CHROMOSOMAL LOCATION OF PLEIOTROPIC NEGATIVE SPORULATION MUTATIONS IN BACILLUS SUBTZLZS

JAMES A. HOCH AND JUDITH L. MATHEWS

Department of Microbiology, Scripps Clinic and Research Foundation, La Jolla, California 92037

Manuscript received August 18, 1972 Revised copy received October 18, 1972

ABSTRACT

Genetic analysis by PBS-1 transduction and transformation of a large group of pleiotropic negative sporulation mutants has shown that mutations of this phenotype may be located in five genetically distinct regions. The first group of mutant sites, spoA mutations, is located in the terminal region of the chromosome and linked to the lys-I marker by PBS-1 transduction. The second group, spoB mutations, is located between phe-I and the attachment site for the lysogenic bacteriophage + 105. Fine structure analysis of the mutant sites within the spoB locus has been accomplished. A third location for mutants of this phenotype, spoE mutants, was found between the metC3 and ura-I mark- ers. Two mutants were found at this site and both were capable of sporulation, in contrast to the rest of the pleiotropic sporulation mutants. A fourth chromo- somal site, spoH mutations, was found near the ribosomal and RNA poly- merase loci. A large group of mutant sites, spoF mutations, was found to be linked to each other by recombination index analysis in transformation but unlinked to any of the known auxotrophic mutations comprising the chromo- somal map. All mutants analyzed showing a pleiotropic negative phenotype were found to map within one of these five regions. Interspecific transforma- tion with Bacillus anyloliquefaciens as donor has shown that all of the pleio- tropic negative sporulation mutations are conserved relative to a selected group of auxotrophic markers. The degree of conservation in decreasing order is: spoH > spoF = spoB > spoA.

HE encystment of a nucleus into an environmentally resistant spore is one Tmeans of survival through differentiation common to single-celled organisms as well as to the more morphologically complex molds and fungi. Sporulation occurs when the organism finds itself in an environment that is no longer con- ducive to continued growth. In liquid cultures the process begins as the readily usable carbon sources are depleted and the organism is forced to use the end products of glycolysis for carbon and energy (HANSON, SRINIVASON and HALVOR- SON 1963). The metamorphosis that changes a cell to a spore follows a distinct series of biochemical and morphological stages (RYTER, SCHAEFFER and IONESCO 1966). The first step in the process appears to be the condensation of the nuclei of a bi-nucleated cell to form an axial filament of chromatin. A septum or forespore membrane is initiated at one end of the cell and this membrane eventually engulfs half of the chromatin, leading to the formation of a spore protoplast Genetics 73: 215-228 Februaly, 1973.

216 JAMES A. HOCH A N D JUDITH L. MATHEWS

within the cell. Maturation of the protoplast by the laying down of cortex and coat completes the process, whereupon the mother cell lyses to release the free spore. The stages of development are characterized by the appearance of new enzymatic activities and the destruction of some preexisting ones ( KORNBERG et al. 1968). Although these modulations in enzymatic activity have been the subject of numerous investigations, the problem of distinguishing between sporu- lation specific functions and the biochemistry of cell growth remains. Attempts to clarify the relationships between the appearance of new biochemical events and a particular stage of the sporulation process have centered around the use of mutants blocked in sporulation.

Mutants defective in sporulation may be categorized into two broad classes: (1) mutants in specific components of the metabolism required for sporulation, e .g . , citric acid cycle mutants, and (2) mutants with no obvious metabolic defect that are, nevertheless, unable to sporulate. Mutants in this latter class have been subjected to genetic analysis (IONESCO et al. 1970; HOCH and SPIZIZEN 1969; ROGOLSKY 1969). These studies have shown that one region of the chromosome is especially enriched in mutant loci giving rise to an asporogenous phenotype; that region is at the terminal end of the chromosome between phe-1 and Zys-1. Mutations in this region account for about two-thirds of the sporulation mutants analysed. Morphological examination revealed that these mutations resulted in blockage of the sporulation process at many different stages. Also mapping in this region are mutations leading to the loss of all known sporulation functions. These are the mutants in which sporulation is blocked at the axial filament stage and no further sporulation specific biochemical events occur-the so-called pleiotropic negative sporulation mutants ( SPIZIZEN 1965; SCHAEFFER 1969; HOCH and SPIZIZEN 1969). I n this study we define additional loci in which a mutation may give rise to this phenotype.

