JOURNAL OF BACTERIOLOGY, June 1973, p. 1254-1263Copyright © 1973 American Society for Microbiology

Vol. 114, No. 3Printed in U.S.A.

Use of Constructed Double Mutants forDetermining the Temporal Order of Expression

of Sporulation Genes in Bacillus subtilisJ. G. COOTE' AND J. MANDELSTAM

Microbiology Unit, Department of Biochemistry, University of Oxford, Oxford, England

Received for publication 5 March 1973

Double mutants containing two Spo mutations concerned with different stagesof sporulation were constructed. In these, the phenotype that is exhibited is thatof the earlier sporulation block. The same procedure was applied to sporulationmutants damaged in the same stage of development. The results provide a basisfor placing in a temporal order different mutations concerned in stage II andstage IV of spore development. In general, the order indicated by the phenotypesof the double mutants is in agreement with the order derived on biochemicalgrounds. Double oligosporogenous mutants have also been constructed. Theirsporulation incidence is roughly equal to the product of the incidences of theparent strains, idicating that separate factors are involved in overcoming eacholigosporogenous block. The number of dependent sequential steps in sporulationis estimated as not less than about 12.

The biochemical and morphological eventsthat accompany sporulation can be subdividedinto several categories (6). In summary, theseare (i) the primary sequences of dependentevents specifically concerned with the process(A, B, C, etc., see diagram below). The termdependent in this context applies to an eventwhich will not occur unless the earlier eventshave been successfully completed. The depend-ent nature of the sequence suggests that sporu-lation mutations are pleiotropic, a fact that wasemphasized even in early studies of sporulationmutants (12, 14). For some stages of develop-ment, several events (E, F, G) might have tooccur simultaneously. Examples of dependentevents are formation of the spore septum, sepa-ration of the spore protoplast from the mem-brane of the mother cell, etc. (ii). The secondcategory is side effects (Bl, Cl, etc.). Anexample is the appearance of dipicolinic acid, aspore-specific compound which shows up at adefinite stage in the process. Nevertheless, laterevents do not seem to be dependent upon itsappearance, and in mutants of Bacillus cereuswhich lack this compound viable spores are stillformed (16). (iii) The third category consists ofchanges in vegetative functions that take placebecause the cultural conditions that induce

1 Present address: Institut de Microbiologie, Universite deParis-Sud, 91 Orsay, France.

sporulation differ from those that promotegrowth. An example is the appearance of aconi-tase, and other examples will be found else-where (6).

C2

A __+B C D F )jH .........SPORE

B1 C1,

It is often not possible to distinguish betweenevents of type (i) and those of type (ii). Forinstance the appearance of alkaline phospha-tase is correlated with the transition of stage IIto stage III of sporulation, but since the functionof the enzyme is unknown it is impossible to tellwhether later events are dependent upon it orwhether it is a side effect. A further complica-tion has been introduced by the observationthat the alkali-soluble protein of the spore coatwhich is incorporated into the structure at avery late stage is, in fact, synthesized severalhours earlier (17). Events of this type (C2) arealso indicated in the diagram.Because events in the main sequence are

dependent, it is easy to distinguish mutantsblocked early in the process from those blockedlater since the mutations give rise to phenotypeswhich are morphologically distinct or whichdiffer in the biochemical marker events thataccompany sporulation (15, 1). It is, however,

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much more difficult to decide which gene ex-pression precedes another when both are ex-pressed at about the same time. One might havethought that the mapping of sporulation geneswould help, but this is not so because mutationsthat block the process at some particular time,for instance stage II, may be widely separatedon the chromosome, and, conversely, genes thatare very close to each other may be associatedwith events that are widely separated in time (4,2).However, when some of these mutants are

induced to sporulate, the process proceeds nor-mally up to a given point and then gives rise toobvious maldevelopment. This is particularlylikely to occur with stage II mutants, andcurious, often bizarre, forms are likely to be seen(see 10, 18, 15). Examples include the produc-tion of multiple septa, abortive disporic forms,overproduction of membrane, etc.

