JouRNAL OF BACrmOLOGY, June 1975, p. 1109-1116 Copyright 0 1975 American Society for Microbiology
Vol. 122, No. 3 Printed in U.S.A.
Genetic Mapping of Sporulation Operons in Bacillus subtilis Using a Thermosensitive Sporulation Mutant M. YOUNG Department of Biochemistry, University of Oxford, Oxford OXI 3QU, United Kingdom Received for publication 27 March 1975
A thermosensitive sporulation mutant was used to determine the order of sporulation operons in the ura region of the Bacillus subtilis chromosome. Data from three-factor transformation crosses and three- and four-factor transduction crosses established the order metC-spo-96(SpoIH)-spo-85(SpoV)-spo-279(SpoII)furA-ura-cysC-spo-NG1.67(SpoI). Previously, furA was thought to lie to the right of ura and cysC to the left (Dubnau, 1970; Young and Wilson, 1972). In a recent genetic analysis of a large number of sporulation mutations in Bacillus subtilis, Piggot (10) grouped mutations together into sporulation operons. If two mutations satisfied one (at least) of the following three criteria, they were considered to be in separate operons: (i) they were separated by an auxotrophic marker; (ii) they were unlinked by transformation; (iii) they caused blocks at different stages of sporulation. About 30 operons have now been described (9), and they are clustered into four main groups on the chromosome. The order of some operons in the cluster occurring near the cysA locus has been established with three-factor transformation crosses (10). The ordering of sporulation operons in the other chromosomal regions has been difficult, mainly because of the lack of linked auxotrophic or other suitable markers. Crosses that involve two sporulation mutations and an auxotrophic marker are often difficult to interpret because the various recombinant classes may not be readily distinguished. However, Hoch(7) has described a reciprocal cross method by which the order of two sporulation mutations may be determined relative to a flanking auxotrophic marker. In crosses that involve two sporulation mutations giving rise to distinct colony types and where the earlier-blocked mutation is also conditional, it is possible to distinguish all classes of recombinants, and hence the order of mutations may be determined from a single cross. In this investigation, a thermosensitive stage II sporulation mutant was used to determine the order of sporulation operons in the ura region of the chromosome.
isolated after an exponentially growing culture of the wild type had been treated with N-methyl-N'-nitroN- nitrosoguanidine as described by Coote (1). The mutagen-treated culture was plated on tryptophansupplemented minimal agar and examined after incubation for 3 days at 42 C. Colonies with a different pigmentation from the normal brown color were picked, streaked on minimal agar, and incubated at 30 and 42 C. The thermosensitive mutant 279 gave colonies that were normal at 30 C but were very light brown and contained few spores at 42 C. The mutant was reisolated from nutrient agar and tested to ensure that the growth rate on lactate as sole carbon source was the same as that of the wild type. Deoxyribonucleic acid (DNA) from the mutant was prepared by the technique of Ephrati-Elizur (5) and used to transform strain MB21 to Ieu+ at 42 C. Some 5% of the transformants also inherited mutation spo-279 by congression, (i.e., the simultaneous transfer of unlinked markers that occurs at saturating concentrations of DNA). The resultant isogenic strain 279.1 was found to give colonies almost indistinguishable from those of the wild type at 37 C; this was chosen as the permissive temperature. As the growth temperature was increased, colonies contained progessively fewer spores. At 42 C, the restrictive temperature used, the colonies contained very few spores ( < < 1%) and displayed a light-brown pigmentation quite distinct from that of the wild type. Spontaneous Spo+ revertants of strains bearing the spo-279 mutation have never been observed, although revertants can be induced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. The characterization of strain 279.1 is to be reported elsewhere. The other sporulation mutants used here have been described previously (9, 10). Transduction and transformation. The methods used were as described by Piggot (10); details of individual crosses are to be found in the text. Media. The media used have been described previously (10). 5-Fluorouracil was added to media, as appropriate, at 1 Ag/ml. MATERIALS AND METHODS Selection of spontaneous prototrophic revertants of uracil auxotrophs. Uracil auxotrophs were Strains. The strains used are listed in Table 1. The thermosensitive sporulation mutant 279 was streaked for single colonies on nutrient agar and an 1109
1110
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TABLE 1. Strains of Bacillus subtilis Strain
Genotype
Sporulation phenotype
168 GSY289 GSY753 GSY1441 MB19a MB21a MB49
trpC2 trpC2 ura-1 trpC2 fur-2 cysC7 trpC2 leu-8 rif metC3 leu-8 tal trpC2 cysC7
Spo; Spo+ Spo+ Spo+ Spo+ Spo+ Spo+
MB50
cysC7 ura-1
Spo+
6.4b 36.1b
leu-8 tal spo-ElO metC3 tal spo-NG1.67
Spo IIC Spo In
41. lb
leu-8 tal spo-NG4.14
Spo ld
55.1b
leu-8 tal spo-NG12.12
Spo IIF
71.1b
trpC2 rif spo-NG20.12
Spo IIe
85.1' 96.2'
metC3 tal spo-85 metC3 tal spo-96 cysC7 spo-96 metC3 tal spo-279 cysC7 spo-279 cysC7 fur-2 spo-279
Spo He Spo II' Spo II' Spo II' Spo II'
96.3 279.1a 279.2 279.6
Spo V
Origin
Laboratory wild-type strain C. Anagnostopoulos C. Anagnostopoulos C. Anagnostopoulos P. J. Piggot P. J. Piggot cysC7 (from GSY1441) transformed into GSY289 by congression with ura+ cysC7 (from GSY1441) transformed into GSY289 by congression with trp+ spo-ElO transformed into MB21 by congression with met+ spo-NG1.67 transformed into MB21 by congression with leu+ spo-NG4.14 transformed into MB21 by congression with met+ spo-NG12.12 transformed into MB21 by congression with met+ spo-NG20.12 transformed into MB19 by congression with leu+ spo-85 transformed into MB21 by congression with leu+ spo-96 transformed into MB21 by congression with leu+ spo-96 transformed into MB49 by congression with trp+ spo-279 transformed into MB21 by congression with leu+ spo-279 transformed into MB50 by congression with ura+ fur-2 (from GSY753) transformed into 279.2
a Isogenic
with the wild type except for the markers listed. bStrains isolated by Piggot (10); isogenic with the wild type except for the markers listed. c Abortively disporic, often with two septa at one end and one at the other. dAbortively disporic, originally designated stage 0 (see text). e Abortively disporic. Strains isolated by Hranueli et al. (9); isogenic with the wild type except for the markers listed. 'Prominent mesosomes at both cell poles, subtended by incomplete septa.
inoculum from one colony was grown overnight in Penassay broth. The bacteria were washed and resuspended in a glucose-salts solution, and revertants to uracil prototrophy were selected on unsupplemented minimal agar. Measurement of sporulation alkaline phosphatase activity. Bacteria were grown and sporulated at 42 C by using the resuspension procedure of Sterlini and Mandelstam (12) with the modification used by Piggot (10). Samples of culture (1.0 ml) were removed at approximately hourly intervals and stored at -20 C. Alkaline phosphatase activity in the entire culture samples was measured by the method of Grant (6).