MATERIALS A N D METHODS

Bacierial strains: Strains used in this study were derived from B. subiilis 168 (Table 1). The pleiotropic sporulation mutants were obtained from strain 168 by either ultraviolet irradiation or treatment with N-methyl-N’-nitro-N-nitrowguanidine. Many of the pleiotropic sporulation mutants were kindly provided by Dr. JOHN SPIZIZEN, as was the Bacillus amyloliquefaciens H strain.

Genetic analysis: Transformation was carried out according to ANAGNOSTOPOULOS and SPIZIZEN (1961). Selection of spof transformants with spo- recipients was accomplished by a

TABLE 1

Basic strains of B. subtilis

Strain Genotype Source

BR149 ilv-1, leu-6, phe-1 B. REILLY BD25 purAlb,leu-S, rnetB3, nic-38 D. DUBNAU SB26 trpC2, meK3 E. NESTER SB5 trpC2, ura-I, hisAI E. NE~TF.R W168 ($105) @IO5 lysogenic L. RUTBERG

PLEIOTROPIC NEGATIVE SPORULATION MUTATIONS 21 7

previously described technique (HOCH 1971 b) . Transduction with PBS-1 also followed previously described procedures (HOCH 1971a).

The spo- character was scored in genetic crosses on the basis of colony morphology. Strains bearing the spo- mutation form enlarged transparent colonies while those of the sporulating strain are smaller, white, and opaque. The differences between the two classes are readily ap- parent on the minimal medium of SPIZIZEN (1958) supplemented with 0.01 mM MnCl,.

RESULTS

Isolation of pleiotropic negative sporulation mutants: One of the most obvious phenotypes of pleiotropic negative sporulation mutants is their enlarged and transparent colony morphology compared to the smaller and opaque colonies formed by their sporulating parent. The mutants examined in this study were isolated on the basis of this enlarged colony morphology by plating mutagen treated cultures on solid media. In earlier studies (HOCH and SPIZIZEN 1969; HOCH 1971a) we examined the properties of those pleiotropic negative sporu- lation mutants which mapped in the terminal spore region between Zys-I and phe-I. These mutations were found to comprise a tightly linked group of sites, and, on the basis of recombination values, are thought to define one or at most two genes. Since genetic complementation is not readily accomplished in this organism the number of loci cannot be established with certainty. Nevertheless, we have chosen to designate the sites as spoA to distinguish sites mapping in this region from others of similar phenotype in other regions. Although many of the mutants isolated on the basis of colony morphology map in this region: others were unlinked to lys-1 by PBS-1 transduction analysis. An extensive search of the chromosome by transduction was undertaken to define the number and location of loci giving rise to the pleiotropic phenotype.

Genetic fine structure of the spoB locus: Two-factor transduction crosses with spo- donors used to transduce various auxotrophic markers on the chromosome revealed that a tight linkage between eight spo- mutants and phe-1 existed. The spo- mutation was cotransduced greater than 90% of the time with phe-1. This linkage suggested that the spo- markers might also be linked to phe-I by trans- formation since PBS-1 transduction transfers a much larger DNA fragment than that transferred in transformation (BARAT, ANAGNOSTOPOULOS and SCHNEIDER 1965), In two-factor coupling transformation crosses the sites were found to map between 23% and 31 % recombination from phe-l (Table 2). Three-factor trans- formation crosses were done to establish the order of the spo- mutations with respect to phe-1 and the adjacent nic-38 marker. A double marked recipient carrying spoB83 and nic-38 was constructed and this recipient was transformed with phe-I DNA. Selection for either nic+ or spo+ showed that the wild-type recombinant was the least frequent of the recombinant classes (Table 3 ) . Assum- ing the double crossover class to be least frequent, the results are consistent with the order spoB83-phe-1-nic-38. Since the order of markers in this region has been shown to be leu-8-phe-I-nic-38, the spoB83 marker was taken to be between leu4 and phe-l on the basis of these results. Subsequent to these results, however, IONESCO et a2. (1970) reported that similar mutants linked to phe-1

21 8 JAMES A. HOCH A N D JUDITH L. MATHEWS

TABLE 2

Two factor transformation crmses with spoB mutations and phe-1

Recipient' phe+ spo+/phe+ Recombination (percent)

phe-I spoB125 phe-I spoBl26 phe-I spoB136 phe-I spoB141 p h - I ~p0B123 p h - I ~p0Bl49 p h - I ~p0B83 p h - I ~p0Bl76

