Bacillus subtilis is a gram-positive bacterium commonly found in soil. It has been well-studied as a model for endospore formation, and as a model for developmental processes such as asymmetric cell division, differential gene expression, and intercellular communication. B. subtilis is also useful as a non-pathogenic close cousin to Bacillus anthracis, whose spores can cause anthrax, a lethal disease in animals and humans when inhaled or ingested. General information about Bacillus subtilis can be found on Microbewiki (http://microbewiki.kenyon.edu/index.php/Bacillus_subtilis) and Wikipedia (http://en.wikipedia.org/wiki/Bacillus_subtilis). Information on individual genes can be found on SubtiWiki: (http://www.subtiwiki.uni-goettingen.de/wiki/index.php/Main_Page)
- Vegetative cells proliferate by cell divisions that produce identical sister cells.
- Starvation initiates sporulation.
- Unequal cell division produces smaller forespore cell and larger mother cell.
- Mother cell encloses forespore, deposits spore coat, eventually dies and lyses. This is an example of a terminally differentiated cell, because it cannot reproduce.
- Forespore cell differentiates into an environmentally-resistant spore.
- Spore is released when mother cell lyses, germinates under conditions favorable for vegetative growth. Thus a forespore, which will eventually give rise to future generations, is equivalent to a germ line cell.
- What signal initiates the switch from vegetative growth to sporulation?
- How is asymmetric cell division achieved? What gives the two unequal daughter cells different cell fates?
- How is differential gene expression regulated in mother cells and forespores?
- What cell-cell interactions coordinate forespore development and mother cell gene expression?
Sporulation defective mutants
The earliest analysis of sporulation began by isolating mutants that were blocked in sporulation at different stages of the sporulation process.
Spo0 – mutants blocked at initiation of sporulation.
SpoI, SpoII, SpoIII, SpoIV, SpoV – mutants blocked at stages I, II, III, IV, & V of sporulation.
Transcription is carried out by RNA polymerase. The core RNA polymerase consists of 4 polypeptide subunits: 2 alpha, a beta, and a beta’. The core polymerase is not very good at initiating transcription, and has no promoter recognition.
Bacterial RNA polymerases recognize promoters with the help of an accessory protein called a sigma factor. Note: such accessory proteins that regulate RNA polymerase initiation with promoters are generally called transcription factors. Sigma factors are a special class of bacterial transcription factors. Sigma factors recognize specific DNA sequences at the -10 and – 35 regions upstream of the transcription start site.
Different sigma factors are present in vegetative cells, early stages of sporulation, and in the mother cell and forespore compartments. The different sets of sigma factors cause different sets of genes (genes that have sporulation-specific, mother cell-specific and forespore-specific promoters) to be expressed in the different cell types, resulting in cell differentiation.
So what regulates the sigma factors?
Initiation of Sporulation
The switch from vegetative growth to sporulation involves a switch from sigmaA, the vegetative sigma factor, to sigmaH and to sigmaF activation in the forespore compartment. Starvation produces unknown signals that initiate a phosphorelay involving two-component signal transduction systems. Two-component signal transduction systems have an integral membrane or cytoplasmic sensor kinase that autophosphorylates at a histidine residue (ATP is the phosphate donor). The phosphate is then transferred to an aspartate residue on a cytoplasmic response regulator, often a transcriptional regulator. Upon starvation, sensor kinases KinA, KinB and KinC transfer a phosphate from ATP to Spo0F (a response regulator). The phosphorylated Spo0F transfers its phosphate to Spo0B, which in turn transfers the phosphate to Spo0A, the master transcriptional regulator (Burbulys et al., 1992). Spo0A~P activates transcription of some genes and represses transcription of other genes. It represses the abrB gene; falling levels of AbrB protein, a repressor of sporulation genes, initiates transcription of sporulation genes. Spo0A~P also activates transcription of the spoIIA operon encoding sigmaF, the spoIIG operon encoding sigmaE, and the spoIIE gene required for activation of sigmaF in the forespore (see below).
The phosphorylation state of Spo0F and Spo0A are also regulated by phosphatases RapA and RapB, which dephosphorylate Spo0F~P, and Spo0E, which dephosphorylates Spo0A~P.
