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Sporulation: SpoIIIE Is the Key to Cell Differentiation
Dispatch R871
Sporulation: SpoIIIE Is the Key to Cell Differentiation Sporulation in Bacillus subtilis requires asymmetric cell division, chromosome transfer into the spore and establishment of differential gene expression patterns. Several recent studies highlight the key roles of the SpoIIIE motor in this process. Ian Grainge In response to a variety of environmental stresses, the bacterium Bacillus subtilis can form spores, keeping its DNA safe to await better times. During sporulation, replication is followed by an asymmetric cell division, resulting in a small ‘forespore’ and a larger ‘mother cell’ (Figure 1). The chromosome origin-proximal regions are localised to the cell poles by the action of several proteins, including RacA, Spo0J and Soj, so that roughly 30% of one of the replicated chromosomes becomes trapped in the developing forespore [1,2]. The DNA translocase SpoIIIE is then required for transfer of the remaining DNA into the forespore [3]. SpoIIIE has four membranespanning domains in its amino terminus, which are sufficient for localising the protein to the division septum [4], while the carboxy-terminal portion encodes an ATP-dependent DNA translocase [5]. This motor domain has homology to the FtsK translocase, which has been crystallised as a hexameric ring with a central pore large enough to accommodate a single DNA duplex [6]. When localised to the septal membrane during sporulation, SpoIIIE pumps the remainder of the chromosome, roughly 3 Mbp, into the forespore over the span of about 15 minutes. During this time, GFP–SpoIIIE is seen to localise as a focus, presumably centering on the DNA. Indeed, focus formation is dependent upon trapped DNA spanning the septum [7]. Gene expression differs in the mother cell and forespore and is mainly governed by different s factors in the two cell types. Establishment and maintenance of this state requires that there be no exchange of cytoplasmic factors between the mother and forespore. This is achieved by full membrane fusion occurring relatively
early, prior to, and independent of, SpoIIIE action [8]. SpoIIIE then pumps the DNA across two closed membranes (Figure 1). The foci seen in the mother cell likely represent at least two hexameric translocase complexes, since, during DNA transfer into the forespore, both chromosome arms are transferred simultaneously [8], suggesting that (at least) two active motors export DNA from the mother cell. In a recent study, Marquis et al. [9] now show that DNA transport through this relatively narrow pore in the membrane also acts like a wire-stripper, removing protein bound to the DNA. Both a fluorescent RNA polymerase and a GFP-tagged form of the tet repressor (TetR–GFP) bound to the tet operator (tetO) were observed to be excluded from the forespore during DNA transfer. Purified SpoIIIE motor protein was also found to displace a stalled RNA polymerase in vitro, confirming that the motor has the necessary power for this proposed action. Importantly, these findings give insight into the mechanism of cell differentiation. SpoIIIE assembles around DNA trapped in the closed membranes of the septum and is the only known link between the cytoplasm of the forespore and mother cell. Then, by stripping protein from DNA during transfer, SpoIIIE delivers naked DNA into the forespore and excludes mother-cell-specific transcription complexes, allowing the developmental differentiation of the forespore. SpoIIIE is, however, found in both the mother cell and the forespore [4,8,10,11] — how, then, does it achieve the directed pumping of the DNA into the forespore, rather than emptying the forespore of its DNA? Placing SpoIIIE under the control of forespore-specific or mother-cell-specific s factors allowed its expression in either the forespore or the mother cell [4]. When expressed in the forespore alone, SpoIIIE was able to export DNA to the
mother cell, albeit inefficiently, whereas expression in the mother cell led to DNA transfer into the forespore as normal. Thus, SpoIIIE can be active in either compartment. Using an elegant technique, Becker, Pogliano and colleagues [10,11] were able to visualise the distribution of SpoIIIE expressed from its natural promoter in a cell-specific manner: SpoIIIE was fused to the leucine zipper of c-Jun (SpoIIIE–JunLZ) and a fusion protein comprising GFP and the c-Fos leucine zipper (FosLZ–GFP) was placed under the control of the appropriate s factor to promote expression in either the mother cell or the forespore. The SpoIIIE–JunLZ—FosLZ–GFP complex was then visualised. SpoIIIE on the mother-cell side forms foci but is de-localised over the septum in the forespore. The foci presumably represent the active hexameric DNA pumps. An indication of how SpoIIIE achieves this activity on one side of the membrane alone came from the examination of spo0J/soj mutants, which often fail to localise the origin-proximal region to the forespore and can trap other regions of the chromosome instead. In this situation DNA is seen to be exported from the forespore. Therefore, a DNA-encoded cue for translocation directionality was suggested. The molecular basis for the directionality of translocation has also recently been revealed [11]. The B. subtilis chromosome contains a number of polarised octamer sequences, and one of these, termed SRS (SpoIIIE recognition sequence, GAGAAGGG), is recognised specifically by the g domain at the very carboxyl terminus of SpoIIIE. In vitro, SpoIIIE and a mutant lacking the g domain (Dg) are both active translocases. However, the Dg mutant failed to produce viable spores in vivo. Without the g domain, SpoIIIE foci were seen on both sides of the septal membrane with equal frequency and DNA was pumped both into and out of the forespore. Therefore, it can be concluded that recognition of the polarised SRS sequences by the g domain is responsible for establishing the active translocase complexes in the mother cell, which pump DNA into the forespore. By analogy to FtsK, this could be controlled exclusively at the motor-loading step [12].