169/236 41 7/542 140/196 373/5 13 149/196

598/817 726/956

164/239

28 23 29 27 24 31 29 24

* Donor = wild type.

were not located between leu-8 and p h - l but rather mapped to the right of phe-l and the sites extended for various lengths into the region between phe-l and Zys-1. Since their results were obtained with transduction we examined the order of our markers with this method. Two recipients, spoBl4l phe-I and spoBl76 phe-I were transduced with phage carrying leu-8 and phe+ was selected. Analysis of the recombinants (Table 4) showed the phe+spo-leu- class to be least frequent, which agrees with the placement of spoB between phe-2 and leu-8. All of the pleiotrophic sporulation mutants isolated that were linked to phe-I by transduc- tion were found to map between phe-l and leu-8.

The attachment site for the lysogenic + 105 bacteriophage has also been mapped between phe-I and leu-8 (RUTBERG 1969). Furthermore, it has been shown that the phe-l marker and some prophage markers are carried on the same transforming fragment (PETERSON and RUTBERG 1969). It was of interest to determine the order of spoB markers with respect to phe-l and attl05. It was reasoned that if the attachment site were between spoB and phe-I, transforma- tion with DNA from a lysogenic donor should disrupt the cotransfer of the two markers because of the presence of the phage. If the phage resided outside of this region, the integrity of linkage should be conserved. A phe-I spoB141 recipient

TABLE 3

Three-factor transformation crosses with spoB83

Recipient Donor

nic-38 spoB83 p h - I

nic-38 spoB83 phe-I

Classes

nic+ spo+ p h F nic+ spo+ phe- nicfspo- phe+ nicf spo- phe-

spof phef nicf spo+ phe+ nic- spof phe- nic+ spofphe- n i c

N U k Order implied

2 17 68 13

13 152 137 111

spoB83 - phe-I - nic-38

spoB83 - phe-I - nic-38

PLEIOTROPIC NEGATIVE SPORULATION MUTATIONS

TABLE 4

Three-factor PBS-1 transduction crosses with spoB mutations

219

Recipient Donor Classes

p h - 1 spoBl4l leu-8 phef spo+ leu+ phe+ spo+ leu- phe+spo- leu+ phe+ spo- leu-

phe-1 spoB176 leu-8 phef spa+ leu+ phef spo+ leu- phefspo- leu+ phe+ spo- leu-

Number Order implied

133 133 p h - 1 - spoB141- leu-8 w 1

79 92 10 1

phe-1 - spB176 - leu-8

was transformed with DNA from lysogenic and non-lysogenic donors and phe+ was selected. The recombination values between phe-1 and spoBI41 were not drastically altered by the presence of the phage (Table 5 ) , suggesting that the order of markers in this region is leu-8-att105-spoBI41-phe-1.

The sites within spoB were ordered with respect to phe-1 by classical tech- niques. Double marked phe-1 spoB strains were transformed with spoB donors and spo+ was selected. The segregation of phe-1 was scored among the sp+ recombinants and a tentative order deduced from the ratio of the two recombi- nant classes. The order was confirmed by reciprocal crosses with the two markers (Table 6). With the exception of spoB123, all of the markers recombined with each other and could be ordered unambiguously. The spoB123 and spoBI26 sites did not recombine with each other and are assumed to be identical. The recombi- nation values between phe-l and the various sites appear to bear no relationship to the position of a particular site within the locus (Figure 1 ) . The distance across the sites is difficult to determine but appears to be about 6% to 8% recombination, which is the size of an average gene in the tryptophan operon of this organism when analyzed by transformation ( CARLTON 1967).

Location of spoE mutations: Among the group of pleiotropic mutations not mapping in either the spoA or spoB region, two mutants were found to be linked to metC3 in two-factor transduction crosses. Further analysis showed that the mutations could also be weakly linked to urtr-1 (Table 7 ) . The order of markers in this region is argC4-metC3-ura-I (DUBNAU et al. 1967). Although these authors reported that metC3 and ura-1 were linked in two-factor transduction crosses, we have not observed this linkage in our studies. Since the spoE sites cotransduce with both metC3 and ura-1 and no linkage was observed to argC4,

