Suppression of Engulfment Defects in Bacillus subtilis by Elevated Expression of the Motility Regulon
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《细菌学杂志》
Division of Biological Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0377
ABSTRACT
During Bacillus subtilis sporulation, the transient engulfment defect of spoIIB strains is enhanced by spoVG null mutations and suppressed by spoVS null mutations. These mutations have opposite effects on expression of the motility regulon, as the spoVG mutation reduces and the spoVS mutation increases D-directed gene expression, cell separation, and autolysis. Elevating D activity by eliminating the anti- factor FlgM also suppresses spoIIB spoVG, and both flgM and spoVS mutations cause continued expression of the D regulon during sporulation. We propose that peptidoglycan hydrolases induced during motility can substitute for sporulation-specific hydrolases during engulfment. We find that sporulating cells are heterogeneous in their expression of the motility regulon, which could result in phenotypic variation between individual sporulating cells.
TEXT
A key step in Bacillus subtilis sporulation is engulfment, which mediates a dramatic rearrangement of the two cells required for sporulation, from two adjacent daughter cells to a sporangium in which one daughter cell (the forespore) is completely enclosed within the cytoplasm of another (the mother cell). This transformation is mediated by the migration of the mother cell membrane around the forespore, which is ultimately released into the mother cell cytoplasm (Fig. 1A). Engulfment depends on three conserved proteins, SpoIID, SpoIIM, and SpoIIP (1, 5, 17, 30), and is facilitated by the less-conserved SpoIIB protein (19, 23). These proteins participate in septal thinning (1, 5, 17, 19, 23, 30), during which septal peptidoglycan is degraded by the SpoIID peptidoglycan hydrolase and perhaps other enzymes (1). While spoIID, spoIIM, and spoIIP mutants are completely defective in septal thinning and membrane migration (Fig. 1B), a spoIIB mutant shows slow and uneven septal thinning but ultimately completes engulfment and produces spores (Fig. 1C). Two additional proteins have been proposed to play a role in septal thinning: SpoVG, whose absence exacerbates the engulfment defect of spoIIB (19, 23), and SpoVS, whose absence suppresses a spoIIB spoVG double mutant, allowing engulfment to proceed (27). Here, we provide evidence that the spoVS mutation causes decreased expression of early-sporulation genes and increased expression of motility genes and that motility-specific genes, likely peptidoglycan hydrolases, can suppress septal thinning defects.
To investigate the mechanism of spoVS-mediated suppression of spoIIB spoVG, we used an in vivo membrane fusion assay that employs two membrane stains, the membrane-impermeable FM 4-64 and the membrane-permeable stain MitoTracker Green (strains of B. subtilis used in this study are shown in Table 1) (29). During engulfment in the wild type, the engulfing membrane smoothly curves around the forespore (Fig. 2A, arrow and arrowhead), with the septal membrane staining twice as brightly as other regions, due to the presence of two membranes (24). Ultimately, the migrating membrane meets and fuses to release the forespore into the mother cell cytoplasm. After membrane fusion, FM 4-64 is excluded from the forespore, resulting in a green forespore enclosed within the red mother cell (Fig. 2A, double arrowhead). In spoIIB spoVG, the growing forespore pushes through the unthinned septum, forming a bulge that is typical of septal thinning defective mutants (Fig. 2C to D). In the spoIIB spoVG spoVS triple mutant, sixfold-fewer bulges were observed than in the spoIIB spoVG mutant (6% versus 35% at t3.0) (Fig. 2G and H; Table 2), and engulfment was complete in 21% of spoVS spoIIB spoVG sporangia versus 0% of spoIIB spoVG sporangia at t4 (Fig. 2; Table 2). The spoVS mutation also weakly suppressed spoIID and spoIIM strains, reducing the frequency of bulges twofold (from 64% to 31% in spoIIM and from 45% to 21% in spoIID) but not supporting the completion of engulfment or increased spore production (see Fig. S1 and Table S1 in the supplemental material). These results suggest that the spoVS mutation is a general suppressor of septal thinning defects.
