Proc. NatI. Acad. Sci. USAVol. 87, pp. 9221-9225, December 1990Genetics

Control of developmental transcription factor F by sporulationregulatory proteins SpoIIAA and SpoIIAB in Bacillus subtilis

(RNA polymerase/r factor/promoter)

RUTH SCHMIDT*, PETER MARGOLIS*, LEONARD DUNCAN*, RAFFAELLA COPPOLECCHIAt,CHARLES P. MORAN, JR.t, AND RICHARD LosICK**Department of Cellular and Developmental Biology, The Biological Laboratories, Harvard University, Cambridge, MA 02138; and tDepartment ofMicrobiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322

Communicated by Ira Herskowitz, September 6, 1990 (received for review July 3, 1990)

ABSTRACT The sporulation operon spolIA of Bacillussubtilis consists of three cistrons called spoIIAA, spollAB, andspollAC. Little is known about the function of spoIIAA andspollAB, but spollAC encodes a a, factor called aF, which iscapable of directing the transcription in vitro of genes that areexpressed in the forespore chamber of the developing spo-rangium. We now report that the products ofthe spollA operonconstitute a regulatory system in which SpoIIAA is an antag-onist of SpoIIAB (or otherwise counteracts the effect of Spoil-AB) and SpoIlAB is, in turn, an antagonist of SpoIIAC (0,F).This conclusion is based on the observations that (i) overex-pression of spoIIAB inhibits crF-directed gene expression, (ii) amutation in spoIIAB stimulates aF-directed gene expression,(iii) a mutation in spoIIAA blocks CF-directed gene expression,and (iv) a mutation in spoIIAB relieves the block in aF-directedgene expression caused by a mutation in spoIIAA. The SpoIl-AA/SpoIIAB/SpoIIAC regulatory system could play a role incontrolling the timing of crF-directed gene expression and/orcould be responsible for restricting aF-directed gene expres-sion to the forespore chamber of the sporangium.

Gene expression during the process of endospore formationin the Gram-positive soil bacterium Bacillus subtilis is gov-erned in part by five developmentally specific RNA poly-merase o- factors, .H, OF, a.E, aG and 0.K (1, 2). Thesefactors come into play in an ordered sequence that is tem-porally and spatially correlated with the morphological stagesof spore formation. Thus 0JH controls gene expression at theonset of sporulation. Next, o- acts during the stage of septumformation, at which time the sporangium is partitioned intoseparate mother-cell and forespore compartments. 0.E, whichis produced after the septum is formed, is, in turn, activeduring the engulfment stage of sporulation. Finally, thecompartment-specific factors 0oG and cuK direct gene expres-sion in the forespore and mother-cell chambers of the spo-rangium, respectively. a.F is of central importance because ithelps to govern the transition from a single-celled sporangiumto one that consists oftwo cellular compartments ofdivergentdevelopmental fates.

a' is encoded by the promoter-distal member of thethree-cistron sporulation operon spoIIA (3-7). The a.F_encoding cistron is designated spoIIAC and the two upstreamcistrons are called spoIIAA and spoIIAB. Although theproduct of spoIIAC (SpoIIAC or o0F) has been characterizedbiochemically (7), little is known about the function of thespoIIAA gene product (SpoIIAA) and the spoIIAB geneproduct (SpoIIAB). SpoIIAA and QF (SpoIIAC) evidentlyplay similar roles in sporulation, since spoIIAA and spoIIACmutations have indistinguishable phenotypic effects (8-10).