RESULTS Sporulation operons in the ura-l region. Piggot (10) and Hranueli et al. (9) have described 11 sporulation mutations that lie in the ura-I region of the B. subtilis chromosome. These mutations were considered to lie in six operons. The cotransduction frequencies of mutations representing each of these operons with
metC3 and with cysC7 are given in Table 2. These mutations were used to establish the linkage relationships of the new mutation, spo279. In transformation crosses with strains bearing the various spo mutations as recipients and DNA bearing the spo-279 mutation as donor, spo-279 was cotransformed with spo-ElO, spoNG4.14, spo-NG12.12, and spo-85 (Table 3). Three of these mutations, originally considered to be in separate operons, now appear to lie within a single stage II operon. Mutations spo-ElO and spo-NG12.12 were originally considered to lie in separate operons because they appeared to be unlinked in transformation crosses. We have repeated this and demonstrated the cotransformation of these two mutations (recombination index = 0.17). (In crosses with strains bearing the spo-ElO mutation, it seems to be particularly difficult to obtain satisfactory selection for Spo+ recombinants by the chloroform method [81; this may arise from the tendency of this mutation to revert to Spo+.)
TABLE 2. Phenotype and linkage by transduction of mutations in the ura-1 region
Linkage to selected Mutation
spo-ElO spo-NG1.67 spo-NG4.14 spo-NG12.12 spo-85 spo-96 spo-279
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markers
Type
II III II II V f II
metC3
cysC7b
3c 1c 2c 5c 6 (48/860) 49d 5 (34/732)
81 (254/314) 42 (210/506) 84 (198/237) 82 (396/486) 77 (111/144) 17 (92/529) 61 (119/195)
a PBS1 lysate was prepared on the wild-type strain (168) and used to transduce the Spo- metC3 strains to methionine prototrophy. Linkage is shown as the percentage of cotransduction with the numbers of transductants in parentheses [Spo+/(Spo+ + Spo-)]. bPBS1 lysate was prepared on the Spo- prototroph and used to tranaduce the Spo+ auxotroph (strain MB49) to cysteine prototrophy. Linkage is shown as the percentage of cotransduction with the numbers of transductants in parentheses [Spo-/(Spo- + Spo+). c Data from Piggot (10). dData from Hranueli et al. (9).
prototrophy by using PBS1 lysates prepared on the cysteine auxotrophs 279.2 and 96.3, respectively, whereas in the other pair the cysteine auxotrophs were transduced to prototrophy by using PBS1 lysates prepared on the methionine auxotrophs. Colonies that were phenotypically Spo+ at 37 C were screened for temperature sensitivity, and the frequencies of the three phenotypic recombinant classes are given in Table 4. (Recombinants bearing both mutations spo-96 and spo-279 were phenotypically indistinguishable from those bearing spo-96 alone.) If metC3 and cysC7 are flanking markers, then in one cross out of each pair (A + B and C + D), the spo mutation in the donor is the middle marker. In this cross, but not in the other cross in each pair, Spo+ recombinants are only generated by multiple crossovers, and hence they should form a rare class (7). On the TABLE 3. Linkage by transformation of the spo-279 mutation to other spo mutations in the ura-1 region Linkage° Mutation
Type
Spo+
selection"
Mutation spo-NG4.14, which was cotransformed with another mutation in the stage II operon represented here by mutation spoNG12.12, was considered to lie in a different operon because it had been classified as a stage O mutation instead of stage II. (When a strain bearing this mutation was originally examined by electron microscopy, most of the cells appeared to be blocked at stage 0-I (axial filament), although a few stage II cells were seen. When it was re-examined some time later, the majority of cells reached stage II [Hranueli, unpublished data].) Mutation spo-279, which lies in the same stage II operon as mutations spoE10, spo-NG4.14, and spo-NG12.12, was used to represent the operon in the three- and fourfactor crosses. The other three operons that lie in this region are represented by mutations spoNG1.67 (stage I), spo-85 (stage V), and spo96 (stage II). The cotransformation of mutations spo-85 and spo-279 indicates that the two operons which they represent map in close proximity (Table 3). Mapping the stage II operons. The cotransduction frequencies of mutations spo-96 and spo-279 with metC3 and cysC7 (Table 2) suggest the order metC3-spo-96-spo-279-cysC7, the two auxotrophic markers being approximately 130 transduction units apart. This order was confirmed in three-factor reciprocal crosses. In one pair of crosses, the methionine auxotrophs 96.2 and 279.1 were transduced to
spo-ElO spo-NG1.67 spo-NG4.14 spo-NG12.12 spo-NG20.12d spo-85 spo-96
II III II II II V I
ND 2 (3/156) 34 (53/154) 35 (53/153) 1 (2/207) 46 (71/155) 3 (1/31)e
Auxotrophic selectionc
68 (71/104) ND ND 52 (54/103) 9 (9/104) ND 8 (8/99)
aLinkage is shown as the percentage of contransformation with the numbers of transformants in parentheses [Spotm/(Spo" + Spo+) ]. ND, not done. "DNA prepared from strain 279.1 was used at a final concentration of 0.016 ug/ml to transform strains bearing the various spo mutations to Spo+ at 37 C (selection was by chloroform treatment [8]). Spo+ recombinants were tested at 37 and 42 C for inheritance of mutation spo-279. cDNA prepared from strain 279.1 was used at a saturating concentration (16 jg/ml) to transform strains bearing the various spo mutations to leucine or tryptophan prototrophy at 37 C. Approximately 8% of the transformants were simultaneously transformed to prototrophy for the unlinked spo marker, for which there was no selection. The resultant Spo+ double transformants were tested at 37 and 42 C for inheritance of mutation spo-279. d Mutation spo-Ng20.12, which maps in the cysA region (10) and is not linked to mutation spo-279, was used as a control. eStrains bearing mutation spo-96 are oligosporogenous, which made it difficult to obtain satisfactory selection for Spo+ recombinants by the chloroform method. In one experiment considerable enrichment for Spo+ recombinants were obtained by early treatment with chloroform (12 to 14 h after plating).
