Statistical Estimate of the Total Number of Operons Specific for ...

JOURNAL OF BACTERIOLOGY, Sept. 1974, p. 684-690 Copyright 0 1974 American Society for Microbiology

Vol. 119, No. 3 Printed in U.S.A.

Statistical Estimate of the Total Number of Operons Specific for Bacillus subtilis Sporulation D. HRANUELI,l P. J. PIGGOT,2 AND J. MANDELSTAM Microbiology Unit, Department of Biochemistry, University of Oxford, Oxford OXI 3QU, United Kingdom

Received for publication 2 May 1974

As an alternative to exhaustive mapping, an attempt has been made to obtain a rough estimate of the total number of sporulation operons by statistical treatment. Sixteen sporulation mutants taken at random were characterized biochemically and morphologically. The mutations they contained were mapped to determine whether they fell into any one of 23 known operons. From the proportion that do so ('0A6), it is calculated that the most probable number of sporulation operons is 37 (68% confidence limits of 31 and 46). If allowance is made for the fact that two of the operons apparently contain mutagenic "hot spots" and the calculation is amended accordingly, the most probable numbers of operons becomes 42 (limits 33 and 59).

In considering the regulation of bacterial spore formation, it is obviously important to have some idea of the complexity of the process as indicated by the number of operons specifically concerned with sporulation. An attempt to obtain a minimal estimate of this number was made by Piggot (19), who mapped 37 characterized sporulation mutations in Bacillus subtilis 168. Sporulation-specific mutations do not affect vegetative growth and are presumed to be in genes that are only expressed during sporulation. Thus, each gene identified by a sporulation mutation must have a "switch-on" mechanism that activates it during spore formation. Several adjacent genes may share the same mechanism. If they are closely linked and concerned with the same stage, we have assumed, to obtain a minimal estimate, that they do share the same mechanism. Each cluster of genes with its own switch-on mechanism is called a sporulation operon (23). To make a minimal estimate of the number of operons, the following three criteria were adopted as indications that two Spo mutations were in separate operons: (i) if they were separated by an auxotrophic marker, (ii) if they were unlinked by transformation, (iii) if they were concerned with different stages of sporulation and, thus, expressed at different times. On the basis of these criteria, the 37 mutations were 'Present address: Research Department, PLIVA Pharmaceutical & Chemical Works, Zagreb, Yugoslavia. 2Present address: Microbiology Division, National Institute for Medical Research, Mill Hill, London, NW7 1AA, United Kingdom. 684

found to fall into 16 separate operons (19). In addition, the mapping data published by others (4, 8, 11, 13, 20, 25, 26) indicate 12 additional operons which were almost certainly different, making a total of 28 in all. At this stage, there seemed to be two ways of following up the work. The first was to continue to isolate and characterize mutants and map the Spo genes until the map was saturated. The alternative approach, which we adopted, was to map a limited number of randomly isolated mutations, ascertain what proportion of them fell into known operons, and hence calculate the probable number of operons that would be discovered by further mapping. It should also be possible from the results of such a limited investigation to calculate how many more mutations would need to be mapped to provide a value for the probable number of operons to a specified degree of accuracy. For this work, we had to disregard the additional operons indicated by the work from other laboratories because the relevant mutants were not immediately available to us. However, those mutants which were isolated by Coote (4) in this laboratory and which lie in distinct operons have been included in the study. This gives a total of 23 "known" operons. Sixteen new mutants were characterized and the Spo mutations were mapped. Of these, 10 fell into the 23 known operons. From the raw data, it can be calculated that the number of operons is 37 (68% confidence limit of 31 and 46). However, account needs to be taken of the fact that some operons are likely to be more

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mutable than others because they contain more ratio of Spo+/leu+ transformants for Spo-2 as donor. because they contain "hot spots." If At the same time, a similar cross is performed but this is done, the estimate has to be revised, and with Spo-3 leu+ as the donor and where Spo-3 is not to Spo-1. the probable number of operons becomes 42 linked The RI is given by the formula RI = [Spo+/leu+(68% confidence limits of 33 and 59). (The (Spo-2 as donor) 1[Spo+/leu+(Spo-3 as donor)]. If theoretical possibility exists that there are oper- Spo-1 and Spo-2 are not linked by transformation, ons that do not mutate at all; if they exist, these then the RI is 1.0. If Spo-1 and Spo-2 are in contiguous obviously have to be disregarded.) Finally, it genes, the RI is about 0.1 (2, 16). can be calculated that to obtain an estimate to A minimum of 100 prototrophic colonies was scored within one operon over 1,000 mutants would in each cross, and in the control crosses a minimum of 100 Spo+ colonies was scored. have to be characterized and mapped. genes or

