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1.
To clarify the function of DivIVA in Streptococcus pneumoniae, we localized this protein in exponentially growing cells by both immunofluorescence microscopy and immunoelectron microscopy and found that S. pneumoniae DivIVA (DivIVA(SPN)) had a unique localization profile: it was present simultaneously both as a ring at the division septum and as dots at the cell poles. Double-immunofluorescence analysis suggested that DivIVA is recruited to the septum at a later stage than FtsZ and is retained at the poles after cell separation. All the other cell division proteins that we tested were localized in the divIVA null mutant, although the percentage of cells having constricted Z rings was significantly reduced. In agreement with its localization profile and consistent with its coiled-coil nature, DivIVA interacted with itself and with a number of known or putative S. pneumoniae cell division proteins. Finally, a missense divIVA mutant, obtained by allelic replacement, allowed us to correlate, at the molecular level, the specific interactions and some of the facets of the divIVA mutant phenotype. Taken together, the results suggest that although the possibility of a direct role in chromosome segregation cannot be ruled out, DivIVA in S. pneumoniae seems to be primarily involved in the formation and maturation of the cell poles. The localization and the interaction properties of DivIVA(SPN) raise the intriguing possibility that a common, MinCD-independent function evolved differently in the various host backgrounds.  相似文献   

2.
The Bacillus subtilis protein DivIVA controls both the positioning of the vegetative cell division site and the polar attachment of the chromosome during sporulation. In vegetative growth DivIVA attracts the bipartite cell division inhibitor MinCD away from the cell centre and towards the cell pole. This process ensures the inactivation of old polar division sites and leaves the cell centre free for the assembly of a new cell division complex. During sporulation MinCD and DivIVA levels fall, but DivIVA remains at the cell poles and becomes involved in the migration of the chromosomes to the pole. In order to investigate polar targeting of DivIVA, we undertook a mutational analysis of the 164-amino-acid protein. These studies identified one mutant (divIVA(R18C)) that could not localize to the cell pole but which retained the ability to support both vegetative growth and 50% sporulation efficiency. Further analysis revealed that, in the absence of polar targeting, DivIVA(R18C) localized to the nucleoid during vegetative growth in a Spo0J/Soj-dependent manner and required Spo0J/Soj and MinD to orientate the chromosomes correctly during sporulation. We demonstrate that polar targeting of DivIVA(R18C) is not essential during vegetative growth because the mutant can recognize the cell division site and influences the localization of MinD. Similarly we show that DivIVA(R18C) can function during sporulation because it can support the Spo0J/Soj orientation of the chromosome. In addition, we establish that both residues 18 and 19 constitute a DivIVA polar targeting determinant.  相似文献   

3.
The divIVA minicell locus of Bacillus subtilis.   总被引:3,自引:1,他引:2       下载免费PDF全文
The Bacillus subtilis divIVA1 mutation causes misplacement of the septum during cell division, resulting in the formation of small, circular, anucleate minicells. This study reports the cloning and sequence analysis of 2.4 kb of the B. subtilis chromosome including the divIVA locus. Three open reading frames were identified: orf, whose function is unknown; divIVA; and isoleucyl tRNA synthetase (ileS). We identified the point mutation in the divIVA1 mutant allele. Inactivation of divIVA produces a minicell phenotype, whereas overproduction of DivIVA results in a filamentation phenotype. Mutants with mutations at both of the minicell loci of B. subtilis, divIVA and divIVB, possess a minicell phenotype identical to that of the DivIVB- mutant. The DivIVA-mutants, but not the DivIVB- mutants, show a decrease in sporulation efficiency and a delay in the kinetics of endospore formation. The data support a model in which divIVA encodes the topological specificity subunit of the minCD system. The model suggests that DivIVA acts as a pilot protein, directing minCD to the polar septation sites. DivIVA also appears to be the interface between a sporulation component and MinCD, freeing up the polar septation sites for use during the asymmetric septation event of the sporulation process.  相似文献   

4.
DivIVA is involved in placement of the division septum and chromosome segregation in Bacillus subtilis and it plays important roles in cell division or morphogenesis in diverse Gram-positive bacteria. In Staphylococcus aureus, DivIVA is localized at the division septum, but it does not colocalize with the chromosomal origin of replication, as labeled with SpoOJ protein. Unexpectedly, a divIVA null mutant is not impaired in growth, nor is it affected in chromosome segregation or cell morphology.  相似文献   