Switch from Medial to Polar Septation
Newly replicated chromosomes are attached near their origins of replication to the cell membrane at opposite poles of the pre-divisional cell. During medial division, FtsZ (a contractile protein similar to eukaryotic tubulin) polymerizes in a ring at the midpoint of the cell. GTP-dependent contraction of FtsZ drives cytokinesis. But in sporulation, FtsZ polymerizes as two rings, at potential division sites near both poles. This switch in FtsZ localization is mediated in part by Spo0IIE interaction with FtsZ, and also by RefZ (Wagner-Herman et al. 2012).
Criss-cross Regulation of Sigma Factors (Losick and Stragier, 1992):
Sigma F – gene expression in forespore cells, produced before septation. Sigma F active only in forespore cell, inhibited in mother cell. Product of spoIIAC gene. Regulated by other proteins encoded in the spoIIA operon: SpoIIAA inhibits SpoIIAB, which inhibits SigmaF (Duncan and Losick, 1993). SpoIIAB phosphorylates SpoIIAA (Min et al., 1993). Partner-switching model (Alper et al. 1994) postulates that at high ATP levels, SpoIIAB phosphorylates SpoIIAA; phosphorylated SpoIIAA (SpoIIAA-P) releases SpoIIAB, which then complexes with sigma F to keep sigma F inactive. At low ATP levels (high ADP levels), SpoIIAA is not phosphorylated; SpoIIAB complexes with SpoIIAA and frees active sigma F.
So how is sigmaF activated only in the forespore? SpoIIE, a phosphatase localized in the septum membrane, dephosphorylates SpoIIAA~P preferentially in the forespore. SpoIIE may be present or active only on the forespore side of the septum, or the asymmetric division creates a higher ratio of surface to volume in the smaller forespore compartment, thus a higher ratio of SpoIIE to cytoplasm. SpoIIAB is also degraded preferentially in the forespore. Transient genetic asymmetry (takes 15 min to complete transfer of chromosome to forespore) may also play a role, as the SpoIIA operon is distal to the ori and therefore is not initially present in the forespore.
Sigma E – gene expression in mother cells, also produced before septation. Product of SpoIIGB gene. Produced in inactive form, as pro-sigmaE with extra 27 amino acids at N-terminus. Cleavage of pro-sigmaE by SpoIIGA membrane protease occurs in mother cells after septation, and activates sigmaE. This requires Sigma F activity in forespore! Hypothesis: SigmaF in forespore activates directional signal that crosses the septation membrane to activate SigmaE in mother cell. SpoIIR is transcribed by sigma F in forespore, and required for activation of sigma E in mother cell (Karow et al. 1995). However, expression of SpoIIR in mother cells has only mild effects, indicating that the signal is not particularly directional. In any case, this mechanism ensures that sigma E is not activated until after septation. SigmaE in the forespore is degraded shortly after septation in the forespore, by an unknown mechanism, ensuring mother cell specificity.
Sigma G – gene expression in forespore. Product of SpoIIIG gene. SpoIIIG gene transcription activated by SigmaF, maintained by SigmaG – autoregulation. Activation of sigmaG is delayed until after engulfment of forespore by mother cell is complete. The anti-sigma factor SpoIIAB binds sigmaG as well as sigmaF. Activation of sigmaG depends on SpoIIIA operon transcription by sigmaE in mother cell, and SpoIIIQ protein in the forespore (transcribed by sigmaF). What signal or molecule from the mother cell is required is not yet known, but does appear to require a channel between the two (http://genesdev.cshlp.org/content/23/8/1014.long) formed by interaction between SpoIIIAA-AH (which resemble a type III secretion system) and SpoQ. The transition from sigmaF to sigmaG in the forespore depends on a small protein named Fin (inhibitor of sigmaF) (Camp et al. 2011).
Sigma K – gene expression in mother cells. Encoded by two different open reading frames – SpoIVCB (N-terminal half) and SpoIIIC (C-terminal half). This split gene is activated by a DNA rearrangement that involves excision of intervening 42 kb DNA element. DNA rearrangement catalysed by SpoIVCA (recombinase) and SpoIIID (small DNA binding protein). Both spoIVCA and spoIIID are regulated by SigmaE, and transcription of SigmaK gene is also regulated by SigmaE. SigmaK synthesized as inactive pro-sigmaK; N-terminal 20 amino acids cleaved by product of spoIVF, which is also transcribed by SigmaE. Transcription maintained by SigmaK – autoregulation. SpoIVF activity depends on SigmaG in forespore: SigmaG directs transcription of spoIVB in forespore, spoIVB activates spoIVF in mother cell membrane.