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basis of this reasoning, the order metC3-spo96-spo-279-cysC7 is established. In the final three-factor cross in Table 4 (cross E), fur-2 was mapped relative to cysC7 and spo-279. Strain 279.6, a 5-fluorouracil-resistant cysteine auxotroph, was transduced to cysteine independence by using a PBS1 lysate prepared on the wild-type strain 168. After incubation at 42 C, colonies were scored for inheritance of the spo-279 mutation and then screened for resistance to 5-fluorouracil. These data (Table 4, cross E) establish the order spo-279-fur-2spo-cysC7, and hence we have metC3 spo-96 279-fur-2-cysC7. According to the genetic map of B. subtilis, cysC7 should lie closer to metC3 than does fur-2 (13). Since the present data suggest an order of markers different from that previously determined, it was important to establish the map position of ura-1. Map position of ura-1. A double auxotroph bearing the cysC7 and ura-1 markers (MB50) was transformed to prototrophy with a strain bearing the fur-2 marker as donor (GSY 753). Transformants to cysteine independence and to uracil independence were selected separately and then screened for inheritance of the unselected markers. From the cross in which cysteine prototrophs were selected, 330 uracil prototrophs and 190 uracil auxotrophs were obtained. Of the 330 uracil protttrophs, 177 -
-
resistant to 5-fluorouracil and 153 were sensitive. The uracil auxotrophs could not be tested directly for 5-fluorouracil resistance, since sensitive strains will grow on media containing uracil in addition to 5-fluorouracil (it has been suggested that analogue resistance at the furA locus is due to enhanced uracil production [11]). The uracil-requiring, 5-fluorouracilresistant recombinants were identified after spontaneous revertants to uracil prototrophy had been obtained. Five ura+ revertants were obtained from each ura- transformant, and when tested for resistance or sensitivity to 5-fluorouracil, all five invariably showed the same phenotype. Of the 190 uracil auxotrophs derived from the cross, only nine gave ura+ revertants that were resistant to 5-fluorouracil. These data are summarized in Table 5 together with the frequencies of recombinants from the cross in which uracil prototrophs were selected and then screened for cysteine requirement and for 5-fluprouracil resistance. These data establish the order fur-2-ura-1-cysC7. Mapping the stage V and stage III operons. Mutation spo-85 (stage V) was mapped in a four-factor transduction cross relative to spo-279, fur-2, and cysC7. A PBS1 lysate prepared on strain 85.1 was used to transduce the 5-fluorouracil-resistant cysteine auxotroph 279.6 to cysteine prototrophy at 42 C. The
were
TABLE 4. Three-factor transduction crosses to map the stage II operons
Donor (111)
Cross
Recipient(000)
Se-
Recombinant classes
Se-e
FrePheno- quency Genotype Clses Reobian
marker
(1- -) A met+ spo-96 spo+ (96.3)a
B met+ spo+ spo-279
(279.2)
C
D cys+ spo+ spo-279 (279.1)
a The
metC3 spo-96 spo+
met+
met+
(96.2)
cys+ spo-96 spo+ (96.2)
E cys + fr (168)
metC3 spo+ spo-279 (279.1)
spoW
cysC7 spo+ spo-279 (279.2)
cys+
cysC7 spo-96 spo+ (96.3)
cys+
cysC7 fur-2 spo-279 (279.6)
strain used is given in parentheses.
cys+
Suggested order
type
Spot Spo+
322 8
Spo:
Spo
22 229
met+ 96 met+ 96 met+ + met+ +
+ Sp 279 Spo
576
100 101 110 111
Cys+ +
279 Spo"
cys+ + cys+ 96 cys+ 96
+
Spo+ 279 Spo+ Spo
139 32 3
100 101 110 111
cys+ 96 cys+ 96 cy8+ + cys+ +
+ Spo279 Spo-
610 6
100 101 110 111
met' met+ met+ met+
100 101 110 111
100 cys+ 101 cys+ 110 cys4 Ill cys+
+ + 96 96
279 + 279 +
+
279
Spo+
SpoU
1
fur-2 279 Spo" fur-2 + SpO fur+ 279 Spo"
46 73
Spo+
-
spo-279
cysC
-
spo-279
-
spo-96
-
fur
71
95
+
spo-96
171 31
Spo+
fur+
-
l
279 Spot
+
metC
73c8 294
-
spo-279
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TABL 5. Three-factor transformation crosses to order the markers ura-1, cysC7 and fur-2 Donor (111) (GSY753)a
Recipient (000)
(MB50)
Selected marker (1- -)
No. of colonies in classes: recombinant 100
101 110 111
Number of transforPossible order mants classesin requiring multiple
Suggested order
crosovers
cys + fur-2 ura+
cysC7fur+ ura-1
cys+
181 153
9 177
cys-fur-ura
fur-cys-ura fur-ura-cys ura+ fur-2 cys+
ura-1 fur+ cysC7
ura+
97 152 79 189
cys-fur-ura
fur-cys-ura fur-ura-cys a The
153 b
fur-ura-cys
152 79 __
fur-ura-cys
9
strain used is given in parentheses. crossovers are undetectable with this order.
