The EMBO Medal for 1991 has been awarded to Dr PatrickStragier of the Institut de Biologie Physico-Chimique, Paris,France. Dr Stragier receives the medal for his work on therole of sigma factors in the control of sporulation in the soilbacterium Bacillus subtilis, about which he writes in the

review beginning on the facing page.

The Medal is sponsored by the following companies:AKZO, Amersham, ASTRA, Becton-Dickinson, Boehringer Ingelheim, Boehringer Mannheim, Carlsberg Bryggerierne, Ciba-Geigy AG,F. Hoffman-La Roche AG, Sandoz AG, EMBL, Farmitalia, Glaxo, ICI, Kontron, LKB, Merieux, NOVO Industri A/S, Pharmacia, Sanofi, Sclavo,Senetek, Tuborgfondet.

'

t

The EMBO Journal vol.10 no.12 pp.3559-3566, 1991

EMBO MEDAL REVIEW

Dances with sigmas

Patrick Stragier

Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie,75005 Paris, France

Sporulation of Bacillus subtilis is a simpledevelopmental system

When I started looking for my first lab just after finishingmy theoretical studies at the University of Paris I was

determined to work on the regulation of transcription. I hadbeen enthralled by the elegance and subtlety of themechanisms governing the control of lysogeny inbacteriophage lambda and I wanted to study a similar system.

With this goal in mind I visited Luisa Hirschbein who hadbeen working previously on RNA polymerase in Escherichiacoli and had recently joined Pierre Schaeffer's laboratoryat the Microbiology Institute of the University of Orsay. Onthat day I heard for the first time about an organism thatlooked even more fascinating than lambda, the sporulatingsoil bacterium Bacillus subtilis (Figure 1). When starved ofnutrients (either carbon, nitrogen or phosphorous), B. subtilisundergoes a series of morphological changes whichultimately lead to the formation of a dormant spore

(Figure 2) (reviewed in Losick et al., 1986). These spores

can be seen under the light microscope as early as 'stageIV' (see Figure 2) and they accumulate a brown pigmentwhich gives the colonies a characteristic appearance on

plates. Because of their unpigmented phenotype many

mutants had been selected which were blocked at specificstages during the sporulation process, and since generalizedtransducing phages were available, in addition to theremarkable property of B. subtilis of being spontaneouslytransformable, these mutations had been mapped on thechromosome and had defined several unlinked spo loci.Presumably these loci encoded products required for theelaboration of the spore and they had to be expressed in a

tightly ordered fashion. Moreover, since the two

compartments present during sporulation contained an

identical chromosome issued from the last round ofreplication but behaved differently, the 'forespore' becomingthe mature spore released by lysis of the 'mother cell' (seeFigure 2), it could be speculated (although no evidence was

then available) that different genes were expressed in the

two compartments of the sporulating cell. Obviously, some

quite complex mechanisms were involved in regulatingtranscription of the spo genes.On the day of my first visit I missed some of these exciting

aspects of sporulation of B.subtilis, but I was immediatelyconvinced that RNA polymerase played a central role in this

developmental process and that it was the enzyme to study.Although I was captivated by Luisa's explanations I could

not help noticing the intense activity of two young American

people who kept running from the lab to the cold room: they

co Oxford University Press

were Rich Losick and Jan Pero, who were spending a fewmonths in P.Schaeffer's department and nervously checkingthe column collector which used to get stuck at the time ofelution of their precious RNA polymerase fractions. Readingthe literature was soon going to tell me who these peoplewere but when I started working in the lab after the summervacation they had gone back to the States and it would bemany years before our paths crossed again.

