Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sharp, M. D. Articles by Pogliano, K. Search for Related Content PubMed PubMed Citation Articles by Sharp, M. D. Articles by Pogliano, K.

 Previous Article  |  Next Article 

Journal of Bacteriology, March 2003, p. 2005-2008, Vol. 185, No. 6
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.6.2005-2008.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Membrane Domain of SpoIIIE Is Required for Membrane Fusion during Bacillus subtilis Sporulation

Marc D. Sharp and Kit Pogliano^*

Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093-0349

Received 2 August 2002/ Accepted 27 November 2002

Top Abstract Text References

 
During Bacillus subtilis sporulation, SpoIIIE is required for both postseptational chromosome segregation and membrane fusion after engulfment. Here we demonstrate that SpoIIIE must be present in the mother cell to promote membrane fusion and that the N-terminal membrane-spanning segments constitute a minimal membrane fusion domain, as well as direct septal localization.

Top Abstract Text References

 
Bacillus subtilis SpoIIIE and Escherichia coli FtsK are well-characterized members of a conserved family of bacterial proteins involved in chromosome segregation. These proteins have similar architectures, with an N-terminal membrane domain, a variable linker, and a C-terminal cytoplasmic domain containing a Walker-type ATP binding site (6, 7). The membrane domains of FtsK and SpoIIIE localize the proteins to the site of cell division (2, 13, 16), whereas the cytoplasmic domains move along DNA in an ATP-dependent manner (1, 2). FtsK is an essential protein required for cell division as well as chromosome decatenation and segregation; under such circumstances, it likely aligns the recombination sites (4) and activates the Xer recombinase (1). In contrast, SpoIIIE is dispensable for growth but plays two crucial roles in sporulation. First, it completes chromosome partitioning during sporulation, which differs from that of vegetative growth since the asymmetrically positioned sporulation septum initially bisects one chromosome, trapping 30% in the forespore (Fig. 1ii) (14). SpoIIIE acts in the mother cell to rescue this trapped chromosome, exporting the DNA into the forespore (12) in a process which requires a functional ATP binding site (10). Second, SpoIIIE is necessary for the membrane fusion event which releases the forespore into the mother cell cytoplasm at the completion of the phagocytosis-like process of engulfment (Fig. 1iii to v) (10). These two roles of SpoIIIE are distinct and genetically separable, as we have isolated mutations that block DNA translocation but produce only a minor membrane fusion defect (10). It is tempting to speculate that the SpoIIIE/FtsK family of proteins also participates in the membrane fusion event at the completion of cell division, perhaps serving to coordinate chromosome segregation with cell division and thereby preventing chromosome damage that would arise if daughter cell separation preceded the completion of chromosome segregation, a proposal consistent with the late division defect of certain FtsK mutants (3, 5).

View larger version (28K): [in this window] [in a new window]

FIG. 1. Stages of forespore engulfment and chromosome translocation. The black circles indicate the localization of SpoIIIE.

The membrane fusion event at the completion of engulfment occurs in the membrane of the migrating mother cell, suggesting that proteins directly involved in membrane fusion should act in the mother cell and localize to the cell pole before membrane fusion. While SpoIIIE localizes to the pole before fusion (10), it is produced prior to polar septation and is predicted to be present in both cells of the sporangium. To test if SpoIIIE is specifically required in the mother cell for membrane fusion, we fused spoIIIE to similarly expressed forespore- and mother cell-specific promoters (P_spoIIQ and P_spoIID, respectively [12]) and tested the ability of these hybrid genes to complement the membrane fusion defects of various spoIIIE mutants (Table 1) using an assay which relies on the membrane-impermeable stain FM 4-64 and the membrane-permeable stain Mitotracker Green (MTG) (10). When these stains were applied to sporangia that had completed membrane fusion, the forespore membranes failed to stain with FM 4-64 and stained only with MTG (Fig. 2A) while the forespores of unfused sporangia were accessible to both stains (Fig. 2A).

View this table: [in this window] [in a new window]

TABLE 1. B. subtilis strains used in this study

View larger version (82K): [in this window] [in a new window]

FIG. 2. Membrane fusion assay at t3, showing the membranes stained with FM 4-64 (red) and MTG (green). Arrowheads indicate sporangia that have completed membrane fusion. (A) Wild type (the arrows indicate an unfused sporangium); (B) spoIIIE sporangia; (C) spoIIIE36 sporangia; (D) spoIIIE36 amyE::spoIIIE-gfp sporangia; (E) spoIIIE36 amyE::PspoIIQ-spoIIIE-gfp sporangia; (F) spoIIIE36 amyE::PspoIID-spoIIIE-gfp sporangia; (G) spoIIIEMSS1-4 sporangia; (H) spoIIIEMSS1-4 amyE::PspoIIQ-spoIIIEMSS1-4 sporangia; (I) spoIIIEMSS1-4 amyE::PspoIID-spoIIIEMSS1-4 sporangia. Scale bar, 2 µm.