Current Biology Vol 18 No 18 R872
Forespore
Mother cell Post-DNA replication
Asymmetric septation
Membrane fusion
SpoIIIE assembly
Chromosome transfer Current Biology
Figure 1. A representation of sporulation in B. subtilis. Upon replication, the cell has two copies of the chromosome (red and blue). The origin region (yellow circle) is localised to the cell poles. Asymmetric septation traps roughly a third of the red chromosome in the forespore and forespore-specific proteins are produced (blue squares). Membranes are fully fused at this point, and cell wall components may also be present between the two membranes. SpoIIIE assembles an active motor on the mother-cell side of the membrane (this may occur concurrently with membrane fusion rather than following it). Polarised SRS sequences in the chromosome determine that the active motor assembles only on the mother-cell side of the membrane. SpoIIIE then pumps DNA into the forespore (grey arrows show direction of DNA movement), stripping off any bound proteins (green circles) as it does so, thus preventing entry of mother-cell-specific transcription complexes into the forespore and ensuring the differentiated state of the forespore.
Several important questions remain to be answered. SpoIIIE pumps both chromosome arms simultaneously [8], probably through two separate pores, producing an ever decreasing loop of chromosome on the mother-cell side. This creates a topological problem: how is this last segment of DNA transferred? Also, the exact nature of the pore through which DNA is transported is not understood. It seems likely that the foci seen on the mother-cell side of the septum are hexameric rings of the motor domains. The prevailing model is that the motors pump DNA through a channel in the membrane formed by the six amino-terminal domains of the active hexameric motor, but other possibilities exist. However, no corresponding foci are seen on the forespore side of the membrane [10,11]. Does SpoIIIE contribute to the formation of a pore through this membrane as well? When SpoIIIE is expressed in the mother cell alone, viable spores are formed at wild-type levels [4], suggesting that SpoIIIE is not required to form a pore in the forespore membrane. Indeed, membrane fusion, which traps DNA in the forespore, occurs even in the complete absence of SpoIIIE [8], showing that DNA can
cross both membranes without SpoIIIE. Further, Ptacin et al. [11] observe that in the Dg mutant reversal of DNA transfer direction following motor stalling is a very slow event (w20 minutes). These data suggest that perhaps pre-formed hexameric rings of a motor-pore complex are not assembled around DNA on both sides of the membrane simultaneously, and that assembly (at least in the absence of g) may be a slow step. What, if anything, contributes to the formation of the pore through which DNA translocates on the forespore side of the septum? The resolution of these tough questions is eagerly awaited. References 1. Ben-Yehuda, S., Rudner, D.Z., and Losick, R. (2003). RacA, a bacterial protein that anchors chromosomes to the cell poles. Science 299, 532–536. 2. Wu, L.J., and Errington, J. (1994). Bacillus subtilis SpoIIIE protein required for DNA segregation during asymmetric cell division. Science 264, 572–575. 3. Wu, L.J., and Errington, J. (1997). Septal localization of the SpoIIIE chromosome partitioning protein in Bacillus subtilis. EMBO J. 16, 2161–2169. 4. Sharp, M.D., and Pogliano, K. (2002). Role of cell-specific SpoIIIE assembly in polarity of DNA transfer. Science 295, 137–139. 5. Bath, J., Wu, L.J., Errington, J., and Wang, J.C. (2000). Role of Bacillus subtilis SpoIIIE in DNA transport across the mother cell-prespore division septum. Science 290, 995–997.