b Multiple
transductants were screened for inheritance of the fur-2 marker. Three colony types were discemible on the plates at 42 C. All transductants bearing the spo-279 mutation gave lightbrown Spo- colonies, those bearing mutation spo-85 alone gave dark-brown Spo- colonies, and those bearing neither mutation gave Spo+ colonies. Transductants bearing both mutations spo-279 and spo-85 were separated from those bearing spo-279 alone by screening at 37 C, when the former gave dark-brown Spo- colonies whereas the latter gave Spo+ colonies. The frequencies of the eight recombinant classes are given in Table 6. Four of the eight classes were rare, and these represent those classes requiring multiple crossovers for the order cysC7-fur-2spo-279-spo-85. The possible orders cysC7spo-85-fur-2-spo-279 and cysC7-fur-2-spo-85spo-279 may be dismissed since the frequency of recombinants in certain of the classes requiring multiple crossovers is too high. The remaining possibility is the order spo-85-cysC7-fur-2spo-279, and this is improbable for two reasons: first, mutations spo-85 and spo-279 are cotransformed (Table 3), and second, two of the observed rare classes would not be expected for this order. The order of markers inferred from these data is cysC7-fur-2-spo-279-spo-85. Mutation spo-NGI .67 was mapped in a fourfactor cross analogous to that described above. The eight recombinant classes given in Table 6 were not all simply identified from their ability to grow on plates containing 5-fluorouracil and their colony morphologies at 37 and 42 C. Unfortunately, recombinants bearing both mutations spo-NG1.67 and spo-279 had a colony type that was identical to those bearing mutation spo-NG1.67 alone, and hence they could not be separated from each other. The distinction was made by monitoring the produc-
tion of alkaline phosphatase during sporulation in liquid medium at 42 C. Synthesis of this enzyme seems to depend upon the successful completion of the spore septum. Mutants blocked at stage HI or later (and, of course, the wild type) show a burst of enzyme synthesis 2 to 3 h after resuspension, whereas mutants blocked at stage II or earlier generally show little or no enzyme production. Thus, recombinants bearing mutation spo-NG1.67 alone showed the normal period of rapid alkaline phosphatase synthesis 2 to 3 h after resuspension at 42 C, whereas recombinants bearing both mutations spo-NG1.67 and spo-279 showed the greatly reduced rate of enzyme production that is characteristic of strains bearing the spo-279 mutation. The only order of markers that is consistent with the data in Table 6 is spo-NG1.67-cysC7-fur-2-spo-279; all other possible orders may be dismissed since the frequencies of recombinants in certain of the classes requiring multiple crossovers is too high. To simplify the treatment of the mapping data in Table 6, it has been assumed throughout that the order of markers in the recipient strain is spo-279-fur-2-cysC7, as was established in Table 4, cross E. Even without this simplifying assumption, the data from the two crosses are not consistent with any other possible order and provide independent confirmation of the previously established order. DISCUSSION The thermosensitive sporulation mutant 279 has been used to determine the orientation of the four sporulation operons known at present in the ura region of the B. subtilis chromosome. There were previously thought to be six sporulation operons in this region (9), but for the reasons given earlier, this number should now
1114
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1115
SPORULATION OPERONS IN B. SUBTILIS
VOL. 122, 1975
Spo-ElO Spo- NG4b Spo-W50 Spo - NG 10.2 Spo-W0l Spo-NG12.12 Spo-96 Spo-85 Spo-279
SpO- 82
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9-' 29 -a64 FIG. 1. Map of the ura region of the B. subtilis chromosome. Sporulation operons are assigned the stage of sporulation reached by strains carrying mutations in the operon. The mutations that lie in each operon appear above it; those mutations not mapped in this paper are denoted (a) Hranueli et al. (9) and (b) Piggot (10). The sporulation operons are approximately positioned according to the cotransduction frequencies of the mutations chosen to represent them (shown in bold type) with metC3, ura-1, and cysC7. Distances are given as recombination values (100 cotransduction frequencies); these data are taken from (c) Hranueli et al. (9), (d) Table 2, (e) Table 6, (f) Young (unpublished data), and (g) Piggot (10). -
be reduced to four (accepting the criteria given by Piggot [10] to define a sporulation operon). The relative positions on the chromosome of these four operons are given in Fig. 1. Although the location of the stage V operon between the two stage II operons has not been confirmed in crosses with the metC3 and spo-96 markers which lie to the left, this assignment seems justified since mutation spo-85 (stage V) is strongly cotransformed with mutation spo-279 (stage II), whereas mutations spo-96 (stage II) and spo-279 are unlinked by transformation (Table 3). The mapping of sporulation operons was facilitated by the conditional sporulation phenotype of strains bearing the spo-279 mutation. At 42 C the cells are blocked at stage II, whereas sporulation is normal on solid media at 37 C. This thermosensitivity was particularly useful in mapping mutations spo-NG1.67 and spo-85 (stages III and V, respectively). Coote and Mandelstam (2) have shown that, in general, the phenotype of a double mutant constructed from parent strains blocked at different stages of sporulation corresponds to that of the earlierblocked parent. In accordance with this finding, strains bearing either mutations spo-NG1 .67 or spo-85 in addition to mutation spo-279 showed the phenotype of the conditional earlier-blocked parent (bearing mutation spo-279, stage II) at the restrictive temperature, whereas at the permissive temperature the phenotype corresponded to that of the later-blocked parent
(bearing mutation spo-NG1.67, stage III, or mutation spo-85, stage V). Conditional earlyblocked sporulation mutants would be a useful tool for ordering the large cluster of sporulation operons that lie in the phe-lys region of the chromosome. Dubnau et al. (4) placed the furA locus on the side of ura distal to metC on the basis of two-factor transduction crosses, and data to support the assignment of cysC to a position between metC and ura have not been published (3). During the course of this work it became apparent that these assignments have been made erroneously. It is suggested, therefore, that the order established here from three- and four-factor transduction crosses and from threefactor transformation crosses (Fig. 1) should now be adopted. ACKNOWLEDGMENTS I thank Sonia Higgs for her skilled technical assistance.
This work was supported by the Science Research Council. LITERATURE CITED 1. 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. 2. Coote, J. G., and J. Mandelstam. 1973. Use of constructed double mutants for determining the temporal order of expression of sporulation genes in Bacillus subtilis. J. Bacteriol. 114:1254-1263. 3. Dubnau, D. 1970. Linkage map of Bacillus subtilis, p. 1-39-I-45. In H. A. Sober (ed.), Handbook of biochemistry, 2nd ed. Chemical Rubber Co., Cleveland, Ohio. 4. Dubnau, D., C. Goldthwaite, I. Smith, and J. Marmur.
1116 5.
6. 7. 8. 9.
YOUNG
1967. Genetic mapping in Bacillus subtilis. J. Mol. Biol. 27:163-185. Ephrati-Elizur, E. 1968. Spontaneous transformation in Bacillus subtilis. Genet. Res. 11:83-96. Grant, W. D. 1974. Sporulation in Bacillus subtilis 168. Control of synthesis of alkaline phosphatase. J. Gen. Microbiol. 82:363-369. Hoch, J. A. 1971. Genetic analysis of pleiotropic negative sporulation mutants in Bacillus subtilis. J. Bacteriol. 105:896-901. Hoch, J. A. 1971. Selection of cells transformed to prototrophy for sporulation markers. J. Bacteriol. 105:1200-1201. Hranueli, D., P. J. Piggot, and J. Mandelstam. 1974. Statistical estimate of the total number of operons specific for Bacillus subtilis sporulation. J. Bacteriol. 119:684-690.
