Vol. 123, No. 1 Printed in U.S.A.

JOURNAL OF BACrERIOLOGY, JUlY 1975, p. 83-95 Copyright 0 1975 American Society for Microbiology

Isolation, Characterization, and Mapping of Bacillus subtilis 168 Germination Mutants J. TROWSDALEl* AND D. A. SMITH Genetics Department, Birmingham University, Birmingham, United Kingdom

Received for publication 14 April 1975

After mutagenesis with nitrosoguanidine, germination mutants of Bacillus subtilis 168 were selected by killing, with heat, spores that germinated at 42 C and collecting survivors at 30 C. The germination properties of nine mutants variously affected in amino acid biosynthesis and sugar utilization were studied in detail. They were divided into two groups: (i) GerALA mutants, failed to germinate in 10 mM L-alanine but germinated in complex media (some of these mutants were temperature sensitive); (ii) GerPAB mutants, germinated poorly, even in complex media, suggesting that they were blocked in important germination functions. All the mutants failed to germinate in L-a-amino-n-butyrate or L-valine (including temperature-sensitive mutants only at the restrictive temperature) showing that there is a step necessary for germination affected by all three amino acids. The mutants had normal growth rates, indicating that the defective gene products were specific for germination functions. These defects were not identified. Eight of the mutants were mapped by transduction with phage PBS-1. The recombinants were scored either by observation, by microscopy of phase darkening of the spores, or by a plate test involving the reduction of tetrazolium by heated colonies of spores. Five of the mutations, of at least three phenotypes, were between thr-5 and cysB3 away from all the sporulation markers that have been previously mapped. A linked ald (alanine dehydrogenase) locus was on the other side of thr-5. The other Ger markers were located in at least two additional positions. Auxotrophic strains that were used for mapping germinated normally, but germination of the Ger mutants differed slightly in different genetic backgrounds.

Spores of Bacillus subtilis 168 germinate well in suitable buffer solutions containing L-alanine or a mixture of L-asparagine plus glucose plus fructose plus potassium ions (39). Some mutants fail to germinate with L-alanine, but do so with the mixture of compounds (4, 40). It has been suggested that in both systems (i.e., L-alanine or the mixture of compounds) germination is induced by supplying three requirements: an amino group; reducing power, as the reduced form of nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide

fructose. Either L-alanine or L-asparagine can act as amino donors (31). There is conflicting evidence for the involvement of an alanine dehydrogenase in germination (5, 6, 23, 25, 27, 28, 30). Little is known about the mechanism of germination, the events that result in the rapid loss of dormancy, or of resistance to chemical and physical agents (9). There have been several reports of the isolation of mutants with altered germination properties (4, 22, 31, 37, 38, 40) that may be useful in studying this mechanism. In most cases however, the specific defects of the mutants have not yet been identified. Some mutants of Bacillus megaterium QM B1551 with defects in vegetative growth (e.g., nutritional requirements, antibiotic resistance, or differences in morphology or pigmentation) were also defective in germination (22). The two phenotypes may not have been due to the same mutation as another group of mutants similarly defective in vegetative growth were normal for germination (36). Temperature-sensitive (ts)

phosphate (NADPH); and fructose-6-phosphate. Apparently, L-alanine produces the reducing power by its metabolism (direct or indirect) by dehydrogenases, and the fructose6-phosphate by a fructoneogenic pathway. When the mixture of compounds is used for germination, NADH (or NADPH) is produced by glucose dehydrogenase using glucose as substrate, and fructose-6-phosphate is supplied by I Present address: Centre de G6nftique Molkculaire, C.N.R.S., 91190 Gif-sur-Yvette, France.

