Institute of Molecular Biology, Slovak Academy of Sciences, 842 51 Bratislava, Slovakia1
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, UK2
Department of Biology, University of York, Heslington YO10 5YW, UK3
Author for correspondence: Imrich Barák. Tel: +421 7 5941 2152. Fax: +421 7 5477 2316. e-mail: umbibara{at}savba.sk
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ABSTRACT |
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Keywords: cell division, blue native electrophoresis, analytical ultracentrifugation, sporulation
Abbreviations: GFP, green fluorescent protein
a Present address: Laboratory of Molecular Biophysics, The Rex Richards Building, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
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INTRODUCTION |
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Although there are many similarities in cell division between E. coli and B. subtilis, it appears that bacilli employ a different strategy to initiate mid-cell division. B. subtilis has MinCD homologues but lacks a MinE counterpart. MinCD inhibition seems to be mediated through the unrelated protein DivIVA. This protein ensures that MinCD specifically inhibits division at the cell poles whilst allowing division at the mid-cell during vegetative growth (Cha & Stewart, 1997 ; Edwards & Errington, 1997
). DivIVA localizes to both cell poles as well as to the mid-cell site (Edwards & Errington, 1997
). Localization of DivIVA to division sites is dependent not only on FtsZ, but also on other cell-division proteins, including DivIB, DivIC and PbpB (Marston et al., 1998
). DivIVA is targeted to the division sites late in their assembly, where it recruits MinD to prevent another division from taking place near the newly formed cell poles. MinD is required both to pilot MinC to the cell poles and to constitute a functional division inhibitor. Sequestration of MinD and subsequently MinC to the cell poles makes available the next mid-cell sites in the newly formed cells for division. Recent results show that the main role of DivIVA is to retain MinCD at the cell poles after the division is complete. After division nears completion, all of the components of the division apparatus except DivIVA seem to disappear from this site. Therefore, it appears that at some point DivIVA must become attached to a more permanent anchor at the mature cell pole (Marston & Errington, 1999
).
The same pattern of DivIVA targeting seen in B. subtilis was observed when a DivIVA-GFP fusion protein was expressed in E. coli (Edwards et al., 2000 ). The subcellular targeting of DivIVA in E. coli is dependent on FtsZ and independent of the MinCD system. However, DivIVA does not seem to be able to substitute for MinE, a topological specificity factor in E. coli. Surprisingly, the DivIVA-GFP fusion protein was also directed to nascent division sites in the yeast Schizosaccharomyces pombe (Edwards et al., 2000
) and it is thus possible that some conserved target for DivIVA exists in both prokaryotes and eukaryotes.
In contrast to E. coli, B. subtilis can undergo an asymmetric septation during the process of sporulation. It has been shown that at the onset of sporulation, assembly of the FtsZ ring shifts from the mid-cell to both polar sites (Levin & Losick, 1996 ), the switch being dependent on the transcription factor Spo0A. The FtsZ ring forms initially at both polar sites, implying that MinCD inhibition of FtsZ assembly at both sites must be overcome early in the sporulation process. MinCD-deficient cells that have begun to sporulate can form sporulation-like septa at both the normal asymmetric position as well as at, or near, the mid-cell site (Barák et al., 1998
). However, the role of DivIVA in sporulation septation is unclear, although it was proposed that an unknown sporulation factor interacts with DivIVA to make polar sites available for septation (Cha & Stewart, 1997
).
DivIVA is a 19·5 kDa cytoplasmic protein that has sequence similarity to homologues from other Gram-positive bacteria and at a lower level to eukaryotic proteins, including myosin (Edwards et al., 2000 ). Computer analysis of the DivIVA amino acid sequence suggested that the central region of DivIVA might form an
-helical coiled-coil structure in vivo (Edwards et al., 2000
). Such structures could be used for oligomerization as in tropomyosin (Lupas, 1996
). To examine the possibility that the predicted
-helical coiled-coil structure in DivIVA is involved in protein oligomerization, we have cloned the B. subtilis divIVA gene into an E. coli expression system. The encoded protein has been purified to homogeneity and analysed by analytical ultracentrifugation and blue native polyacrylamide gel electrophoresis. We have also characterized the oligomerization state of three different mutant proteins: DivIVA1, DivIVA2 and DivIVA9.
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METHODS |
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Expression, purification and electrophoresis of proteins.
Protein overexpression was performed by using E. coli strain IB706. Cell cultures were grown in LB medium containing 30 µg kanamycin ml-1 to an OD600 of 0·6, when protein expression was induced by the addition of IPTG to a final concentration of 1 mM. After 3 h further growth, the cells were harvested by centrifugation and frozen at -80 °C. Then 3·5 g cells were resuspended on ice in 10 ml buffer A (50 mM Tris/HCl pH 8·0, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF), lysed by sonication and clarified by centrifugation at 30000 g for 30 min. The soluble cell lysate was applied to a 13 ml Q Sepharose Fast Flow column (Pharmacia Biotech) pre-equilibrated in buffer A. DivIVA9 was eluted with a linear gradient of 0·10·5 M NaCl. Fractions containing DivIVA9 were diluted fivefold with buffer A and the procedure was repeated using an FPLC Mono Q HR 10/16 column (Pharmacia Biotech). Fractions containing DivIVA9, which eluted at 300 mM NaCl, were concentrated to a volume of 1 ml and loaded onto a Pharmacia Superose 12 gel filtration column.