We noted that spoVS strains showed fewer polar septa than the wild type (Fig. 2E and F; Table 2). This was due to decreased expression of early sporulation genes such as spoIIAC (Fig. 3F) and spoIIE (see Fig. S3 in the supplemental material), which require the Spo0AP transcription factor that governs entry into sporulation (7). Expression of Spo0AP-dependent genes is regulated by kinases and phosphatases that modulate the level of phosphorylated Spo0A (22, 32) and by various transcription factors that inhibit Spo0AP-dependent gene expression (7-9). We found that the defect in polar septation and Spo0AP-dependent gene expression could be substantially rescued by a sinR mutation, which eliminates one of these transcription factors, but not by spo0Asad, which encodes a Spo0A protein active without phosphorylation (11), rvtA11, which encodes a Spo0A protein that can be phosphorylated by an alternative kinase (16), or abrB or spo0J-soj mutations, which eliminate repressors of Spo0AP-dependent gene expression (25, 34; see Fig. S3 in the supplemental material). These results suggest that the spoVS mutant has increased SinR activity.
SinR is a transcription factor that directly regulates biofilm formation (13), inhibits Spo0AP-dependent gene expression, and activates D-dependent motility genes (4, 6, 13, 18, 26), perhaps indirectly (13). Thus, if the spoVS strain has elevated SinR activity, it should also show increased expression of motility genes. Indeed, the spoVS strain showed approximately twofold elevated expression of the flagellin gene (hag-lacZ) relative to the wild type, while the spoVG mutation (enhancer of spoIIB) showed approximately fivefold reduced D activity (Fig. 3G). This suggests that SpoVS directly or indirectly governs SinR activity, similar to two other recently described proteins, YlbF and YmcA (13). Strains lacking these proteins share some phenotypes with spoVS strains, with continued SinR activity, a failure to form biofilms, and growth that is slightly slower than that of the wild type (spoVS data are not shown) (13).
It is unclear if SpoVS directly or indirectly modulates SinR or D activity, and it remains possible that spoVS mutation has additional direct or indirect effects on gene expression. We therefore tested if elevated D activity was sufficient to suppress spoIIB spoVG by inactivating the anti-sigma factor for D, FlgM (21). As expected, flgM strains have increased D-directed gene expression (Fig. 3G). Further, like the spoVS mutation, the flgM mutation increased spore production by spoIIB spoVG 20-fold (from 9 x 104 to 2 x106) (Table 2), reduced the frequency of bulges 7-fold (from 35% to 5%), and increased the proportion of sporangia that completed the final step of engulfment, membrane fusion, from 0% to 9% (Fig. 2I and J; Table 2). Thus, increased D activity is sufficient to suppress the engulfment defect of spoIIB spoVG to a similar extent as the spoVS mutation.
B. subtilis cells often grow in short chains of cells that remain connected by septal peptidoglycan (Fig. 3A and C), which ultimately is split by peptidoglycan hydrolases to allow daughter cell separation (31). We noted that spoVS strains rarely grew in chains (Fig. 3B), suggesting an increased activity of peptidoglycan hydrolases involved in cell separation. Indeed, the D regulon includes genes that encode peptidoglycan hydrolases that mediate cell separation (14, 15, 20), which likely facilitates motility by the generation of single cells (3). The overexpression of peptidoglycan hydrolases in spoVS and flgM strains was confirmed by an autolysis assay, which measures the lysis of bacteria whose growth has been arrested (3, 12). Both strains lysed faster than the wild type; after 4 h, optical density at 600 nm was reduced by 60% in the wild type versus 84% in spoVS and 94% in flgM (Fig. 3D). The elevated autolysis of flgM and spoVS strains was eliminated by a sigD mutation, which inactivates D (Fig. 3E), and by a lytABC mutation, which eliminates the major D-directed autolysin (see Fig. S2 in the supplemental material). The lytABC mutation also reduced but did not eliminate spoVS- and flgM-mediated suppression of spoIIB spoVG, increasing bulges and reducing spore formation to 25% of suppressed levels (Table 2). We propose that spoVS and flgM suppress septal thinning defects by increasing expression of peptidoglycan hydrolases involved in cell separation and speculate that several such hydrolases are required for full suppression.
