Endospore Formation in Bacillus subtilis: An Example of Cell ...

Modern Microbial Genetics, Second Edition. Edited by Uldis N. Streips, Ronald E. YasbinCopyright # 2002 Wiley-Liss, Inc.ISBNs: 0-471-38665-0 Hardback); 0-471-22197-X Electronic)11Endospore Formation in Bacillus subtilis:An Example of Cell Differentiation by aBacteriumCHARLES P. MORAN JR.Department of Microbiology and Immunology, Emory University School of Medicine,Atlanta, Georgia 30322I. Introduction ................................... 273A. Morphological Stages of Development. . ........ 273B. Developmentally Regulated Gene Expression .... 274C. The Cascade of RNA Polymerase Sigma Factor. . 275II. Signal Transduction and the Initiation of Sporulation 276A. Phosphorylation of the TranscriptionalActivator Spo0A ............................ 276B. The Phosphorelay System, Phosphatases,and Integration of Multiple Systems............ 276III. Determination of a Cell's Developmental Fateby a Phosphatase and Anti-sigma Factor . . . ........ 277IV. Coordination of Two Cells' Developmental Programsby Crisscross Regulation of Sigma Factor Activity . . . 278A. Regulation of s E Activity..................... 278B. Regulation of s K Activity. .................... 279C. Regulation of s G Activity. .................... 279V. More Unanswered Questions ..................... 280I. INTRODUCTIONA. Morphological Stages ofDevelopmentMany species of bacteria are capable ofundergoing remarkably complex cellulardifferentiations. In each case the process involvesthe activation and sequential expressionof a large number of genes. One type ofcellular differentiation is the formation ofendospores by Clostridium and Bacillusspecies. In no example of bacterial differentiationis it completely understood how geneexpression is regulated, but the most extensivelycharacterized system, and probably theone most ameanable to genetic analysis, isthat of endospore formation in Bacillus subtilisfor reviews on sporulation, see Errington,1993; Stragier and Losick, 1996).Bacillus subtilis is a rod-shaped Grampositivebacterium, which like most bacteria,multiplies by binary fission. However, in responseto nutrient depletion, the bacteriumdifferentiates in a complex developmentalprocess that culminates in the production of

274 MORAN JR.a new cell type, the endospore. The endosporeis a dormant cell that can survivenumerous environmental insults e.g., dessication,heat, chemical stresses, and nutrientdepletion). Although the endospore is a dormantcell, it responds to specific nutrientsignals by germinating to produce a vegetativecell that resumes multiplication bybinary fission.Endospore development is a morphologicallycomplex process that proceeds through awell-defined series of morphological stagesFig. 1). Multiplication of vegetative cellsby binary fission involves a cell divisionthat produces two identical daughter cells.However, at the onset of sporulation thecell divides asymetrically to produce asmall cell and a large cell. These two cellshave different developmental fates. Thesmaller cell, forespore, develops into themature endospore. The larger cell, knownas the mother cell, develops into a terminallydifferentiated cell that nurtures the developingendospore, providing to it a largenumber of products. Ultimately the mothercell dies when the endospore is mature.In the next most conspicuous morphologicalevent after the asymmetric cell divisionthe peptidoglycan layer between theforespore and mother cells is hydrolyzed,and the mother cell engulfs the forespore ina phagocytic-like process in which themother cell cytoplasmic membrane surroundsthe membrane of the forespore. Theengulfed forespore protoplast floats withinthe mother cell cytoplasm where it is surroundedby both its own original membraneand a second membrane that originatedfrom the mother cell cytoplasmic membrane.This cell-within-a-cell form of developmentis why this type of bacterial spore is called anendospore. At this stage both cells, foresporeand mother cell, are metabolically active.Therefore the developing endospore is builtfrom the inside and outside simultaneously,with some products synthesized withinthe forespore where they accumulate e.g.,proteins that bind and protect the sporeDNA), while other products are synthesizedin the mother cell cytoplasm from wherethey are donated to the endospore. In thelater stages of development a thick layerof peptidoglycan accumulates betweenthe two membranes of the forespore, and athick layer of proteins forms a coat thatsurrounds and protects the peptidoglycanlayer.B. Developmentally Regulated GeneExpressionEndospore formation requires the expressionof over 100 genes that are not requiredFig. 1. Morphological stages of endospore formation in B. subtilis. The most conspicuous morphologicalchanges include an asymmetric cell division c), engulfment of the forespore by the mother cell membrane dand e), synthesis of the cortex f), assembly of the coat surrounding the forespore g), and lysis of the mothercell to release the dormant spore h). Also shown are the sigma factors s) active during sporulation Thedashed arrows represent signals from one cell that signal activation of a s in the other cell. The symbol ?)indicates the anti-sigma factor SpoIIAB that represses the activity of s F and s G .