Part of the excitement about RNA polymerase in the early1970s was due to the recent discovery of sigma, a proteinfactor binding to E. coli core RNA polymerase and conferringon it template specificity. In their seminal paper Burgesset al. (1969) had conjectured that several sigma factors couldco-exist in bacteria (or in phage-infected bacteria) andactivate specific sets of genes. Soon afterwards, the discoverythat B. subtilis RNA polymerase lost its ability to transcribephage 4be DNA when purified from sporulating cells led tothe intriguing suggestion that the onset of sporulation inB. subtilis was accompanied by a change of templatespecificity of RNA polymerase which in turn led to theexpression of a new class of genes (Losick and Sonenshein,1969). Either a covalent modification of core RNApolymerase or the inactivation of vegetative sigma factorseemed to be required for such a change of templatespecificity in order to allow core RNA polymerase to binda putative new sigma factor. Encouraged by the report ofan alteration of the a subunits of core RNA polymerase afterinfection of E. coli by bacteriophage T4 (Goff and Weber,1970), I decided to look for a similar phenomenon insporulating B. subtilis. It was my turn to watch suspiciouslyover the column collector before analysing RNA polymerasefractions by in vitro transcription and SDS -polyacrylamidegel electrophoresis. Inspired by the T4 example I wasspecifically looking for the covalent binding of somephosphorous-containing compound but, after some transienthopes, I had to admit that core RNA polymerase was notphosphorylated during sporulation in B. subtilis.Meanwhile, some evidence had been provided that the

vegetative sigma factor was inhibited early during sporulation(Tjian and Losick, 1974) and various polypeptides had beenfound that co-purified with core RNA polymerase fromsporulating cells (Fukuda et al., 1975; Linn et al., 1975).I then chose to address a somewhat neglected problem, themolecular basis for the supposed compartmentalization ofgene expression during sporulation. Isolation of foresporesfrom Bacillus cereus and Bacillus megaterium had beenreported (Andreoli et al., 1973; Ellar and Postgate, 1974)and I decided to follow similar approaches for comparingRNA polymerase composition in forespore and mother cellfrom B.subtilis. Purified RNA polymerase would then beused to transcribe nucleoids in vitro and the RNA productswould be analysed in hybridization-competition experiments.My secret dream was to be able to purify a polypeptide withthe characteristics of a forespore-specific sigma factor. Allmy efforts were then devoted to setting up a protocol for

3559

P.Stragier

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Fig. 1. Electron micrograph of a sporulating B. subtilis cell at an intermediate to late stage of sporulation. The forespore appears as an organelle ( 0.5 -Irm) within the mother cell. The white area is the cortex, a peptidoglycan-like material, which is deposited between the two membranes of the forespore.The lamellar structures depositing on the outside of the forespore are the coats. Photograph kindly provided by A.Ryter.

purifying forespores in sufficient amounts and as free aspossible from mother-cell material. The forespore fractionswere examined by electron microscopy by Claude Frehelwho was working in Antoinette Ryter's laboratory at thePasteur Institute. Antoinette had been one of the pioneerswho defined the morphological stages of sporulation inB.subtilis (Ryter et al., 1966) and I could not have founda better place for checking my samples. However, despiteintensive and multiple attempts I was never able to obtainclean enough forespore fractions and Claude got used togreeting me with a slightly embarrassed smile beforeshowing me her pictures of the latest experiment. I felt allthe more frustrated since it had just been demonstrated bya very clever genetic approach that some sporulation geneswere required only in the mother cell and others exclusivelyin the forespore (Lencastre and Piggot, 1979). Thisdisappointment was also shared by many people who hadstarted using the newly available cloning techniques to studyB. subtilis. For many years it appeared almost impossible topropagate a recombined plasmid in B.subtilis without theinsert being rearranged, which precluded any reliablecomplementation analysis. The remarkable recombinationefficiency of B.subtilis was then a handicap which delayedthe era of B. subtilis molecular genetics and led many peopleto switch to another field. Therefore the cloning of the firstsporulation gene was a real tour deforce (Segall and Losick,

3560

1977). Many years would go by before other spo genes werecloned and they were a prerequisite for assaying thespecificity of a putative sporulation sigma factor. It is ironicthat the molecular genetics of B. subtilis are now so welldeveloped, essentially as a result of the efficiency ofhomologous recombination: it is very easy to createmutations by reverse genetics, cloned DNA can beintroduced back into the chromosome either at its originallocus or at some other desired location and expression ofany gene can be monitored throughout sporulation by fusingits promoter region to the E. coli lacZ gene. I was not in aposition to wait for this golden age and, having to get a Ph.D.as soon as possible, I betrayed B.subtilis and moved to theother end of the corridor where I joined the lab of Jean-Claude Patte who was working on regulation of lysinebiosynthesis in E.coli.