The increased D activity in spoVS and flgM strains could be due to either an increased D activity in all sporangia or an increased fraction of sporangia with D activity. To test these possibilities, we used immunofluorescence microscopy to visualize Spo0A and D activity in sporulating cultures. To detect Spo0A activity, we used antibodies to F, the production of which requires Spo0AP; to detect D activity, we used antibodies to -galactosidase produced from the hag-lacZ fusion. After 2.5 h of sporulation, D activity was observed in 16% of wild-type sporangia (cells containing a sporulation septum visualized by FM 4-64 membrane staining) (Fig. 4A; Table 3), compared to 36% of spoVS sporangia and 72% of flgM80 sporangia (Fig. 4B to C; Table 3). Thus, spoVS and flgM strains have two to four times more sporangia expressing motility genes than the wild type, which would likely result in an increased number of sporangia with elevated levels of peptidoglycan hydrolases involved in cell separation. Clearly, even a wild-type population of sporulating cells displays heterogeneity in the activity of the motility regulon.
(DAPI; blue) and using antibodies specific for F (magenta) and for -galactosidase produced by hag-lacZ (green). The wild-type strain (KP818) (A), the spoVS strain (KP821) (B), the flgM80 strain (KP819) (C), and the negative control strain KP174 (spoIIAA-AC) which has a deletion in the gene encoding F and no lacZ fusion (D) are shown.
These results demonstrate an unanticipated link between expression of the D-directed motility regulon and the ability of sporulating cells to complete the phagocytosis-like process of engulfment during sporulation. Apparently, the continued expression of D-directed genes during sporulation allows engulfment in the septal thinning defective spoIIB spoVG strain, perhaps by providing additional hydrolases that contribute to septal thinning. Because there is cell-to-cell variation in the level of motility gene expression in individual sporulating cells, these observations might explain why there is also cell-to-cell variation in the phenotypes of engulfment mutants, with variations in whether they divided at the second site in the mother cell and whether they have a flat or bulged septum (24). We anticipate the discovery of additional examples in which a cell's proteomic "memory" of past gene expression alters its behavior during subsequent transcriptional responses or developmental pathways.
ACKNOWLEDGMENTS
We thank Stefan Aung, Dan Broder, Rich Losick, Jonathan Shum, and Joe Pogliano for providing comments on the manuscript; Marta Perego, John D. Helmann, and the Bacillus Genetics Stock Center (BGSC) for providing strains; and Rich Losick for providing F-specific antibodies.
REFERENCES
Abanes-De Mello, A., Y. L. Sun, S. Aung, and K. Pogliano. 2002. A cytoskeleton-like role for the bacterial cell wall during engulfment of the Bacillus subtilis forespore. Genes Dev. 16:3253-3264.
Arigoni, F., L. Duncan, S. Alper, R. Losick, and P. Stragier. 1996. SpoIIE governs the phosphorylation state of a protein regulating transcription factor sigma F during sporulation in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 93:3238-3242.
Blackman, S. A., T. J. Smith, and S. J. Foster. 1998. The role of autolysins during vegetative growth of Bacillus subtilis 168. Microbiology 144:73-82.
Cervin, M. A., R. J. Lewis, J. A. Brannigan, and G. B. Spiegelman. 1998. The Bacillus subtilis regulator SinR inhibits spoIIG promoter transcription in vitro without displacing RNA polymerase. Nucleic Acids Res. 26:3806-3812.
Frandsen, N., and P. Stragier. 1995. Identification and characterization of the Bacillus subtilis spoIIP locus. J. Bacteriol. 177:716-722.
Fredrick, K., and J. D. Helmann. 1996. FlgM is a primary regulator of D activity, and its absence restores motility to a sinR mutant. J. Bacteriol. 178:7010-7013.
Grossman, A. D. 1995. Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis. Annu. Rev. Genet. 29:477-508.
Hamoen, L. W., G. Venema, and O. P. Kuipers. 2003. Controlling competence in Bacillus subtilis: shared use of regulators. Microbiology 149:9-17.
Hamon, M. A., N. R. Stanley, R. A. Britton, A. D. Grossman, and B. A. Lazazzera. 2004. Identification of AbrB-regulated genes involved in biofilm formation by Bacillus subtilis. Mol. Microbiol. 52:847-860.