TABLE 5 Determination of the location of attlO5

&e+ spo-/phe+ Recombination (percent) Recipient Donor

p h - 1 spoBl41 W168 118/391 30 p h - 1 spoB141 W168 (@105) 180/417 43

220 JAMES A. HOCH A N D JUDITH L. MATHEWS

TABLE 6

Three-factor transformation crosses ordering spoB mutant sites

Recipient

phe-I s p o B X F phe-I spoB176

phe-I spoB141 phe-1 spoBl36 phe-I spoBl41 phe-I spoB136

phe-I ~p0Bl76

phe-I ~ p B 1 2 5 phe-I ~p0Bl49 phe-I ~p0B125 phe-I spoBl49 phe-I spoB126

Donor

spoBl76 spoBX3 spoBl41 spoBl76 spoB141 spoB136 spoB125 spoBl36 spoB125 spoBl49 spoBl26 spoBl49

Classes spotphe- spo+ phef Order implied by results

34 60 176- 83-phe

58 72 141 -176-pphe 159 23

125 108

41 51 66 194 125 - 136 - phe 46 10

126 24 149 - 125 - phe 51 24Q 61 21 3 126 - 149 - p h

105 20

84 14 136 - 141 - p h e

the order of sites in this region is most probably argC4-metC3-spoE-ura-1. Also, mapping in this region between metC3 and ura-1 is a locus giving rise to conditional streptomycin resistance (STAAL and HOCH 1972) and a mutant site resulting in decreased dihydrolipoyl dehydrogenase activity (HOCH and MATHEWS 1972). Unfortunately, neither the streptomycin resistance nor the spoE mutations are selectible markers. Both the spoE mutations sporulate at frequencies which make selection or analysis by recombination index impossible. Therefore, the relative order of markers within this region is unknown; whether the spoE mutant sites are close enough to be considered part of a single locus is also unknown.

FIGURE 1.-Fine structure map of the spoB locus. The numbers are recombination values in transformation.

PLEIOTROPIC NEGATIVE SPORULATION MUTATIONS

TABLE 7

Two-factor transduction crosses of spoE mutants with metC3 and ura-l

221

Recombination Recipient Donor Class Number (percent)

ura-l spoEIl ura+ spof 183 96 ura+ spo- 7

ura+ spo- 5

met+ spo- 19

metfspo- 52

ura-1 spoEI6l ura+ spof 213 98

m t c 3 spoElI met+ spof 83 81

mtc3 spo E l 61 met+ spo+ 144 73

Mapping of spoH mutations: Two-factor PBS-1 transduction crosses revealed that two spo- mutant sites were linked to the cysA14 marker located adjacent to the ribosomal and RNA polymerase loci (HARFORD and SUEOKA 1970; GOLDTH- WAITE, DUBNAU and SMITH 1970). Both markers are co-transduced with cysAl4 more than 90% of the time. Three-factor transduction crosses to order the spo- markers with respect to the adjacent strAl and rfm-1 markers gave ambiguous results. The discrepancy in crosses performed in this region was noted earlier by GOLDTHWAITE et al. (1970). Transformation analysis with a strAl donor and spoH recipient with selection for spo+ also gave unreproducible results. In some experiments of this type, the two markers were co-transformed 50% of the time, whereas the same experiment performed on a different day would give 10% cotransfer. Similar results were obtained with a rfm-1 donor. I n view of these anomalies we did not attempt to characterize further the genetic position of the spoH locus. Two-factor transformation crosses by the recombinant index method of the two markers found here, spoH75 and spoH81, gave less than 1 % recombi- nation between the markers, suggesting that the markers are alleles of the same gene.

Genetic analysis of spoF mutations: A large group of the pleiotropic negative mutations could not be linked to any of the chromosomal regions described above. We have carried out extensive two-factor transduction crosses using these mutants as donors to transduce virtually all of the known auxotrophic markers on the chromosome. In no case have we found even a weak linkage. The shiki- mate kinase mutants of the aromatic pathway of amino acids have also been found to have this characteristic (HOCH and NESTER, in preparation). In addition to the non-linkage of these sites to the chromosome, the different loci are unlinked to each other in two-factor transduction o r transformation crosses.

This group of unlinkable pleiotropic negative mutations was subjected to recombination index analysis by transformation. DNA from the spo- mutants was used as donor to transform spo-phc recipients and both spof and phe+ were selected. The results of this analysis (Table 8) showed that the group of spo- mutations were linked to each other. The recombination values obtained in this analysis are quite low and of the order found for recombination within a gene.