A spo gene encodes a sigma factorThe lysA gene, encoding the enzyme convertingdiaminopimelate (DAP) to lysine, had just been cloned andsomebody had to sequence it. In those days sequencing wasstill an adventure and I could not afford to lose more time.So I spent the summer of 1980 in Moshe Yaniv's lab at thePasteur Institute where I learned how to play Lego withrestriction enzymes and how to survive the Maxam and

4'

Dances with sigmas

IP--spoOH-I

Lri0 starvation I

2 chromosomes

germinationI

free spore

IE

t--II J spoIIGBII \ GE

asymmetricseptation

'V

Am01~--I_ ~~~ _ _ _ ,~~1-

mother cell

(aK)

sigK

Fig. 2. The sporulation cycle. The various stages are indicated by roman numerals. The five sigma factors involved in sporulation are indicated with the

names of their encoding genes at the time of their activation. Adapted from Losick et al. (1986).

Gilbert technique. Back in Orsay I dismantled the 4.4 kbfragment that I had inherited and found two genes in it, bothrequired for lysA activity (Stragier et al., 1983b). One was

lysA itself, and the second one, located upstream and in theopposite orientation of lysA, encoded a protein absolutelyrequired for lysA transcription (Stragier et al., 1983a;Stragier and Patte, 1983). I called this regulatory gene lysR.I had no idea that this obscure LysR protein would later haveso many cousins and become the paradigm for a family ofbacterial regulators (Henikoff et al., 1988).

Expression of biosynthetic pathways in bacteria is usuallyrepressed by the final product of the pathway acting inconjunction with an aporepressor. In the case of amino acidbiosynthesis the recent discovery of attenuation had shownthat complex cis-acting sequences could induce prematuretranscription termination if the final product was present inexcess (Lee and Yanofsky, 1977). Therefore it was quitea surprise to find that the final step of lysine biosynthesisin E.coli was controlled by an activator. This result could,however, be rationalized because DAP, the substrate of thelysA product, is also a major constituent of the bacterial cellwall. Since lysA expression was induced by DAP andrepressed by lysine, the simplest interpretation was to assume

that DAP and lysine modulated the activation of lysAtranscription by LysR in opposite ways. In other words,building the cell wall had priority over making new proteins.That was my first contribution to regulation of transcriptionin bacteria and it allowed me to finally become a 'Doctor'.

I was then seriously thinking about finding a post-doctoralposition in the States and switching to some fancy eukaryoticsystem. But a conjunction of circumstances made me delaythat project. First, several genes involved in DAP

biosynthesis had now been cloned in the lab and none showed

any attenuation-like sequence, which left the basis of their

regulation by lysine mysterious. Secondly, J.-C.Patte was

moving to Marseilles, in the south of France, where he was

going to work on Pseudomonas and, because of some

administrative mix-up, no substitute teacher was appointedby the University to take up the lab. Third, I had enjoyeda cheerful and productive association with my colleague JeanBouvier during the last year and we both wished to carry

on for a while. So we were given some space and supportto stay at the Microbiology Institute and to extend our

investigations on the dap genes. We were joined by CatherineRichaud who had played a major role in cloning andanalysing the genes of the DAP - lysine pathway and whowas now hunting for the last missing dap genes. Weanticipated completing the whole story in a couple of years.

Before he left for Marseilles in June 1983, J.-C.Patte gave

a party at the Microbiology Institute and many of hiscolleagues were present. While I was enjoying the food andwine I was approached by Jekisiel Szulmajster. He was thelast scientist in France to be still working on sporulation inB. subtilis. In his lab at the CNRS campus of Gif-sur-Yvette,a few kilometres from Orsay, Celine Bonamy had clonedthe spoOB gene. He knew that I was a sequencing addictand he wanted me to sequence that piece of DNA. Thatseemed an easy way of settling an old score with B.subtilisand I agreed to do it with J.Bouvier. When we brought thespoOB sequence to Gif 3 months later, J.Szulmajsterconfessed that C.Bonamy had actually cloned a second gene,

spoIIG, and suggested that we could also spend some timeon its sequencing. We agreed but made it clear that therewould be no third gene... After finishing some experimentson the expression of spoOB (Bouvier et al., 1984) and stillworking mainly on the dap genes, we sequenced spolIG inMarch 1984. I had kept my connections with M.Yaniv's laband one of his students, Olivier Danos, advised me to