ENDOSPORE FORMATION IN BACILLUS SUBTILIS 275for growth or survival of vegetative cells.These genes are regulated primarily atthe level of transcription, and are transcribedin a specific temporal sequence.Transcription of sporulation genes is alsoregulated in a cell-type specific manner;some genes are transcribed exclusively inthe forespore, whereas other genes are transcribedexclusively in the mother cell.C. The Cascade of RNA PolymeraseSigma FactorsRegulation of both the temporal patternof transcription and the cell-type specifictranscription of genes during endosporedevelopment is primarily governed by afamily of transcription factors known asRNA polymerase sigma factors reviewedin Losick and Stragier, 1992; Kroos et al.,1999). RNA polymerase in bacteria ismade of several protein subunits. Thecore enzyme is composed of two alpha, onebeta, and one beta 0 subunits. The sigma subunit,or sigma factor, associates with thecore RNA polymerase to form the holoenzyme.Association of the sigma withRNA polymerase confers on the polymeraseits ability to bind to and utilize specificpromoters. All bacteria have a primarysigma factor called sigma A, s A ,inB. subtilis)that directs transcription of most housekeepinggenes. RNA polymerase containings A Es A ) recognizes specific promoters becausethe sigma interacts with two specificsequences of DNA located 35 and 10 basepairs upstream from the start point of transcription,the 35 and 10 regions, respectively,of the promoter. Most bacteria containsecondary sigma factors that enable RNApolymerase to utilize promoters that aredefined by different sequences of DNA.Five secondary sigma factors, each conferinga different specificity for promoter recognitionon RNA polymerase, direct transcriptionduring endospore formation in B.subtilis see Helmann, this volume).One of the secondary sigma factors requiredfor endospore development, s H , ispresent in vegetative cells. However, theother secondary sigma factors required forendospore development are synthesized afterthe onset of sporulation in the order s F , s E ,s G , and s K . The sequential appearance ofthese sigma factors produces the temporallyregulated pattern of gene transcription.Therefore genes whose transcription isdirected by s F are transcribed before geneswhose transcription is dependent on s E , s G ,or s K . Although these secondary sigmafactors define the major temporal classes ofgene transcription during sporulation, eachclass is subdivided by ancillary transcriptionfactors that work with the sigma factors.These are DNA-binding proteins that repressor activate transcription of specficgenes. For example, s K is active during thefinal stages of development. When s K becomesactive, it directs the transcription ofseveral genes, one of which encodes a DNAbindingprotein called GerE. When GerEaccumulates, it binds to and represses transcriptionfrom a subset of s K dependent promoters.GerEalso binds to and activates adifferent subset of s K -dependent promotersthat are only active when both s K andGerEare present. Therefore GerEsubdividesthe s K regulon into genes that are transcribedthroughout the period of s K activity,genes that are transcribed early but are shutoff when GerEaccumulates, and geneswhose transcription is activated only in thefinal stage of s K activity when GerEis present.The secondary sigma factors also play thecentral role in the cell-type specific transcriptionof genes. s F and s G are active exclusivelyin the forespore, whereas, s E and s Kare active exclusively in the mother cell.Therefore genes whose transcription is dependenton one of these sigma factors canonly occur in one cell type, either the foresporeor mother cell. Transcription of genesis coordinated between the two cell types.The mechanisms that coordinate the geneexpression in the two cells types involveregulation of sigma factor activity. Thesemechanisms are discussed below in SectionIV.)