It has been uncertain whether SpoIIAB is required forsporulation, because no lesions in spoIIAB were found in theextensive collection of traditionally isolated mutations in thespoIfA operon. Recently, a spoIIAB mutation of a specialkind was obtained by a screen that did not depend upon adefect in spore formation (11). As we will show here, spoIIABdoes have an important role in sporulation, but spoIIABmutants were not discovered in traditional searches for spomutants because such mutants lose viability during stationaryphase.Here we present genetic evidence that the products of the

spoIIA operon constitute a regulatory system in which SpoIl-AA is an antagonist of SpoIIAB (or otherwise counteracts theeffect of SpoIIAB), and SpoIIAB is, in turn, an antagonist ofSpoIIAC (o0F). 0,F directs the transcription in vitro of theforespore regulatory gene spolfIG and of other genes (e.g.,gpr) that are preferentially expressed in the forespore (refs.7 and 12; D. Sun, R. M. Cabrera-Martinez, and P. Setlow,personal communication). The use of an isopropyl 8-D-thiogalactopyranoside (IPTG)-inducible promoter to causeexpression of spoIIAC in vegetative cells has enabled us toshow that oQF can drive the transcription of spoIIIG (thepresent paper) and gpr (unpublished results) in vivo. Wediscuss the possibility that the SpoIIAA/SpoIIAB/SpoIIACregulatory system plays a role in the timing of o-F-directedgene expression and/or is responsible for restricting 0.F_directed gene expression to the forespore chamber of thesporangium.

RESULTSUse of the P Promoter to Induce oF-Directed Transcrip-

tion of spolliG During Growth. To study the capacity of 0.Fto direct transcription of the forespore regulatory gene spo-IIG in vivo, we fused spoIIAC to the IPTG-inducible pro-moter Pap,, (13). Plasmid pRS11 contains a segment of thespofIA operon, extending from the 3' end ofspoIIAB into themiddle of spoIIAC, fused to PSp,,, . Integration of this plasmidby single-reciprocal (Campbell) recombination into the chro-mosome brings the crF-encoding spoIIAC under the control ofPsPal but leaves spoIIAA and spoIIAB under the control ofthespoIIA operon's normal promoter (Fig. la). To monitorspoIIIG expression, we used a transcriptional fusion ofspoIIIG to the IacZ gene, inserted into the chromosome at theamyE locus (15).Because spoIIIG is subject to autoregulation, we carried

out our experiments in cells containing the mutation spo-HIGIM (15) to ensure that any spoIIIG-lacZ expression wasnot an indirect consequence of the induction of 0.G synthesis.Addition of IPTG to spoIIIGAI mutant cells bearing thePspa5-spoIIAC fusion caused a rapid induction of spoIIIG-lacZ expression (Fig. 2a). B-Galactosidase reached maxi-

Abbreviation: IPTG, isopropyl 8-D-thiogalactopyranoside.

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a

Pp.pIIA

FFHA B Ic'

b

PJ,,JIA--~ ,Psp a c

FPjP/ r- I C

C

NcoI BgII PstI

d

PspoI[A _Ai 169 VA233 erm blo

NcoI Bgll PstI NcoI BglI[ Pstl

FIG. 1. Molecular genetic manipulations ofthe spoIIA operon. (aand b) Structures of fusions of the IPTG-inducible promoter Pspac tospoIIAC and to the entire spoIIA operon, respectively. The PspacspollAC fusion was created by the insertion of plasmid pRS11 intothe chromosome, whereas the PspacspollA operon fusion wascreated by the insertion of pRS7 into the chromosome. The diagramsare not drawn to scale except for the relative sizes of spoIIAA, -AB,and -AC. Restriction maps of Pspac and the vector region have beenreported (13, 14). (c) Recombination between hybrid plasmid pPM23or pPM24, bearing a segment ofspollA DNA extending from the NcoI site within spollAB to a Pst I site just downstream of spoJIAC, andthe corresponding region of spollA DNA in the chromosome. Therecombination results in the replacement of sequences downstreamof the recombination site by corresponding plasmid-borne se-quences. (d) Structure of chromosomal DNA in the vicinity ofintegrated plasmid. The vector backbone (thick line) is not drawn toscale. A, B, and C are abbreviations for spollAA, spoIIAB, andspollAC, respectively. A', B', and C' are abbreviations for truncated

copies of spollAA, spollAB, and spollAC, respectively.

mum accumulation within 2 hr after the addition of theinducer and was then rapidly depleted from the cells. Weconclude that o.F has the capacity to direct efficient tran-scription from the spoIJIG promoter when its synthesis isartificially induced in vegetative cells.