MATERIALS AND METHODS B. subtilis 168 trpC2 was used as the standard strain and is referred to as the wild type. The other auxotrophic strains used are listed in Table 1. All these strains were Spo+. Strains that carry sporulation mutations used to identify known operons are listed in Table 2. New asporogenous mutants were obtained after mutagenesis of exponentially growing cultures of the wild type or the isogenic derivative MB21 with ethyl methane sulfonate or N-methyl-N'-nitro-N nitrosoguanidine (3). Mutagenized cultures were plated on minimal agar with the appropriate amino acid supplement and incubated for 4 days at 37 C. Colonies having abnormal pigmentation were picked and examined with a phase-contrast microscope. Those colonies containing few, or no, free spores were reisolated on nutrient agar. If this did not enhance their sporulation, they were considered, presumptively, to be Spo mutants. They were tested on lactate as the sole carbon source to ensure that they were not defective in the tricarboxylic acid cycle (11). In addition, any that did not have the same growth rate as the wild type were rejected'. The strains were made isogenic as described previously (19). All of the strains were transformable to Spo+ at frequencies, indicating that each strain had a single Spo mutation, although the possibility that they carried two Spo mutations very closely linked by transformation cannot be excluded. The 16 mutants with their sporulation phenotypes and the regions of the map in which they are located by transduction are listed in Table 3. Genetic mapping. Genetic mapping was done by transduction with phage PBS1 and by transformation as described previously (19). Crosses between strains carrying Spo mutations. Where possible, crosses between Spo mutations were carried out as described previously (19). The oligosporogenous strains could often only be used as donors, and then it was necessary to use deoxyribonucleic acid purified from the donor (17), rather than deoxyribonucleic acid released by stationary-phase cultures (7). Under these conditions, the selection with chloroform (9) was satisfactory. The distance between adjacent mutations Spo-1 and Spo-2 was established by the recombination index (RI) method (15). An unlinked auxotrophic marker, leu-8, metC3, or trpC2, was used as internal standard. For example, a recipient Spo-1 leu-8 is transformed by a donor Spo-2 leu+. Spo+ and leu+ transformants are selected separately. This leads to a

Procedure for obtaining sporulation. The procedure for obtaining sporulation was the resuspension TABLE 1. Auxotrophic strains used Genotype

Strain

Origin

BD70 metC3 BD112 cysA14 GSY254 lys-1 trpC2 MB3 phe-12 MB21 metC3 leu-8 thialaR

D. Dubnau D. Dubnau C. Anagnostopoulos This laboratory This laboratory

TABLE 2. Phenotype and linkage by transduction of Spo mutations: previously described mutations that identify known operons Strain

Type

E10

II

E13 E22 E31 E34 NG1.67

IV 0 IV III III

NG6.21 NG7.17 NG12.12

0 0 II

NG14.7 NG17.17 NG17.23 NG20.12 P7 PlO W5 W10

III III IV II IV 0 Late, abnormal V

X8

IV

Y13 Z5

0

Z33A

IV

IV

Linkedmarker auxotrophic

Source

metC3 (3) ura-1 (83)a lys-1 (11) cysA14 (95) phe-12 (13) lys-1 (50) metC3 (1) ura-1 (36) lys-1 (43) cysA14 (96) metC3 (5) ura-1 (83) lys-1 (48) lys-1 (53) trpC2 (93) cysA14 (95) Iys-1 (66) lys-1 (54) metC3 (11) ura-1 (55) metC3 (19) ura-1 (46) leu-8 (61) phe-12 (97) Iys-1 (70) leu-8 (57) phe-12 (90) leu-8 (49)

Piggot (19) Piggot Piggot Piggot Piggot Piggot Piggot Piggot Piggot

Piggot Piggot Piggot Piggot Coote (4) Coote Coote Coote Coote Coote Coote

Coote

phe-12 (89) a Linkage, expressed as percent co-transduction with the auxotrophic marker, is given in parenthesis.