6. Massey, T.H., Mercogliano, C.P., Yates, J., Sherratt, D.J., and Lowe, J. (2006). Double-stranded DNA translocation: structure and mechanism of hexameric FtsK. Mol. Cell 23, 457–469. 7. Ben-Yehuda, S., Rudner, D.Z., and Losick, R. (2003). Assembly of the SpoIIIE DNA translocase depends on chromosome trapping in Bacillus subtilis. Curr. Biol. 13, 2196–2200. 8. Burton, B.M., Marquis, K.A., Sullivan, N.L., Rapoport, T.A., and Rudner, D.Z. (2007). The ATPase SpoIIIE transports DNA across fused septal membranes during sporulation in Bacillus subtilis. Cell 131, 1301–1312. 9. Marquis, K.A., Burton, B.M., Nollmann, M., Ptacin, J.L., Bustamante, C., Ben-Yehuda, S., and Rudner, D.Z. (2008). SpoIIIE strips proteins off the DNA during chromosome translocation. Genes Dev. 22, 1786–1795. 10. Becker, E.C., and Pogliano, K. (2007). Cell-specific SpoIIIE assembly and DNA translocation polarity are dictated by chromosome orientation. Mol. Microbiol. 66, 1066–1079. 11. Ptacin, J.L., Nollmann, M., Becker, E.C., Cozzarelli, N.R., Pogliano, K., and Bustamante, C. (2008). Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis. Nat. Struct. Mol. Biol. 15, 485–493. 12. Lo¨we, J., Ellonen, A., Allen, M.D., Atkinson, C., Sherratt, D.J., and Grainge, I. (2008). Molecular mechanism of sequence-directed DNA loading and translocation by FtsK. Mol. Cell 31, 498–509.
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK. E-mail: [email protected]
DOI: 10.1016/j.cub.2008.07.047
Sporulation: SpoIIIE Is the Key to Cell Differentiation Sporulation in Bacillus subtilis requires asymmetric cell division, chromosome transfer into the spore and establishment of differential gene expression patterns. Several recent studies highlight the key roles of the SpoIIIE motor in this process. Ian Grainge In response to a variety of environmental stresses, the bacterium Bacillus subtilis can form spores, keeping its DNA safe to await better times. During sporulation, replication is followed by an asymmetric cell division, resulting in a small ‘forespore’ and a larger ‘mother cell’ (Figure 1). The chromosome origin-proximal regions are localised to the cell poles by the action of several proteins, including RacA, Spo0J and Soj, so that roughly 30% of one of the replicated chromosomes becomes trapped in the developing forespore [1,2]. The DNA translocase SpoIIIE is then required for transfer of the remaining DNA into the forespore [3]. SpoIIIE has four membranespanning domains in its amino terminus, which are sufficient for localising the protein to the division septum [4], while the carboxy-terminal portion encodes an ATP-dependent DNA translocase [5]. This motor domain has homology to the FtsK translocase, which has been crystallised as a hexameric ring with a central pore large enough to accommodate a single DNA duplex [6]. When localised to the septal membrane during sporulation, SpoIIIE pumps the remainder of the chromosome, roughly 3 Mbp, into the forespore over the span of about 15 minutes. During this time, GFP–SpoIIIE is seen to localise as a focus, presumably centering on the DNA. Indeed, focus formation is dependent upon trapped DNA spanning the septum [7]. Gene expression differs in the mother cell and forespore and is mainly governed by different s factors in the two cell types. Establishment and maintenance of this state requires that there be no exchange of cytoplasmic factors between the mother and forespore. This is achieved by full membrane fusion occurring relatively
early, prior to, and independent of, SpoIIIE action [8]. SpoIIIE then pumps the DNA across two closed membranes (Figure 1). The foci seen in the mother cell likely represent at least two hexameric translocase complexes, since, during DNA transfer into the forespore, both chromosome arms are transferred simultaneously [8], suggesting that (at least) two active motors export DNA from the mother cell. In a recent study, Marquis et al. [9] now show that DNA transport through this relatively narrow pore in the membrane also acts like a wire-stripper, removing protein bound to the DNA. Both a fluorescent RNA polymerase and a GFP-tagged form of the tet repressor (TetR–GFP) bound to the tet operator (tetO) were observed to be excluded from the forespore during DNA transfer. Purified SpoIIIE motor protein was also found to displace a stalled RNA polymerase in