Interestingly, cells containing the Pspac-spoIIAC fusionform small colonies that lyse after 1-2 days when grown onsolid medium in the presence of IPTG. We attribute thistoxicity to overexpression of spolIAC because (i) colonyformation in the absence of IPTG was normal, (ii) a controlstrain in which Pspac, was inserted in the opposite orientationto that of spoJIAC was fully viable (though Spo-) in thepresence of IPTG, and (iii) the introduction of pRS11 intocells containing a spoliAC mutation (spoliACI) gave rise totransformants that were insensitive to IPTG.SpoIIAB Is an Inhibitor of oF-Directed Gene Expression.

We also attempted to induce oF-directed transcription of

10-60-

8-

6 40

20-

2~~~~~0

*10A

300 A

>20L 2ol000~~~~~~0

12345 1 3 57Time, hr

FIG. 2. Gene fusion-directed /3-galactosidase synthesis. (a-c)IPTG-dependent induction of spoIIIG-directed /3-galactosidase syn-thesis. Cells were grown to an 0D595 of 0.3 in DS medium (16, 17).Parallel cultures of induced and uninduced cells were then estab-lished by splitting each culture into two portions and adding IPTG (1mM) to one portion. Samples were removed and assayed (18) forj3-galactosidase at the indicated times after IPTG addition. The levelof /3-galactosidase activity in the uninduced cells was subtracted asbackground from the /3-galactosidase activity of the cells that wereexposed to IPTG. Strains used in the experiments of a-c wereisogenic except as indicated and contained the mutation spoJIJGAl,a spoIJJG-acZ transcriptional fusion at the amyE locus, and anintegrated copy of pRS11 to create a Pspac fusion to spollAC or anintegrated copy ofpRS7 to create a Pspac fusion to the spoflA operon.The strains of a contained pRS11 and pRS7 integrated into thechromosomes of spoIfA + bacteria to create a Pspac^ fusion to spoJIAC(A, strain RS79) and a Pspac fusion to the spoJIA operon (0, strainRS78), respectively. The strains of b contained pRS7 integrated intospoffA+ and spolfA mutant bacteria to create P5,,0c fusions to thewild-type spolfA operon (e, strain RS78) and to operons bearing themutations spoJIAA69 (A, strain RS11O) or spoJJABAl (a, strainR5150). The strains of c contained pRS7 integrated into spoIlABAlmutant cells that harbored pHDAB (in, strain RS165) or pJ89 (0,strain RS162). (d-f ) sspB-directed (3-galactosidase synthesis duringsporulation of a aF altered-specificity mutant. Cells were grown inDS medium and /3-galactosidase specific activities were determinedat the indicated times after the end of the exponential phase ofgrowth. The strains contained spoJIJGAJ, sspB-lacZ, and spollAoperons of the following genotypes: spoJJAA+ spoJJAB+ spoJJAC+(e, strain PM1O) and spoIIAA+ spoIIAB+ spoJJAC-VA233 (0, strainPM73) in d; spoJJAA+ spoIIAB+ spoIJAC-VA233 (0, strain PM276),spoJJAA69 spoJIAB+ spolJAC-VA233 (U, strain PM282), and spoil-AA spolIABAl spolJAC-VA233 (A, strain PM274) in e; and spoil-AA69 spoIIAB+ spoIJAC-VA233 (U, strain PM282) and spoIIAA69spoIIABAl spoJIAC-VA233 (A, strain PM279) in f.

spoIIIG-IacZ by use of a fusion of Pspac to the entire spoflAoperon. The Pspa,-spoIIA operon fusion was created by useof the plasmid pRS7, which contains a fusion of Pspac to afragment of spoIIAA that extends from just upstream of thespoIIA transcription start site (position -31) to codon 59 of

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the spoIJAA open reading frame. Integration ofpRS7 into thechromosome by single-reciprocal recombination created afusion of Pspac to the entire spoIIA operon, thereby leavingonly a truncated copy of spoIIAA under the control of thenormal spoIfA promoter (Fig. lb). Addition of IPTG to cellsbearing the Pspa`SPoIIA operon fusion was therefore ex-pected to induce transcription of the entire operon, includingthe promoter-distal genes. To our surprise, however, littlespoIIIG-directed p-galactosidase synthesis was observed fol-lowing the addition of IPTG to cells bearing the Pspac-SPoIIAoperon fusion (Fig. 2a). Among the possible explanations forthe observed inability of the Pspac-spoIJA operon fusion todirect spoIIG-IacZ expression is that the induction of SpoIl-AA or SpoIIAB synthesis by IPTG addition was interferingwith o-F-directed gene expression.