276 MORAN JR.II. SIGNAL TRANSDUCTIONAND THE INITIATION OFSPORULATIONA. Phosphorylation of theTranscriptionalActivator Spo0AMost of the developmental gene expressionrequired for endspore formation is the resultof the production of RNA polymerase sigmafactors. But how is the synthesis of the firstsporulation-induced sigma factors regulated?s F and s E are the first two sigma factorsproduced after the initiation of sporulation.The structural genes encoding these sigmafactors are part of the spoIIA and spoIIGoperons, respectively. Transcription of bothoperons is activated within minutes after theinitiation of sporulation. The promoter forthe spoIIG operon is used by RNA polymerasecontaining s A , the primary sigma factorin vegetative cells. The promoter for thespoIIA operon is used by RNA polymerasecontaining s H . s H , like s A , is present invegetative cells; therefore these sigma factorscannot account for the activation of thespoIIA and spoIIG promoters at the onsetof sporulation. Activation of these promotersrequires an additional factor, aDNA-binding protein known as Spo0A.Spo0A is similar to a large family of proteinsthat form the response regulator class oftwo-component regulatory systems. Likeother regulators of this class, Spo0A is activeonly when phosphorylated on a specific aspartateresidue. Once phosphorylated, itbinds to specific sites on the spoIIA andspoIIG promoters where it interacts withRNA polymerase to activate transcription.Binding of phosphorylated Spo0A to othersites on the chromosome activates, or somecases represses, transcription of other genes.B. The Phosphorelay System,Phosphatases, and Integration ofMultiple SystemsSpo0A is the key to the initiation of sporulation.Spo0A is present in growing cells, andits phosphorylation triggers the initiation ofsporulation. Phosphorylation of Spo0A iscontrolled by a phosphorelay system that ismore complex than most other two-componentsystems see the chapter by Bayles andFujimoto, this volume) Fig. 2) reviewed inFabret et al., 1999; Hoch, 2000; Perego,1998). Unlike simple two-component systemsin which a kinase phosphorylates the responseregulator, the phosphoryl group thatis added to Spo0A is first passed from one oftwo kinases to another response regulatorlikeprotein called Spo0F. The phosphorylgroup is then tranfered from Spo0FPto Spo0A by a phosphotransferase calledSpo0B. The concentration of Spo0AP isthe key determinant of whether the cell willsporulate or continue to divide. The concentrationof Spo0AP depends on the rate offlow of phosphoryl groups through the phosphorelay.The rate of flow of phosphorylgroups to Spo0A is modulated by the controlof kinase activity, and by phosphatases thatremove phosphoryl groups from the streamFig. 2).The complexity of the network of regulatorsthat control of Spo0AP concentrationallows the cell to integrate several signals, aFig. 2. The phosphorelay system that controls theinitiation of sporulation. Kinases KinA and KinB feedphosphate into the phosphorelay. The flow of phosphorylgroups to Spo0A is contolled by KipI, whichcontols KinA activity, and by phosphatases RapB andSpo0E, which remove phosphoryl groups fromSpo0F and Spo0A, respectively. PhosphorylatedSpo0A Spo0AP) acts as a DNA-binding proteinto directly activate some promoters and represstranscription from other promoters.

ENDOSPORE FORMATION IN BACILLUS SUBTILIS 277way of considering other responses to nutrientdepletion, before committing to the mostdrastic response of producing a dormant cell.Other responses, such as chemotaxis and thedevelopment of the competent state forDNA uptake, are discussed in other chapters.Nutrient depletion may affect phosphateflow to Spo0A by modulating kinaseactivity. KipI, an inhibitor of KinA, themajor kinase of the phosphorelay, andKipA, an anti-kinase inhibitor, are encodedin an operon that is responsive to glucoseand nitrogen levels. Recently, A. L. Sonensheinand his colleagues found that CodY, aDNA-binding protein, is also a GTP-bindingprotein, and CodY acts as an intracellularsensor of GTP levels Ratnayake-Lecamwasamet al., 2001). Under conditions of nutrientexcess the intracellular level of GTP ishigh so CodY binds GTP and acts as a transcriptionalrepressor of several genes. Underconditions of nutrient depletion the intracellularlevel of GTP decreases. In the absenceof bound GTP CodY fails to acts as a transcriptionalrepressor. CodY may regulate theinitiation of sporulation in response to nutrientconditions by regulating the transcriptionof