosome condensation is seen, whereas cells lacking ${sigma}$ ^H show decondensed chromosomes that appear distinct from both the bilobed chromosomes of growing cells and the more-condensed chromosomes of sporulating bacteria.

Surprisingly, asymmetric chromosome condensation not only precedesasymmetric cell division but also occurs when asymmetric divisionis completely abolished by the depletion of cell division proteinFtsZ. This suggests that asymmetric chromosome condensationis independent of the known members of the cell division machinery,since FtsZ is likely to be required for the recruitment of theseproteins to potential division sites (7, 20, 42). Our resultsimply the existence of a novel determinant of cellular asymmetrythat is independent of the known cell division machinery andthat directs the reorganization of the chromosome in preparationfor asymmetric division.

ACKNOWLEDGMENTS

We thank Richard Losick for his gift of ${sigma}$ ^F-specific antibodiesand strains and Petra Levin and Richard Losick for the FtsZ-specificantibodies. We thank Aileen Rubio, Petra Levin, and RichardLosick for comments on the manuscript.

This work was supported by awards from the Arnold and MabelBeckman Foundation and the Searle Scholars Program/The ChicagoCommunity Trust and by an NSF predoctoral fellowship awardedto M.D.S.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biology, University of California at San Diego, La Jolla, CA 92093-0349. Phone: (858) 822-1314. Fax: (858) 822-1431. E-mail: kpogliano{at}ucsd.edu.

REFERENCES

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