83

84

J. BAcToL.

TROWSDALE AND SMITH

germination mutants of the same organism have been isolated, some of which were also ts for vegetative growth (37). Mutations in vegetative growth or sporulation may affect germination indirectly as the conditions during sporulation can influence subsequent germination (16, 21, 23). We would expect genes responsible for the normal germination mechanism to be transcribed during sporulation, as germination is a rapid, lytic process, insensitive to antibiotics that block transcription and translation (12, 15). From a genetic point of view there are several questions concerning germination, the answers to which would be valuable for understanding the sporulation cycle and the initiation of growth. These questions concern the number of specific genes involved, at what stage they are

transcribed and translated, and the nature of their products. To start a genetic analysis of germination we have isolated and mapped several germination mutants. A preliminary report of this work has been published (34). MATERIALS AND METHODS Bacterial strains. Strains of B. subtilis 168 used in this study are listed in Table 1. The germination mutants have only been given a phenotypic designation, (either GerA or GerPAB, according to their germination properties) as they are mutations resulting in altered development (42). The recommendation that the symbol Gsp should be used to designate both germination and outgrowth mutants (42) has not been followed, at present, to avoid confusion between the two stages. Media. The following media were used: PGA, potato-glucose-yeast extract agar (4); TBAB, tryptose

TABLE 1. Strains of B. subtilis No.a

Genotype and phenotypeb

Origin and remarks

A

I. Takahashi N. Sueoka N. Sueoka D. Dubnau D. Dubnau D. Dubnau D. Dubnau D. Dubnau C. Anagnostopoulos C. Anagnostopoulos E. Freese J.-A. Lepesant

168 Mu8u5u5 Mu8u5u6 BD40 BD70 BD111 BD112 BD163 GSY254 GSY 289 60229 QB637

trpC2 leu-8 metB5 thr-5 leu-8 metB5 purB6 phe-12 argA3 metC3 trpC2 thr-5 cysB3 trpC2 cysA14 hisAl argC4 lys-1 trpC2

1484 1485 1486

GerALA-84 trpC2 Ger PAB-85 trpC2 GerALA_86ts trpC2 thi (ts) GerPAB 87 trpC2

NGc

GerALA-88ts trpC2 Wrd GerALA-97ts trpCt, smo thi (ts) GerPAB-98 trpC2smo GerPAB-3 trpC2 GerPAB-98 thr-5 trpC2 GerALA_97ts thr-5 trpC2 GerPAB-3 thr-5 trpC2 GerALA-58ts trpC2 Wrd Ger PAB-87 thr-5 trpC2 Ger+ thr-5 trpC2

Unstable, NG NG NG NG 1498-_BD111e

ura-3 trpC2

aid trpC2 sacQ36 thr-5 trpC2

B

1487d 1488 1497 1498 1503 1524 1549 1554 1558 1559 1562

NG NG NG

1497_,BD111e

1503-_BD111e Unstable, NG

1487-oBD11e 168 -_BD111e

Strains with four-figure numbers were isolated in this laboratory. A, Basic strains carrying reference markers; B, germination mutants and their derivatives. bald, Lacks alanine dehydrogenase (6); smo, smooth colonies (tentatively assigned this designation because of the similarity to smo mutants which map in the same region) (10, 41, 42); thi, requires thiamine (1 pg/ml); sacQ36, high levan sucrase production (screening tests for this phenotype were done by J. LepesantKejzlarovi according to a published method [181). c NG, Mutagenesis (of strain 168) with N-methyl-N'-nitro-N-nitrosoguanidine. d Grew slowly on minimal medium, but defect not identified. e Strains obtained by transduction. Arrows point to recipient strain. a