5.
The cell division protein DivIVA influences protein transport via the accessory SecA2 secretion route in Listeria monocytogenes. In contrast, DivIVA from the closely related bacterium Bacillus subtilis contributes to division site selection via the MinCDJ system. However, no classical min phenotype, i.e. filamentation and minicell production was observed with a listerial ΔdivIVA mutant. This has prompted the speculation that division site selection is DivIVA‐independent in L. monocytogenes. We addressed this question with genetic, cytological and bacterial two‐hybrid experiments and the data obtained correct this view. DivIVA not only binds to MinJ but also directly interacts with MinD. Experiments with fluorescently tagged proteins showed that localization of MinC and MinD was clearly DivIVA‐dependent, whereas localization of MinJ was not. An impact of DivIVA on cell division was confirmed by careful comparisons of cell size distributions of divIVA and secA2 mutants. Gene deletion studies and epistasis experiments consistently reinforced these findings, and also revealed that MinJ must have a DivIVA‐independent function. The frequency of minicell formation is low in L. monocytogenes min mutants. However, since listerial minicells might be useful as carriers for the introduction of therapeutic compounds into eukaryotic cells, we present a strategy how minicell frequency can be increased.  相似文献   

6.
Bacterial cell division commences with the assembly of the tubulin-like protein, FtsZ, at midcell to form a ring. Division site selection in rod-shaped bacteria is mediated by MinC and MinD, which form a division inhibitor. Bacillus subtilis DivIVA protein ensures that MinCD specifically inhibits division close to the cell poles, while allowing division at midcell. We have examined the localization of MinC protein and show that it is targeted to midcell and retained at the mature cell poles. This localization is reminiscent of the pattern previously described for MinD. Localization of MinC requires both early (FtsZ) and late (PbpB) division proteins, and it is completely dependent on MinD. The effects of a divIVA mutation on localization of MinC now suggest that the main role of DivIVA is to retain MinCD at the cell poles after division, rather than recruitment to nascent division sites. By overexpressing minC or minD, we show that both proteins are required to block division, but that only MinD needs to be in excess of wild-type levels. The results suggest a mechanism whereby MinD is required both to pilot MinC to the cell poles and to constitute a functional division inhibitor.  相似文献   

7.
The Bacillus subtilis divIVA gene, first defined by a mutation giving rise to anucleate minicells, has been cloned and characterized. Depletion of DivIVA leads to inhibition of the initiation of cell division. The residual divisions that do occur are abnormally placed and sometimes misorientated relative to the long axis of the cell. The DivIVA phenotype can be suppressed by disruption of the MinCD division inhibitor, suggesting that DivIVA controls the topological specificity of MinCD action and thus septum positioning. A DivIVA–GFP fusion targets to new and used sites of cell division, consistent with it having a direct role in topological specification.  相似文献   

8.
The Bacillus subtilis divIVA gene encodes a coiled-coil protein that shows weak similarity to eukaryotic tropomyosins. The protein is targeted to the sites of cell division and mature cell poles where, in B.subtilis, it controls the site specificity of cell division. Although clear homologues of DivIVA are present only in Gram-positive bacteria, and its role in division site selection is not conserved in the Gram-negative bacterium, Escherichia coli, a DivIVA-green fluorescent protein (GFP) fusion was targeted accurately to division sites and retained at the cell pole in this organism. Remarkably, the same fusion protein was also targeted to nascent division sites and growth zones in the fission yeast Schizosaccharomyces pombe, mimicking the localization of the endogenous tropomyosin-like cell division protein Cdc8p, and F-actin. The results show that a targeting signal for division sites is conserved across the eukaryote-prokaryote divide.  相似文献   

9.
Sporulating cells of Bacillus subtilis undergo a highly polarized cell division and possess a specialized mechanism to move the oriC region of the chromosome close to the cell pole before septation. DivIVA protein, which localizes to the cell pole, and the Soj and Spo0J proteins, which associate with the chromosome, are part of the mechanism that delivers the chromosome to the cell pole. A sporulation-specific protein, RacA, encodes a third DNA-binding protein, which acts in conjunction with Soj and Spo0J to effect efficient polar chromosome segregation. divIVA mutants and soj racA double mutants have an unexpected phenotype in which specific markers to the left and right of oriC can be captured in the prespore compartment but the central oriC region is efficiently excluded. This 'residual' trapping requires Spo0J protein. We suggest that the Soj RacA DivIVA system is required to extract the oriC region from its position determined by the vegetative chromosome segregation machinery and anchor it to the cell pole.  相似文献   