Western blot analysis of B. subtilis cell lysates fractionated by blue native gel electrophoresis was performed as follows. B. subtilis cell cultures were grown in DSM medium (Harwood & Cutting, 1988 ) at 37 °C until the onset of stationary phase. The cells were harvested by centrifugation and resuspended in buffer A containing 0·25 mg lysozyme ml-1. The cells were lysed by sonication and the suspension clarified by centrifugation at 30000 g for 15 min. An equal amount (75 µg) of protein from each sample was mixed with glycerol (15%, v/v) and electrophoresed through a blue native 518% linear gradient polyacrylamide gel. The proteins were blotted onto a nitrocellulose membrane for immunodetection of DivIVA.
Analytical ultracentrifugation.
All analytical ultracentrifugation experiments were performed in a Beckman XL-A analytical ultracentrifuge (Beckman-Coulter) using an AN-Ti 60 rotor. The molecular mass averages were determined using the program BIOSPIN and were as follows:
The number average:
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The weight average:
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The z-average molecular mass:
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M, molecular mass; c, concentration (mg ml-1).
Light and fluorescence microscopy.
Cells were prepared for fluorescence microscopy as described previously (Barák et al., 1996 ). An Olympus BX60 microscope and MicroImage software was used for microscopy observations, using a filter set for FM 4-64 visualization (550583 nm excitation and 617690 nm emission).
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RESULTS AND DISCUSSION |
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Based on the observation that the last seven amino acid residues of the protein are not necessary for the function of DivIVA (Cha & Stewart, 1997 ), we chose DivIVA9 for our next biochemical experiments. The divIVA9 gene contains a single base transition at nucleotide 484, which results in a glutamate to lysine amino acid substitution at residue 162 (the third residue from the C terminus) in the protein product. Protein was overexpressed in E. coli BL21(DE3) transformed with pETIVA9. SDS-PAGE analysis of cell extracts showed a high level of DivIVA9 production (not shown). Although large amounts of DivIVA9 partitioned into the membrane fraction, sufficient DivIVA9 remained in the soluble fraction to allow protein purification. FPLC purified protein was loaded on a Superose 12 gel filtration column (see Methods). DivIVA9 exhibits a low retention volume, suggesting it exists as a high molecular mass species. To determine the molecular mass of this oligomer, blue native polyacrylamide gradient gel electrophoresis and analytical ultracentrifugation studies were performed.
Blue native gel electrophoresis
The negative charge of protein-bound Coomassie blue G-250 dye is the main determinant of sample electrophoretic mobility in this method, which is suitable for determining the apparent molecular masses of proteins with a pI below 8·6 (Schägger et al., 1994 ). The theoretical pI of DivIVA is 4·6, thus DivIVA was resolved by blue native gel electrophoresis with a 518% linear acrylamide gradient. The electrophoretic mobility of protein standards is linearly dependent on log Mr, allowing the molecular mass of DivIVA9 to be estimated at 200 kDa, corresponding to a decamer (Fig. 1a
).
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Western blot analysis of B. subtilis cell lysates revealed the presence of the same 1012 mer in the divIVA1 mutant strain (Fig. 1b; lane 3). This allele contains a G to A transition, resulting in an alanine to threonine substitution at amino acid residue 78. It is predicted that this amino acid is located in the hydrophobic core of the proposed coiled-coil structure. This suggests that the loss of DivIVA function in this mutant strain is not due to altered oligomerization and that oligomerization per se is not sufficient for correct DivIVA function. An alternative interpretation is that this mutation affects the interaction of DivIVA with other components of the division machinery. The single DivIVA band corresponding to the 1012 mer with no trace of smaller or higher order oligomeric forms is in contrast to the analytical ultracentrifugation observations, which suggest oligomers ranging from dimers to 1012 mers. We assume that in native blue electrophoresis experiments the 1012 mer of DivIVA is stabilized by the glycerol present in the sample buffer in these experiments.
In contrast to the above, the protein DivIVA2 behaves differently, with 56 mer complexes prominent in addition to the 1012 mers (Fig. 1b; lane 5). The B. subtilis IB704 strain harbouring the divIVA2 mutation grows so poorly that we were unable to load enough cell lysate to clearly detect the DivIVA2 protein. Fig. 1(b)
shows recombinant DivIVA2 in an E. coli cell lysate (lane 5). The divIVA2 mutation causes a Leu to Pro substitution at amino acid residue 120, which is also predicted to be located in the hydrophobic core, but at the end of the proposed coiled-coil structure. This mutation is probably not compatible with the proposed coiled-coil structure in this region (Edwards et al., 2000
).
Biological effect of divIVA mutations
The divIVA1 mutation causes a misplacement of the septum during vegetative growth, resulting in the formation of mini-cells and filaments (Cha & Stewart, 1997 ; Edwards & Errington, 1997
). To check the phenotype of the divIVA9 and divIVA2 mutants, we transformed derivatives of plasmid pUK19 containing the cloned mutant genes into the wild-type B. subtilis strain PY79 selecting for kanamycin resistance. Correct recombination by a single crossover event to replace the wild-type copy of divIVA with the two mutant genes was checked by Southern blot hybridization analysis (not shown). Light microscopy revealed that the divIVA9 mutation has no significant effect on cell length or morphology, and no mini-cells were observed (Fig. 3a, c
). In contrast, the divIVA2 mutant has a similar phenotype to divIVA1 mutant cells, characterized by longer cells and easily detectable mini-cells (Fig. 3b, d
). The formation of septa in the wild-type and mutant divIVA strains was observed by fluorescence microscopy using the vital stain FM 4-64 (Fig. 3a*d*
). This stain specifically labels the lipid bilayer of the plasma membrane and is useful for the detection of septa (Pogliano et al., 1999
). Normal septa are formed in wild-type and divIVA9 mutant strains, in contrast to the divIVA1 and divIVA2 mutant strains in which many partially formed septa are visible (Fig. 3a*d*
).
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ACKNOWLEDGEMENTS |
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Received 25 June 2001;
revised 13 September 2001;
accepted 1 November 2001.