It follows, again from the dependent nature ofthe system, that if one introduced into a singleorganism two mutant genes, each of which givesrise to a distinct sporulation phenotype, oneshould be able to distinguish the earlier muta-tion from the later because, in such a mutant,sporulation would proceed normally until thetime came for expression of the first alteredgene. This would produce its damaging effectand the process would go wrong, so that theorganism would exhibit the characteristic phe-notype for that gene. The second gene, beingconcerned with a later event, would not beexpressed at all.The work to be described was carried out with

the following objectives: (i) to establish inprinciple that constructed double mutantscould be used to distinguish early and lategenes; (To this end they were constructed fromsingle mutants blocked at obviously differentstages of development [e.g., II and IV].); (ii)to order mutations affecting events con-nected with the same stage of sporulationwhich are morphologically distinguishable; (iii)to determine whether constructed double mu-tants could afford any further insight into thenature of oligosporogeny which might well becaused by leaky genes (2, 12). Even if this is so,it is still a puzzle to know why, within an oligo-sporogenous population, a small proportion ofthe cells are able to sporulate successfully.This success could be due to some generalphenotypic factor (e.g., age of the cell or an un-usually high content of adenosine 5'-triphos-phate) that allowed a few cells to overcome theblock in the process. If such a general factorenabled cells to overcome more than one typeof oligosporogeny, it follows that if one con-

structed a double mutant from strains sporu-lating with a frequency of, for example, 1% and10%, any cell in the population having enoughof the general factor to overcome the moresevere block would even more easily overcomethe less severe one. The double mutant shouldaccordingly sporulate at 1%. If, however, thefactors involved in overcoming the two typesof block are independent, then the probabilityof sporulation in the double mutant would bethe product of the separate probabilities or0.1%.The results to be described show that double

mutants exhibit the phenotype of the earliergene so that the method is, in principle, appli-cable to mutations affecting the same stage ofsporulation. In addition, it appears that what-ever factor allows a cell to overcome oneoligosporogenous block does not help it over-come another.

MATERIALS AND METHODSOrganism. Bacillus subtilis 168 (trpC2) was used.

It forms spores normally in resuspension mediumsupplemented with tryptophan (see below) and isreferred to as the wild type. The Spo and Osp mutants(see below) derived from the wild type and theirmorphological and biochemical properties have beendescribed elsewhere (1, 15). We are indebted to S. R.Ayad for a prototrophic strain of B. subtilis 168. Allmutations in the strain except DG2, N25, andNG17.29 were transformed into the standard 168strain to make them isogenic. The exceptions weretested for the absence of multiple spore mutations byusing Spo+, trp+ deoxyribonucleic acid (DNA) at asaturating concentration (2 ,ug/ml) to transform eachof them to prototrophy. All yielded between 5 and 10%Spo+ colonies among the trp+ transformants as aresult of the integration of a second DNA fragmentpossessing the unlinked wild-type allele of the sporemutation. If the mutants had carried a second un-linked spore mutation, the expected proportion ofSpo+ transformants obtained would have been about0.5%. A list of the strains carrying auxotrophic mark-ers which were used for transduction analysis hasbeen given elsewhere (2), and the map positions of thesporulation mutations had already been determinedearlier by Piggot (8) and Coote (2) by using transduc-tion mapping with phage PBSl and also in someinstances by transformation.

Media. PAB, antibiotic assay medium no. 3 (Difco,Detroit, Mich.), was used. Lactate-glutamate mini-mal agar plates were prepared as already described(1).Nomenclature. The asporogenous and oligo-

sporogenous phenotypes will be referred to as Spoand Osp, respectively.Growth and sporulation. The organisms were

grown with shaking at 37 C in a medium containinghydrolyzed casein, L-tryptophan, and inorganic ions(13). Growth was measured spectrophotometricallyby using a calibration curve relating extinction at 600

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nm to bacterial dry weight. To initiate sporulation, aculture of 0.25 mg (dry wt)/ml was centrifuged, andthe cells were transferred at the same density to aresuspension medium containing L-glutamate, L-tryp-tophan, and inorganic ions (13). With the wild typethis procedure produces about 80% refractile spores in7 to 8 h. Time in hours after transfer of cells toresuspension medium is indicated by to, tI, etc. Stages0 to VI are the generally recognized cytological stagesof spore formation (9).Biochemical events during sporulation. As-

sociated with the morphological stages of develop-ment are a number of biochemical marker eventswhich occur at characteristic times during sporulation(15). Those used for the work described here areexo-protease (stage 0), alkaline phosphatase (stageII-III), glucose dehydrogenase (late stage III), andheat resistance (stage VI). They were assayed insamples removed from the resuspension cultures (seereference 1).