Helmann, J. D., L. M. Marquez, and M. J. Chamberlin. 1988. Cloning, sequencing, and disruption of the Bacillus subtilis 28 gene. J. Bacteriol. 170:1568-1574.
Ireton, K., D. Z. Rudner, K. J. Siranosian, and A. D. Grossman. 1993. Integration of multiple developmental signals in Bacillus subtilis through the Spo0A transcription factor. Genes Dev. 7:283-294.
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ABSTRACT
During Bacillus subtilis sporulation, the transient engulfment defect of spoIIB strains is enhanced by spoVG null mutations and suppressed by spoVS null mutations. These mutations have opposite effects on expression of the motility regulon, as the spoVG mutation reduces and the spoVS mutation increases D-directed gene expression, cell separation, and autolysis. Elevating D activity by eliminating the anti- factor FlgM also suppresses spoIIB spoVG, and both flgM and spoVS mutations cause continued expression of the D regulon during sporulation. We propose that peptidoglycan hydrolases induced during motility can substitute for sporulation-specific hydrolases during engulfment. We find that sporulating cells are heterogeneous in their expression of the motility regulon, which could result in phenotypic variation between individual sporulating cells.
TEXT
A key step in Bacillus subtilis sporulation is engulfment, which mediates a dramatic rearrangement of the two cells required for sporulation, from two adjacent daughter cells to a sporangium in which one daughter cell (the forespore) is completely enclosed within the cytoplasm of another (the mother cell). This transformation is mediated by the migration of the mother cell membrane around the forespore, which is ultimately released into the mother cell cytoplasm (Fig. 1A). Engulfment depends on three conserved proteins, SpoIID, SpoIIM, and SpoIIP (1, 5, 17, 30), and is facilitated by the less-conserved SpoIIB protein (19, 23). These proteins participate in septal thinning (1, 5, 17, 19, 23, 30), during which septal peptidoglycan is degraded by the SpoIID peptidoglycan hydrolase and perhaps other enzymes (1). While spoIID, spoIIM, and spoIIP mutants are completely defective in septal thinning and membrane migration (Fig. 1B), a spoIIB mutant shows slow and uneven septal thinning but ultimately completes engulfment and produces spores (Fig. 1C). Two additional proteins have been proposed to play a role in septal thinning: SpoVG, whose absence exacerbates the engulfment defect of spoIIB (19, 23), and SpoVS, whose absence suppresses a spoIIB spoVG double mutant, allowing engulfment to proceed (27). Here, we provide evidence that the spoVS mutation causes decreased expression of early-sporulation genes and increased expression of motility genes and that motility-specific genes, likely peptidoglycan hydrolases, can suppress septal thinning defects.
To investigate the mechanism of spoVS-mediated suppression of spoIIB spoVG, we used an in vivo membrane fusion assay that employs two membrane stains, the membrane-impermeable FM 4-64 and the membrane-permeable stain MitoTracker Green (strains of B. subtilis used in this study are shown in Table 1) (29). During engulfment in the wild type, the engulfing membrane smoothly curves around the forespore (Fig. 2A, arrow and arrowhead), with the septal membrane staining twice as brightly as other regions, due to the presence of two membranes (24). Ultimately, the migrating membrane meets and fuses to release the forespore into the mother cell cytoplasm. After membrane fusion, FM 4-64 is excluded from the forespore, resulting in a green forespore enclosed within the red mother cell (Fig. 2A, double arrowhead). In spoIIB spoVG, the growing forespore pushes through the unthinned septum, forming a bulge that is typical of septal thinning defective mutants (Fig. 2C to D). In the spoIIB spoVG spoVS triple mutant, sixfold-fewer bulges were observed than in the spoIIB spoVG mutant (6% versus 35% at t3.0) (Fig. 2G and H; Table 2), and engulfment was complete in 21% of spoVS spoIIB spoVG sporangia versus 0% of spoIIB spoVG sporangia at t4 (Fig. 2; Table 2). The spoVS mutation also weakly suppressed spoIID and spoIIM strains, reducing the frequency of bulges twofold (from 64% to 31% in spoIIM and from 45% to 21% in spoIID) but not supporting the completion of engulfment or increased spore production (see Fig. S1 and Table S1 in the supplemental material). These results suggest that the spoVS mutation is a general suppressor of septal thinning defects.