VOL. 123, 1975

GERMINATION MUTANTS OF B. SUBTILIS

blood agar base (Difco); soft TBAB, the constituents of TBAB with 1% Oxoid agar no. 1 for the normal agar content; PAB, penassay broth (Difco); and transduction medium (14). Minimal medium (1), containing glucose (5 mg/ml), was solidified by the addition of Oxoid agar no. 1 (15 mg/ml) when used in plates. Alanine (1 mg/ml) replaced glucose when selection was for aid+ (6). Other amino acids were added at a final concentration of 50 Ag/ml, bases at 100 ,ug/ml, and thiamine at 1 ,gg/ml. Preparation of spores. Spores were produced on PGA plates incubated for 60 h at 37 C. They were scraped from the agar and washed three times by centrifugation in distilled water at 4 C. After lysozyme treatment (200 ,g/ml in 0.85% saline at 20 C for 30 min), the spores were washed an additional 10 times. They were stored as suspensions in water at -20 C. Whenever spores were collected from a mutant they were also similarly collected from strain 168. The 168 spores were then used as a control in all germination experiments, as length of storage of spores and conditions during sporulation can affect germination. Spore preparations usually contained less than 1% germinated (phase dark) spores and no vegetative cells. Germination. To study germination the fall in optical density (OD,.0) of suspensions of spores was followed in a Hilger Watts "Biochem" spectrophotometer. The initial OD6.0 was adjusted to about 0.4 (about 1.2 x 108 spores/ml). Germination was confirmed by observation of the percentage of phase-dark spores after 60 min. The spores were heat activated at 80 C for 10 min before use. Except for germination in PAB medium, all germination preparations were in 0.1 M Tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.4) and contained 10 mM 0-carbamyl-D-serine as an amino acid racemase inhibitor (8). L-Alanine (ALA), L-asparagine (ASN), L-valine, L-a-amino-n-butyric acid, and D-alanine were used at a concentration of 10 mM in this buffer. D-Glucose (GLC), D-fructose (FRU), and 2-deoxyglucose were used at 1 mg/ml, and KCl at 10 mg/ml. In the text ALA buffer, ASN plus GLC buffer, etc., indicate the amino acids or sugars at the above concentrations in 0.1 M tris(hydroxymethyl)aminomethane - hydro ride buffer (pH 7.4) plus 10 mM O-carbamyl-D-serine. Readings of OD,60 have been normalized to the initial value (relative OD..0 = OD,../OD,., initial). Isolation of germination mutants. Log-phase cultures of strain 168 were mutagenized with N-methylN'-nitro-N-nitrosoguanidine (75 ug/ml) for 1 h at 37 C (about 0.1% survival). Samples of the mutagenized culture were spread onto PGA plates and spores were collected. A suspension of spores (about 108/ml) in PAB medium was heat activated (80 C for 10 min) and incubated immediately at 42 C for 2 h. Germinated spores were killed by heating (80 C for 10 min) and incubation was continued for 16 h. After further heat treatment (80 C for 10 min) survivors (about 2 x 10- 6) were detected by incubating samples of the culture on TBAB plates at 30 C for 24 h. Colonies were picked and purified by streaking from single colonies at least five times. Mutants 1484 and 1485 were isolated using a slightly modified tech-