222 JAMES A. HOCH A N D JUDITH L. MATHEWS

TABLE 8

Recombination index analysis of spoF mutations

Recipient Donor spo-221 spo-187 spo-138

spo-111 SPO-124 SPO-128 S ~ O - 1 29 SPO-I38 spo-167 spo-170 spo-187 SPO-191 SPO-197 spo-223

10 47 N.R.' 0.5 0.2 1.5 3.3

10 0.1

N.R. N.R.

4 11

7.8 4.4 3.5 3.9

N.R. N.R. 21 11 7.4

3.9 14 N.R. 7.4

. .

3.5

N.R. N.R.

* No recombination

The spo-124 marker seems to be an exception since the recombination values to the three recipients are higher than other donors. There is insufficient data to attempt an estimation of the number of genes covered by these sites. Again we have chosen to designate all of the sites as spoF to indicate their linkage group.

Conseruation of the pleiotropic loci: On the basis of interspecific transformation and hybridization studies, it has been shown that sequence homologies exist among the species of the genus Bacillus (Do1 and IGARASHI 1965; DUBNAU et al. 1965). Relative to the majority of the chromosome. the ribosomal and transfer RNA loci seem to be highly conserved. Do1 and IGARASHI (1965), using inter- specific hybridization of messenger RNA, were unable to show that any of the sporulation genes were conserved among the species. In order to test this possi- bility by more sensitive genetic techniques, we have attempted to transform the pleiotropic negative sporulation mutants with DNA from related species of the genus. In this study we have utilized the closely related Bacillus amylolique- faciens H strain (WELKER and CAMPBELL 1967) and a more distant relative, Bacillus pumulis (LOVETT and YOUNG 1970). Isogenic recipient strains were constructed with representatives of each pleiotropic locus, with the exception of spoE since the spontaneous sporulation of these strains is too high. The recipients were brought to competence and transformed in the usual manner with satur- ating levels of DNA. Samples of the transformation tube were plated for the selection of trp+ and spo+.

Transformation of any of the pleiotropic strains with B. pumulis NRRL B-3275 DNA did not lead to either t rp f or spo+ transformants. The donor strain used also carried a rifampicin resistance marker and selection for this character gave about 1 x lo3 rfm. transformants/ml at the concentration of DNA employed. Thus, none of the spo- mutations or the trpC2 mutation is sufficiently conserved in this distant relative of B. subtilis to transform the markers to prototrophy. The conservation of the ifmr locus has been observed before (LOVETT and YOUNG,