Therefore, to examine the possible effects of SpoIIAA andSpoIIAB on 0.F-directed spoIIIG-lacZ expression, we con-structed fusions of Ppac to operons bearing the missensemutation spoIIAA69 (19) or the in-frame deletion spoIIABAI(11). To create fusions ofPspac to the mutant spoIIA operons,plasmid pRS7 (see Fig. lb) was inserted into the chromo-somes of a spoIIAA69 mutant and a spoIJABAI mutant. Tomonitor o.F-directed gene expression, the fusions of Pspac tothe mutant spoIIA operons were introduced by DNA-mediated transformation into spoIIIGAJ mutant cells bearinga spoIIG-IacZ transcriptional fusion at the amyE locus.spoIIAA69 had little effect on spoIIIG-lacZ expression

(Fig. 2b); thus, addition of IPTG to cells containing the Pspacfusion to the mutant operon bearing spoIIAA69 caused thesame low level of /3-galactosidase synthesis as was observedfollowing induction of the Pspac-spoIIA wild-type operonfusion. In contrast, however, the addition of IPTG to cellsbearing the PspacfspoIIABA1 mutant operon fusion caused adramatic induction of f3-galactosidase synthesis (Fig. 2b). Weconclude that IPTG-induced expression of spoIIAB in thePspac-spoIJA operon fusion is responsible for the impairedexpression of spoIIJG-lacZ.Next, we asked whether the spofIAB gene product acts in

trans to block oF-directed gene expression. This questionwas investigated by introducing plasmid pHDAB into cellscontaining the Pspa, fusion to the spoIIABAI mutant operon.pHDAB is a multicopy plasmid bearing wild-type copies ofthe A and B cistrons, which are transcribed from a vegeta-tively expressed promoter (11). The level of 3-galactosidasein cells bearing pHDAB was much lower than that observedin otherwise identical cells containing in place ofpHDAB theplasmid vector pJ89 (Fig. 2c). We conclude that spoIIABencodes a diffusible product (SpoIIAB) that acts in trans toinhibit o.F-directed gene expression.The results of Fig. 2a showed that the level of 8-galacto-

sidase in cells containing Pspa,-spoHIAC began to decreaserapidly 2 hr after induction by IPTG. We can now explain thisobservation by hypothesizing a spoIIAB-dependent shutoffin spoIIJG-lacZ expression shortly after the cells begin tosporulate (the drop in 83-galactosidase levels presumablybeing due to proteolysis of preexisting enzyme). In cellscontaining a direct fusion of Pspac to spoIIAC, spoIIAB (andspoIJAA) remains under the control of the spoIlA operonpromoter (Fig. la) and hence expression of spoIJAB isexpected to switch on in early stationary phase. In confir-mation of our hypothesis, f3-galactosidase in PspaspoJIAC-containing cells ofa spoIIABAl mutant was found to continueto accumulate for several hours after the addition of IPTG(data not shown).

Toxicity Due to the spoIIABAl Mutation. While manipulat-ing spoIIABAI, we discovered that cells containing thedeletion mutation readily accumulated suppressor mutationsin spolIAC that blocked o0F function. When separated fromthese second-site mutations by backcross experiments, thespoIIABAI mutation was found to cause the formation of

colonies that lysed after 1-2 days. We attribute the toxiceffect of spoIIABAI to the stimulation of a.F activity causedby the absence of the spoIIAB product.SpoIIAB Inhibits aF-Directed Gene Expression During