one of more structural genes forcomponents of the phosphorelay.Other responses to nutrient depletion andother physiological signals compete with thesporulation response by affecting the flow ofphosphate through the phosphorelay. Thedevelopment of competence affects the activityof a phosphatase, RapA, which dephosphorylatesSpo0F. A second Spo0FPphosphatase, RapB, is expressed in growingcells. Another phosphatase Spo0EdephophorylatesSpo0AP directly, but it isunknown how its activity is regulated. PresumablySpo0Ephosphatase activity is usedto integrate another, as yet unknown, inputinto the decision to sporulate. Another levelof complexity was revealed by the discoveryof peptides that regulate phosphatase activity.PhrA is a 44 amino acid peptide thatinhibits RapA activity. This peptide is secretedand then a peptide consisting of atleast the five amino acyl residues at the carboxyterminal end PhrA is internalizedwhere it may act directly on the phosphatase.It is not known whether the main role ofPhrA is to serve as a timing device or as anintercellular signal. However, it is evidentthat PhrA is essential for inhibition of theRapA phosphatase and thereby for the initiationof sporulation.III. DETERMINATION OF ACELL'S DEVELOPMENTAL FATEBY A PHOSPHATASE ANDANTI-SIGMA FACTOREarly during endospore formation the asymetriccell division produces two cells thathave different developmental fates. Differentsets of genes are transcribed in each of thesetwo cells because the activities of specificsigma factors are restricted to specific cellsfor review on the control of sigma activityduring sporulation, see Kroos et al., 1999;Losick, 1994; Losick and Stragier, 1992).Specific genes are transcribed exclusively inthe forespore within minutes after the asymmetricdivision. Transcription of these genesin the forespore is directed by s F , one of thesigma factors synthesized early after the initiationof sporulation. Although s F is producedin the predivisional cell within minutesafter the onset of sporulation, s F is notactive in the predivisional cell. After theasymmetric cell division, s F remains inactivein the mother cell but becomes active in theforespore. It is the activity of s F in the foresporethat leads to expression of foresporespecific genes, and as is discussed in the nextsection, s F activity in the forespore also signalsthe activation of mother cell specificgene expression. Since activation of s F inthe forespore determines its developmentalfate, it is important to understand how s Factivity is restricted to the forespore.In order to discuss the regulation of s Factivity three additional proteins must beintroduced Fig. 3). The first is SpoIIAB,an anti-sigma factor that is capable of bindingto s F and keeping it inactive. SpoIIAB isproduced in the predivisional cell at the same

278 MORAN JR.Fig. 3. Regulation of s F activity by the anti-sigmafactor SpoIIAB. SpoIIAB binds to s F inhibiting itsfunction. SpoIIAB can also bind to SpoIIAA but cannotstably bind s F and SpoIIAA simultaneously. SpoIIABalso is a kinase that phosphorylates SpoIIAA. However,SpoIIAAP does not bind SpoIIAB. In the predivisionalcell Fig. 1, panel b) almost all of SpoIIAA isfound in its phosphorlated form SpoIIAAP). ThereforeSpoIIAB is free to bind and inhibit s F . After theasymmetric septation Fig. 1, panel c) SpoIIAAP isdephosphorylated by the phosphatase SpoIIE exclusivelyin the forespore compartment. The dephosphorylatedSpoIIAA binds SpoIIAB releasing s F ,which is therefore active exclusively in the forespore.time that s F is produced. SpoIIAB binds tos F in the predivisional cell antagonizing itsactivity. SpoIIAB also binds and antagonizess F in the mother cell after cell division.SpoIIAB releases s F in the forespore becauseof the action of an anti-anti-sigma,SpoIIAA. SpoIIAA binds to SpoIIAB forcingit to release s F . The SpoIIAA protein ispresent in the predivisional cell, and after theasymmetric cell division it is present in boththe mother cell and forespore. However,SpoIIAA disrupts the SpoIIAB-s F complexexclusively in the forespore because inthe predivisional cell and mother cellSpoIIAA remains phosphorylated. SpoIIABis the kinase that phosphorylates SpoIIAA.The phosphorylated form of SpoIIAASpoIIAAP) does not bind to SpoIIAB.Therefore SpoIIAAP leaves SpoIIAB freeto bind and inhibit s F . SpoIIAAP is dephosphorylatedin the forespore by a membranebound phosphatase called SpoIIE. SpoIIE issynthesized in the predivisional cell, but afterthe assymmetric cell division