3561

spoIIACcyF

/ forespore(aG)

spoIIIG

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III

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P.Stragier

compare the sequence of the spoIIG product with all theavailable protein sequences that were stored in the PasteurInstitute computer in order to get some hint about itsfunction. I had never heard of such an approach before andfound the idea attractive. On April I 1 I introduced the spoIIGsequence into the Pasteur computer and ran the comparisonprogram. And two hours later I had the answer: the spoIIGproduct contained a region which showed a highly significantsimilarity with part of the E.coli a70 factor! The similaritywas such that it could be predicted that the spoIIG productwas itself a sigma factor. During the preceding years thefield of sigma factors in B.subtilis had moved quite fast.First, a new sigma factor, called a29, had been found to bepresent exclusively in sporulating cells (Haldenwang et al.,1981). Secondly, spoVG, the first sporulation gene to becloned, was induced at the onset of sporulation and dependedon minor vegetative sigma factors for its transcription in vitro(Johnson et al., 1983). Thirdly, the program of transcriptionafter infection of B. subtilis with phage SPO 1 was controlledby phage-encoded sigma factors acting sequentially andrecognizing highly conserved promoter sequences (Tjian andPero, 1976; Talkington and Pero, 1979). Altogether theseresults had led to the proposal that sporulation was controlledby a cascade of sigma factors (Losick and Pero, 1981). Thismodel was so attractive that it was almost immediatelyadmitted as being correct and we had a hard time convincingthe Editor of Nature that there was still no genetic evidencefor it and that the spolIG sequence provided it (Stragieret al., 1984).Having found what appeared to be the first spo gene

encoding a sigma factor after so many years of frustratingwork on B. subtilis, I was not just going to forget all aboutit and I decided to share my time between E. coli andB.subtilis. The next question to address was the exactfunction of the spoIIG product. The obvious candidate was(X29 (which would soon be renamed aE) and with J.BouvierI chose an in vivo approach. The idea was to express spoIIGin E. coli (since we were familiar with physiologicalexperiments using that organism) and to monitor expressionof a reporter gene fused to a aE-controlled promoter. Sucha promoter was provided by Linc Sonenshein (whom I hadknown when he was a post-doctoral fellow in P.Schaeffer'slab) as the promoter of the spoIID gene which was efficientlyrecognized by e in vitro. We constructed the requiredplasmids and E. coli strains but could not find any inductionof spoIID in the presence of the spoIIG product. A possibleunexpected explanation was provided when we heard aboutthe results of Bill Haldenwang, the discoverer of EJE,reporting that aE was synthesized as an inactive largerprecursor, pro-uE (Trempy et al., 1985b). Therefore westarted making a series of deletions in the proximal part ofthe spoIIG gene, selecting directly for the ability to induceexpression of the spoIID promoter in E. coli. Many monthswould be necessary before we had truncated versions of thespolIG gene that showed JE activity in E. coli and, in themeantime, it was demonstrated by immunological analysisthat our hypothesis was actually correct and that aE was theproduct of spoIIG (Trempy et al., 1985a).

Conserved domains in sigma factorsJust a few weeks after we obtained the spoIIG sequence itwas reported that the product of htpR, the gene controlling

the heat shock response in E. coli, had all the characteristicsof a sigma factor in vitro (Grossman et al., 1984) and thatit contained regions of high similarity with a70 (Landicket al., 1984). The sequence of ciA, the B.subtilis majorvegetative sigma factor, also became available (Gitt et al.,1985) so it was then possible to analyse more thoroughlythe conserved regions in these four sigma factors. With theexpert help of Claude Parsot we found three regions ofconservation. The most striking one, which was the oneinitially found in spolIG, covered about 80 amino acids andwas located roughly in the first third of the proteins. Wespeculated that its proximal part was involved in binding ofthe sigmas to the core RNA polymerase, while its morevariable distal region could modulate that interaction. Thetwo other conserved regions appeared to contain potentiallya similar secondary structure, the helix-turn-helix motifwhich was becoming well known as a putative DNA-bindingdomain. One of these regions was close to the carboxy-terminal end of the sigmas and the other was internal. Weproposed that these two regions were involved in recognitionof the two highly conserved sequences found in bacterialpromoters and reported these hypotheses in a paper that wasvery difficult to get published because of the absence of anyexperimental data (Stragier et al., 1985). Since then,biochemical and genetic evidence have been provided thatsupport part of our model: the most conserved intemal regionis actually involved in binding to core RNA polymerase(Lesley and Burgess, 1989) and the carboxy-terminalhelix-turn-helix motif interacts with DNA sequenceslocated 35 bp upstream of the transcription start (Gardellaet al., 1989; Siegele et al., 1989). No function has beenfound for the other putative helix -turn -helix region andrecognition of DNA sequences located 10 bp upstream ofthe transcription start is provided by the more variable regionadjacent to the core-binding domain (Siegele et al., 1989;Zuber et al., 1989). Today 30 sequences of bacterial sigmafactors are known which fit the modular scheme shown inFigure 3, while a few others (the 'aJ54 family' (Kustu et al.,1989) and the phage-encoded sigmas) have a completelydifferent organization.

