Bacillus subtilis sporulation

Bacillus subtilis

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). Another short Wikipedia article has a nice figure summarizing the regulatory network for initiation of sporulation in B. subtilis: (http://en.wikipedia.org/wiki/Sporulation_in_Bacillus_subtilis).  Information on individual genes can be found on SubtiWiki: (http://www.subtiwiki.uni-goettingen.de/wiki/index.php/Main_Page)

Life cycle:

B. subtilis life cycle

Sporulation cycle of B. subtilis. Errington 2003 Fig 1

  • 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.

 

Developmental questions

  • 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.

Sigma factors

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

phosphorelay activates sporulation in B. subitlis

Shapiro and Losick 1997 Fig. 3A The phosphorelay that activates the transcriptional regulator Spo0A in B. subtilis 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.

A diagram of the regulatory circuits for sporulation initiation can be found here:

http://upload.wikimedia.org/wikipedia/commons/a/aa/Gene_Regulatory_Network_for_Initiation_of_Sporulation_in_Bacillus_subtilis.png

Switch from Medial to Polar Septation

Shapiro & Losick 1997 Fig. 2

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 SpoIIE interaction with FtsZ, and also by the RefZ protein (Wagner-Herman et al. 2012).

Criss-cross Regulation of Sigma Factors (Losick and Stragier, 1992):

Shapiro and Losick 1997 Fig. 4A. Determination of cell fate by protein localization during sporulation in B. subtilis. Spo0A induces the synthesis of AA, AB, sigmaF, and pro-sigmaE.

Sigma F – drives gene expression in forespore cells, produced before septation.

Sigma F is made before septation, but is active only in the forespore cell; it is 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).  The 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.

Hilbert & Piggot 2004 Fig. 4 Models of sigmaF regulation. A) basic model; B) integrative model

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 – drives 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 the N-terminus.  Cleavage of pro-sigmaE by SpoIIGA membrane protease (imamura et al. 2008) 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. Recently published findings by Chary et al. (2010) indicate that activation of sigma E in the foreposre has only minor effects on sporulation efficiency. SigmaE  in the forespore is degraded shortly after septation in the forespore, by an unknown mechanism, ensuring mother cell specificity.

Imamura et al. 2008 Figure 1. Model of pro-sigmaE processing

Sigma G – drives late gene expression in forespore.  Product of SpoIIIG gene.  SpoIIIG gene transcription is activated by SigmaF, and 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 – drives late 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 is synthesized as inactive pro-sigmaK; the N-terminal 20 amino acids are cleaved by the product of spoIVF, which gene is also transcribed by SigmaE.  Transcription of the rearranged sigmaK gene is maintained by SigmaK – autoregulation.  The SpoIVF protease activity depends on SigmaG in the forespore:  SigmaG directs transcription of spoIVB in forespore, spoIVB activates spoIVF in the mother cell membrane (Mastny et al. 2013).

Engulfment

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.

Morlot et al. 2010 Figure 7 Coordinated activities in the engulfment complex drive membrane migration around the forespore. (A) Schematic representation of the complementary and sequential activities of IIP and IID. (1) In the diagram, IIP cleaves the stem peptide from the glycan strand and the cross-links between the tetrapeptides. (2) Following peptide cleavage by IIP, IID cleaves the denuded glycan strands into anhydrodisaccharides. (B) Comparison of the amide bonds cleaved by IIP. (C) Schematic diagram of the proposed catalytic cycle of the engulfment complex. (1) IIP (P) and IID (D) in complex with IIM (M) bind the PG. (2) IID stimulates the amidase activity of IIP, resulting in cleavage of the stem peptides and peptide cross-links (black). (3) IIP is released from the PG and rebinds at a nearby site. (4) IID cleaves the denuded glycan strands (green). (5) IID is released from the PG and rebinds adjacent to IIP. The leading edge of the engulfing mother cell membrane (dark purple) moves toward the forespore pole (to the right). (D) Circumferentially distributed engulfment complexes drive membrane movement around the forespore. Shown is a schematic diagram of a sporangium during the morphological processes of engulfment. The membranes of the mother cell (purple) are migrating around the forespore (blue). The glycan strands (green) are shown running perpendicular to the long axis of the cell, as has been proposed for E. coli and C. crescentus (Holtje 1998; Gan et al. 2008). The hoops of glycan strands are stitched together by the cross-linked tetrapeptides. IID (yellow) and IIP (red) are anchored in the leading edge of the mother cell membrane. Processive degradation of the PG drives the mother cell membranes toward the cell pole. For simplicity, we show the engulfment complex acting on the basement layer of the PG meshwork. It is possible that the complex degrades more than one layer, and not necessarily the layer adjacent to the forespore.

Topics for further exploration:

The Losick lab has gone on to explore communal growth: bacterial biofilm formation.

References:

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://www.ncbi.nlm.nih.gov/pmc/articles/PMC3019954/

Chary VK, Xenopoulos P, Eldar A, Piggot PJ 2010. Loss of compartmentalization of σ(E) activity need not prevent formation of spores by Bacillus subtilis. J Bacteriol. 192:5616-5624. doi: 10.1128/JB.00572-10. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2953694/

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.

Mastny M, Heuck A, Kurzbauer R, Heiduk A, Boisguerin P, Volkmer R, Ehrmann M, Rodrigues CD, Rudner DZ, Clausen T 2013. CtpB assembles a gated protease tunnel regulating cell-cell signaling during spore formation in Bacillus subtilis. Cell 155:647-58. doi: 10.1016/j.cell.2013.09.050. PubMed Central PMCID: PMC3808539.

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.

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