We noted that spoVS strains showed fewer polar septa than the wild type (Fig. 2E and F; Table 2). This was due to decreased expression of early sporulation genes such as spoIIAC (Fig. 3F) and spoIIE (see Fig. S3 in the supplemental material), which require the Spo0AP transcription factor that governs entry into sporulation (7). Expression of Spo0AP-dependent genes is regulated by kinases and phosphatases that modulate the level of phosphorylated Spo0A (22, 32) and by various transcription factors that inhibit Spo0AP-dependent gene expression (7-9). We found that the defect in polar septation and Spo0AP-dependent gene expression could be substantially rescued by a sinR mutation, which eliminates one of these transcription factors, but not by spo0Asad, which encodes a Spo0A protein active without phosphorylation (11), rvtA11, which encodes a Spo0A protein that can be phosphorylated by an alternative kinase (16), or abrB or spo0J-soj mutations, which eliminate repressors of Spo0AP-dependent gene expression (25, 34; see Fig. S3 in the supplemental material). These results suggest that the spoVS mutant has increased SinR activity.
SinR is a transcription factor that directly regulates biofilm formation (13), inhibits Spo0AP-dependent gene expression, and activates D-dependent motility genes (4, 6, 13, 18, 26), perhaps indirectly (13). Thus, if the spoVS strain has elevated SinR activity, it should also show increased expression of motility genes. Indeed, the spoVS strain showed approximately twofold elevated expression of the flagellin gene (hag-lacZ) relative to the wild type, while the spoVG mutation (enhancer of spoIIB) showed approximately fivefold reduced D activity (Fig. 3G). This suggests that SpoVS directly or indirectly governs SinR activity, similar to two other recently described proteins, YlbF and YmcA (13). Strains lacking these proteins share some phenotypes with spoVS strains, with continued SinR activity, a failure to form biofilms, and growth that is slightly slower than that of the wild type (spoVS data are not shown) (13).
It is unclear if SpoVS directly or indirectly modulates SinR or D activity, and it remains possible that spoVS mutation has additional direct or indirect effects on gene expression. We therefore tested if elevated D activity was sufficient to suppress spoIIB spoVG by inactivating the anti-sigma factor for D, FlgM (21). As expected, flgM strains have increased D-directed gene expression (Fig. 3G). Further, like the spoVS mutation, the flgM mutation increased spore production by spoIIB spoVG 20-fold (from 9 x 104 to 2 x106) (Table 2), reduced the frequency of bulges 7-fold (from 35% to 5%), and increased the proportion of sporangia that completed the final step of engulfment, membrane fusion, from 0% to 9% (Fig. 2I and J; Table 2). Thus, increased D activity is sufficient to suppress the engulfment defect of spoIIB spoVG to a similar extent as the spoVS mutation.
B. subtilis cells often grow in short chains of cells that remain connected by septal peptidoglycan (Fig. 3A and C), which ultimately is split by peptidoglycan hydrolases to allow daughter cell separation (31). We noted that spoVS strains rarely grew in chains (Fig. 3B), suggesting an increased activity of peptidoglycan hydrolases involved in cell separation. Indeed, the D regulon includes genes that encode peptidoglycan hydrolases that mediate cell separation (14, 15, 20), which likely facilitates motility by the generation of single cells (3). The overexpression of peptidoglycan hydrolases in spoVS and flgM strains was confirmed by an autolysis assay, which measures the lysis of bacteria whose growth has been arrested (3, 12). Both strains lysed faster than the wild type; after 4 h, optical density at 600 nm was reduced by 60% in the wild type versus 84% in spoVS and 94% in flgM (Fig. 3D). The elevated autolysis of flgM and spoVS strains was eliminated by a sigD mutation, which inactivates D (Fig. 3E), and by a lytABC mutation, which eliminates the major D-directed autolysin (see Fig. S2 in the supplemental material). The lytABC mutation also reduced but did not eliminate spoVS- and flgM-mediated suppression of spoIIB spoVG, increasing bulges and reducing spore formation to 25% of suppressed levels (Table 2). We propose that spoVS and flgM suppress septal thinning defects by increasing expression of peptidoglycan hydrolases involved in cell separation and speculate that several such hydrolases are required for full suppression.