85

nique, substituting 10 mM ALA plus 10 mM 0-carbamyl-D-serine plus 0.1 M tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.4) for PAB medium and repeating the whole procedure twice. The following groups of mutants were isolated in different experiments, using separately mutagenized cultures: (i) 1484 and 1485; (ii) 1488 and 1558; and (iii) 1486, 1487, 1497, 1498, and 1503. Scoring Ger+ and Ger recombinants. Two methods were used to distinguish Ger+ and Ger recombinants. (i) After purifying the transductants, by streaking on selective media, inoculations were made about 10 mm apart on PGA plates. The plates were incubated for 5 days at 37 C to allow maximum sporulation. A loopful of spores from each colony was suspended in a drop of PAB medium on a microscope slide and examined immediately for the presence of phase-dark spores (these were usually less than 1%). After a 1-h incubation at 37 C the slides were reexamined. Ger+ clones contained over 80% phase-dark spores, and GerPAB less than 10%. The reliability of this technique was confirmed by testing clones (usually five) of each phenotype for germination. This was done by following the fall in OD,80 of washed suspensions of spores. GerA" and Ger+ clones were not readily distinguishable by this method. (ii) Colonies of transductants were produced on PGA plates, as above. After 7 days of incubation the plates were heated in an oven at 80 C for 30 min, allowed to cool, then overlain with 10 ml of a solution of triphenyl tetrazolium chloride (2:3:5) (British Drug Houses), at a concentration of 50 jsg/ml in soft TBAB agar at 50 C. The plates were incubated at 30 C and observed for the red coloration of the colonies after 16 and 72 h. In general, colonies of Ger mutants had less red coloration than those of Ger+. However, there were some important differences in the level of coloration of some strains which will be referred to later. Owing to the difficulties in testing large numbers of recombinants by these techniques the samples taken from each transduction experiment were small (usually \ 1484, 1486, and 1497. ;\\ In PAB medium (Fig. 2), strains 1484 and l497(42-_. 30°) 1488 germinated almost as well as strain 168. u,\ \ All the other mutants responded poorly (strain 0\. 1558 was not tested in PAB, but it is probably o0 1488 similar to strain 1488 as both mutants had \A similar ts germination properties in ALA X0 A buffer). Similar results were obtained at 42, 37, 1 168 and 30 C. However, mutants 1486 and 1497 30 60 germinated rapidly on shifting the temperature from 42 to 30 C after 30 min. Without this Time(min) preincubation at 42 C germination in PAB meFIG. 2. Germination of the Ger mutants and the dium at 30 C was poor (as for 37 C, Fig. 2). The parental strain (168) in PAB medium at 37 C. Similar graphs show the results of the experiments results were obtained at 42 or 30 C. 1497 is also shown where the fall in OD was maximum, and this with a temperature shift from 42 to 30 C after 30 min, the 1487 mutants sometimes especirepresented less, less, was sometimes especially if the spores were are included byonthethedashed figure: line. the Not curvesall for and used within a few days of preparation. 1503 were identical to that shown for 1498; similarly, It is convenient to consider the mutants in that for 1486 was identical to 1497; and 1484 to 1488. two phenotypic groups: (i) GerAIA, which failed 1558 was not tested in PAB medium. 1.0 < to germinate in ALA buffer but germinated in °\- l484 30. PAB medium (1484, 1558, 1488, 1486, and 1497, a the last two of which germinated in PAB £ 1486 30Q \\,4 medium on shifting the temperature from 42 to U* 0.91497- 0 30 C); (ii) GerPAB, which germinated poorly, \\ \\ >8_ even in PAB medium (1485, 1487, 1498 and 0 o 1503). o 0 The mutants were next tested for germination 0\ 0 1488 37 ui o compounds previously shown to be important in > 0.8-1842 0 for B. subtilis 168 spore germination (31). The 168-42' results \ are summarized in Table 2. As with U.j \ oa previous work (31) the parental strain (168) was ° germinated by the mixture of ASN plus GLC 0.7N plus FRU. If any component of the mixture was 14 883left out germination did not take place. (Con0,L , , X , 63 trary to previous studies [31] KCl was stimula30 60 tory in our experiments but never obligatory. Time(min) When germination took place its presence usually increased in relative OD580 after 60 FIG. 1. Germination of the Ger mutants and the m*n by abot1thet fall0) ). parental strain (168) in ALA buffer at 42, 37 and 30 C. Y The curves for 1558 were identical to those of 1488 and Of the GerArA mutants, strains 1486 and 1497 have not been shown. The dashed line indicates all were similar to strain 168 in the mixtures of the other mutants without separate curves: 1488 and compounds tested. They differed from this 1558 at 42 C; 1484, 1486, and 1497 at 37 and 42 C; and strain in that they were germinated poorly by 1485, 1487, 1498, and 1503 at 30, 37, and 42 C. ALA plus GLC or ALA plus FRU, although 0

was

InX

168-3081 -o0168-37-1

GERMINATION MUTANTS OF B. SUBTILIS

VOL. 123, 1975

87

TABLE 2. Summary of the germination properties of the Ger mutants. The results are shown for experiments at 37 Ca

j AL Stran

PAB

168

+

Strain

+

1486

-b

1497

-b

1484

1558 1488

PAB

ALA

FRU +

b

b

ALA+ FRU

ALA+ GLC +

GLC+ ASN+ ASN+ FRU GLC ASN+ FRU GLC+

b

+

BU AB

VAL VL

FRU

-

+

+

-

-

+

±

-

-

-

-

ND -c

ND -

ND

+

b+

+

+

+

+

+

- (ts)

ND

ND

- (ts)

+

+

ND ND 4b

-

-

-

-

ND _C

ND +

4

(tS)

ND

-(tS)