I was very confident in our model and I was eagerlywaiting for other sigma sequences that would confirm it. So,when the rumour came by mid-1985 that another spo gene,spolIAC, also encoded a sporulation sigma factor (Erringtonet al., 1985) I feverishly studied its sequence that had beenpublished a few months before without any mention of aputative function of the spoIIAC product because comparinga new sequence with others in the database was not yetnormal practice. The spoIIAC sequence aligned very wellwith the other sigmas, but it stopped before the conservedcarboxy-terminal domain. I could not believe it and sincethe data did not fit the model, the data had to be incorrect!Examining closely the nucleotide sequence of spoIIAC, Ifound that the missing similarity was actually present, butdownstream of the stop codon and in another reading frame.

NH2 CORE-KCO2H

Fig. 3. Schematic structure of bacterial sigma factors. Boxes indicate regionsof sigma factors involved in binding to core RNA polymerase or to the- 10 and -35 regions of promoters. The sigma factors belonging to the4 family and the bacteriophage-encoded sigma factors have a different

organization.

3562

Dances with sigmas

It was just a matter of adding one nucleotide here and onethere and the spoIIAC product would be quite a decent sigmafactor. This was diplomatically suggested to the authors andthe missing nucleotides were found where they had to be.Then I knew with certitude that our alignments of sigmasconveyed powerful information that could be very useful ifcorrectly interpreted (Stragier, 1986). And 3 years later thesealignments would give me an astonishing reward.

Controlling the activation of a sigma factorThe identification of the spolIG product as aE leftunexplained the significance of the synthesis of oE as aninactive precursor pro-oE. Physiological and geneticexperiments had to be done in B.subtilis and my ownexperience was limited to growing bacteria before breakingthem to prepare an extract. I desperately needed someonewith a good knowledge of B.subtilis and I found the rightperson on the next floor, Celine Karmazyn-Campelli, whowas a former student of P.Schaeffer. A spoIID-lacZ fusionwas introduced into B. subtilis and could be induced duringvegetative growth by a truncated form of aE that was underthe control of an IPTG-inducible promoter. Conversely, theintact spoIIG gene was inefficient during growth but workedvery well if induced after the onset of sporulation.Interestingly, the amino-terminal part of pro-aE showed theability to form an amphiphilic helix and, by analogy withthe targeting sequences of imported mitochondrial proteins,I fancied the idea that the aE pro-sequence could play asimilar role and that pro-oE processing took place while thepro-rE molecules were crossing the septum doublemembrane. Such a mechanism could be used to segregatethe active aE molecules in one of the two compartments ofthe cell. According to that model the pro-oE processingmachinery was expected to contain transmembrane andcytoplasmic domains (or subunits), playing the role of thepore and of the peptidase respectively. There was anexcellent candidate for the pro-oE processing enzyme andthat was the product(s) of the spoIIE locus. This was theonly locus known in which mutations did not interfere withpro-oE synthesis but prevented its conversion to rE (Trempyet al., 1985b). Internal fragments of spollE were providedto us by Mike Young and we started cloning the remainingpart of this locus as a first step towards sequencing it. Havinglearned that sequencing of spoIIE was already well advancedin Phil Youngman's laboratory, we focused our efforts onstudying spollE expression in relation to spolIG. Both lociwere found to be expressed at the same time, about one hourbefore spolID which was used as an indicator of aE activityand therefore of pro-oE processing. Thus the situation wasquite confused by mid-1986 when C.Bonamy decided tospend one year in my laboratory. She had found that oneof the classical spolG mutations was not in the geneencoding pro-oE but in another gene located immediatelyupstream and I thought that it could be informative tosequence this other gene. The spolIG locus was found tocontain two genes organized in an operon and, followingthe usual nomenclature, we called the first one spoIIGA andthe second one, which encoded pro-orE, spoIIGB. It wasquite a surprise when I realized in October 1986 that thesequence of the spoIlGA product had all the features thatI had predicted for the pro-uE processing enzyme and ittook only one night to decide that everybody was wrong