PLEIOTROPIC NEGATIVE SPORULATION M U T A T I O N S

TABLE 9

Heterologous transformation of spo markers by Bacillus amyloliquifaciens DNA

223

Recipient

trpC2 phe-I spoB14I

trpC2 phe-I spoFZ21

trpC2 phe-1 spoHS1

trpC.2 pht-1 spoAl2

DNA

H' W H W H W H W

Transfonnants per ml

trp+ spo+

0 1.0 x 104 1.5 x 106

0 2.0 x 104 3.0 x 106

0 5.6 x 104 1.1 x 106

9.3 x 105

9.0 x 1 0 5

7.1 x 104 0 1.2 x 102

1.3 X 106 7.4 x 104

Ratio n m spa+ 0.0067

0.0067

0.051

0.000092

~~~~~~

* Abbreviations: H = B. amyloliquifaciens H and W = B. subtilis Wi68.

unpublished). Transformation by B. amyloliquefaciens DNA gave a different picture (Table 9). Although the DNA was unable to transform the trpC2 marker, it was effective with all of the spo markers tested. The most effectively trans- formed locus was spoH, where the heterologous DNA transformed about 5% as well as the homologous DNA. This is not unexpected since the spoH locus maps in the ribosomal and RNA polymerase region of the chromosome. Both spoBI4l and spoF221 were transformed about ten-fold less well than the spoH8l marker. Least efficient of all was the transformation of the spoAl2 marker, which was almost 100-fold lower than either spoBl42 or .spoF21. The inability of B. amylo- liquefaciens DNA to transform trpC2 was not restricted to this marker. No trans- formation of l e u 4 or metB3 (less than 4 X per homologous transformant) or metC3 (less than 1 X per homologous transformant) was observed. The purB6 marker was transformed at a frequency of 5 x

These results indicate that the pleiotropic negative sporulation loci are most probably less subject to change than the auxotrophic loci but not as conserved as those regions coding for ribosomal functions. The conservation of the spoB region was expected since this locus is very near to the attachment site for the lysogenic +lo5 and this phage can lysogenize B. amyloliquefaciens as well as B. subtilis (REILLY 1965). Furthermore, CHILTON and MCCARTHY (1969) have shown that sequence homology between Bacillus globigii and B. subtilis not only at the ribo- somal region but also in the leucine region which is adjacent to spoB.

DISCUSSION

The genetic analysis of the pleiotropic negative sporulation mutants has shown that mutations in five genetically distinct regions can lead to the classical pleio- tropic negative phenotype. The location of the spoA mutations in the terminal spore gene region has been described before (HOCH 1971a; IONESCO et al. 1970; ITO and SPIZIZEN 1971). The spoB mutations linked to pheA were also found by IONESCO et al. (1970) and ITO and SPIZIZEN (1971), although our extended genetic analyses suggest that the order of mutations in this region is Zeu-8-

224 J A M E S A. HOCH A N D JUDITH L. MATHEWS

spoB-pheA. Our analysis has also shown that the spoB locus is located between the attachment site for the lysogenic phage $105 and the pheA locus. Fine struc- ture analysis of the spoB mutant sites by transformation has shown that the mutant sites can be ordered with respect to pheA, and the recombination values observed in transformation do not correlate to that order. Furthermore. the recombination values obtained in transformation for the spoB locus suggest that it is composed of one or at most two genes. This same conclusion was reached for the spoA locus (HOCH 1971a). In all cases, the assignment of one o r more genes to a linked group of markers is based solely upon the recombination values between the markers and not upon genetic complementation tests.

Although a sizable proportion of the mutants isolated in this study was found to map in either the spoA or spoB region, two further chromosomal sites were found for mutations of this type. One of these. the spoE locus, is located between metC3 and ura-2 on the chromosome. These spoE sites differ from the majority of the pleiotropic negative sporulation mutants in that they both are able to sporulate above reversion levels. The second site is the spoH locus which is linked to the cysA24 marker. Because of instability in linkage values between this marker and the antibiotic resistance markers previously shown to be located in this region (HARFORD and SUEOKA 1970; GOLDTHWAITE et al. 1970), we have not been able to characterize the exact genetic location of the spoH mutant sites with respect to these markers.

All of the mutations not linked to any of these four chromosomal locations were found to be linked to each other by the recombination index method of transfor- mation. These sites comprised the spoF locus and the properties of this locus are of interest. I n an extensive two-factor analysis by PBS-1 transduction we have been unable to link the spoF sites to any of the markers known to be on the chromo- some of Bacillus subtilis (DUBNAU 1970; YOUNG and WILSON 1972). Using the spoF strains as donors, we discovered no linkage between any of the ends of the transducing fragments nor between selected markers in the middle of the known transducing fragments. This situation resembles that found for the aconitase markers (RUTBERG and HOCH 1970) and also the aroI markers in the aromatic pathway (HOCH and NESTER, in preparation). In both of these latter cases, no linkage was found to any known chromosomal marker, whether the aconitase or aroI strains were used as donors or recipients in PBS-1 transduction. Subsequent- ly, ZAHLER (cited in YOUNG and WILSON 1972) has shown that the aconitase markers are located in a heretofore uncharted region of the chromosome. The most likely hypothesis for this genetic behavior is that the known chromosome map of Bacillus subtilis is incomplete. Thus. there may be extensive regions where no auxotrophic markers have been found, except for the aroI or spoF markers. A second possibility for this behavior is suggested from the work of YAMAGISHI and TAKAHASHI (1968). These authors have found that the transducing DNA con- tained within the PBS-1 particle is of various sizes in a sucrose density gradient. If the smaller transducing fragments were unique in that they contained only specific regions and these markers were a part of those regions, then a map expansion would be affected by the bacteriophage or the events required to pack-