4

1503

1485 1487

iGALA+ GLC+

_

_

a ABU, L-a-amino-n-butyrate; VAL, L-valine; ND, not determined; +, relative OD.80 after 60 min less than 0.7 at 37 C; 4, relative OD,80 after 60 min between 0.7 and 0.85 at 37 C; -, relative OD6.0 after 60 min greater than 0.85 at 37 C. All samples were in 0.1 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.4) and contained 10 mM O-carbamyl-D-serine. b Responded to a temperature shift from 42 to 30 C after 30 min. c Some germination, probably due to contaminating amino acids in the spore preparation.

normally by the mixture of all three compounds. If incubated at 42 C for 30 min, followed by 30 C, they were germinated more rapidly by ALA, ALA plus GLC, or ALA plus FRU than if the spores had simply been incubated at 30 C throughout the experiment. This effect was most pronounced in ALA plus FRU buffer where there was a twofold increase in the maximum rate of fall in relative OD580 at 30 C, if incubated at 42 C for 30 min first. Mutant 1484 was normal in combinations of ASN, GLC, and FRU, but it needed the addition of only GLC for germination in ALA buffer. Unlike strains 1486 and 1497, described above, 1484 spores were not germinated by ALA plus FRU, even with a 42 to 30 C temperature shift after 30 min. 2-Deoxyglucose could replace the GLC requirement for germination with ALA or ASN plus FRU for this mutant. D-Alanine inhibited germination of 1484 spores induced by ALA plus GLC but was without effect with ALA (or ASN) plus GLC plus FRU (relative 0D680 after 60 min at 37 C was 0.93 and 0.58, respectively). Of the two ts mutants, 1488 and 1558, only the former was studied in detail. Unfortunately these two mutants sporulated poorly and spore preparations contained up to 10% phase-dark (germinated) spores. The exudate from these spores probably contaminated the preparation with traces of germinants. This may have been the reason why even with GLC plus FRU (i.e., no amino acid) there was considerable germination. At the nonpermissive temperature (42 C)

1488 spores germinated best in ALA (or ASN) plus GLC plus FRU buffer. Like spores 1486 and 1497, germination was stimulated by preincubation in ALA plus FRU buffer at 42 C followed by a drop to 30 C after 30 min. When 1488 spores were incubated with L-a-amino-n-butyrate or L-valine (two other amino acids which alone germinated spores of B. subtilis to some degree; 39) germination was still ts. The results are shown for L-a-amino-n-butyrate in Fig. 3. D-Alanine inhibited germination stimulated by any of the three amino acids. The GerPAB mutants were germinated slowly 427

Time (min)

FIG. 3. Germination of mutant 1488 in L-aamino-n-butyrate buffer at 42, 37, and 30 C, and with a temperature shift from 42 to 30 C after 30 min. Strain 168 is also shown at 37 C. The dashed line indicates all the other mutants (except 1558 which was not tested) at all three temperatures.

88

TROWSDALE AND SMITH

J.BACTFJOL.

by PAB medium and by the mixture of ALA (or ASN) plus GLC plus FRU (plus KCl). However, some of these mutants did respond weakly to certain germination conditions. For example, spores of strain 1485 were germinated slowly by ALA plus GLC plus FRU, or by ALA plus FRU, after a temperature shift from 42 to 30 C after 30 min (the maximum rate of fall in relative OD,680 was 0.004 U/min). Mutant 1503 also germinated to some extent in ALA plus GLC plus FRU buffer (relative OD,80 after 60 min, 0.8 at 37 C). To check that measurement of OD was a reliable indicator of the failure of GerP" spores to germinate, spores of one of these mutants were tested for their ability to form colonies on TBAB plates. Suspensions of spores of strains 168 and 1498 were adjusted to the same OD5,0 (they presumably contained similar numbers of spores). After heat activation (80 C for 10 min) they were diluted and plated on TBAB, and the colonies were counted after incubation for periods of 14 to 48 h. The numbers of colonies arising from strain 168 spores remained constant (5.9 x 108/ml) over this time period. On the contrary, the numbers of colonies from strain 1498 spores increased from 1.6 x 107/ml after 14 h of incubation to the maximum of 2.6 x 107/ml after 37 h. These results show that germination of strain 1498 was less than 5% of that of 168 when measured in this way. The 1498 spores that did germinate in these conditions were not revertants; when five colonies were picked and the spores were collected and tested for germination in PAB medium, they retained their Gerp^ phenotype. Other properties of the Ger mutants. Some of the mutants had altered growth properties in the vegetative phase. In all but two cases these