Sporulation. Having demonstrated that SpoIIAB inhibitsa'-directed gene expression in cells that had been engineeredto transcribe the spoIlA operon during growth, we next askedwhether SpoIIAB is also an inhibitor of a F-directed geneexpression in cells undergoing sporulation-that is, in cells inwhich spoIIA is induced during sporulation under the controlof its normal promoter, PSP0JJA. Since spoJIIG-IacZ is ex-pressed at only a low level during sporulation, we tookadvantage of the finding (P.M., unpublished results) that thesubstitution of alanine for valine at residue 233 of aF (as aconsequence of mutation spoIIAC-VA233) converts thespoIIAC product to the promoter specificity of aG. (crF andoGare very similar to each other in their predicted amino acidsequences, and the valine to alanine substitution increasesthe similarity of the putative "-35" recognition helix of aFto that of aG; refs. 15 and 20.) As a result of the valine toalanine substitution, mutant aF is able to direct transcriptionefficiently from the promoter for sspB, a strongly expressedgene that is normally under the control of a G (7).

Fig. 2d shows an experiment in which the spoIIAC-VA233mutant gene was used to replace the wild-type spoIIAC genein the chromosome of a spoIIIG mutant containing an sspB-lacZ fusion. (The spoIIIG mutation was included to preventaG-directed transcription of the gene fusion.) The replace-ment was carried out by recombinational insertion of plasmidpPM7 to create strain PM73. pPM7 contains a 967-base-pairsegment of spoIIA DNA extending from a Bgl II site in themiddle of spoIIAB to a Pst I site immediately downstream ofthe mutant spoIIAC gene. A control strain (PM10) wasconstructed by inserting into the chromosome a plasmid(pPM3) that was identical to pPM7 except that it contained awild-type spoIIAC gene. Active expression of the sspB-IacZfusion commenced at about hour 2 of sporulation in spoIIIGmutant cells bearing the spoIIAC-VA233 mutation (Fig. 2d).This activity was dependent on the mutant a.F protein, as littlesspP-lacZ expression was detected in spoIIIG mutant cellsbearing a wild-type copy of spoIIAC. An advantage of thespoIIAC-VA233/sspB-IacZ system is that it ensured in thesporulation experiments to follow that we were monitoringthe expression of a gene under the direct control of a.To investigate the effect of SpoIIAB on aF-directed gene

expression during sporulation, we constructed a spoIlIGmutant strain (PM274) containing the spoIIABAI mutationand the aF altered-specificity mutation spoIIAC-VA233.PM274 was constructed by inserting into the chromosome(Fig. 1 c and d) a plasmid (pPM23) that contains a segment ofspoIIA DNA (extending from the Nco I site near the begin-ning ofspoIIAB to the Pst I site just downstream ofspoIJAC)bearing spoIIABAl and spoIIAC-VA233. As a control, strainPM276 was constructed by inserting into the chromosome aplasmid (pPM24) that was identical to pPM23 except for theabsence of the spolfABAl mutation. We then monitoredo.F-directed gene expression in the mutant cells by use of thesspB-lacZ fusion. The spolIABAl mutation markedly in-creased the rate of sspB-directed ,B-galactosidase synthesisabove that observed in otherwise isogenic cells that werespoIIAB+ (Fig. 2e). This finding agrees with the view thatSpoIIAB interferes with the synthesis or activity ofaF in cellsundergoing sporulation.SpoIIAA Is an Inhibitor of the Synthesis or Activity of

SpoIIAB. We next wished to investigate the role of SpoIIAA,the product of the promoter-proximal member of the spoIlAoperon, on ar'directed gene expression during sporulation.We therefore constructed a spoIlIG mutant strain (PM282)containing spolIAA69 and the 0.F altered-specificity mutationspoIIAC-VA233. PM282 was constructed by inserting pPM24