PLEIOTROPIC NEGATIVE SPORULATION M U T A T I O N S 225

age the transducing particle. At this time there is no evidence to favor either hypothesis and further work is required to understand these markers as well as to complete the chromosome map of this organism.

An abbreviated map of the chromosome of Bacillus subtilis showing the location of the spore markers used in this study is shown in Figure 2. Mapping of sporulation mutants by IONESCO et al. (1970) and ROGOLSKY (1969) has shown that at least 2/3 of the mutants isolated as being unable to sporulate map in the terminal spore region between the lys-1 and aroD4 markers. Included in the terminal spore gene region is the spoA locus of the pleiotropic negative sporula- tion mutants (HOCH 1971a). None of the other pleiotropic negative sporulation loci maps in an established region of spore genes. In most cases, however, since the biochemical defect of sporulation mutants is unknown, the definition of a spore gene is tenuous. That is, mutations affecting a biochemical function which results in a dimunition of the rate of sporulation but is in itself not directly con- nected with the sporulation process will be designated as a spore gene mutant in

FIGURE 2.-Chromosomal map of Bacillus subtilis showing the location of sporulation muta- tions.

226 J A M E S A. HOCH A N D J U D I T H L. MATHEWS

the absence of knowledge of its normal biochemical function. Therefore, it is risky to try to assign certain regions of the chromosome as sporulation specific. Al- though all of the mutants studied in this investigation are characterized by the enlarged transparent colony of morphology of the classical pleiotropic negative sporulation mutants ( SPIZIZEN 1965) there are certain phenotypic differences be- tween the different classes of mutants. These differences will be dealt with in more detail in another paper.

The conservation of pleiotropic negative sporulation loci relative to other re- gions of the chromosome may indicate that the products of these genes are suffi- ciently important to be retained in a stable sequence. This argument has been proposed for the ability of B . subtilis to be transformed for antibiotic resistance from a variety of sources of heterologous DNA (DUBNAU, SMITH and MURMUR 1965). The structure of the ribosome is assumed to have evolved early and the number of acceptable alterations in the sequence of its components is restricted. The same rationale has been put forward for conservation of the rifampicin re- sistance or RNA polymerase markers (LOVETT and YOUNG, personal communi- cation). CHILTON and MCCARTHY (1969) have suggested that the conservation of auxotrophic markers in certain regions between B. globigii and B. subtilis is due to the fact that these markers are located adjacent to conserved loci, e.g., ribosomal RNA, and that the conservation of the auxotrophic sites is a reflection of their proximity to these loci. Since the functions of the pleiotropic negative sporulation loci are unknown it is difficult to assess the importance of their conservation. These loci are conserved in the closely related B . amyloliquefaciens strains but not in a more distant relative, B. pumulis. The rfm" marker, however, is con- served in this latter strain. This suggests that some selective pressure to conserve the sequence of the pleiotropic loci must exist but this pressure is not as strong as that exerted on the RNA polymerase. The proximity hypothesis does not hold in the B. pumulis case since the spol-181 marker is relatively closely linked to rfm" and this latter marker was transfered while spoH81 was not transformed to pro- totrophy by B. pumulis DNA. The hypothesis is suggested that the pleiotropic loci reprecent an intermediate stage between the non-conserved sequences of simple catalytic proteins and the conserved sequences of proteins involved in com- plex structural entities. One interesting facet of this analysis is the fact that the most pleiotropic of these loci, the spoA locus, is the least conserved of the loci examined.

This research was supported, in part, by grants HD 02807 from the National Institute of Child Health and Human Development and GB 15602 from the National Science Foundation. J. A. HOCH is a Faculty Research Associate Awardee (PRA-66) of the American Cancer Society.

LITERATURE CITED

ANAGNOSTOWULOS, C. and J. SPIZIZEN, 1961 Requirements for transformation in Bacillus subtilis. J . Bacteriol. 81: 741-746.

BARAT, M., C. ANAGNOSTOPOULOS and A. M. SCHNEIDER, 1965 Linkage relationships of genes controlling isoleucine, valine, and leucine biosynthesis in Bacillus subtilis. J . Bacteriol. 90 : 35 7-369.

PLEIOTROPIC NEGATIVE SPORULATION M U T A T I O N S 227

CARLTON, B. C., 1967

CHILTON, M. D., and B. J. MCCARTHY, 1969

DOI, R. H. and R. T. IGARASHI, 1965

DUBNAU, D., 1970

DUBNAU, D., C. GOLDTHWAITE, I. SMITH and J. MARMUR, 1967

DUBNAU, D., I. SMITH, P. MORELL, and J. MARMUR, 1965

Transformation mapping of the genes controlling tryptophan biosynthesis

Genetic and base sequence homologies in Bacilli. Genetics 62: 697-710.

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