J. BACTERIOL.
10. Piggot, P. J. 1973. Mapping of asporogenous mutations of Bacillus subtilis: a minimum estimate of the number of sporulation operons. J. Bacteriol. 114:1241-1253. 11. Polsinelli, M. 1965. Linkage relationship between genes for amino acid or nitrogenous base biosynthesis and genes controlling resistance to structurally correlated analogues. G. Microbiol. 13:99-110. 12. Sterlini, J. M., and J. Mandelstam. 1969. Commitment to sporulation in Bacillus subtilis and its relationship to the development of actinomycin resistance. Biochem. J. 113:29-37. 13. Young, F. E., and G. A. Wilson. 1972. Genetics of Bacillus subtilis and other Gram-positive sporulating bacilli, p. 77-106. In H. 0. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores V. American Society for Microbiology, Washington, D.C.
Vol. 122, No. 3 Printed in U.S.A.
Genetic Mapping of Sporulation Operons in Bacillus subtilis Using a Thermosensitive Sporulation Mutant M. YOUNG Department of Biochemistry, University of Oxford, Oxford OXI 3QU, United Kingdom Received for publication 27 March 1975
A thermosensitive sporulation mutant was used to determine the order of sporulation operons in the ura region of the Bacillus subtilis chromosome. Data from three-factor transformation crosses and three- and four-factor transduction crosses established the order metC-spo-96(SpoIH)-spo-85(SpoV)-spo-279(SpoII)furA-ura-cysC-spo-NG1.67(SpoI). Previously, furA was thought to lie to the right of ura and cysC to the left (Dubnau, 1970; Young and Wilson, 1972). In a recent genetic analysis of a large number of sporulation mutations in Bacillus subtilis, Piggot (10) grouped mutations together into sporulation operons. If two mutations satisfied one (at least) of the following three criteria, they were considered to be in separate operons: (i) they were separated by an auxotrophic marker; (ii) they were unlinked by transformation; (iii) they caused blocks at different stages of sporulation. About 30 operons have now been described (9), and they are clustered into four main groups on the chromosome. The order of some operons in the cluster occurring near the cysA locus has been established with three-factor transformation crosses (10). The ordering of sporulation operons in the other chromosomal regions has been difficult, mainly because of the lack of linked auxotrophic or other suitable markers. Crosses that involve two sporulation mutations and an auxotrophic marker are often difficult to interpret because the various recombinant classes may not be readily distinguished. However, Hoch(7) has described a reciprocal cross method by which the order of two sporulation mutations may be determined relative to a flanking auxotrophic marker. In crosses that involve two sporulation mutations giving rise to distinct colony types and where the earlier-blocked mutation is also conditional, it is possible to distinguish all classes of recombinants, and hence the order of mutations may be determined from a single cross. In this investigation, a thermosensitive stage II sporulation mutant was used to determine the order of sporulation operons in the ura region of the chromosome.
isolated after an exponentially growing culture of the wild type had been treated with N-methyl-N'-nitroN- nitrosoguanidine as described by Coote (1). The mutagen-treated culture was plated on tryptophansupplemented minimal agar and examined after incubation for 3 days at 42 C. Colonies with a different pigmentation from the normal brown color were picked, streaked on minimal agar, and incubated at 30 and 42 C. The thermosensitive mutant 279 gave colonies that were normal at 30 C but were very light brown and contained few spores at 42 C. The mutant was reisolated from nutrient agar and tested to ensure that the growth rate on lactate as sole carbon source was the same as that of the wild type. Deoxyribonucleic acid (DNA) from the mutant was prepared by the technique of Ephrati-Elizur (5) and used to transform strain MB21 to Ieu+ at 42 C. Some 5% of the transformants also inherited mutation spo-279 by congression, (i.e., the simultaneous transfer of unlinked markers that occurs at saturating concentrations of DNA). The resultant isogenic strain 279.1 was found to give colonies almost indistinguishable from those of the wild type at 37 C; this was chosen as the permissive temperature. As the growth temperature was increased, colonies contained progessively fewer spores. At 42 C, the restrictive temperature used, the colonies contained very few spores ( < < 1%) and displayed a light-brown pigmentation quite distinct from that of the wild type. Spontaneous Spo+ revertants of strains bearing the spo-279 mutation have never been observed, although revertants can be induced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. The characterization of strain 279.1 is to be reported elsewhere. The other sporulation mutants used here have been described previously (9, 10). Transduction and transformation. The methods used were as described by Piggot (10); details of individual crosses are to be found in the text. Media. The media used have been described previously (10). 5-Fluorouracil was added to media, as appropriate, at 1 Ag/ml. MATERIALS AND METHODS Selection of spontaneous prototrophic revertants of uracil auxotrophs. Uracil auxotrophs were Strains. The strains used are listed in Table 1. The thermosensitive sporulation mutant 279 was streaked for single colonies on nutrient agar and an 1109
1110
J. BACTERIOL.
YOUNG
TABLE 1. Strains of Bacillus subtilis Strain
Genotype
Sporulation phenotype
168 GSY289 GSY753 GSY1441 MB19a MB21a MB49
trpC2 trpC2 ura-1 trpC2 fur-2 cysC7 trpC2 leu-8 rif metC3 leu-8 tal trpC2 cysC7
Spo; Spo+ Spo+ Spo+ Spo+ Spo+ Spo+
MB50
cysC7 ura-1
Spo+
6.4b 36.1b
leu-8 tal spo-ElO metC3 tal spo-NG1.67
Spo IIC Spo In
41. lb
leu-8 tal spo-NG4.14
Spo ld
55.1b
leu-8 tal spo-NG12.12
Spo IIF
71.1b
trpC2 rif spo-NG20.12
Spo IIe
85.1' 96.2'
metC3 tal spo-85 metC3 tal spo-96 cysC7 spo-96 metC3 tal spo-279 cysC7 spo-279 cysC7 fur-2 spo-279
Spo He Spo II' Spo II' Spo II' Spo II'
96.3 279.1a 279.2 279.6
Spo V
Origin
Laboratory wild-type strain C. Anagnostopoulos C. Anagnostopoulos C. Anagnostopoulos P. J. Piggot P. J. Piggot cysC7 (from GSY1441) transformed into GSY289 by congression with ura+ cysC7 (from GSY1441) transformed into GSY289 by congression with trp+ spo-ElO transformed into MB21 by congression with met+ spo-NG1.67 transformed into MB21 by congression with leu+ spo-NG4.14 transformed into MB21 by congression with met+ spo-NG12.12 transformed into MB21 by congression with met+ spo-NG20.12 transformed into MB19 by congression with leu+ spo-85 transformed into MB21 by congression with leu+ spo-96 transformed into MB21 by congression with leu+ spo-96 transformed into MB49 by congression with trp+ spo-279 transformed into MB21 by congression with leu+ spo-279 transformed into MB50 by congression with ura+ fur-2 (from GSY753) transformed into 279.2
a Isogenic
with the wild type except for the markers listed. bStrains isolated by Piggot (10); isogenic with the wild type except for the markers listed. c Abortively disporic, often with two septa at one end and one at the other. dAbortively disporic, originally designated stage 0 (see text). e Abortively disporic. Strains isolated by Hranueli et al. (9); isogenic with the wild type except for the markers listed. 'Prominent mesosomes at both cell poles, subtended by incomplete septa.