Electron microscopy. Cells initiated to sporula-tion in the resuspension medium were sampled at t7and fixed, and thin sections were prepared (5).Morphological assessment was made only on com-plete cells in longitudinal sections.

Transduction. The preparation of transducing ly-sates with phage PBS1 and the procedure used fortransuction were as already described (2).

Transformation. DNA was extracted with minormodifications by the method of Marmur (7), and thetransformation procedure has been described previ-ously (2). For the construction of double mutants,DNA was used at a concentration of 0.025 /ig/ml.

Construction of double sporulation mutants.Most of the double mutants were constructed by usingtransduction. The most convenient procedure in-volved donor strains carrying spore mutations linkedto trpC2. The recipient (also trpC2) carried a differentspore gene mutation not linked to trpC2, but toanother auxotrophic marker elsewhere on the chromo-some map. Each donor strain was first transformed toprototrophy by using DNA prepared from the trp+strain of B. subtilis. Lysates prepared from each trp+derivative were then used to infect the recipientstrain, selection being made for trp+ transductants. Apercentage of these, depending on the degree oflinkage, took up the sporulation mutation of the donorand became double-sporulation mutants.

In one donor, X8, the sporulation mutation was soclosely linked to the auxotrophic marker phe-12 thattransformation could be more conveniently used. Inthis case the recipients were previously constructedphe- derivatives carrying spore mutations unlinked tophe-12. Each was transformed to prototrophy byusing DNA prepared from X8, phe+.

Selection and screening of double mutants.Transductants and transformants obtained in thecrosses just described were plated on lactate-gluta-mate minimal agar and incubated at 37 C for 2 to 3days to allow development of the pigment associatedwith sporulation in this strain (11). Spo+ colonieswere dark brown, whereas Spo or Osp colonies wereeither translucent, white, or light brown. In someinstances, in the crosses involving two sporulation

mutants, it was possible to distinguish by color twotypes of spore-defective colony, one exhibiting thephenotype of the recipient and the other the pheno-type associated with the double mutant.

Whether two colony types were distinguishable bycolor or not, it was essential to know that the straintaken for investigation contained both sporulationmutations. This was checked by back-crossing asfollows: eight single colonies, presumptive doublemutants, from each cross were picked at random andsubcultured on nutrient agar. A phage lysate was thenprepared from each of the eight separate cultures andused to transduce to prototrophy two recipient strainscarrying the auxotrophic markers to which each of thetwo sporulation mutations was known to be linked.The resulting prototrophic transductants in each casewere then checked to ensure that the expected per-centage of cells had acquired the appropriate sporula-tion phenotype.

RESULTS

The phenotypes of the four donor mutants aresummarized in Table 1. In resuspension cul-tures, mutant Y13 produced long elongated cellswhich were blocked at stage 0. It was includedas an obvious control because any recipienttaking up its mutation should give a doublemutant in which the majority of the cells(>99.9%) remained blocked at stage 0. MutantP18 was damaged at spore septum formation(stage II). It developed septa at both ends of thecell and also laid down cell wall materialbetween the double membranes of both septa(Fig. 2). Mutant P20 was characterized bydeposition of spore coat material in the cyto-plasm of the mother cell instead of around thedeveloping prespore (Fig. 7). Although, for bio-chemical reasons, this mutant would appear tobe blocked between stages III and IV, its ap-pearance in electron micrographs suggestedthat it might be blocked at a much later stagesince coat proteins do not normally becomevisible until stage V. It was included in theseries to determine whether the double mutantmethod would help to place this gene in thetemporal sequence. Finally, mutant X8 wasused as an example of an organism blocked at alater stage. In these cells, cortex formation wasinitiated but no coat material was laid down(Fig. 4).The phenotypes of the recipients are summa-

rized in Table 2 and will be referred to below.Phenotypic characteristics of double mu-

tants containing mutation Y13 (stage 0).Double mutants were constructed from Y13 andeach of the following: P14 (stage II), P9 (stageII), X8 (stage IV), W10 (stage V). All the doublemutants exhibited the stage 0 phenotype char-acteristic of mutant Y13.

