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Journal of Bacteriology, August 2006, p. 6039-6043, Vol. 188, No. 16
0021-9193/06/$08.00+0     doi:10.1128/JB.01750-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The Bacillus subtilis DivIVA Protein Has a Sporulation-Specific Proximity to Spo0J

S. E. Perry and D. H. Edwards^*

Division of Pathology & Neuroscience, University of Dundee, Ninewells Medical School, Dundee, DD1 9SY, United Kingdom

Received 16 November 2005/ Accepted 8 June 2006

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The Bacillus subtilis DivIVA protein controls the positioning of the division site and the relocation of the chromosome during sporulation. By performing coimmunoprecipitation experiments, we demonstrated that a myc-DivIVA protein is in proximity to FtsZ and MinD during vegetative growth and Spo0J during the first 120 min of sporulation.

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The process by which Bacillus subtilis changes from vegetative growth into a distinct developmental pathway that results in spore formation has been studied extensively (5, 9). A key event in the initial stages of spore development is the movement of the two chromosomes away from the cell center and their eventual attachment to the cell poles (axial filament formation). This occurs in two distinct steps, beginning with the extraction of the chromosomal origin from its vegetative position and movement towards the cell pole (18). In a second step, the chromosome is decorated with the DNA binding protein RacA and is attached to the cell pole (1, 17). Interestingly, both of these events require the vegetative cell division positioning protein DivIVA (2, 4). DivIVA is a small, predominantly coiled-coil protein that is recruited to the vegetative cell division site after the assembly of FtsZ (12) and the incorporation of FtsW (data not shown). DivIVA remains associated with the division site as it matures and eventually splits to form the two new cell poles (4). The subcellular localization of DivIVA is crucial for the correct distribution of a bipartite cell division inhibitor complex consisting of the MinC and MinD proteins (MinCD) (12). By maintaining MinCD at the cell pole, DivIVA prevents the assembly of an FtsZ ring in the chromosome free space at the cell pole and promotes vegetative division at the midcell. Recently we identified a polar targeting mutant of DivIVA that functions in both vegetative growth and sporulation (14). DivIVA_R18C localizes to the chromosome in the presence of Spo0J/Soj and can be observed to occur transiently at the cell division site. It appears that the temporary association of this mutant protein with the division site is sufficient to partially localize MinCD. More intriguingly, the association of the mutant protein with the chromosome is sufficient to allow the relocation of the chromosome by MinD and Spo0J/Soj at the onset of sporulation. To identify proteins in proximity to DivIVA and DivIVA_R18C, we have developed a coimmunoprecipitation (co-IP) protocol for the isolation of myc-DivIVA-containing complexes.

Immunoprecipitation of DivIVA interacting proteins. To precipitate a DivIVA complex from B. subtilis, we utilized a myc-DivIVA protein that could support both normal vegetative growth and wild-type levels of sporulation (Table 1). For our preliminary co-IP experiments, we used strain SE65, which contains a divIVA deletion (divIVA) and an IPTG (isopropyl-ß-D-thiogalactopyranoside)-inducible myc-divIVA (P_spac myc-divIVA::amyE) (for descriptions of strains and plasmids used in this study, see Tables 2 and 3, respectively ). This strain is viable and when grown in the presence of 0.5 mM IPTG has a phenotype similar to that of the isogenic strain SE85 (divIVA P_spac divIVA::amyE) (Table 1). In order to validate our protocol, we also chose to work with two partially active mutant proteins. The first, DivIVA_R18C, is unable to target the cell poles and localizes to the chromosome in a Spo0J/Soj-dependent manner (14). The second, DivIVA_W148A, has not been described previously and contains a mutation in a highly conserved tryptophan that is located close to the C terminus of the protein. In complementation experiments, this mutant was able to support vegetative growth but produced a phenotype similar to that of the original divIVA1 mutant (16) (Table 1). The two alleles were cloned to enable the controllable expression of the epitope-tagged derivatives myc-divIVA_R18C and myc-divIVA_W148A in a divIVA deletion background (Table 1).