SpoIIEis locatedin the septum that divides the foresporeand mother cell. The reason that SpoIIEisactive exclusively in the forespore is a topicthat is currently being investigated. One hypothesisstates that SpoIIEmay be locatedprimarily on the forespore side of the septum.However, even if SpoIIEis located on bothsides of the septum, its concentration wouldbe much higher in the forespore because thevolume of the forespore is much smaller thanthe mother cell. One hypothesis suggests thatthis difference in volume may be sufficient toaccount for the forespore-specific activationof s F through the action of the SpoIIEphosphatase.In summary, s F activation in the foresporedetermines the developmental fate ofthis cell, and as described below, dictates thefate of the mother cell. s F activity is regulatedby an anti-sigma factor SpoIIAB) andan anti-anti-sigma factor SpoIIAA), whosephosphorylation state is controlled by aphosphatase SpoIIE) whose activity is restrictedto the forespore.IV. COORDINATION OF TWOCELLS' DEVELOPMENTALPROGRAMS BY CRISSCROSSREGULATION OF SIGMAFACTOR ACTIVITYA. Regulation of s E ActivityLike s F , s E is synthesized in the predivisionalcell but is not active until after theasymmetric cell division during endspore development.However, the mechanism controllings E activity is very different fromthat controlling s F . s E , is initially producedas an inactive precursor pro-s E ) that contains27 amino acids at its amino terminus,-which must be removed to produce the activeform of s E . The protease SpoIIGA) thatprocesses pro-s E to its mature form is membranebound and located in the septum thatseparates the mother cell and forespore. Activationof the protease requires the productof the spoIIR locus, which is transcribed inthe forespore under the direction of s F . ThespoIIR product is a small peptide that issecreted through the forespore membraneso that it can interact with the membranebound SpoIIGA protease to activate processingof pro-s E to form active s E . s Eis active exclusively in the mother cell. It isclear that its activation depends on processingby SpoIIGA, but it is not known whether

ENDOSPORE FORMATION IN BACILLUS SUBTILIS 279this is the mechanism of control is responsiblefor restricting s E activity to the mothercell. Some evidence supports a model inwhich s E is degraded in the forespore by anunidentified protease.Activation of s E requires spoIIR expressionthat is dependent on s F ; therefore s Eactivation is dependent on s F activation,which in turn is coupled to the asymmetriccell division. In this way both s F and s Eactivation is coupled to completion of thedivision septum. Coupling of sigma activationto a morphological landmark in thiscase, the completion of the division septum)appears to be a reoccurring theme throughoutdevelopment of the endospore seebelow). A second theme is illustrated by thefact that activation of s E in the mother cell isregulated by events in the forespore the s F -dependent expression of spoIIR). Gene expressionin the two cells may be coordinatedto ensure the proper assembly of spore componentsfrom the inside and outside of theforespore during development. The coordinationof gene expression in the two cells isregulated by a crisscross pattern of regulationof sigma factor activity Fig. 1). s F inthe forespore is an essential prerequisite foractivation of s E in the mother cell. s E activityin the mother cell is required for activationof s G in the forespore. Finally, s Gactivity in the forespore is required for theactivation of s K in the mother cell. Themechanisms controlling these sigma factorsare described below.B. Regulation of s K ActivityWe will examine regulation of s K activityfirst because its mechanism seems superficallysimilar to that regulating s E activity.The structural gene encoding s K is transcribedin the mother cell by RNA polymerasecontaining s E . The primary product prosK contains 22 amino acids at its aminoterminal end, which must be proteolyticallyremoved to produce the active s K . However,here the similarity ends, since the mechanismsof processing for these two mother cellsigma factors are not homologous. The proteasethat activates s E appears to be an aspartateprotease, whereas the protease thatactivates s K is a member of a subset of zincmetalloproteases. Processing of s K in themother cell is dependent on events inthe forespore, but the mechanism that transducesthe signal from forespore to the proteaseis not homologous to the SpoIIRpathway that signals