HRANUELI, PIGGOT, AND MANDELSTAM

686

J. BACTERIOL.

TABLE 3. Phenotype and linkage by transduction of of these, 81 and 83, were co-transformed with Spo mutations: newly isolated mutations cysA14 (75 and 73%, respectively) and were Strain

Type

34 79 81 82 83 85

0 III 0 III 0 Late, abnormal III 0 IV V III IV IV 0 II" IIP

86 87 88 89 90 91 92 93 95 96

Linked auxotrophic markers

lys-1 (50) a lys-1 (54) trpC2 (25) cysA14 (96) ura-1 (47) cysA14 (98) ura-1 (83)

lys-1 (42) trpC2 (16) cysA14 (49) leu-8 (70) phe-12 (95) lys-1 (61) lys-1 (52) trpC2 (29) leu-8 (51) phe-12 (84) leu-8 (12) phe-12 (30) cysA14 (48) cysA14 (86) metC3 (49) ura-1 (21)

a Linkage, expressed as percent co-transduction with the auxotrophic marker, is given in parenthesis. b Abortively disporic.

method of Sterlini and Mandelstam (24), except that L-alanine (1.58 g per liter) replaced DL-alanine. Electron microscopy. After the organisms had been in sporulation medium for 6 to 7 h, the cells were fixed and sections were prepared as described by Kay and Warren (14). Stages II to V are the recognized morphological stages of spore formation (21). It is often difficult to distinguish between stages 0 and I (3); in this paper we have designated as stage 0 mutants those that are blocked before the formation of the spore septum. Media. Media have been described previously (19).

RESULTS The 16 newly isolated strains were all shown to have single Spo mutations linked by PBS1mediated transduction to one of four auxotrophic markers, cysA14, ura-1, phe-12, or lys-1 (Table 3). Each strain was then tested to determine whether the mutation fell into a Spo operon previously shown to be linked by transduction to the same auxotrophic marker. Spo mutations linked to cysA. Five mutations were linked by transduction to cysA14 (Table 3). One of these, 95, caused a block at stage II, giving the abortively disporic phenotype. This mutation was 48% co-transformed with cysA14. It was crossed by transformation with NG20.12 as recipient. This latter is a mutant with a similar phenotype; the mutation maps near cysA14 (19). The RI was 0.017, and mutation 95 thus is presumed to lie in the same operon as NG20.12. The other four mutations, 81, 83, 87, and 93, caused a block at stage 0. Two

linked to each other by transformation (RI = 0.04). The other two mutations, 87 and 93, were less strongly linked by transduction to cysA14, and were not linked by transformation. They were linked to each other in transformation crosses (RI = 0.39), but not to the first pair. One stage 0 operon has been located in this region (19) and is identified by mutations E22 and NG717. These last mutations had RI values of 0.11 and 0, respectively, with mutation 81. Thus, mutations 81 and 83 are considered to lie in a known operon, whereas mutations 87 and 93 define a new stage 0 operon. Spo mutations linked to ura. Three mutations, 96, 82, and 85, were linked to ura-1 by trafnsduction (Table 3). Mutation 96 caused a block at stage II, resulting in the abortively disporic morphology. By RI values it lies in a third stage II operon, distinct from the two other stage II operons identified previously in this region (19). Mutation 82 (stage III) lies in the known stage III operon (RI = 0.095 with NG1.67). Mutation 85 (abnormal, late) was not linked by transformation to the stage III mutations. Rather, it was linked (RI = 0.053) to the mutation W5 described by Coote (3) as having a similar phenotype. Both mutations were also linked (less strongly; RI = 0.40) to mutation W10, being blocked at stage V (3). All three are considered to lie in a single late operon designated V. Spo mutations linked to phe. Two stage IV mutations, 88 and 91, were strongly co-transduced with phe-12 (Table 3). Both mutations were also co-transformed with phe-12, 88 to 73% and 91 to 18%, respectively. These were crossed by transformation with the three strains blocked at this stage and shown by Coote (4) to carry mutations strongly co-transduced with phe-12; these mutations are, by our criteria, in a single operon. Mutation 88 probably also lies in this operon, and mutation 91 identifies a new operon (Table 4). Since both mutations were linked to phe-12 by transformation, it seemed likely that they lay on opposite sides of phe-12. This was confirmed by three-factor transduction crosses (Table 5), which indicated that the order is: leu-8-Spo-88-phe-12-Spo-91. This gives a different position for the stage IV operon represented by mutation 88 from that indicated by two-factor transduction crosses (4). A third stage IV mutation, 92, was weakly linked to phe-12 by transduction (Table 3) and was not linked to it by transformation. Threefactor transduction crosses were consistent with the order leu-8-phe-12-Spo-92. However, in