vitro, confirming that the motor has the necessary power for this proposed action. Importantly, these findings give insight into the mechanism of cell differentiation. SpoIIIE assembles around DNA trapped in the closed membranes of the septum and is the only known link between the cytoplasm of the forespore and mother cell. Then, by stripping protein from DNA during transfer, SpoIIIE delivers naked DNA into the forespore and excludes mother-cell-specific transcription complexes, allowing the developmental differentiation of the forespore. SpoIIIE is, however, found in both the mother cell and the forespore [4,8,10,11] — how, then, does it achieve the directed pumping of the DNA into the forespore, rather than emptying the forespore of its DNA? Placing SpoIIIE under the control of forespore-specific or mother-cell-specific s factors allowed its expression in either the forespore or the mother cell [4]. When expressed in the forespore alone, SpoIIIE was able to export DNA to the
mother cell, albeit inefficiently, whereas expression in the mother cell led to DNA transfer into the forespore as normal. Thus, SpoIIIE can be active in either compartment. Using an elegant technique, Becker, Pogliano and colleagues [10,11] were able to visualise the distribution of SpoIIIE expressed from its natural promoter in a cell-specific manner: SpoIIIE was fused to the leucine zipper of c-Jun (SpoIIIE–JunLZ) and a fusion protein comprising GFP and the c-Fos leucine zipper (FosLZ–GFP) was placed under the control of the appropriate s factor to promote expression in either the mother cell or the forespore. The SpoIIIE–JunLZ—FosLZ–GFP complex was then visualised. SpoIIIE on the mother-cell side forms foci but is de-localised over the septum in the forespore. The foci presumably represent the active hexameric DNA pumps. An indication of how SpoIIIE achieves this activity on one side of the membrane alone came from the examination of spo0J/soj mutants, which often fail to localise the origin-proximal region to the forespore and can trap other regions of the chromosome instead. In this situation DNA is seen to be exported from the forespore. Therefore, a DNA-encoded cue for translocation directionality was suggested. The molecular basis for the directionality of translocation has also recently been revealed [11]. The B. subtilis chromosome contains a number of polarised octamer sequences, and one of these, termed SRS (SpoIIIE recognition sequence, GAGAAGGG), is recognised specifically by the g domain at the very carboxyl terminus of SpoIIIE. In vitro, SpoIIIE and a mutant lacking the g domain (Dg) are both active translocases. However, the Dg mutant failed to produce viable spores in vivo. Without the g domain, SpoIIIE foci were seen on both sides of the septal membrane with equal frequency and DNA was pumped both into and out of the forespore. Therefore, it can be concluded that recognition of the polarised SRS sequences by the g domain is responsible for establishing the active translocase complexes in the mother cell, which pump DNA into the forespore. By analogy to FtsK, this could be controlled exclusively at the motor-loading step [12].
Current Biology Vol 18 No 18 R872
Forespore
Mother cell Post-DNA replication
Asymmetric septation
Membrane fusion
SpoIIIE assembly
Chromosome transfer Current Biology
Figure 1. A representation of sporulation in B. subtilis. Upon replication, the cell has two copies of the chromosome (red and blue). The origin region (yellow circle) is localised to the cell poles. Asymmetric septation traps roughly a third of the red chromosome in the forespore and forespore-specific proteins are produced (blue squares). Membranes are fully fused at this point, and cell wall components may also be present between the two membranes. SpoIIIE assembles an active motor on the mother-cell side of the membrane (this may occur concurrently with membrane fusion rather than following it). Polarised SRS sequences in the chromosome determine that the active motor assembles only on the mother-cell side of the membrane. SpoIIIE then pumps DNA into the forespore (grey arrows show direction of DNA movement), stripping off any bound proteins (green circles) as it does so, thus preventing entry of mother-cell-specific transcription complexes into the forespore and ensuring the differentiated state of the forespore.