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TABLE 1. Phenotypes of the strains used as donors in constructing double mutantsa

Stage Pr ~~~~~~~~Alkaline Glucose HeatGetiStrain T Morphologic al description Pro- -phospha- dehydro- resist- Geneticblocked tease ~~~~~tase genase ance linkage

Y13 Osp 0 Long vegetative cell 5.0 3.0 0.06 trpC2 (61)P18 Osp II Septa at both poles of cell + 3.0 1.5 0.005 trpC2 (33)P20 Osp III-IV Incomplete cortex, coat mate- + 78.0 58.0 0.15 trpC2 (12)

rial deposited in mother cellX8 Osp IV Incomplete cortex, no coat ma- + 83.0 80.0 1.0 phe-12 (3)

terial

a Alkaline phosphatase and glucose dehydrogenase were measured in samples taken from resuspensioncultures at t5, and heat resistance was measured at t7. Values are expressed as percentages of those obtainedwith the wild type. A fuller description of the donor phenotypes has been given previously (1). The last columnlists the percentage recombination values obtained by two-factor transduction crosses with the auxotrophicmarker indicated (2).

Phenotypic characteristics of double mu-tants containing mutation P18 (stage II).The sporulation phenotypes of the double mu-tants resulting from the introduction of this mu-tation are summarized in Table 3. It was to beexpected that, since mutation P18 affects someprocess at stage II, expression of the genecontaining this mutation would precede theexpression of genes concerned in later stages.The table shows, in fact, that all the doublemutants involving recipients blocked at laterstages exhibited a phenotype which was charac-teristic of P18. In addition, it was clear that thegene containing mutation P18 also preceded inexpression the genes containing mutationsNG17.29 and P9. Both of these mutations blocksporulation after a single septum at one end ofthe cell has been formed in the normal manner.Development in mutant NG17.29 goes no fur-ther, but in mutant P9 the septum proceeds tobulge into the cytoplasm of the mother cell (Fig.3). Both produce alkaline phosphatase inamounts comparable to those of the wild type(Table 2). The double mutants (P9/P18 andNG17.29/P18) in keeping with their morphologi-cal resemblance to P18 both produced smallamounts of phosphatase.However, when mutation P18 was introduced

into organisms carrying two other stage II muta-tions (DG2 and N25), both of which blocksporulation before production of alkaline phos-phatase, the resulting phenotype was indistin-guishable from that of the recipient. It seemsclear then that expression of these two muta-tions precedes that of P18 (Table 3 and see Fig.9). The two recipients are recognizable becausemutation N25 causes production of a singleseptum at one pole of the cell and in somecircumstances an overproduction of septummembrane (15). Mutation DG2 was morpholog-ically recognizable because it caused a single

septum to be laid down at one pole of the cellwith an exceptionally thick layer of cell wallbetween the twin membranes (Fig. 1). In addi-tion, in some cells several cross walls were laiddown apparently in a random way.The results shown in Table 3 do not help one

to establish the temporal order of the genecontaining mutation DG2 relative to that con-taining N25, nor to establish the relative orderof NG17.29 and P9. To do this it would benecessary to construct double mutants contain-ing these pairs of mutations.Phenotypic characteristics of double mu-