10.
The earliest event in bacterial cell division is the assembly of a tubulin-like protein, FtsZ, at mid-cell to form a ring. In rod-shaped bacteria, the Min system plays an important role in division site placement by inhibiting FtsZ ring formation specifically at the polar regions of the cell. The Min system comprises MinD and MinC, which form an inhibitor complex and, in Bacillus subtilis, DivIVA, which ensures that division is inhibited only in the polar regions. All three proteins localize to the division site at mid-cell and to cell poles. Their recruitment to the division site is dependent on localization of both 'early' and 'late' division proteins. We have examined the temporal and spatial localization of DivIVA relative to that of FtsZ during the first and second cell division after germination and outgrowth of B. subtilis spores. We show that, although the FtsZ ring assembles at mid-cell about halfway through the cell cycle, DivIVA assembles at this site immediately before cell division and persists there during Z-ring constriction and completion of division. We also show that both DivIVA and MinD localize to the cell poles immediately upon spore germination, well before a Z ring forms at mid-cell. Furthermore, these proteins were found to be present in mature, dormant spores. These results suggest that targeting of Min proteins to division sites does not depend directly on the assembly of the division apparatus, as suggested previously, and that potential polar division sites are blocked at the earliest possible stage in the cell cycle in germinated spores as a mechanism to ensure that equal-sized daughter cells are produced upon cell division.  相似文献   

11.
DivIVA proteins and their GpsB homologues are late cell division proteins found in Gram‐positive bacteria. DivIVA/GpsB proteins associate with the inner leaflet of the cytosolic membrane and act as scaffolds for other proteins required for cell growth and division. DivIVA/GpsB proteins comprise an N‐terminal lipid‐binding domain for membrane association fused to C‐terminal domains supporting oligomerization. Despite sharing the same domain organization, DivIVA and GpsB serve different cellular functions: DivIVA plays diverse roles in division site selection, chromosome segregation and controlling peptidoglycan homeostasis, whereas GpsB contributes to the spatiotemporal control of penicillin‐binding protein activity. The crystal structures of the lipid‐binding domains of DivIVA from Bacillus subtilis and GpsB from several species share a fold unique to this group of proteins, whereas the C‐terminal domains of DivIVA and GpsB are radically different. A number of pivotal features identified from the crystal structures explain the functional differences between the proteins. Herein we discuss these structural and functional relationships and recent advances in our understanding of how DivIVA/GpsB proteins bind and recruit their interaction partners, knowledge that might be useful for future structure‐based DivIVA/GpsB inhibitor design.  相似文献   

12.
DivIVA proteins are curvature-sensitive membrane binding proteins that recruit other proteins to the poles and the division septum. They consist of a conserved N-terminal lipid binding domain fused to a less conserved C-terminal domain. DivIVA homologues interact with different proteins involved in cell division, chromosome segregation, genetic competence, or cell wall synthesis. It is unknown how DivIVA interacts with these proteins, and we used the interaction of Bacillus subtilis DivIVA with MinJ and RacA to investigate this. MinJ is a transmembrane protein controlling division site selection, and the DNA-binding protein RacA is crucial for chromosome segregation during sporulation. Initial bacterial two-hybrid experiments revealed that the C terminus of DivIVA appears to be important for recruiting both proteins. However, the interpretation of these results is limited since it appeared that C-terminal truncations also interfere with DivIVA oligomerization. Therefore, a chimera approach was followed, making use of the fact that Listeria monocytogenes DivIVA shows normal polar localization but is not biologically active when expressed in B. subtilis. Complementation experiments with different chimeras of B. subtilis and L. monocytogenes DivIVA suggest that MinJ and RacA bind to separate DivIVA domains. Fluorescence microscopy of green fluorescent protein-tagged RacA and MinJ corroborated this conclusion and suggests that MinJ recruitment operates via the N-terminal lipid binding domain, whereas RacA interacts with the C-terminal domain. We speculate that this difference is related to the cellular compartments in which MinJ and RacA are active: the cell membrane and the cytoplasm, respectively.  相似文献   

13.
DivIVA is a conserved protein in Gram-positive bacteria and involved in various processes related to cell growth, cell division and spore formation. DivIVA is specifically targeted to cell division sites and cell poles. In Bacillus subtilis, DivIVA helps to localise other proteins, such as the conserved cell division inhibitor proteins, MinC/MinD, and the chromosome segregation protein, RacA. Little is known about the mechanism that localises DivIVA. Here we show that DivIVA binds to liposomes, and that the N terminus harbours the membrane targeting sequence. The purified protein can stimulate binding of RacA to membranes. In mutants with aberrant cell shapes, DivIVA accumulates where the cell membrane is most strongly curved. On the basis of electron microscopic studies and other data, we propose that this is due to molecular bridging of the curvature by DivIVA multimers. This model may explain why DivIVA localises at cell division sites. A Monte-Carlo simulation study showed that molecular bridging can be a general mechanism for binding of proteins to negatively curved membranes.  相似文献   