properties

were shown to be due to separate mutations. Strains 1486 and 1497 required thiamine (1 gg/ml) at 37 C, but not at 30 C. Five spontaneous thiamine independent revertants retained their Ger phenotype. Strains 1497 and 1498 had a smooth colony morphology (smo). As will be shown later, both thi and smo behaved independently of the Ger characters in transduction mapping experiments. Mutant 1487 grew poorly on minimal medium. Again, spontaneous (large-colony) revertants retained the Ger'" phenotype. Some of the mutants were unstable on isolation and produced irregularly sized colonies. After repeated purification stable clones were obtained for all the mutants except two, 1488 and 1558. These were the only cases where it was not clear whether this additional phenotype was due to the Ger mutation or a separate locus. The growth rates of the mutants (1486 and 1558 were not tested) measured as the time for a doubling of OD580 in minimal medium at 37 C were all similar to that of strain 168 (87 + 2 min). For these determinations, 1 Ag of thiamine per ml was added for mutant 1497, and a revertant of mutant 1487 that produced normal sized colonies was used. Thus as far as was tested the Ger mutations (except possibly 1488 and 1558) did not have associated phenotypes in the vegetative phase. Mapping of Ger mutations in the thr-5cysB3 region. An alanine dehydrogenase (ald) mutant was mapped first, as this enzyme has been implicated in germination. A PBS-1 lysate of strain 60229 (ald trpC2) was used for transduction of recipients with auxotrophic markers of known location (Table 1). Linkage of ald to thr-5 was established in this way and a series of three-factor crosses (Table 3) located ald in the

TABLE 3. Orientation of sacQ36, aid, thr-5, and cysB3 by three-factor transduction crosses Donor

Selection

Recombinant classes

No.

thr-5 cysB3

aid+

aId+ thr+ cys+ aid+ thr+ cys ald+ thr cys+ ald+ thr cys

31 1 46 9

aId+ thr+ cys+ aId+ thr cys+ aid thr+ cys+ aId thr cys+

10 45 25 0

saca a1d+ thr+ sac+ aId+ thr+

100 0 26 162

Recipient

aid

thr-5 cysB3

sacQ36 thr-5

aId

ald

cys+

thr+

sac aid thr+ sac+ ald thr+ a

The sacQ36 phenotype was scored as high levan sucrase production (18).

Order suggested

ald-thr-5-cysB3

aid-thr-5-cysB3

sacQ36-ald-thr-5

GERMINATION MUTANTS OF B. SUBTILIS

VOL. 123, 1975

order sacQ36-ald-thr-5-cysB3. Before exploring linkage of the Ger mutants to thr-5 a suitable phenotype had to be found for scoring the recombinants. Vary and Kornberg (37) showed that colonies of their germination mutants could be recognized by replicaplating heat-treated colonies (mainly spores) onto fresh medium. Replicas of normally germinating colonies grew under these conditions and those of the mutants failed to do so. A similar technique applied to the GerPAB mutants in the present study was not effective, as some spores from the GerP" clones did germinate and these grew into colonies that were not readily distinguishable from Ger+. A more reliable difference was noted if each clone was transferred individually with a loop, instead of by replicating with a velvet pad. The microscope technique as outlined above was finally used, as it proved to be more sensitive. Five of the Ger markers were linked by transduction to aid, thr-5, and cysB3. The first of these, GerP" -87, is shown in Table 4. The low linkage of this marker to thr-5 and ald (Table 4) indicated that it was not between these two markers. A three-factor cross (Table 4) suggested the order thr-5-GerP" -87-cysB3. Control crosses, of a marker (leu-8) unlinked to ald, thr-5, or cysB3 in the recipient or of a donor Ger+ strain when selection was for a1d+, thr+ or cys+, did not result in transductants of the GerP^ phenotype. In each of these crosses at least 50 transductants were scored by the microscope technique. All the GerP^ transductants grew normally on minimal medium, showing that the poor growth of strain 1487 was not due to the GerPB -87 mutation, as also indicated by