Four hours after the initiation of sporulation (t₄), approximately 85% of wild-type sporangia had completed membrane fusion while only 1% of spoIIIE-null sporangia had fused (Table 2; Fig. 2A and B show t3). We tested the effects of cell-specific SpoIIIE expression in both a spoIIIE strain and a spoIIIE36 strain, the latter of which had a less severe membrane fusion defect (24% of sporangia fused by t₄) (Fig. 2C; Table 2) but normal compartmentalization of cell-specific gene expression, unlike the null mutant (9, 14). The maintenance of cell-specific gene expression in the spoIIIE36 background highlighted the differences between mother cell- and forespore-expressed spoIIIE genes. Mother cell-expressed spoIIIE complemented the membrane fusion defect of spoIIIE36 as well as the expression of spoIIIE from its native promoter (79% of sporangia with P_spoIIIE-spoIIIE fused versus 78% with P_spoIID-spoIIIE fused) (Fig. 2D and F; Table 2). In contrast, expression of spoIIIE in the forespore had no effect on membrane fusion (23% of sporangia with P_spoIIQ-spoIIIE fused) (Fig. 2E; Table 2). Similar effects were seen in the spoIIIE-null strain (Table 2), although forespore-produced SpoIIIE supported some membrane fusion (albeit less than mother cell-produced SpoIIIE), likely because of the compartmentalization defect. Thus, SpoIIIE is required in the mother cell to promote fusion of the engulfing mother cell membrane, suggesting that it might be directly involved in membrane fusion.

View this table: [in this window] [in a new window]

TABLE 2. Complementation of the membrane fusion defects of various spoIIIE mutants with forespore- or mother cell-produced SpoIIIE

Mutagenesis of the ATP binding site in the cytoplasmic domain of SpoIIIE abolishes DNA translocation while only modestly effecting membrane fusion (10), suggesting that this domain may be dispensable for membrane fusion. To test this hypothesis, we deleted the cytoplasmic domain of SpoIIIE, truncating the protein after amino acid 192 and leaving intact the four membrane-spanning segments and a short linker region before green fluorescent protein. This truncated protein (SpoIIIEMSS1-4) was completely inactive for chromosome segregation (data not shown) but was able to support some membrane fusion; by t₄, 13% of spoIIIEMSS1-4 sporangia completed membrane fusion (Fig. 2G, Table 2), compared to 1% of spoIIIE sporangia (Fig. 2B; Table 2). While 13% was significantly lower than the 84% membrane fusion of the wild type, it was more than 10-fold higher than the percent membrane fusion of the spoIIIE null strain. In contrast, strains expressing only the cytoplasmic domain of spoIIIE were defective in both DNA translocation and membrane fusion (12). Overproduction of SpoIIIEMSS1-4 in the mother cells of spoIIIEMSS1-4 sporangia (from P_spoIID-spoIIIEMSS1-4, which should produce about 100-fold more SpoIIIEMSS1-4 than P_spoIIIE [12]) increased the fusion efficiency from 13 to 24% (Fig. 2I; Table 2), whereas overproduction of SpoIIIEMSS1-4 in the forespores (P_spoIIQ-spoIIIEMSS1-4) of spoIIIEMSS1-4 sporangia had no effect (Fig. 2H; Table 2), suggesting that the membrane-spanning segments alone are capable of promoting membrane fusion. The relatively low efficiency of membrane fusion supported by SpoIIIEMSS1-4 is likely caused its inefficient relocalization to the cell pole (2), where membrane fusion occurs (10).

These results demonstrate that SpoIIIE is required in the mother cell to mediate membrane fusion at the completion of engulfment, as well as to translocate the forespore chromosome across the sporulation septum (12). Interestingly, the membrane fusion domain of SpoIIIE comprises the membrane-spanning segments, which are conserved even in distantly related bacteria that do not produce endospores (7). Such bacteria would not require a protein to catalyze membrane fusion at the completion of engulfment, supporting speculation that these proteins might be able to participate in membrane fusion during cell division.

 
This work was supported by the National Institutes of Health (grant GM57045).

 
* Corresponding author. Mailing address: Division of Biological Sciences, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0349. Phone: (858) 822-1314. Fax: (858) 822-1431. E-mail: kpogliano{at}ucsd.edu.