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(above) into the chromosome of a spoIIAA69 mutant. Thepresenc# of the spoIIAA69 mutation caused a complete blockin sspBkdirected 8-galactosidase synthesis (Fig. 2e). Thus,SpoIIAA is required for aF-directed gene expression, theeffect of the spoIIAA69 mutation being opposite to that ofspoIIAAJw.A simple hypothesis to explain the observation that spoIJAA

and spoIIAB mutations had opposite effects on a -directedgene expression is that SpoIIAA is an antagonist of SpoIIABor otherwise reverses the inhibitory effect of SpoIIAB oncF directed gene expression. Thus, in the absence of func-tional SpoIIAA, o,'-directed gene expression is blocked by theinhibitory action of SpoIIAB. As a test of this hypothesis wecreated a spoIIAA69 spoIIABAJ double mutant (PM279) withthe expectation that the spoIIABAJ mutation should relievethe block in crF-directed gene expression caused by thespoIIAA69 mutation. In other words, if SpoIIAA is an antag-onist of SpoIIAB, then spoIIABAl should be epistatic tospoIIAA69. PM279 was constructed by inserting thespoIIA$AJ-bearing plasmid pPM23 (above) into the chromo-some of a spoIIAA69 mutant. A high level of P-galactosidasesynthesis was observed in cells of the spoIIAA69 spoIIABAJdouble mutant (Fig. 21) and, indeed, the pattern of sspB-lacZexpression in the double mutant was indistinguishable fromthat observed in spoIIAA' cells containing the spoIIABAJmutation (Fig. 2e).

Finally, to address the question of whether the effects ofspoIIAA and spoIlAB mutations on oF-directed transcriptionof sspB-lacZ are a general feature of ayF-directed geneexpression, we constructed strains similar to those describedabove, except that they contained lacZ fusions to spoIIIG (7)and to gpr (12): Our results demonstrated that spoHIIG-IacZand gpr-lacZ expression was (i) enhanced in spoIIABAJmutant cells, (ii) prevented in spoIIAA69 mutant cells, and(iii) restored by the introduction of spoIIABAI into spoII-AA69 mutant cells. Thus, the observation that spoIlAA andspoIIAB mutations have opposite effects and that spoIIABI3Ais epistatic to spoIIAA69 applies to the capacity of cyF todirect the transcription of three sporulation genes.SpoIIAA and SpoIIAB Do Not Exert their Effects on cOF_

Directed Gene Expression at the Level of Transcription orTranslation of spollAC. We interpret our results to indicatethat SpoIIAB is an inhibitor of &'-directed gene expressionand that SpoIIAA blocks or otherwise reverses the effect ofSpoIIAB. Are the effects of SpoIIAA and SpoIIAB exertedat the level of the expression of spolIAC? Northern hybrid-ization experiments (ref. 21; R.S., unpublished results) dem-onstrate that the accumulation of the 1.6-kilobase spoIIAoperon mRNA is unimpaired in spoIIAA69 mutant cells. IfSpoIIAB were an inhibitor of spoIIAC transcription (forexample, if it blocked read-through transcription from thepreceding gene in the operon), a marked decrease in theamount or size of the polycistronic message should have beendetected. As an independent way to monitor spoJIAC expres-sion, we constructed a gene fusion in which spolIAC wasjoined i" frame at its 180th codon to lacZ. Fig. 3 shows thatspoIIAA69 caused no detectable impairment in the expres-sion of the gene fusion.

DISCUSSIONWe have monitored aF-directed gene expression duringgrowth in cells that had been engineered to transcribe spoIIAin response to IPTG and during sporulation in cells in whichtranscription of spollA was under its normal sporulationcontrol. Our principal observations are that (i) overexpres-sion of spoIIAB during growth causes strong inhibition ofyF.-directed gene expression, (ii) a mutation in spolIAB

stimulates a--directed gene expression during sporulation,(iii) a mutation in spoIIAA prevents oF-directed gene expres-

>~ 40

t~Dig_CA

C!,& 1

1 2 3Time, hr

FIG. 3. spoJIAC-lacZ-directed f3-galactosidase synthesis. ,-Ga-lactosidase activity was measured during sporulation in DS mediumof spoIIAC-4acZ fusion-bearing cells of strain LD1 (o), of thespolIAA69 mutant strain LD2 (e), and of the spoOH mutant strainLD4 (o). The strains were constructed by integrating into thechromosomes of otherwise isogenic spo+ and spo& cells plasmidpLD1, which contained a 730-base-pair fragment of spollA DNA,extending from a Bgi II site in spolIAB to a Bcl I site at the 180thcodon of spollAC, that was joined in-frame (at the Bcl I site) to E.coli lacZ.