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TABLE 1. Complementation of a divIVA deletion by ectopic expression of myc-tagged versions of divIVA, divIVAR18C, and divIVAW148A

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TABLE 2. Bacterial strains used in this study

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TABLE 3. Plasmids used in this study

In preliminary studies, we performed co-IP experiments with mid-exponential-phase cultures of test strain SE65 (myc-divIVA::amyE) and control strain SE85 (divIVA::amyE). All cells were grown in LB supplemented with 0.5 mM IPTG to an optical density at 600 nm of 0.1. A 20-ml aliquot of the culture was harvested by centrifugation at 4,000 rpm for 5 min and protoplasts produced by incubation for 20 min at 37°C in 1x SMM (0.5 M sucrose, 20 mM maleic acid, 20 mM MgCl₂, pH 6.5) with 1 mg/ml lysozyme and 1 U/ml DNase I. The protein concentration of each sample was determined using the Bradford assay (1a). Dithiobis(succinimidyl propionate) (11) (20 µmol per 100 µg protein) was added to each aliquot to cross-link proteins within the protoplasts. The cross-linking reactions were allowed to proceed for 5 min at 37°C. Excess dithiobis(succinimidyl propionate) was inactivated by the addition of 20 mM Tris-HCl, pH 7.5 (final concentration), and the protoplasts lysed by the addition of 0.02% to 0.05% Triton X-100 (final concentration). Dynabeads protein G coated magnetic beads (Dynal Biotech Ltd.), which had been cross-linked to mouse monoclonal anti-c-myc antibody 9E10, were then added to each sample. The magnetic bead-cell extract mix was then incubated at 25°C on a rotary shaker for 1 h. The beads were washed four times with 1 ml phosphate-buffered saline. Next, the myc-DivIVA-containing complexes were eluted in 1x Laemmli sample buffer supplemented with dithiothreitol, and the cross-links holding the complexes together were reduced. Eluted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting. This approach confirmed that myc-DivIVA could be precipitated from strain SE65 (myc-divIVA::amyE) but not from strain SE85 (divIVA::amyE) (data not shown). We repeated the procedure with several strains and screened for the precipitation of four candidate proteins, MinD, FtsZ, Spo0J, and YpsB. MinD is known to require the presence of DivIVA for its correct localization, and FtsZ is required for the recruitment of DivIVA to the cell division site (12). Spo0J is required for the proper organization and trapping of the chromosome origin at the onset of sporulation (18) and is essential for the localization of a DivIVA_R18C mutant to the chromosome (14). The fourth protein, YpsB, localizes to the cell division site in an FtsZ-dependent manner but does not interact with either DivIVA or MinD (data not shown) and was therefore intended to act as a negative control.

The FtsZ and MinD proteins were precipitated with myc-divIVA, but Spo0J and YpsB were not (Fig. 1, lane 1). The precipitation of MinD was expected, and the identification of FtsZ is consistent with the subcellular location of DivIVA at the cell division site. The myc-DivIVA_R18C and myc-DivIVA proteins precipitated similar levels of FtsZ (Fig. 1, lane 2), but myc-DivIVA_R18C precipitated lower levels of MinD (Fig. 1, compare lane 1 and lane 2). myc-DivIVA_R18C does not localize to the cell pole (14) and therefore would be expected to precipitate less MinD. However, since myc-DivIVA_R18C is rarely observed to occur at the cell division site, the amount of FtsZ precipitated by this mutant protein was surprising. A possible explanation may be provided by the observation that a ring of DivIVA molecules is visualized only rarely in close proximity to FtsZ and the majority of DivIVA at the midcell is actually incorporated into a coating that lines the invaginating septum (7). One hypothesis would be that the rarely observed coincidence of DivIVA and FtsZ represents the initial recruitment of the protein and that this is the same point in division when DivIVA_R18C is in proximity to the division apparatus. We then envisage that, once recruited, DivIVA goes on to form the coating that is ultimately responsible for the polar cap, a process that DivIVA_R18C cannot follow. While this hypothesis would explain why both proteins precipitate similar amounts of FtsZ, we cannot exclude the possibility that myc-DivIVA_R18C is locked in a conformation that remains associated with FtsZ molecules after it has left the division site or that the mutant protein may interact with FtsZ molecules that are not part of the cell division ring. More importantly, myc-DivIVA_R18C also precipitated Spo0J, and the isolation of this protein was not dependent on MinD (Fig. 1, lane 2 and lane 4). This set of biochemical data is consistent with genetic and cell biology data which indicate that DivIVA_R18C and Spo0J are in close proximity during vegetative growth (14). To understand the biological significance of this result, we turned our attention to sporulation, specifically, the initial 90 min when DivIVA, MinD, and Spo0J/Soj are all required for the relocation of the origin (14).