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SPORULATION OPERONS IN B. SUBTILIS

687

transformation crosses, mutation 92 was linked neither to mutation 91, nor to the other group of stage IV mutations (represented by E31) mapping on the same side of phe-12 (Table 6). Therefore, mutation 92 identifies a new stage IV operon. One mutation, E13, had been shown to be co-transducible with lys-1 but not with phe-12 and so had been considered to lie in a separate operon from E31 (19). However, the transformation data (Table 6) clearly indicate that these mutations are closely linked and probably lie in the same operon. Spo mutations linked to lys. One stage 0 mutation, 34, was linked to lys-1 by transduction (Table 3). It was closely linked by transformation (RI = 0.19) to the stage 0 mutation NG6.21 previously mapped in this region (19).

The stage 0 mutations P10 and Y13, described by Coote (3), had RI values of 0.04 and 0.03, respectively, with NG6.21, so that all are considered to lie in a single stage 0 operon. (A large group of stage 0 mutations, including P10 and Y13, was originally considered to be in a different operon from the stage 0 mutation NG6.21 because of the latter's distinctive phenotype [19]; the RI values reported here show that NG6.21 and the other stage 0 mutations could easily lie in one operon.) Three of the mutations that were co-transducible with Iys-1 caused a block at stage III (Table 3). In crosses by transformation, all were linked to mutations E34 and NG14.7 which are in a known stage II operon (Table 7). One mutation, 89, found to be co-transducible with lys-1, was blocked at stage V. No stage

TABLE 4. Recombination indexes (RI) from transformation crosses between Spo mutations (stage IV) mapping in the phe region

TABLE 6. Recombination indexes (RI) from transformation crosses between late-blocked Spo mutations mapping in the phe-lys region

RI

Donor 88a (metC3)"

91a

Donor

91 (trpC2)

trpC?

X8 Z5 Z33A

0.028 0.031 0.033

1.1 1.2 1.0

91

0.99

0.0

89 92 E13

1.2

E31 trpC2

RI E13 trpC2

P7 NG17.23 trpC2 metC3

0.99 0.89 0.004

1.4 0.93 0.0

0.95 1.26 1.08

0.87

a Recipient. b Auxotrophic marker used in the cross.

a Recipient. b Auxotrophic marker used in the cross.

TABLE 5. Three-factor transduction crosses of Spo mutations with phe-12 and leu-8 Selected Donor marker Lsonor 111w neclplent tuw) marKer (111) (000) Recipient

No. of colonies in recombination classes

(1--)

100 101 110 111

leu+phe+Spo-88

leu-8 phe-12 Spo+

leu+

37

phe+leu+Spo-88

phe-12 leu-8 Spo+

phe+

5

leu+phe+Spo-91

leu-8 phe-12 Spo+

leu+

51

phe+leu+Spo-91

phe-12 leu-8 Spo+

phe+

7

leu+phe+Spo-92

leu-8 phe-12 Spo+

leu+

19

phe+leu+Spo-92

phe-12 leu-8 Spo+

phe+

24

Possible ordera oraera

leu-phe-Spo (5) leu-Spo-phe (2) phe-leu-Spo (-) 34 2 89 phe-leu-Spo (34) phe-Spo-leu (2) leu-phe-Spo (-) 1 16 62 leu-phe-Spo (1) leu-Spo-phe (16) phe-leu-Spo (-) 37 14 70 phe-leu-Spo (37) phe-Spo-leu (14) leu-phe-Spo (-) 0 62 16 leu-phe-Spo (0) leu-Spo-phe (62) phe-leu-Spo (-) 14 69 22 phe-leu-Spo (14) phe-Spo-leu (69) leu-phe-Spo (-) 5