sion during sporulation, and (iv) a mutation in spoIIABrelieves the block in aF-directed gene expression caused bya mutation in spoIIAA. We infer from these observations thatthe products of the spoIIA operon constitute a regulatorysystem in which SpoIIAB inhibits a -directed gene expres-sion and SpoIIAA prevents or reverses the inhibition ofaF-directed gene expression caused by SpoIIAB.How does the regulatory system work? Because a mutation

in spoIIAA does not block the accumulation ofspoIIA operontranscripts or the expression of a spoIIAC-lacZ gene fusion,we infer that the regulatory system does not operate at thelevel of the transcription of the aF structural gene or thetranslation of its mRNA, but rather at the level of aF action.Thus, SpoIIAA and SpoIIAB could modulate the activity ofa F protein or affect the capacity of a`-RNA polymerase tointeract with cognate promoters. For example, SpoIIABcould inhibit a-F by direct interaction to form an inactivecomplex or by chemical modification. Alternatively, SpoII-AB could be a repressor that blocks the capacity ofaF-RNApolymerase to initiate transcription from a-F recognized pro-moters. Similarly, SpoIIAA could inhibit SpoIIAB by directinteraction to form an inactive complex or by chemicalmodification. Alternatively, SpoIIAA could interact with a-Fto protect it from or reverse the effect of SpoIIAB.These considerations suggest two possible relationships

among the products of the spolIA operon. One is a hierar-chical cascade in which SpoIIAA antagonizes the action ofSpoIIAB, and SpoIIAB, in turn, blocks the capacity of aFtoswitch on transcription from its target promoters:

SpoIIAA SpoIIAB a-

An alternative possibility is that SpoIIAB inhibits a.F byconverting it to an inactive form, a-F*, and that SpoIIAAreverses (or counteracts) the effect ofSpoIIAB by convertinga F* back to the active form of the a factor:

F SpoIIAAIrr ? o

SpolIAB

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Kalman et al. (22) have discovered that the structural genefor the non-sporulation o- factor 0oB is preceded in the sameoperon by two genes, V and W, whose products bear astriking similarity in their predicted amino acid sequences tothose of SpoIIAA and SpoIIAB, respectively. V and W aretheorized to control the activity of cyB. By analogy with therole of SpoIIAA and SpoIIAB in o-F-directed gene expres-sion, it seems likely that V and W constitute a system ofregulation of o.B-directed gene expression in which V inhibits(or reverses) the action of W, which, in turn, inhibits theaction of o-

An important contribution of our current work is thediscovery that the spoIIABAJ mutation causes impaired cellviability. Cell death is presumably due to the enhanced level(or prolonged duration) of 0.F activity, because spoIIABmutant cells readily accumulate second-site mutations inspoIIAC that block 0.F activity and that suppress the lethaleffect of the spoIIABAI mutation. This is consistent withearlier work (23) indicating that expression of a cloned copyofspoIIAC is toxic in E. coli and with our present finding thatoverexpression ofspoIIAC causes death in B. subtilis. Theseobservations explain the absence ofspoIIAB mutations in thelarge collection of spoIIA operon alleles that were identifiedin traditional screens for sporulation mutants; spoIIAB mu-tants would have been lost because of their inviability orbecause they had accumulated spo mutations in spoIIAC.Moreover, the compensatory effect of spoIIAC mutations inspoIIABAJ mutant cells suggests that o0F activity is theultimate target of SpoIIAB.