tants containing mutations P20 (stage III-IV) and X8 (stage IV). The sporulationphenotypes of double mutants containing P20are also shown in Table 3. It is manifest that theexpression of the gene containing P20 precedesthose containing the late mutations W10 andW5, as the phenotypes of the double mutantswere identical with that of the donor. Bothmutants W10 and W5 formed spores almostnormally to a late stage, but in W10 the cortexhad an unusual, streaked appearance (Fig. 5),and in W5 the cortex was almost entirelymissing (Fig. 6). A somewhat curious result wasobtained with mutant X8 in which the recipientmutation was presumed to concern a slightlyearlier stage, i.e., the initiation of cortex forma-tion (see Fig. 4). The double mutant (X8/P20)exhibited the phenotype of the donor but in amodified way. For example, coat material, in-stead of being deposited in concentric layers inthe cytoplasm of the mother cell, tended towrap itself around the developing prespore (Fig.8). The remaining double mutants described inTable 3 all exhibited the morphology ch#racter-istic of the recipient, and it would thus appearthat the expression of mutation P20 could beplaced temporally after that of Y10 (stage III)and before that of X8 (Fig. 9).

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3

4

FIG. 1. Mutant DG2 is blocked at stage II and is aberrant in that it lays down a thick layer of cell wallbetween the two membranes of the spore septum.

FIG. 2. Mutant P18 forms spore septa at both poles of the cell and lays down cell wall between the septummembranes.

FIG. 3. Mutant P9 forms a single septum in the normal way which bulges into the mother cell, although itremains attached to the original cell wall invaginations.

FIG. 4. Mutant X8 develops as far as the first stage of cortex formation, the primordial germ cell wall. This isseen as an electron-dense band between the prespore membranes. The bars represent 0.2 jm.

Mutant X8 (stage IV, Fig. 4) was used as

donor with four recipient strains, P14 and P9(both stage LI), and W5 and W10 (stage IV andV) (see Table 2 and Fig. 3, 5, and 6). Thephenotype of the double mutant P14/X8 is

discussed below. Double mutant P9/X8, asexpected, had a phenotype indistinguishablefrom that of P9. However, the double mutantsW1O/X8 and W5/X8 were both blocked at stageIII, i.e., at an earlier stage than either of the

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7

FIG. 5. In mutant W10 the cortex has an unusual, striated appearance and development of the coat layers isonly complete at the poles of the spore.

FIG. 6. Mutant W5 shows well-developed coat layers, but growth of the cortex which normally occurs

between the two membranes of the prespore is almost entirely absent.FIG. 7. Mutant P20 develops normally up to the free prespore stage, but the coat material is deposited in

concentric layers in the mother cell cytoplasm instead of around the prespore.FIG. 8. Double mutant X8/P20 exhibits a modified P20 phenotype where the coat material is attempting to

wrap itself around the prespore in a more normal way instead of forming a mass in the mother cell cytoplasm.The bars represent 0.2 ,gm.

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TABLE 2. Phenotypes of strains used as recipients in constructing double mutantsa

I Stage Pro- Alkaline Glucose Ha eeiStrain Type blocked Morphological description tease phospha- dehydro- rHeat Geneticblocked tease tase genase ~~~resistance linkagetase genase

DG2 Spo II Single septum with thick + 2.0 1.0 <0.0001 cysA 14 (5)cell wall betweenmembranes

N25 Osp II Single septum and over- + 3.0 1.5 0.01 cysA14 (7)production of mem-brane

P14 Osp II Septa at both poles of + 23.0 19.0 20.0 cysA14 (27)cell

NG171.29 Spo II Single septum + 47.0 2.0 <0.0001 hisA I (85)P9 Osp JI Single septum that + 70.0 11.0 0.008 hisAl (57)

bulges into mother cellY1O Osp III Free prespore + 25.0 24.0 0.03 ura-1 (84)W5 Osp IV-V Incomplete cortex, nor- + 87.0 69.0 0.001 ura-1 (45)

mal coat layersWio Osp V Striated cortex, normal + 85.0 84.5 0.0001 ura-1 (54)

coat layers

a For descriptivepreviously (1, 14).

details see Table 1. A fuller description of the recipient phenotypes has been given

TABLE 3. Phenotypes of double mutants produced by introduction of mutations P18 and P20 into recipientstrains blocked at various stagesa

Recipient Donor P18 (II) Donor P20 (III-IV)