2

87

Suggested

~~~~~~order

leu- (Spo) phe phe-Spo-leu leu-phe-Spo leu-phe-Spo leu-phe-Spo

leu-phe-Spo

a Numbers in parenthesis indicate the number of transductants in the class requiring multiple crossovers for the particular order. Where multiple crossovers are undetectable, this is indicated by a dash.

688

HRANUELI, PIGGOT, AND MANDELSTAM

TABLE 7. Recombination indexes (RI) from transformation crosses between stage III Spo mutations mapping in the lys-1 region RI

Donor

79 86 90

E34a trpC2b

NG14.7 trpC2

0.062 0.035 0.14

0.41 0.36 0.29

a Recipient.

Auxotrophic marker used in the cross.

V mutation has previously been mapped in this region, so that by our criteria this represents a new operon. However, since the morphological state reached by late mutants can be difficult to classify as a particular stage (see 3, 5), mutation 89 was crossed by transformation with stage IV mutations mapping in the region. It was not linked to any of them (Table 6) and, therefore, identifies a new (stage V) operon.

DISCUSSION From the mapping data it appears that of the 16 mutations mapped, 10 fall into known operons and 6 appear in new positions. The probability, p, of a new mutation mapping within one of the known operons is thus 0.625, and the probability, q, of falling in a new operon is 0.375. The stadard error is given by:

J. BACTERIOL.

after) are not as easily distinguished from the wild type as colonies of early-blocked mutants, so the possibility had to be considered that our sample might be biased against late mutants. However, Balassa (1) has reported that in a large screening of colonies with the same pigmentation as the wild type no Spo- colonies were detected; this is also our experience. The second assumption is that all mutations occur with equal probability in any operon. In practice, this is likely to be untrue for at least two reasons. First, the greater the number of nucleotide pairs in an operon, the more likely it is to undergo mutation; second, it is well known from other genetic systems that there are hot spots at which mutation is more frequent than elsewhere. With these possibilities in mind, we have combined the original data reported by Piggot (19) for 37 mutations and the information relating to the 16 new mutations. Figure 2 shows a plot of the relative incidence of mutations in sporulation operons. (The data of Coote [4] have been ommitted because the mutations have not been studied in sufficient detail for 95

81 84

85 82 W5

NG717

C20 12

TT

v

E22

O

\ 1/

W10

n11 11

nv

hiSA

I~

y

cysB

v

o

II

87 93

0.375 =

A

NGI 67 E10 NG1212 n fi o ICU

96

0.121

n1

34

88

where n is the number of new mutations studied. If we assume that the true value of P lies between 0.625 a (for confidence limits of 68%), the upper and lower estimates for p become 0.504 and 0.746. The number of known operons is 23. Of these, 22 are identified by Spo mutations that have been linked to a marker on the genetic map of B. subtilis (Fig. 1). An additional operon is identified by mutations showing no linkage to any marker (4, 10). The corresponding estimates for the total number of operons are that the most probable number is 37 (equal to 23/0.625), with 68% confidence limits of 31 and 46. This calculation is based on two assumptions. The first is that the 16 mutations studied are a random sample of all the possible Spo mutations. In practice, the Spo- strains were detected as colonies that had a different pigmentation from the wild type on minimal agar (12, 22). Millet and Ryter (18) have noted that colonies of late-blocked mutants (stage IV and

79 86

90

B

X88

Z5 Z33A Ea

P1O

E13 E31

-II

,V

1i

Y13 NG147 NG621 E34 P7 O FF,, -v

NG17 23

B,

rv

I~~~~~~~~y5 lV 91

lY

Y

92

89

FIG. 1. Location on the B. subtilis genome of sporulation operons considered in this study. For clarity, the linkage map of B. subtilis (6, 27) has been divided into two portions: (A) purA to leu and (B) leu to trpC. Operons are assigned the stage of sporulation reached by strains carrying mutations in the operon. The mutations used are also indicated. The 22 operons previously mapped and used as the basis for this study are placed above the line. The five new operons are placed below the line. In (B), the phe-lys region, the positioning of the operons is based on co-transduction frequency with phe-12 or lys-1. Where the orientation of a sporulation operon relative to the linked auxotrophic markers is not known, it is drawn as mapping at that marker. For clarity, portion (B) has been considerably expanded, and the distances between markers are not comparable with those in (A).