The increased D activity in spoVS and flgM strains could be due to either an increased D activity in all sporangia or an increased fraction of sporangia with D activity. To test these possibilities, we used immunofluorescence microscopy to visualize Spo0A and D activity in sporulating cultures. To detect Spo0A activity, we used antibodies to F, the production of which requires Spo0AP; to detect D activity, we used antibodies to -galactosidase produced from the hag-lacZ fusion. After 2.5 h of sporulation, D activity was observed in 16% of wild-type sporangia (cells containing a sporulation septum visualized by FM 4-64 membrane staining) (Fig. 4A; Table 3), compared to 36% of spoVS sporangia and 72% of flgM80 sporangia (Fig. 4B to C; Table 3). Thus, spoVS and flgM strains have two to four times more sporangia expressing motility genes than the wild type, which would likely result in an increased number of sporangia with elevated levels of peptidoglycan hydrolases involved in cell separation. Clearly, even a wild-type population of sporulating cells displays heterogeneity in the activity of the motility regulon.
(DAPI; blue) and using antibodies specific for F (magenta) and for -galactosidase produced by hag-lacZ (green). The wild-type strain (KP818) (A), the spoVS strain (KP821) (B), the flgM80 strain (KP819) (C), and the negative control strain KP174 (spoIIAA-AC) which has a deletion in the gene encoding F and no lacZ fusion (D) are shown.
These results demonstrate an unanticipated link between expression of the D-directed motility regulon and the ability of sporulating cells to complete the phagocytosis-like process of engulfment during sporulation. Apparently, the continued expression of D-directed genes during sporulation allows engulfment in the septal thinning defective spoIIB spoVG strain, perhaps by providing additional hydrolases that contribute to septal thinning. Because there is cell-to-cell variation in the level of motility gene expression in individual sporulating cells, these observations might explain why there is also cell-to-cell variation in the phenotypes of engulfment mutants, with variations in whether they divided at the second site in the mother cell and whether they have a flat or bulged septum (24). We anticipate the discovery of additional examples in which a cell's proteomic "memory" of past gene expression alters its behavior during subsequent transcriptional responses or developmental pathways.
ACKNOWLEDGMENTS
We thank Stefan Aung, Dan Broder, Rich Losick, Jonathan Shum, and Joe Pogliano for providing comments on the manuscript; Marta Perego, John D. Helmann, and the Bacillus Genetics Stock Center (BGSC) for providing strains; and Rich Losick for providing F-specific antibodies.
REFERENCES
Abanes-De Mello, A., Y. L. Sun, S. Aung, and K. Pogliano. 2002. A cytoskeleton-like role for the bacterial cell wall during engulfment of the Bacillus subtilis forespore. Genes Dev. 16:3253-3264.
Arigoni, F., L. Duncan, S. Alper, R. Losick, and P. Stragier. 1996. SpoIIE governs the phosphorylation state of a protein regulating transcription factor sigma F during sporulation in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 93:3238-3242.
Blackman, S. A., T. J. Smith, and S. J. Foster. 1998. The role of autolysins during vegetative growth of Bacillus subtilis 168. Microbiology 144:73-82.
Cervin, M. A., R. J. Lewis, J. A. Brannigan, and G. B. Spiegelman. 1998. The Bacillus subtilis regulator SinR inhibits spoIIG promoter transcription in vitro without displacing RNA polymerase. Nucleic Acids Res. 26:3806-3812.
Frandsen, N., and P. Stragier. 1995. Identification and characterization of the Bacillus subtilis spoIIP locus. J. Bacteriol. 177:716-722.
Fredrick, K., and J. D. Helmann. 1996. FlgM is a primary regulator of D activity, and its absence restores motility to a sinR mutant. J. Bacteriol. 178:7010-7013.
Grossman, A. D. 1995. Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis. Annu. Rev. Genet. 29:477-508.
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