Top Abstract Text References

 

1
1. Aussel, L., F. X. Barre, M. Aroyo, A. Stasiak, A. Z. Stasiak, and D. Sherratt. 2002. FtsK is a DNA motor protein that activates chromosome dimer resolution by switching the catalytic state of the XerC and XerD recombinases. Cell 108:195-205.[CrossRef][Medline]
2
2. Bath, J., L. J. Wu, J. Errington, and C. Robinson. 2000. Role of Bacillus subtilis SpoIIIE in DNA transport across the mother cell-prespore division septum. Science 290:995-997.[Abstract/Free Full Text]
3
3. Begg, K. J., S. J. Dewar, and W. D. Donachie. 1995. A new Escherichia coli cell division gene, ftsK. J. Bacteriol. 177:6211-6222.[Abstract/Free Full Text]
4
4. Corre, J., and J.-M. Louarn. 2002. Evidence from terminal recombination gradients that FtsK uses replichore polarity to control chromosome terminus positioning at division in Escherichia coli. J. Bacteriol. 184:3801-3807.[Abstract/Free Full Text]
5
5. Diez, A. A., A. Farewell, U. Nannmark, and T. Nyström. 1997. A mutation in the ftsK gene of Escherichia coli affects cell-cell separation, stationary-phase survival, stress adaptation, and expression of the gene encoding the stress protein UspA. J. Bacteriol. 179:5878-5883.[Abstract/Free Full Text]
6
6. Donachie, W. D. 2002. FtsK: Maxwell's demon? Mol. Cell 9:206-207.[CrossRef][Medline]
7
7. Errington, J., J. Bath, and L.-J. Wu. 2001. DNA transport in bacteria. Nat. Rev. Mol. Cell Biol. 2:538-545.[CrossRef][Medline]
8
8. Guerout-Fleury, A. M., N. Fransdsen, and P. Stragier. 1996. Plasmids for ectopic integration in Bacillus subtilis. Gene 180:57-61.[CrossRef][Medline]
9
9. Pogliano, K., A. E. M. Hofmeister, and R. Losick. 1997. Disappearance of the ^E transcription factor from the forespore and the SpoIIE phosphatase from the mother cell contributes to the establishment of cell-specific gene expression during sporulation in Bacillus subtilis. J. Bacteriol. 179:3331-3341.[Abstract/Free Full Text]
10
10. Sharp, M. D., and K. Pogliano. 1999. An in vivo membrane fusion assay implicates SpoIIIE in the final stages of engulfment during Bacillus subtilis sporulation. Proc. Natl. Acad. Sci. USA 96:14553-14558.[Abstract/Free Full Text]
11
11. Sharp, M. D., and K. Pogliano. 2002. MinCD-dependent regulation of the polarity of SpoIIIE assembly and DNA transfer. EMBO J. 22:6267-6274.[CrossRef]
12
12. Sharp, M. D., and K. Pogliano. 2002. Role of cell specific assembly of SpoIIIE in polarity of DNA transfer. Science 295:137-139.[Abstract/Free Full Text]
13
13. Wang, L., and J. Lutkenhaus. 1998. FtsK is an esential cell division protein that is localized to the septum and induced as part of the SOS response. Mol. Microbiol. 29:731-740.[CrossRef][Medline]
14
14. Wu, L. J., and J. Errington. 1994. Bacillus subtilis SpoIIIE protein required for DNA segregation during asymmetric cell division. Science 264:572-575.[Abstract/Free Full Text]
15
15. Youngman, P., J. B. Perkins, and K. Sandman. 1984. New genetic methods, molecular cloning strategies, and gene fusion techniques for Bacillus subtilis which take advantage of Tn917 insertional mutagenesis, p. 103-111. In J. A. Hoch and A. T. Ganesan (ed.), Genetics and bio/technology of bacilli. Academic Press, New York, N.Y.
16
16. Yu, X., A. H. Tran, Q. Sun, and W. Margolin. 1998. Localization of cell division protein FtsK to the Escherichia coli septum and identification of a potential N-terminal targeting domain. J. Bacteriol. 180:1296-1304.[Abstract/Free Full Text]

---

Journal of Bacteriology, March 2003, p. 2005-2008, Vol. 185, No. 6
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.6.2005-2008.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Bogush, M., Xenopoulos, P., Piggot, P. J. (2007). Separation of Chromosome Termini during Sporulation of Bacillus subtilis Depends on SpoIIIE. J. Bacteriol. 189: 3564-3572 [Abstract] [Full Text]  
• Wang, L., Yu, Y., He, X., Zhou, X., Deng, Z., Chater, K. F., Tao, M. (2007). Role of an FtsK-Like Protein in Genetic Stability in Streptomyces coelicolor A3(2). J. Bacteriol. 189: 2310-2318 [Abstract] [Full Text]  
• Gunton, J. E., Gilmour, M. W., Baptista, K. P., Lawley, T. D., Taylor, D. E. (2007). Interaction between the co-inherited TraG coupling protein and the TraJ membrane-associated protein of the H-plasmid conjugative DNA transfer system resembles chromosomal DNA translocases. Microbiology 153: 428-441 [Abstract] [Full Text]  
• Hilbert, D. W., Piggot, P. J. (2004). Compartmentalization of Gene Expression during Bacillus subtilis Spore Formation. Microbiol. Mol. Biol. Rev. 68: 234-262 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sharp, M. D. Articles by Pogliano, K. Search for Related Content PubMed PubMed Citation Articles by Sharp, M. D. Articles by Pogliano, K.