processing of s E .In its default state the SpoIIGA proteasethat processes pro-s E is inactive. It is activatedby the spoIIR product, which is producedin the forespore. In its default state theprotease that processes pro-s K SpoIVFB) isactive. It resides in the forespore membranewhere it is held inactive by two other proteins.The forespore-expressed gene productthat signals pro-s K processing spoIVB) isnot homologous to SpoIIR, and it acts byantagonizing the inhibitors of the SpoIVFBprotease, thus activating the proteolytic activationof s K . Transcription of the spoIVBgene in the forespore, which signals pro-s Kprocessing, is directed by s G . Therefore s Kactivation in the mother cell is tied to theactivation of s G activation in the forespore,which, as discussed below, may be coupledto another morphological landmark.C. Regulation of s G Activitys G activity also is regulated. s F directs transcriptionof spoIIIG, the structural gene fors G . However, s G does not become activeuntil after the forespore protoplast isengulfed by the mother cell Fig. 1). Activationof s G in the forespore is dependenton the activity of s E in the mother cell. It isnot known how s G activity is regulated inthe forespore, or how s E dependent events inthe mother cell affect this regulation.According to one hypothesis, the anti-sigmafactor SpoIIAB may regulate s G activity.However, this model raises a number of unansweredquestions. For example, in orderfor SpoIIAB to sequentially regulate both s Fand s G , the SpoIIAA-dependent inhibitionof SpoIIAB's anti-s activity must be temporaryso that, after s F directs expression of s G ,SpoIIAB will be active to inhibit the newly

280 MORAN JR.Fig. 4. Endospore structure. Shown is an electronmicrographof a thin cross section of a B. subtilisendospore. The proteinaeous coat forms threelayers: an straited electron-dense outer coat Oc),a lamellar inner coat Ic), and diffuse undercoat Uc).A thick layer of peptidoglycan known as the cortexCx) lies beneath the coat. The core of the spore isalso indicated Cr). The radius of this spore is approximately0.5 mm.synthesized s G . It is known that the unphosphorylatedform of SpoIIAA disappearsafter s F is activated; however, it is notknown how SpoIIAB is inactivated duringthis later stage to release s G activity.V. MORE UNANSWEREDQUESTIONSThe assembly of structures in bacteria requiresthat proteins be targeted to specificsubcellular locations. For example, proteinsare targeted to the division septum duringbacterial cell division. Morphogenesis of theendospore is a complex process in whichmany proteins are targeted to specific locations.For example, the mature spore is surroundedby coat composed of over twodozen proteins. These proteins are arrangedinto at least three layers around the foresporemembrane Fig. 4). Most of the sporecoat proteins are synthesized in the mothercell. However, it is not known how theyare targeted to their final locations. Futurestudies of spore coat assembly may lead toimportant insights into how bacteria buildcomplex cellular structures for reviews onthe the spore coat, see Driks, 1999; Henriquesand Moran, 2000).REFERENCESDriks A 1999): Bacillus subtilis spore coat. MicrobiolMol Biol Rev 63:1±20.Errington J 1993): Bacillus subtilis sporulation: Regulationof gene expression and control of morphogenesis.Microbiol Rev 57:1±33.Fabret C, Feher VA, Hoch JA 1999): Two-componentsignal transduction in Bacillus subtilis: how one organismsees its world. J Bacteriol 181:1975±1983.Henriques AO, Moran CP, Jr 2000): Structure andassembly of the bacterial endospore coat. Methods20:95±110.Hoch JA 2000): Two-component and phosphorelaysignal transduction. Curr Opin Microbiol 3:165±170.Kroos L, Zhang B, Ichikawa H, Yu YT 1999): Controlof sigma factor activity during Bacillus subtilis sporulation.Mol Microbiol 31:1285±1294.Losick R 1994): RNA polymerase sigma factors andcell-specific gene transcription in a simple developingorganism. Harvey Lect 90:1±17.Losick R, Stragier P 1992): Crisscross regulation of celltype-specificgene expression during development inB. subtilis. Nature 355:601±604.Perego M 1998): Kinase-phosphatase competition regulatesBacillus subtilis development. Trends Microbiol6:366±370.Ratnayake-Lecamwasam M, Serror P, Wong KW, SonensheinAL 2001): Bacillus subtilis CodY repressesearly-stationary-phase genes by sensing GTP levels.Genes Dev 15:1093±1003.Stragier P, Losick R 1996): Molecular genetics of sporulationin Bacillus subtilis. Annu Rev Genet 30:297±341.