about the role of spoIIE and that we had the true pro-aEprocessing enzyme in our hands.The following months were frantic. I did not want to

publish the spoIIGA sequence without providing someevidence about its function. An in-frame deletion was createdin spoIIGA and found to block spoIID transcription,suggesting that pro-uE was not processed in that mutant.More direct proof was obtained by constructing a geneencoding an hybrid pro-aE-3-galactosidase protein whichcould be partially processed in a wild type strain but not inthe spoIIGA mutant. There seemed to be no doubt thatSpoIlGA was required for pro-uE processing. But was itsufficient? To answer that question we induced the synthesisof both SpoIIGA and pro-uE during vegetative growth andfound a significant induction of spoIID-lacZ. Therefore weconcluded that the spoIIGA product was likely to be the pro-aE processing enzyme. But how could we explain why pro-aE accumulated for about one hour before getting processedif the processing enzyme was synthesized simultaneously?And what was the role of the spoIIE product(s)? The situationwas even more complicated because mutations in the spoIIAoperon (which encoded the other putative sporulation sigmafactor soon to be known as oF) also blocked spoIID-lacZtranscription, although both the spoIIG and the spoIIE operonwere normally expressed. In fact too many gene products

S p1., I IA A

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rnSp0 I I1

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I.I

M(i K(1iliR(''-( E-13

Fig. 4. A model for activation of aE. The SpoIlGA protein is shown asa box partly embedded in the membranes of the sporulation septum (stippledarea). Light arrows indicate the involvement of accessory gene productsin pro-aE processing (symbolized by the thick arrows). Part A is adaptedfrom Stragier et al. (1988) when it was believed that aE and aF were activein both compartments. Part B is an updated version where pro-oEprocessing takes place only in the mother cell in response to a cascade ofinteractions leading to activation of aF in the forespore.

3563

P.Stragier

were required to get active oE in the sporulating cell! It wasfinally possible to build a coherent model from all these databy assuming that SpoIIGA became active only when insertedin the septum membrane, because of some specific propertieswhich required the spoIIE product(s) and the products ofsome other genes controlled by (F. The existing datasuggested that spoIID was transcribed in both compartmentsand it was then concluded that activation of SpoIIGAoccurred on both sides of the septum (Figure 4A). In thismodel, activation of the aE-controlled regulon was strictlydependent on the presence of the sporulation asymmetricseptum and gene expression at an intermediate stage ofsporulation appeared to be coupled to the successfulcompletion of a previous morphological step (Stragier et al.,1988). Very recently new data have been obtained whichstrongly indicate that fF becomes active only in theforespore compartment in response to a cascade ofinteractions initiated by the spolIE products (Margolis et al.,1991) and that fE becomes active only in the mother cell,suggesting that pro-urE processing takes place only in thatcompartment (Driks and Losick, 1991). An updated modelis shown in Figure 4B where the septum still plays a crucialrole in the vectorial transfer of activation from aF to (E.

The forespore sigma factorThe sequence of the spolIG locus showed the presence ofan incomplete open reading frame located only 140 bpdownstream of spoIIGB. Could it be the third gene of thespolIG operon? By mid-1987, in order to address thisquestion, we engineered a disruption of this gene and foundthat the mutated cells were asporogenous, their sporulationbeing arrested at stage IH (see Figure 2). Therefore the openreading frame downstream of the spoIIG operon defined anew sporulation gene which we called spoIIIG. On June 29,looking with a renewed interest at the partial sequence ofthe spoIIIG product, I suddenly recognized motifs that hadbeen posted above my desk for the past 3 years: this wasa sigma! And a very interesting one since it was requiredat a stage of sporulation where gene expression was knownto be compartmentalized. This sigma, which we called o,could be forespore or mother-cell specific. Since only partof the spoIIG gene was present in the insert cloned byC.Bonamy we 'walked' on the B.subtilis chromosome toclone the missing part and the sequence was completed bythe end of October. Moreover, we had a plasmid carryingthe complete spoIIIG sequence under the control of an IPTG-inducible promoter and I could now address the question ofthe physiological role of aG.During all these years we had been handicapped by the