14.
In rod-shaped bacteria such as Bacillus subtilis, division site selection is mediated by MinC and MinD, which together function as a division inhibitor. Topological specificity is imposed by DivIVA, which ensures that MinCD specifically inhibits division close to the cell poles, while allowing division at mid-cell. MinD plays a central role in this process, as it positions and activates MinC and is dependent on DivIVA for its own positioning at the poles. To investigate MinD activities further, we have constructed and analysed a collection of minD mutants. Mutations in the conserved ATPase motifs lead to an inactive protein, possibly unable to oligomerize, but which nevertheless retains some affinity for the cell membrane. Several mutations affecting the mid- to C-terminal parts of MinD led to a protein probably unable to interact with DivIVA, but that could still stimulate division inhibition by MinC. These findings suggest that the ATPase activity of MinD is necessary for all its functions (possibly in part by controlling the oligomerization state of the protein). The other mutations may identify a surface of MinD involved in its interactions with DivIVA and a possible mechanism for control of MinD by DivIVA.  相似文献   

15.
We investigate a mechanism for the polar localization of proteins in bacteria. We focus on the MinCD/DivIVA system regulating division site placement in the rod-shaped bacterium Bacillus subtilis. Our model relies on a combination of geometric effects and reaction-diffusion dynamics to direct proteins to both cell poles, where division is then blocked. We discuss similarities and differences with related division models in Escherichia coli and also develop extensions of the model to asymmetric polar protein localization. We propose that our mechanism for polar localization may be employed more widely in bacteria, especially in outgrowing spores, which do not possess any pre-existing polar division apparatus from prior division events.  相似文献   

16.
17.
18.
Despite years of intensive research, much remains to be discovered to understand the regulatory networks coordinating bacterial cell growth and division. The mechanisms by which Streptococcus pneumoniae achieves its characteristic ellipsoid-cell shape remain largely unknown. In this study, we analyzed the interplay of the cell division paralogs DivIVA and GpsB with the ser/thr kinase StkP. We observed that the deletion of divIVA hindered cell elongation and resulted in cell shortening and rounding. By contrast, the absence of GpsB resulted in hampered cell division and triggered cell elongation. Remarkably, ΔgpsB elongated cells exhibited a helical FtsZ pattern instead of a Z-ring, accompanied by helical patterns for DivIVA and peptidoglycan synthesis. Strikingly, divIVA deletion suppressed the elongated phenotype of ΔgpsB cells. These data suggest that DivIVA promotes cell elongation and that GpsB counteracts it. Analysis of protein-protein interactions revealed that GpsB and DivIVA do not interact with FtsZ but with the cell division protein EzrA, which itself interacts with FtsZ. In addition, GpsB interacts directly with DivIVA. These results are consistent with DivIVA and GpsB acting as a molecular switch to orchestrate peripheral and septal PG synthesis and connecting them with the Z-ring via EzrA. The cellular co-localization of the transpeptidases PBP2x and PBP2b as well as the lipid-flippases FtsW and RodA in ΔgpsB cells further suggest the existence of a single large PG assembly complex. Finally, we show that GpsB is required for septal localization and kinase activity of StkP, and therefore for StkP-dependent phosphorylation of DivIVA. Altogether, we propose that the StkP/DivIVA/GpsB triad finely tunes the two modes of peptidoglycan (peripheral and septal) synthesis responsible for the pneumococcal ellipsoid cell shape.  相似文献   

19.
Cell division in bacteria is governed by a complex cytokinetic machinery in which the key player is a tubulin homologue, FtsZ. Most rod‐shaped bacteria divide precisely at mid‐cell between segregated sister chromosomes. Selection of the correct site for cell division is thought to be determined by two negative regulatory systems: the nucleoid occlusion system, which prevents division in the vicinity of the chromosomes, and the Min system, which prevents inappropriate division at the cell poles. In Bacillus subtilis recruitment of the division inhibitor MinCD to cell poles depends on DivIVA, and these proteins were thought to be sufficient for Min function. We have now identified a novel component of the division‐site selection system, MinJ, which bridges DivIVA and MinD. minJ mutants are impaired in division because MinCD activity is no longer restricted to cell poles. Although MinCD was thought to act specifically on FtsZ assembly, analysis of minJ and divIVA mutants showed that their block in division occurs downstream of FtsZ. The results support a model in which the main function of the Min system lies in allowing only a single round of division per cell cycle, and that MinCD acts at multiple levels to prevent inappropriate division.  相似文献   

20.
In Bacillus subtilis, FtsZ ring formation and cell division is favoured at the midcell because the inhibitor proteins MinC and MinD are indirectly restricted to the cell poles by the protein DivIVA. Here we identify MinJ, a topological determinant of medial FtsZ positioning that acts as an intermediary between DivIVA and MinD. Due to unrestricted MinD activity, cells mutated for minJ exhibited pleiotropic defects in homologous recombination, swarming motility and cell division. MinJ restricted MinD activity by localizing MinD to the cell poles through direct protein-protein interaction. MinJ itself localized to cell poles in a manner that was dependent on DivIVA. MinJ is conserved in other low G+C Gram-positive bacteria and may be an important component of cell division site selection in these organisms.  相似文献   

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