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FIG. 1. Identification of candidate proteins from co-IP experiments performed with myc-DivIVA variants during vegetative growth. Proteins were eluted after co-IP experiments using strains expressing DivIVA, myc-DivIVA, myc-DivIVAR18C, or myc-DivIVAw148A. Samples were separated by SDS-PAGE and Western blotting. Western blots were probed with (A) anti-FtsZ, (B), anti-MinD, (C), anti-Spo0J, or (D) anti-YpsB. Lanes C, proteins precipitated from strain SE85 (divIVA); lanes L, loading control consisting of whole-cell extract from strain SE65 (myc-divIVA); lanes 1, proteins precipitated from strain SE65 (myc-divIVA); lanes 2, proteins precipitated from strain SE66 (myc-divIVAR18C); lanes 3 (for panels A and B), proteins precipitated from strain SE68 (myc-divIVAW148A); lane 4 (for panel C), proteins precipitated from strain SE75 (minD myc-divIVAR18C). Bars show the sizes and positions of molecular size markers. nd, not determined.

Strains expressing myc-DivIVA in the presence (SE65) or absence (SE74) of MinD were harvested at time points from t₁₀ (10 min after the initiation of sporulation) to t₁₈₀ (13). Spo0J was found to precipitate with myc-DivIVA in a MinD-independent manner during the first 120 min of sporulation (Fig. 2A and B ). Notably, the amount of Spo0J precipitated remained constant from t₁₀ to t₁₂₀; however, by t₁₈₀ the amount of Spo0J pulled down with myc-DivIVA decreased dramatically. We confirmed by Western blotting that this reduction was not caused by a change in the levels of either Spo0J or myc-DivIVA (Fig. 2C and D). We also determined the amount of myc-DivIVA precipitated at t₁₈₀ and noted that the efficiency of our co-IP experiments did not alter (data not shown). Therefore, we propose that an association between myc-DivIVA and Spo0J occurs at a specific point during development and that myc-DivIVA_R18C is trapped in a conformation that mimics this developmental change.

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FIG. 2. myc-DivIVA is in proximity to Spo0J during the early stages of sporulation. (A and B) Western blots of SDS-PAGE gels probed with anti-Spo0J. For panel B, proteins were isolated from strain SE75 (minD myc-divIVAR18C). Lanes C, whole-cell extract from strain SE65 (myc-divIVA), lanes 10 to 180, proteins precipitated from strain SE65 (myc-divIVA) 10, 90, 120, or 180 min after inducing sporulation. (C and D) Western blots of SE65 whole-cell extracts separated by SDS-PAGE and probed with (C) anti-Spo0J or (D) anti-DivIVA. Lanes from left to right represent samples taken immediately prior to resuspension (0 min) and then at 10, 90, 120, and 180 min after inducing sporulation, respectively.

divIVA can be deleted in the absence of spo0J-soj. We were unable to inactivate divIVA in a wild-type background and could obtain a viable deletion strain only in the absence of either minD or minC (4, 14; also data not shown). Since myc-DivIVA could be demonstrated to precipitate with both MinD and Spo0J, we determined whether divIVA could be deleted in the absence of spo0J-soj. Strain SE78 [(spo0J-soj)] was transformed with the divIVA deletion plasmid pSP22 (14), and 28 Tet^r Cam^s colonies were identified. For one transformant (SE79), we confirmed the deletion of divIVA by PCR and the genotype [divIVA (spo0J-soj) minD⁺] by Western blotting (data not shown). We considered it necessary to repeat this result by transforming the spo0J-soj deletion strain (SE78) with chromosomal DNA isolated from SE39 (minD::erm divIVA::tet). Once again we confirmed that a deletion of divIVA could be tolerated in the presence of minC and minD (SE80). Analysis of strains SE79 and SE80 revealed an identical phenotype that was characterized by a mixture of filamentous cells and minicells (Table 4 and Fig. 3A and B). Although their phenotype was similar to the original divIVA1 phenotype (4, 16), SE79 and SE80 produced a significantly higher percentage of minicells. Unusually, these minicells often occurred in pairs or short chains that suggested successive rounds of polar cell division (Fig. 3C).