Alka- Glu-No. and stage line cose Heat Alkaline Glucose Heat

blocked Morphology phos- dehy- resistance Morphology phospha- dehydro- resistancepha- dro- tase genasetase genase

DG2 (II) II (recipient) 2.0 1.0 <0.0001 II (recipient) 3.0 1.0 <0.0001N25 (II) II (recipient) 3.0 1.5 <0.0001 II (recipient) 2.0 1.0 <0.0001NG17.29 (II) II (donor) 4.0 2.0 <0.0001 II (recipient) 51.0 2.0 <0.0001P9 (Il) II (donor) 3.0 1.0 <0.0001 II (recipient) 91.0 7.0 <0.0001Y10 (III) II (donor) 2.0 3.0 <0.0001 III (recipient) 33.0 14.0 <0.0001X8 (IV) II (donor)' 2.0 NRe <0.0001 III-IV (modified 84.0 71.0 <0.0001

donor, see Fig.8)b

W5 (IV-V) II (donor) 2.0 NR <0.0001 III-IV (donor) 93.0 65.0 <0.0001W1o (V) II (donor) 3.0 NR <0.0001 III-IV (donor) 88.0 82.0 <0.0001a Details are as in Table 1."The double mutants X8/P18 and X8/P20 were constructed in two ways. Firstly, using a lysate of P18 or P20,

trp+ to transduce X8, trpC2 to protrophy, and secondly, transforming P18 or P20, phe-12 to prototrophy usingX8, phe+ DNA. The same double mutants, constructed by either procedure, had indistinguishable phenotypes.

e Not recorded; the values were presumed to be similar to those of the donor.

parent organisms. Both double mutants formeda complete spore protoplast, free within themother cell, but no cortex of coat formation wasinitiated (see Discussion).Phenotypic properties of double Osp mu-

tants. A double mutant was constructed byusing as recipient mutant P14. In this strain80% of the cells are blocked at stage II, whereasthe remainder go on to form heat-resistantspores (Table 2). The donor was another Ospmutant, X8, which sporulated at an incidence

of 1%, the remaining cells being blocked at stageIV (Fig. 4).

Electron micrographs of the double mutantprepared from resuspension cultures at t7showed that the majority (about 80%) of thecells were blocked at stage II, and that a muchsmaller proportion (about 20%) were blocked atstage IV. The incidence of heat-resistant sporeswas 0.12%. Similarly, when P14 (spore inci-dence 20%) was used as recipient with mutantY13 as donor (spore incidence 0.06%), the re-

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sulting double mutant sporulated at 0.003%.When X8 (1%) was used as recipient with Y13(0.06%) as donor, the double mutant had aspore incidence of about 0.001%.

DISCUSSIONThe oligosporogenous double mutants will be

considered first. Previous studies of oligo-sporogeny have shown that in sister cells theinitiation of sporulation is highly correlated;i.e., if a cell has been induced to start sporulat-ing the probability is high that its sister cell willhave been initiated at the same time (1). This isalso what is found with sibling wild-type cells(3). This synchrony, however, is lost as soon asthe cells encounter an oligosporogenous blockwhen the occurrence of a spore in one cell doesnot help to indicate the probable behavior of itssibling (1). This must be taken to mean thatovercoming the block results from the posses-sion of some factor, presumably biochemical,which was either distributed asymetrically atthe time of cell division or which has beengenerated randomly in a small proportion ofcells sometime after cell division.The present experiments were undertaken to

discover whether possession of this factor,whatever it might be, would help an organism toovercome oligosporogenous blocks in general. Ifthere were such a general factor the doublemutants should sporulate at the incidence char-acteristic of the more severely damaged of thetwo parents (see Introduction). If, on the otherhand, there is no general factor, the probabilityof sporulation should be the product of theseparate probabilities. Reference to the resultswill show that the latter is in fact what is found,and it seems to show that independent factorsare involved in overcoming different oligo-sporogenous blocks at least in the pairs ofmutants we have examined.The results with the double mutants gener-

ally support the validity of the contention thatif such a strain is constructed, the sporulationphenotype will be that of the earlier mutation.Thus, all the double mutants which carried thestage 0 mutation Y13 exhibited the stage 0phenotype. Similarly, when the double mutantcontained a mutated stage II gene and a muta-tion producing a block at any later stage, theprevailing phenotype was stage II (see Table 3).