inoculum from one colony was grown overnight in Penassay broth. The bacteria were washed and resuspended in a glucose-salts solution, and revertants to uracil prototrophy were selected on unsupplemented minimal agar. Measurement of sporulation alkaline phosphatase activity. Bacteria were grown and sporulated at 42 C by using the resuspension procedure of Sterlini and Mandelstam (12) with the modification used by Piggot (10). Samples of culture (1.0 ml) were removed at approximately hourly intervals and stored at -20 C. Alkaline phosphatase activity in the entire culture samples was measured by the method of Grant (6).
RESULTS Sporulation operons in the ura-l region. Piggot (10) and Hranueli et al. (9) have described 11 sporulation mutations that lie in the ura-I region of the B. subtilis chromosome. These mutations were considered to lie in six operons. The cotransduction frequencies of mutations representing each of these operons with
metC3 and with cysC7 are given in Table 2. These mutations were used to establish the linkage relationships of the new mutation, spo279. In transformation crosses with strains bearing the various spo mutations as recipients and DNA bearing the spo-279 mutation as donor, spo-279 was cotransformed with spo-ElO, spoNG4.14, spo-NG12.12, and spo-85 (Table 3). Three of these mutations, originally considered to be in separate operons, now appear to lie within a single stage II operon. Mutations spo-ElO and spo-NG12.12 were originally considered to lie in separate operons because they appeared to be unlinked in transformation crosses. We have repeated this and demonstrated the cotransformation of these two mutations (recombination index = 0.17). (In crosses with strains bearing the spo-ElO mutation, it seems to be particularly difficult to obtain satisfactory selection for Spo+ recombinants by the chloroform method [81; this may arise from the tendency of this mutation to revert to Spo+.)
TABLE 2. Phenotype and linkage by transduction of mutations in the ura-1 region
Linkage to selected Mutation
spo-ElO spo-NG1.67 spo-NG4.14 spo-NG12.12 spo-85 spo-96 spo-279
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SPORULATION OPERONS IN B. SUBTILIS
VOL. 122, 1975
markers
Type
II III II II V f II
metC3
cysC7b
3c 1c 2c 5c 6 (48/860) 49d 5 (34/732)
81 (254/314) 42 (210/506) 84 (198/237) 82 (396/486) 77 (111/144) 17 (92/529) 61 (119/195)
a PBS1 lysate was prepared on the wild-type strain (168) and used to transduce the Spo- metC3 strains to methionine prototrophy. Linkage is shown as the percentage of cotransduction with the numbers of transductants in parentheses [Spo+/(Spo+ + Spo-)]. bPBS1 lysate was prepared on the Spo- prototroph and used to tranaduce the Spo+ auxotroph (strain MB49) to cysteine prototrophy. Linkage is shown as the percentage of cotransduction with the numbers of transductants in parentheses [Spo-/(Spo- + Spo+). c Data from Piggot (10). dData from Hranueli et al. (9).
prototrophy by using PBS1 lysates prepared on the cysteine auxotrophs 279.2 and 96.3, respectively, whereas in the other pair the cysteine auxotrophs were transduced to prototrophy by using PBS1 lysates prepared on the methionine auxotrophs. Colonies that were phenotypically Spo+ at 37 C were screened for temperature sensitivity, and the frequencies of the three phenotypic recombinant classes are given in Table 4. (Recombinants bearing both mutations spo-96 and spo-279 were phenotypically indistinguishable from those bearing spo-96 alone.) If metC3 and cysC7 are flanking markers, then in one cross out of each pair (A + B and C + D), the spo mutation in the donor is the middle marker. In this cross, but not in the other cross in each pair, Spo+ recombinants are only generated by multiple crossovers, and hence they should form a rare class (7). On the TABLE 3. Linkage by transformation of the spo-279 mutation to other spo mutations in the ura-1 region Linkage° Mutation
Type
Spo+
selection"
Mutation spo-NG4.14, which was cotransformed with another mutation in the stage II operon represented here by mutation spoNG12.12, was considered to lie in a different operon because it had been classified as a stage O mutation instead of stage II. (When a strain bearing this mutation was originally examined by electron microscopy, most of the cells appeared to be blocked at stage 0-I (axial filament), although a few stage II cells were seen. When it was re-examined some time later, the majority of cells reached stage II [Hranueli, unpublished data].) Mutation spo-279, which lies in the same stage II operon as mutations spoE10, spo-NG4.14, and spo-NG12.12, was used to represent the operon in the three- and fourfactor crosses. The other three operons that lie in this region are represented by mutations spoNG1.67 (stage I), spo-85 (stage V), and spo96 (stage II). The cotransformation of mutations spo-85 and spo-279 indicates that the two operons which they represent map in close proximity (Table 3). Mapping the stage II operons. The cotransduction frequencies of mutations spo-96 and spo-279 with metC3 and cysC7 (Table 2) suggest the order metC3-spo-96-spo-279-cysC7, the two auxotrophic markers being approximately 130 transduction units apart. This order was confirmed in three-factor reciprocal crosses. In one pair of crosses, the methionine auxotrophs 96.2 and 279.1 were transduced to
spo-ElO spo-NG1.67 spo-NG4.14 spo-NG12.12 spo-NG20.12d spo-85 spo-96
II III II II II V I
ND 2 (3/156) 34 (53/154) 35 (53/153) 1 (2/207) 46 (71/155) 3 (1/31)e
Auxotrophic selectionc
68 (71/104) ND ND 52 (54/103) 9 (9/104) ND 8 (8/99)
aLinkage is shown as the percentage of contransformation with the numbers of transformants in parentheses [Spotm/(Spo" + Spo+) ]. ND, not done. "DNA prepared from strain 279.1 was used at a final concentration of 0.016 ug/ml to transform strains bearing the various spo mutations to Spo+ at 37 C (selection was by chloroform treatment [8]). Spo+ recombinants were tested at 37 and 42 C for inheritance of mutation spo-279. cDNA prepared from strain 279.1 was used at a saturating concentration (16 jg/ml) to transform strains bearing the various spo mutations to leucine or tryptophan prototrophy at 37 C. Approximately 8% of the transformants were simultaneously transformed to prototrophy for the unlinked spo marker, for which there was no selection. The resultant Spo+ double transformants were tested at 37 and 42 C for inheritance of mutation spo-279. d Mutation spo-Ng20.12, which maps in the cysA region (10) and is not linked to mutation spo-279, was used as a control. eStrains bearing mutation spo-96 are oligosporogenous, which made it difficult to obtain satisfactory selection for Spo+ recombinants by the chloroform method. In one experiment considerable enrichment for Spo+ recombinants were obtained by early treatment with chloroform (12 to 14 h after plating).