Another question that has been addressed is how the mother cell engulfs the forespore. The process involves degradation of the petidoglycan of the septum, migration of the membrane-cell wall attachment point around the circumference of the forespore, and fusion of the membrane at the pole to produce a spore cell enclosed in both its own membrane and the mother cell membrane. Proteins localized to the septum (SpoIIM, SpoIID and SpoIIP) form a cell-wall degrading complex (Morlot et al 2010). Surprisingly, SpoIIIE, the membrane protein that functions as the chromosome pump earlier, travels along the leading edge of the membrane migration and is required for membrane fusion.
Topics for further exploration:
The Losick lab has gone on to explore communal growth: bacterial biofilm formation.
Alper et al., 1994. An adenosine nucleotide switch controlling the activity of a cell type-specific transcription factor in B. subtilis. Cell 77:195-205.
Arigoni, F., L. Duncan, S. Alper, R. Losick & 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.
Burbulys et al., 1992. Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell 64:545-552.
Camp, AH and R Losick, 2009. A feeding tube model for activation of a cell-specific transcription factor during sporulation in Bacillus subtilis. Genes & Dev. 23:1014-1024 http://dx.doi.org/10.1101/gad.1781709
Camp, AH, AF Wang and R Losick, 2011. A small protein required for the switch from sigmaF to sigmaG during sporulation in Bacillus subtilis. J. Bacteriol. 193:116-124 http://dx.doi.org/10.1128/JB.00949-10
Duncan, L. and R. Losick, 1993. SpoIIAB is an anti-sigma factor that binds to and inhibits transcription by regulatory protein sigmaF from Bacillus subtilis. Proc. Natl. Acad. Sci. USA 90:2325-2329.
Errington, J 2003. Regulation of endospore formation in Bacillus subtilis. Nature Reviews Microbiol 1:117-126 http://dx.doi.org/10.1038/nrmicro750
Hilbert, DW and PJ Piggot 2004. Compartmentalization of Gene Expression during Bacillus subtilis Spore Formation. Microbiol. Molec. Biol. Reviews 68:234-262 http://dx.doi.org/10.1128/MMBR.68.2.234-262.2004
Imamura, D, R Zhou, M Feig, L Kroos 2008. Evidence that the Bacillus subtilis SpoIIGA protein Is a Novel Type of Signal-Transducing Aspartic Protease. J. Biol. Chem. 283:15287-15299 http://www.jbc.org/content/283/22/15287.full
Karow et al., 1995. Identification of a gene, spoIIR, that links the activation of sigmaE to the transcriptional activity of sigmaF during sporulation in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 92:2012-2016.
Levin, P.A. and Losick, R., 1996. Transcription factor SpoOA switches the localization of the cell division protein FtsZ from a medial to a bipolar pattern in Bacillus subtilis. Genes Dev. 10:478-488.
Losick & Stragier, 1992. Crisscross regulation of cell-type specific gene expression during development in B. subtilis. Nature 355:601-604.
Min et al., 1993. SigmaF, the first compartment-specific transcription factor of B. subtilis, is regulated by an anti-sigma factor that is also a protein kinase. Cell 74:735-742.
Morlot, C, T Uehara, KA Marquis, TG Bernhardt, DZ Rudner 2010 A highly coordinated cell wall degradation machine governs spore morphogenesis in Bacillus subtilis. Genes Dev. 24:411-422 http://dx.doi.org/10.1101/gad.1878110
Perego & Hoch, 1996. Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 93:1459-1553.
Rudner, D.Z., P. Fawcett and R. Losick, 1999. A family of membrane-embedded metalloproteases involved in regulated proteolysis of membrane-associated transcription factors. Proc. Natl. Acad. Sci. USA 96:14765-14770.
Shapiro, L. and R. Losick, 1997. Protein localization and cell fate in bacteria. Science 276:712-718.
Wagner-Herman JK, Bernard R, Dunne R, Bisson-Filho AW, Kumar K, Nguyen T, Mulcahy L, Koullias J, Gueiros-Filho FJ, Rudner DZ. 2012. RefZ facilitates the switch from medial to polar division during spore formation in Bacillus subtilis. J Bacteriol. 194:4608-18. doi: 10.1128/JB.00378-12.
Zhang, L., M.L. Higgins, P.J. Piggot & M.L. Karow, 1996. Analysis of the role of prespore gene expression in the compartmentalization of mother cell-specific gene expression during sporulation of Bacillus subtilis. J. Bacteriol. 178:2813-2817.