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TABLE 4. Summary of phenotypes of strains SE79, SE78, and 1751

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FIG. 3. Phenotype of strains SE79 and SE80 [divIVA (spo0J-soj)] and localization of GFP-MinD in the absence of DivIVA and Spo0J/Soj. (A) Phase-contrast image of mid-exponential-growth-phase SE79 cells fixed in 70% ethanol for measurement (8). (B) Corresponding fluorescent image. Chromosomes were stained with DAPI (4',6'-diamidino-2-phenylindole) to reveal the positions of the nucleoids. (C) Minicells of SE79 and SE80 [divIVA (spo0J-soj)] strains. All differential interference contrast (DIC) images were obtained using a 100x objective, 1.6x Optivar, Hamamatsu Orca ERII charge-coupled-device camera and Openlab 3.0.7 (Improvision). (i) Oblique minicell observed in SE79. The arrow indicates the production of an oblique minicell, a characteristic shared with the original divIVA1 mutant (4, 16). (ii) Different-sized minicells observed in SE80. The arrow indicates a larger-than-usual minicell that has arisen from a second cell division close to the cell pole. (iii) Pairs of SE80 minicells arising from the cell pole. The arrow indicates a small minicell that is the result of an oblique cell division. (D and E) Expression of GFP-MinD in (D) SE83 (divIVA minD) and (E) SE82 [divIVA minD (spo0J-soj)]. (i) Phase-contrast images of living cells induced to express GFP-MinD by the addition of 0.1% xylose for 30 min. (ii) Corresponding fluorescent images, revealing (for panel D) localization of GFP-MinD as transverse bands and (for panel E) punctate localization of GFP-MinD. Arrows indicate the positions of obvious cell division sites. Bar, 10 µm.

GFP-MinD does not recognize the septum in the absence of DivIVA and Spo0J/Soj. The lethality of a divIVA deletion is proposed to result from the uncontrolled activity of the bipartite cell division inhibitor complex MinCD (2, 4). Therefore, to understand the viability of SE79 and SE80 we determined the localization of MinCD in the absence of divIVA and spo0J-soj. For this work, we constructed a strain (SE82) that combined the spo0J-soj, divIVA, and minD deletions with a xylose-inducible green fluorescent protein (GFP)-MinD fusion protein. In the absence of xylose, SE82 exhibited the minicell phenotype characteristic of a minD deletion strain. In the presence of 0.1% xylose, GFP-MinD was expressed and after 30 min the cells began to filament. When we examined these cells by epifluorescence microscopy, we observed discrete patches of fluorescence but no bands of GFP-MinD at potential cell division sites (Fig. 3E, panel ii). This pattern contrasted with that of our control strain SE83 (divIVA minD P_xyl gfp-minD), which under identical conditions produced a diffuse GFP-MinD signal that appeared to coat the cytoplasmic membrane and form a series of transverse bands that corresponded to the location of septa or presumed cell division sites (Fig. 3D, panel ii). Therefore, we conclude that in the absence of both spo0J-soj and divIVA MinD assembles into patches on the cell membrane and that this is sufficient to titrate the level of MinCD inhibitor and allow a small number of FtsZ rings to assemble.

In conclusion, by using a fully functional epitope-tagged version of DivIVA, we demonstrated in co-IP experiments that myc-DivIVA is in proximity to FtsZ and MinD during vegetative growth and Spo0J during sporulation. This proximity to Spo0J does not exist prior to resuspension but occurs rapidly upon resuspension and is independent of MinD. Therefore, the key question of how DivIVA switches from controlling the bipartite cell division inhibitor MinCD during vegetative growth to controlling the positioning of the chromosome during the initial stages of sporulation appears to involve an increased proximity to Spo0J. This association between DivIVA and Spo0J may explain the chromosome partition function assigned to other members of the DivIVA protein family (6, 15).

 
This work was supported by a Wellcome Trust project grant (064258/Z/01/Z), with additional pieces of equipment funded by TENOVUS Tayside and The University of Dundee Medical School Anonymous Trust.

We thank Isabella Clottey and Ian Caithness for their assistance in the laboratory and Jeff Errington, Richard Daniel, Ling Wu, and Francis Fuller-Pace for support, comments, and the kind gifts of strains, plasmids, and antibodies.

 
* Corresponding author. Mailing address: Division of Pathology & Neuroscience, University of Dundee, Ninewells Medical School, Dundee, DD1 9SY, United Kingdom. Phone: (0)1382496388. Fax: (0)1382633952. E-mail: d.h.edwards{at}dundee.ac.uk.

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Journal of Bacteriology, August 2006, p. 6039-6043, Vol. 188, No. 16
0021-9193/06/$08.00+0     doi:10.1128/JB.01750-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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