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c 0

0-

C

0

c

SPORULATION OPERONS IN B. SUBTILIS

total number of operons are 41 anfd 43. For these limits, cr is defined as: 21/(0.5 + a) > 41 and 21/(0.5 - a) < 43, where 0.5 + a < 0.513 and 0.5 - a > 0.488, respectively. Thus, for the specified accuracy, a < 0.012. Now 0.5 x 0.5 < 0.012 =

12 11 10 -

987

0

a L-

o a Ln

n

65

so that n 1,700. Alternatively, the question that could be asked is to what extent the accuracy of the estimate would have been improved if double the number of mutations had been examined and mapped. If the calculation is made, the probable limit (68% confidence) becomes 32 to 43 for the first calculation, and 35 to 53 for the calculation that allows for hot spots. It is apparent that, although the mapping of a much larger number of mutations would improve the accuracy somewhat, the estimation of the number of operons by exhaustive mapping is not practical. To summarize, the most probable number of operons specifically concerned with sporulation appears from this work to be about 40. Of these, 23 have been identified by combining the data of Piggot (19) and Coote (4), and five new operons are identified in this paper (Fig. 1). Five additional operons, identified by the mapping data of other laboratories, have been listed by Piggot (19). The total is 33, and we can thus hope that not many more sporulation operons remain to be described. -

432-

E

D

z

689

1-~

I I

I

1 2 3 4 5 6 7 8 9 Number of mutations in each Sporulation Operon

FIG. 2. Bar diagram indicating the relative incidence of mutations in sporulation operons.

inclusion in this graph, although they clearly establish several distinct operons.) The operons in the largest class (12) were each "hit" once. The number hit more frequently then falls off rapidly, leaving, however, one class representing two operons which were very unusual in having nine hits in each. It is likely that these belong to a separate category that is particularly susceptible to mutation. If, among the 16 new mutations that we are considering, we exclude those which fall in the two highly mutable operons (mutants 79, 86, 90, and 95), we are left with a total of 12 mutations, 6 of which map within the remaining 21 known operons.

On this basis, the working value for p then becomes 0.50 (i.e., 6/12), and the value of a becomes 0..144. The corresponding estimates for the number of operons give the most probable value as 42 (21/0.5), with upper and lower limits of 33 and 59. It will be seen that the exclusion of mutations falling in highly mutable regions has, in fact, not affected the estimate unduly, the 68% confidence limits having changed from 31-46 to 33-59. A more sophisticated mathematical treatment would not seem to be justified. The question that remains is how many mutants (n) would have to be examined to establish the number of operons to the nearest whole number. For the purposes of calculation, we shall assume that the real number is 42 and that, after mapping n mutations, the proportion mapping in 21 known operons is 0.50. The question then is how large must n be so that the limits (68% confidence) for the estimate of the

ACKNOWLEDGMENTS We are very grateful to W. F. Bodmer, R. W. Hiorns, and M. Young for advice and criticism. We thank T. Hranueli for the electron microscope characterization of many of the mutants, and D. Torgerson for skilled assistance with the fine structure mapping. This work was supported by the Science Research Council, and D. Hranueli was supported by a grant from the Republic Council for Scientific Research, S. R. Croatia. LITERATURE CITED 1. Balassa, G. 1971. The genetic control of spore formation in bacilli. Curr. Top. Microbiol. Immunol. 56:99-192. 2. Carlton, B. C. 1966. Fine-structure mapping by transformation in the tryptophan region of Bacillus subtilis. J.

Bacteriol. 91:1795-1803. 3. Coote, J. G. 1972. Sporulation in Bacillus subtilis. Characterization of oligosporogenous mutants and comparison of their phenotypes with those of asporogenous mutants. J. Gen. Microbiol. 71:1-15. 4. Coote, J. G. 1972. Sporulation in Bacillus subtilis. Genetic analysis of oligosporogenous mutants. J. Gen. Microbiol. 71:17-27. 5. Coote, J. G., and J. Mandelstam. 1973. Use of constructed double mutants for determining the temporal order of expression of sporulation genes in Bacillu.s subtilis. J. Bacteriol. 114:1254-1263.

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HRANUELI, PIGGOT, AND MANDELSTAM

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10. Hoch, J. A., location of in Bacillus 11. Hoch, J. A.,

12.

13. 14.

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