poor communication with our colleagues on the other sideof the Atlantic and to change that situation I decided to spendone month in R.Losick's lab at Harvard University. I wantedto look for genes controlled by aE and aG by screening alibrary of transposon-generated lacZ insertions using ourplasmids in which rE and aG synthesis was IPTG inducible.Alan Grossman was using the same strategy for identifyinggenes controlled by uH, a recently identified sigma factorinvolved in the transition to post-exponential phase and Iwould greatly benefit from his advice. Thus, in November1987, I was carefully looking at my X-gal agar plates whenR.Losick got a phone call from Peter Setlow, the worldauthority on SASPs, the small proteins that accumulate in

the forespore and are degraded during germination (Setlow,1988). The ssp genes encoding the SASPs had been clonedin P.Setlow's lab and had been used as in vitro templatesto purify a factor allowing core RNA polymerase torecognize their promoters. P.Setlow was telling R.Losickthat an amino-terminal sequence of the factor had beendetermined but it did not match any known spo gene.R.Losick's reaction was quick and a few minutes later I wason the phone dictating the amino-terminal sequence of a0Gand getting enthusiastic approvals from P.Setlow: the aGsequence matched exactly the sequence of his putativeforespore-specific sigma factor ! Afterwards it was just amatter of weeks before we could demonstrate that synthesisof a0 during vegetative growth induced expression of allknown forespore-specific genes (Sun et al., 1989). Thefunction of oG being unravelled C.Karmazyn-Campellicould concentrate on the regulation of its synthesis and shewas to find multiple and complex levels of controls(Karmazyn-Campelli et al., 1989; Stragier, 1991). It appearsnow that o is itself synthesized exclusively in the foresporeat stage II, but becomes active only later at stage III, inresponse to the end of engulfment.

A sigma factor in piecesAfter such an exciting visit to Harvard I decided to comeback one year later. I had no special experiments in mindbut I knew it was a good time to be there: Lee Kroos hadpurified a sigma factor (to be called aK) which allowedRNA polymerase to transcribe in vitro genes expressed ata late stage in the mother cell. From its amino-terminalsequence an oligonucleotide probe had been designed anda putative sigK gene had been cloned. On October 12 BarbaraKunkel showed me her first sequence of sigK whichcontained an excellent match with the conserved core bindingregion. Looking further up on the autoradiogram it waspossible to guess the region corresponding to theoligonucleotide probe, but no typical pyrimidine-rich regionthat would be the ribosome binding site on thecomplementary strand could be seen immediately upstream.This suggested that aK might also be synthesized as aninactive larger precursor, pro-aK. We were thenencouraged to look back to all the available sequences ofspo genes and to search for the presence of the amino-terminal sequence of aK in an internal position. And wesoon found it in spoIVCB, a gene known to be expressedonly in the mother cell and from which only the first 40codons had been sequenced. But there was still a mystery:the coding sequence for aK was interrupted by a stop codonat what should have been only half of the sigma and thesimilarity with the other sigma factors could not be restoredby shifting the reading frame and reading furtherdownstream. This was the symmetrical situation to thatreported for the spoIIIC gene, which had been found toencode a product showing similarity to the carboxy-terminalpart of sigma factors (Errington, 1987). And spoIlC wasquite close to spoIVCB on the chromosome... It was thentempting to imagine that the two genes spoIVCB and spoIIICwere brought together by a DNA rearrangement that wouldcreate the intact sigK gene in the mother cell. A similarphenomenon was known to occur in some nitrogen-fixingbacteria during the differentiation process that leads to theactivation of the nifoperons (Golden et al., 1985). We were

3564

Dances with sigmas

a __...__... nutrientstarvation

asymmeticn%,r- septation

f

b

C_A

-..........