In light of these results, it seems justifiable touse this method to order, at least tentatively,the mutations affecting stage II. The resultssummarized in Table 3 indicate that the muta-tion which blocks sporulation after the forma-tion of a single spore septum (e.g., N25) pre-

cedes the expression of the mutation P18 whichcauses septa and cell wall to be laid down atboth ends of the cell. From the data a compositediagram (Fig. 9) has been drawn to indicate thetemporal order of expression of sporulationgenes. For mutations blocking sporulation be-fore stage III the order is unambiguous and canbe readily inferred, but the placing of latersporulation genes is more difficult. The results(Table 3) indicate that the gene containing mu-tation P20 should be placed after that contain-ing Y10 (stage III). This is because the doublemutant exhibits the phenotype of Y1O both mor-phologically and biochemically. However, thetemporal position of P20 relative to X8 (stageIV) is not clear. In the double mutant the coatmaterial, instead of forming a mass in the cyto-plasm of the mother cell as it does in mutantP20 (Fig. 7) has now apparently settled outaround the developing prespore so that the re-sulting product differs from both parent pheno-types and is more nearly "normal" than either(Fig. 8). The failure of the double mutant to re-produce the phenotype of either one parent orthe other might be attributed to the fact thatthe coat protein is not part of the primarysequence of events. Instead, and this wouldappear to follow from the work of Wood (17), itis a side product, formed during stage III, whichre-enters the main sequence after a time (seeIntroduction). The result in a double mutantmight then be a phenotype in which bothaberrations are expressed, but expressed inaltered form. For morphological reasons, how-ever, expression of P20 is shown as precedingthat of X8 in Fig. 9.An interesting result was obtained with the

double mutant W5/X8 in which the phenotypewas typically stage III, i.e., an earlier stage thaneither of the parent strains. This is what wouldbe anticipated if there were simultaneous ex-pressions of two mutated genes (see Introduc-tion). Thus, it is very probable that several geneproducts are necessary for the successful transi-tion from stage III to stage IV. Failure toproduce one of these might lead to the appear-ance of feebly refractile, stage IV mutants,whereas failure of two products simultaneouslywould prevent even this amount of developmentand the phenotypic appearance would be that ofa stage III mutant. We have therefore beenunable to assign an order of expression for genescontaining mutations W5 and X8 and haveassumed tentatively that they are expressed atthe same time (Fig. 9).The double mutant W1O/X8 behaved simi-

larly, but in this case a clear distinction could bemade because W10 produces dipicolinic acid.

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FIG. 9. Composite schematic diagram indicating the probable order of expression of sporulation genes. It wasobtained by considering the phenotypes of constructed double mutants and also the biochemical properties ofthe parent strains. The parent strains are described in Tables 1 and 2 and the double mutants in Table 3 (seealso Fig. 1-8). Stages of spore development are indiated by roman numerals. Numbers in brackets refer tobiochemical or morphological properties as follows: (1) protease, (2) phosphatase, (3) glucose dehydrogenase,(4) refractility (note that none of the mutants developed more than feeble refractility and appeared gray ratherthan bright in the phase contrast microscope), (5) dipicolinate, (0) indicates that the mutant did not exhibitany of the indicators of sporulation.

This characterizes it as being blocked later thaneither W5 or X8.

It will be apparent from this discussion thatthe method of constructed double mutants isprobably of limited value in assigning an orderto late sporulation genes. However, for muta-tions producing blocks up to stage III or IV theresults are much more clear-cut and the assign-ment of an order of gene expression is inagreement with the order indicated on biochem-ical grounds.

Finally, if we accept that exclusion of onemutant sporulation phenotype by another indi-cates the sequential order of gene expression, wecan use Fig. 9 to obtain an estimate of thenumber of dependent sequential steps involvedin the process. The order of expression is as

follows: (i) Y13, (ii) DG2, (iii) N25, (iv) P18,etc., and the number of sequential steps isabout 12. It should be noted that this is aminimum estimate which might be greatlyincreased by examining the properties of moreconstructed double mutants. Nevertheless, forreasons given in this paper and also by Piggot(8), the total number of sequential steps isalmost certain to be substantially less than thetotal number of operons concerned in sporula-tion.