Several important questions remain to be answered. SpoIIIE pumps both chromosome arms simultaneously [8], probably through two separate pores, producing an ever decreasing loop of chromosome on the mother-cell side. This creates a topological problem: how is this last segment of DNA transferred? Also, the exact nature of the pore through which DNA is transported is not understood. It seems likely that the foci seen on the mother-cell side of the septum are hexameric rings of the motor domains. The prevailing model is that the motors pump DNA through a channel in the membrane formed by the six amino-terminal domains of the active hexameric motor, but other possibilities exist. However, no corresponding foci are seen on the forespore side of the membrane [10,11]. Does SpoIIIE contribute to the formation of a pore through this membrane as well? When SpoIIIE is expressed in the mother cell alone, viable spores are formed at wild-type levels [4], suggesting that SpoIIIE is not required to form a pore in the forespore membrane. Indeed, membrane fusion, which traps DNA in the forespore, occurs even in the complete absence of SpoIIIE [8], showing that DNA can
cross both membranes without SpoIIIE. Further, Ptacin et al. [11] observe that in the Dg mutant reversal of DNA transfer direction following motor stalling is a very slow event (w20 minutes). These data suggest that perhaps pre-formed hexameric rings of a motor-pore complex are not assembled around DNA on both sides of the membrane simultaneously, and that assembly (at least in the absence of g) may be a slow step. What, if anything, contributes to the formation of the pore through which DNA translocates on the forespore side of the septum? The resolution of these tough questions is eagerly awaited. References 1. Ben-Yehuda, S., Rudner, D.Z., and Losick, R. (2003). RacA, a bacterial protein that anchors chromosomes to the cell poles. Science 299, 532–536. 2. Wu, L.J., and Errington, J. (1994). Bacillus subtilis SpoIIIE protein required for DNA segregation during asymmetric cell division. Science 264, 572–575. 3. Wu, L.J., and Errington, J. (1997). Septal localization of the SpoIIIE chromosome partitioning protein in Bacillus subtilis. EMBO J. 16, 2161–2169. 4. Sharp, M.D., and Pogliano, K. (2002). Role of cell-specific SpoIIIE assembly in polarity of DNA transfer. Science 295, 137–139. 5. Bath, J., Wu, L.J., Errington, J., and Wang, J.C. (2000). Role of Bacillus subtilis SpoIIIE in DNA transport across the mother cell-prespore division septum. Science 290, 995–997.
6. Massey, T.H., Mercogliano, C.P., Yates, J., Sherratt, D.J., and Lowe, J. (2006). Double-stranded DNA translocation: structure and mechanism of hexameric FtsK. Mol. Cell 23, 457–469. 7. Ben-Yehuda, S., Rudner, D.Z., and Losick, R. (2003). Assembly of the SpoIIIE DNA translocase depends on chromosome trapping in Bacillus subtilis. Curr. Biol. 13, 2196–2200. 8. Burton, B.M., Marquis, K.A., Sullivan, N.L., Rapoport, T.A., and Rudner, D.Z. (2007). The ATPase SpoIIIE transports DNA across fused septal membranes during sporulation in Bacillus subtilis. Cell 131, 1301–1312. 9. Marquis, K.A., Burton, B.M., Nollmann, M., Ptacin, J.L., Bustamante, C., Ben-Yehuda, S., and Rudner, D.Z. (2008). SpoIIIE strips proteins off the DNA during chromosome translocation. Genes Dev. 22, 1786–1795. 10. Becker, E.C., and Pogliano, K. (2007). Cell-specific SpoIIIE assembly and DNA translocation polarity are dictated by chromosome orientation. Mol. Microbiol. 66, 1066–1079. 11. Ptacin, J.L., Nollmann, M., Becker, E.C., Cozzarelli, N.R., Pogliano, K., and Bustamante, C. (2008). Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis. Nat. Struct. Mol. Biol. 15, 485–493. 12. Lo¨we, J., Ellonen, A., Allen, M.D., Atkinson, C., Sherratt, D.J., and Grainge, I. (2008). Molecular mechanism of sequence-directed DNA loading and translocation by FtsK. Mol. Cell 31, 498–509.
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK. E-mail: [email protected]
DOI: 10.1016/j.cub.2008.07.047