J. BACTERIOL.
YOUNG
1112
basis of this reasoning, the order metC3-spo96-spo-279-cysC7 is established. In the final three-factor cross in Table 4 (cross E), fur-2 was mapped relative to cysC7 and spo-279. Strain 279.6, a 5-fluorouracil-resistant cysteine auxotroph, was transduced to cysteine independence by using a PBS1 lysate prepared on the wild-type strain 168. After incubation at 42 C, colonies were scored for inheritance of the spo-279 mutation and then screened for resistance to 5-fluorouracil. These data (Table 4, cross E) establish the order spo-279-fur-2spo-cysC7, and hence we have metC3 spo-96 279-fur-2-cysC7. According to the genetic map of B. subtilis, cysC7 should lie closer to metC3 than does fur-2 (13). Since the present data suggest an order of markers different from that previously determined, it was important to establish the map position of ura-1. Map position of ura-1. A double auxotroph bearing the cysC7 and ura-1 markers (MB50) was transformed to prototrophy with a strain bearing the fur-2 marker as donor (GSY 753). Transformants to cysteine independence and to uracil independence were selected separately and then screened for inheritance of the unselected markers. From the cross in which cysteine prototrophs were selected, 330 uracil prototrophs and 190 uracil auxotrophs were obtained. Of the 330 uracil protttrophs, 177 -
-
resistant to 5-fluorouracil and 153 were sensitive. The uracil auxotrophs could not be tested directly for 5-fluorouracil resistance, since sensitive strains will grow on media containing uracil in addition to 5-fluorouracil (it has been suggested that analogue resistance at the furA locus is due to enhanced uracil production [11]). The uracil-requiring, 5-fluorouracilresistant recombinants were identified after spontaneous revertants to uracil prototrophy had been obtained. Five ura+ revertants were obtained from each ura- transformant, and when tested for resistance or sensitivity to 5-fluorouracil, all five invariably showed the same phenotype. Of the 190 uracil auxotrophs derived from the cross, only nine gave ura+ revertants that were resistant to 5-fluorouracil. These data are summarized in Table 5 together with the frequencies of recombinants from the cross in which uracil prototrophs were selected and then screened for cysteine requirement and for 5-fluprouracil resistance. These data establish the order fur-2-ura-1-cysC7. Mapping the stage V and stage III operons. Mutation spo-85 (stage V) was mapped in a four-factor transduction cross relative to spo-279, fur-2, and cysC7. A PBS1 lysate prepared on strain 85.1 was used to transduce the 5-fluorouracil-resistant cysteine auxotroph 279.6 to cysteine prototrophy at 42 C. The
were
TABLE 4. Three-factor transduction crosses to map the stage II operons
Donor (111)
Cross
Recipient(000)
Se-
Recombinant classes
Se-e
FrePheno- quency Genotype Clses Reobian
marker
(1- -) A met+ spo-96 spo+ (96.3)a
B met+ spo+ spo-279
(279.2)
C
D cys+ spo+ spo-279 (279.1)
a The
metC3 spo-96 spo+
met+
met+
(96.2)
cys+ spo-96 spo+ (96.2)
E cys + fr (168)
metC3 spo+ spo-279 (279.1)
spoW
cysC7 spo+ spo-279 (279.2)
cys+
cysC7 spo-96 spo+ (96.3)
cys+
cysC7 fur-2 spo-279 (279.6)
strain used is given in parentheses.
cys+
Suggested order
type
Spot Spo+
322 8
Spo:
Spo
22 229
met+ 96 met+ 96 met+ + met+ +
+ Sp 279 Spo
576
100 101 110 111
Cys+ +
279 Spo"
cys+ + cys+ 96 cys+ 96
+
Spo+ 279 Spo+ Spo
139 32 3
100 101 110 111
cys+ 96 cys+ 96 cy8+ + cys+ +
+ Spo279 Spo-
610 6
100 101 110 111
met' met+ met+ met+
100 101 110 111
100 cys+ 101 cys+ 110 cys4 Ill cys+
+ + 96 96
279 + 279 +
+
279
Spo+
SpoU
1
fur-2 279 Spo" fur-2 + SpO fur+ 279 Spo"
46 73
Spo+
-
spo-279
cysC
-
spo-279
-
spo-96
-
fur
71
95
+
spo-96
171 31
Spo+
fur+
-
l
279 Spot
+
metC
73c8 294
-
spo-279
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SPORULATION OPERONS IN B. SUBTILIS
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TABL 5. Three-factor transformation crosses to order the markers ura-1, cysC7 and fur-2 Donor (111) (GSY753)a
Recipient (000)
(MB50)
Selected marker (1- -)
No. of colonies in classes: recombinant 100
101 110 111
Number of transforPossible order mants classesin requiring multiple
Suggested order
crosovers
cys + fur-2 ura+
cysC7fur+ ura-1
cys+
181 153
9 177
cys-fur-ura
fur-cys-ura fur-ura-cys ura+ fur-2 cys+
ura-1 fur+ cysC7
ura+
97 152 79 189
cys-fur-ura
fur-cys-ura fur-ura-cys a The
153 b
fur-ura-cys
152 79 __
fur-ura-cys
9
strain used is given in parentheses. crossovers are undetectable with this order.