ACKNOWLEDGMENTSWe thank D. Kay for advice on electron microscopy and P.

J. Piggot for many helpful discussions. We are greateful to D.Torgersen for skilled technical assistance. The work wassupported by the Science Research Council of England.

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TEMPORAL ORDER OF EXPRESSION OF SPORE GENES

LITERATURE CITED

1. Coote, J. G. 1972. Sporulation in Bacillus subtilis.Characterization of oligosporogenous mutants andcomparison of their phenotypes with those of asporoge-nous mutants. J. Gen. Microbiol. 71:1-15.

2. Coote, J. G. 1972. Sporulation in Bacillus subtilis.Genetic analysis of oligosporogenous mutants. J. Gen.Microbiol. 71:17-27.

3. Dawes, I. W., D. Kay, and J. Mandelstam. 1971. Deter-mining effect of growth medium on the shape andposition of daughter chromosomes and on sporulationin Bacillus subtilis. Nature (London) 230:567-569.

4. Ionesco, H., J. Michel, B. Cami, and P. Schaeffer. 1970.Genetics of sporulation in Bacillus subtilis Marburg. J.Appl. Bacteriol. 33:13-24.

5. Kay, D., and S. C. Warren. 1968. Sporulation in Bacillussubtilis. Morphological changes. Biochem. J.109:819-824.

6. Mandelstam. J. 1969. Regulation of bacterial spore for-mation. Symp. Soc. Gen. Microbiol. 19:377-402.

7. Marmur, J. 1961. A procedure for the isolation of deoxyri-bonucleic acid from micro-organisms. J. Mol. Biol.3:208-218.

8. Piggot, P. J. 1973. Mapping of asporogenous mutations ofBacillus subtilis: a minimum estimate of the number ofsporulation operons. J. Bacteriol. 114:1241-1253.

9. Ryter, A. 1965. Etude morphologique de la sporulation deBacillus subtilis. Ann. Inst. Pasteur 108:40-60.

10. Ryter, A., P. Schaeffer, and H. Ionesco. 1966. Classifica-tion cytologique, par leur stade de blocage, des mutantsde sporulation de Bacillus subtilis Marburg. Ann. Inst.

Pasteur 110:305-315.11. Schaeffer, P., and H. Ionesco. 1960. Contribution a

l1'tude genetique de la sporogenese bacterienne. C. R.Acad. Sci. 251:3125-3127.

12. Schaeffer, P., H. Ionesco, A. Ryter, and G. Balassa. 1965.La sporulation de Bacillus subtilis: etude genetique etphysiologique. Colloq. Int. Centre Nat. Rech. Sci., p.553-563.

13. Sterlini, J. M., and J. Mandelstam. 1969. Commitmentto sporulation in Bacillus subtilis and its relationshipto development of actinomycin resistance. Biochem. J.113:29-37.

14. Spizizen, J. 1965. Analysis of asporogenic mutants inBacillus subtilis by genetic transformation, p. 125-137.In L. L. Campbell and H. 0. Halvorson (ed.), SporesIII. American Society for Microbiology, Ann Arbor,Michigan.

15. Waites, W. M., D. Kay, I. W. Dawes, D. A. Wood, S. C.Warren, and J. Mandelstam. 1970. Sporulation inBacillus subtilis. Correlation of biochemical eventswith morphological changes in asporogenous mutants.Biochem. J. 118:667-676.

16. Wise, J., A. Swanson, and H. 0. Halvorson. 1967.Dipicolinic acid-less mutants of Bacillus cereus. J.Bacteriol. 94:2075-2076.

17. Wood, D. A. 1972. Properties and time of synthesis ofalkali-soluble protein of the spore coat. Biochem. J.130:505-514.

18. Young, I. E. 1964. Characteristics of an abortively di-sporic variant of Bacillus cereus. J. Bacteriol. 88:242-254.

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