Fig. 5. The chromosomal rearrangement creating sigK. The sigK gene isa composite of the spoIVCB and spoIlC genes which are joined in frameby excision of an intervening sequence. The rearrangement occurs only inthe mother cell and is dependent on the spoIVCA product. Reproduced withpermission from Kunkel (1991).

all excited about this possibility and R.Losick agreed thatI should start immediately with B.Kunkel the experimentsthat would assess that specific hypothesis. DNA was

prepared from vegetative cells as well as from latesporulating cells and digested with various restrictionenzymes. The physical map of the spoIVCB and of thespollIC regions was then determined by Southern blottingusing specific probes for each of the two genes. And 10 daysafter our first examination of the sigK sequencing gel we

had an autoradiogram demonstrating that the two loci were

actually rearranged during sporulation. During the followingweek the rearranged gene was cloned from sporulating cellsand I had one of my best birthday presents when I read thesequence across the rearrangement breakpoint whichconfirmed all our hypotheses (Stragier et al., 1989).

In the following months these experiments were continuedon both sides of the Atlantic. The rearrangement was foundto be due to the excision as a closed circle of an interveningsequence of about 42 kb and to occur only in the mothercell. This was the expected result since the mother cellcompartment is lost at the end of sporulation while theintervening element has to be still present in the spore tobe transmitted to the next generation of bacteria. Interestinglythe rearrangement is controlled by the spoIVCA gene whichis carried by the element itself (Figure 5). At this stage ofthe results the rearrangement was considered to be a keyelement in establishing compartmentalized gene expressionin the mother cell by creating the gene for the mother cellspecific sigma factor aK. However, it was possible to

end ofengulfment

cortexsynthesis

coatsynthesis

H

SpoIIAA-dependentinactivation of SpoIIAB

in the forespore

aF

SpoIIGA-mediatedpro-GE processingin the mother cell

47(YESpolIIA-dependent

activation of aGin the forespore

G

forespore-dependentpro-aK processingin the mother cell

4KaK

Fig. 6. Cascade activation of sporulation sigma factors. Specific requirementsfor activation of each sigma factor are boxed and depend themselves onthe action of a previous sigma factor. The morphological stages of sporulationare indicated at their approximate position according to the transcriptionalcascade. Adapted from Stragier and Losick (1990) where the role of thevarious spo genes involved in the cascade is detailed.

construct a strain in which the interrupted sigK region wasreplaced by an intact rearranged sigK gene. That strain grewand sporulated normally although a rearranged copy of thesigK gene was present in the forespore (Kunkel et al., 1990).Therefore the rearrangement per se is not required forallowing aK synthesis exclusively in the mother cell.Restriction of transcription of sigK and of processing of pro-UK to the mother cell are the actual mechanisms thatestablish mother cell specific gene expression (reviewed byKunkel, 1991).

A new challengeSince 1987 I had definitely quit E. coli and the study of thedap genes and I had progressively attracted new people towork on sporulation of B.subtilis. So, when MarianneGrunberg-Manago offered me a spacious lab in herdepartment at the IBPC, I did not hesitate and in April1989 my lab moved to Paris. Since then other avenues ofsporulation have been explored and the sigma factor sagahas expanded. It is now clear that for all the five sigmafactors known to be required during sporulation (as indicatedin Figure 2) the ultimate and most important control isexerted at the level of their activity. The cascade of sigmafactors predicted 10 years ago is a cascade of sequential

3565

post-transcriptional

induction

P.Stragier

activations in which each sigma depends upon a previousone for becoming active as summarized in Figure 6 (Stragierand Losick, 1990). Most interestingly, the major part of thecascade is compartmentalized and there appears to be aconversation between the forespore and the mother cell, eachcompartment telling the other one when to proceed to thenext developmental step by sending signals conveyed throughthe completion of specific morphological structures.As I tried to make clear in this review I went myself

through a cascade of bits of luck and, thanks to the freedomgiven by CNRS and my successive advisors, I was able tograsp all the opportunities. Maybe Mother Nature decidedto pay me back for all the lost years of my youth and shetaught me little by little how to tame the mythical sigmas.They became familiar inhabitants of the landscape along thereceding frontier where I stayed posted. Now I want to goacross and to explore a new world where cells talk to eachother and secret messages are buried in morphologicalstructures. It should also be fun.During all these years I have had the privilege to work

with many good friends and colleagues, among whom I havea special debt to Jean Bouvier, Celine Karmazyn-Campelliand Rich Losick. I am grateful to David Popham for his helpduring the preparation of this manuscript.

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