Recently, Rather et al. (11) isolated a spoIIAB missensemutation (spolIABI) of a special kind while screening formutants with enhanced expression ofa gene (gdh) that is underthe direct control of the forespore factor o-G Unlike thedeletion mutation spoIIABAl, the spoIIABI missense muta-tion does not impair viability or sporulation or significantlystimulate o.F-directed gene expression (unpublished results).These observations suggest that, in addition to being aninhibitor of 0.F, SpoIIAB may be an antagonist ofthe synthesisor activity of o.. If so, the nature of the inhibitory effect onoFmay be qualitatively different than the inhibitory effect on(70, since spoIIABJ-altered SpoIIAB protein is fully capable ofantagonizing crF-directed gene expression but is impaired in itscapacity to prevent o-G-directed gene expression.There are several possible roles for the SpoIIAA/

SpoIIAB/SpoIIAC regulatory system in the program ofsporulation gene expression. B. subtilis cells enter sporula-tion as a response to conditions of nutritional deprivation.SpoIIAA may be a sensor that blocks or reverses the actionof SpoIIAB in response to certain nutritional signals, therebyensuring that .F (and eOG; ref. 11) becomes active only whenconditions appropriate for sporulation are met.A second, more intriguing possibility is that the regulatory

system plays a role in the establishment of compartment-specific gene expression. Setlow and coworkers (refs. 7 and12; D. Sun, R. M. Cabrera-Martinez, and P. Setlow, personalcommunication) have shown that 0.F directs the transcriptionin vitro of spoIIIG and certain other genes (e.g., gpr) that areknown to be selectively expressed in the forespore. The useof the IPTG-inducible promoter Pspa, has enabled us to showthat 0F can drive the transcription of spoIIIG (presentresults) and gpr (R.S., unpublished results) in vivo. ThespoIIA operon is known to be induced prior to the formationof the septum that partitions the sporangium into mother-cell

and forespore compartments (24). We speculate that SpoIl-AB partially or completely inhibits crF-directed transcriptionin the predivisional cell and then in the mother-cell after thesporulation septum is formed. In these cell types SpoIIAAmight be in an inactive state. Some feature of the foresporemight activate SpoIIAA, causing it to interfere with theaction of SpoIIAB and thereby relieve the inhibition of (7Ffunction. In support ofour model, recent experiments (P.M.,unpublished results) indicate that o.F-directed gene expres-sion is prevented by a mutation in the forespore regulatorygene spoIIIE (25) and that the block in crF-directed geneexpression caused by the spoIIIE mutation is relieved byspoIIABAJ. Finally, we note that since SpoIIAB may be aninhibitor of v.G, as well as a.F, forespore-specific inactivationof SpoIIAB could also be a mechanism for restricting o-G_directed gene expression to the forespore.

We thank J. Errington, P. Piggot, C. Price, P. Setlow, A. L.Sonenshein, P. Stragier, and M. Yudkin for helpful discussions, forcommunicating results prior to publication and for providing plas-mids and gene fusions. P.M. was a National Science FoundationPredoctoral Fellow. L.D. is a Howard Hughes Medical InstitutePredoctoral Fellow. This work was supported by National Institutesof Health Grants GM18568 and A120319 to R.L. and C.P.M.,respectively.

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2. Moran, C. P., Jr. (1989) in Regulation ofProcaryotic Development,eds. Smith, I., Slepecky, R. & Setlow, P. (Am. Soc. Microbiol.,Washington), pp. 167-184.

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Microbiol. 130, 2123-2126.6. Stragier, P. (1986) FEBS Lett. 195, 9-11.7. Sun, D., Stragier, P. & Setlow, P. (1989) Genes Dev. 3, 141-149.8. Errington, J. & Mandelstam, J. (1983) J. Gen. Microbiol. 129,

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Proc. Natl. Acad. Sci. USA 85, 7637-7641.21. Savva, D. & Mandelstam, J. (1986) J. Gen. Microbiol. 132, 3005-

3011.22. Kalman, S., Duncan, M., Thomas, S. & Price, C. W. (1990) J.

Bacteriol. 172, 5575-5585.23. Yudkin, M. (1986) Mol. Gen. Genet. 202, 55-57.24. Gholamhoseinian, A. & Piggot, P. J. (1989) J. Bacteriol. 171,

5747-5749.25. Foulger, D. & Errington, J. (1989) Mol. Microbiol. 3, 1247-1255.

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