b Multiple
transductants were screened for inheritance of the fur-2 marker. Three colony types were discemible on the plates at 42 C. All transductants bearing the spo-279 mutation gave lightbrown Spo- colonies, those bearing mutation spo-85 alone gave dark-brown Spo- colonies, and those bearing neither mutation gave Spo+ colonies. Transductants bearing both mutations spo-279 and spo-85 were separated from those bearing spo-279 alone by screening at 37 C, when the former gave dark-brown Spo- colonies whereas the latter gave Spo+ colonies. The frequencies of the eight recombinant classes are given in Table 6. Four of the eight classes were rare, and these represent those classes requiring multiple crossovers for the order cysC7-fur-2spo-279-spo-85. The possible orders cysC7spo-85-fur-2-spo-279 and cysC7-fur-2-spo-85spo-279 may be dismissed since the frequency of recombinants in certain of the classes requiring multiple crossovers is too high. The remaining possibility is the order spo-85-cysC7-fur-2spo-279, and this is improbable for two reasons: first, mutations spo-85 and spo-279 are cotransformed (Table 3), and second, two of the observed rare classes would not be expected for this order. The order of markers inferred from these data is cysC7-fur-2-spo-279-spo-85. Mutation spo-NGI .67 was mapped in a fourfactor cross analogous to that described above. The eight recombinant classes given in Table 6 were not all simply identified from their ability to grow on plates containing 5-fluorouracil and their colony morphologies at 37 and 42 C. Unfortunately, recombinants bearing both mutations spo-NG1.67 and spo-279 had a colony type that was identical to those bearing mutation spo-NG1.67 alone, and hence they could not be separated from each other. The distinction was made by monitoring the produc-
tion of alkaline phosphatase during sporulation in liquid medium at 42 C. Synthesis of this enzyme seems to depend upon the successful completion of the spore septum. Mutants blocked at stage HI or later (and, of course, the wild type) show a burst of enzyme synthesis 2 to 3 h after resuspension, whereas mutants blocked at stage II or earlier generally show little or no enzyme production. Thus, recombinants bearing mutation spo-NG1.67 alone showed the normal period of rapid alkaline phosphatase synthesis 2 to 3 h after resuspension at 42 C, whereas recombinants bearing both mutations spo-NG1.67 and spo-279 showed the greatly reduced rate of enzyme production that is characteristic of strains bearing the spo-279 mutation. The only order of markers that is consistent with the data in Table 6 is spo-NG1.67-cysC7-fur-2-spo-279; all other possible orders may be dismissed since the frequencies of recombinants in certain of the classes requiring multiple crossovers is too high. To simplify the treatment of the mapping data in Table 6, it has been assumed throughout that the order of markers in the recipient strain is spo-279-fur-2-cysC7, as was established in Table 4, cross E. Even without this simplifying assumption, the data from the two crosses are not consistent with any other possible order and provide independent confirmation of the previously established order. DISCUSSION The thermosensitive sporulation mutant 279 has been used to determine the orientation of the four sporulation operons known at present in the ura region of the B. subtilis chromosome. There were previously thought to be six sporulation operons in this region (9), but for the reasons given earlier, this number should now
1114
YOUNG
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1115
SPORULATION OPERONS IN B. SUBTILIS
VOL. 122, 1975
Spo-ElO Spo- NG4b Spo-W50 Spo - NG 10.2 Spo-W0l Spo-NG12.12 Spo-96 Spo-85 Spo-279
SpO- 82
Spof
Sp
Spo- NG8J7 Spo- NGI.67
SpofI
Spo
met C
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51C
83 d
-, ,
94d 95
23
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39d
Ll1-iJ
7C7e
5 wtd
-
9-' 29 -a64 FIG. 1. Map of the ura region of the B. subtilis chromosome. Sporulation operons are assigned the stage of sporulation reached by strains carrying mutations in the operon. The mutations that lie in each operon appear above it; those mutations not mapped in this paper are denoted (a) Hranueli et al. (9) and (b) Piggot (10). The sporulation operons are approximately positioned according to the cotransduction frequencies of the mutations chosen to represent them (shown in bold type) with metC3, ura-1, and cysC7. Distances are given as recombination values (100 cotransduction frequencies); these data are taken from (c) Hranueli et al. (9), (d) Table 2, (e) Table 6, (f) Young (unpublished data), and (g) Piggot (10). -
be reduced to four (accepting the criteria given by Piggot [10] to define a sporulation operon). The relative positions on the chromosome of these four operons are given in Fig. 1. Although the location of the stage V operon between the two stage II operons has not been confirmed in crosses with the metC3 and spo-96 markers which lie to the left, this assignment seems justified since mutation spo-85 (stage V) is strongly cotransformed with mutation spo-279 (stage II), whereas mutations spo-96 (stage II) and spo-279 are unlinked by transformation (Table 3). The mapping of sporulation operons was facilitated by the conditional sporulation phenotype of strains bearing the spo-279 mutation. At 42 C the cells are blocked at stage II, whereas sporulation is normal on solid media at 37 C. This thermosensitivity was particularly useful in mapping mutations spo-NG1.67 and spo-85 (stages III and V, respectively). Coote and Mandelstam (2) have shown that, in general, the phenotype of a double mutant constructed from parent strains blocked at different stages of sporulation corresponds to that of the earlierblocked parent. In accordance with this finding, strains bearing either mutations spo-NG1 .67 or spo-85 in addition to mutation spo-279 showed the phenotype of the conditional earlier-blocked parent (bearing mutation spo-279, stage II) at the restrictive temperature, whereas at the permissive temperature the phenotype corresponded to that of the later-blocked parent
(bearing mutation spo-NG1.67, stage III, or mutation spo-85, stage V). Conditional earlyblocked sporulation mutants would be a useful tool for ordering the large cluster of sporulation operons that lie in the phe-lys region of the chromosome. Dubnau et al. (4) placed the furA locus on the side of ura distal to metC on the basis of two-factor transduction crosses, and data to support the assignment of cysC to a position between metC and ura have not been published (3). During the course of this work it became apparent that these assignments have been made erroneously. It is suggested, therefore, that the order established here from three- and four-factor transduction crosses and from threefactor transformation crosses (Fig. 1) should now be adopted. ACKNOWLEDGMENTS I thank Sonia Higgs for her skilled technical assistance.
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