89

the reversion studies described earlier. GerP" -98 and -3 were similarly located (Tables 5 and 6). The smooth colony morphology (smo) of strain 1498 appeared, in these experiments, to be due to a separate mutation on the other side of cysB3. The two other germination markers in this region were of Ger ALA mutants (strains 1486 and 1497). Even though germination of these two mutants in PAB medium was not unlike that of the GerP" mutants at 37 C (Fig. 2), in practice it proved difficult to distinquish their GerALA phenotypes from Ger+ by the microscope test. If ALA buffer was used in this test, instead of PAB

medium, the distinction was again difficult, due to the large numbers of Ger+ spores which remained phase bright under these conditions. Thus, small numbers of transductants were tested for germination directly, by measuring the fall in OD of spore suspensions. The results for GerA-97 ts are shown in Table 7. Even with the rather limited tests done this marker also appeared to be between thr-5 and cysB3. Out of the six thr+ cys+ clones examined none were found corresponding to classes requiring four recombination events (thr+ Ger+ cys+ smo+ and thr+ Ger+ cys+ smo), assuming the order thr-5Ger--97 ts-cysB3-smo. GerA-86 ts was mapped similarly (Table 8). None of the transductants in the crosses of Tables 7 and 8 had the ts thiamine requirement of the donors, confirming that in both cases this was due to separate mutations. Mapping of Ger mutations in other regions. As well as being ts for germination in ALA buffer, mutants 1488 and 1558 were unstable. This was particularly noticeable when they were

TABLE 4. Mapping GerPAB-87 Recipienta ] Donor Recipienta

Selection

~~~~Recombinant classes

No.

Cotransduction

Order suggested

A

thr-5

GerPAB_87

thr+

thr+ Ger+PABb

9

46

16

aid

GerPAB-87

aId+

thr+ Ger+ aId+ GerPAB aid+ Ger+

10 40

20

B

thr5-GerPAB-87 0 thr+ Ger+ cys+ cysB3 11 thr+ GerPAIW cys+ 36 thr Ger+ cys+ 33 thr GerPAB cys+ a A, By two-factor transduction with aid and thr-5; B, by a three-factor cross with thr-5 and cysB3. b The Ger+ and GerPAB phenotypes were scored by the microscope technique. Five clones of each phenotype were then tested for germination in ALA buffer and in PAB medium, after collecting spores, and showed that the initial scoring was accurate. thr-5 cysB3

Ger PAB-87

cys+

90

TROWSDALE AND SMITH

J. BACTERIOL.

TABLE 5. Mapping GerPAB-98 and smo by transduction Recipienta

thr-5

Donor

Selection

GerPAB-98 smo

thr+

Recombinant classes

No.

thr+Ger+smo+b

95

thr+ GerPA smo+ thr+ Ger?A8 smo

GerPAS-98 smo

thr-5 cysB3

Order suggested

thr-5-GerP B-98-smo

4

1

cys+

thr+ Ger+ cys+ smo 1 thr+ GerPAB cys+ smo 5 thr Ger+ cys+ smo 18 thr GerPAB cys+ smo 19 thr-5-GerPAB-98thr+ Ger+ cys+ smo+ 0 cysB3-smo thr+ GerPAB cys+ smo+ 17 thr Ger+ cys+ smo+ 11 thr Ger PAB cys+ smo+ 9 a thr-5, Orientation by a three-factor cross; thr-5 cysB3, orientation by a four-factor cross. b,As in Table 4, the Ger+ and Ger^AB phenotypes were scored by the microscope technique and five clones of each phenotype were then checked by germination in ALA buffer and in PAB medium after collecting spores. TABLE 6. Mapping GerPAB-3 Recipienta

Donor

Selection

Recombinant classes

No.

Cotransduction

Order suggested

(%) GerPAB_3

thr-5

GerPAS_3

thr-5 cysB3

thr+

cys+

thr+ GerPABs

thr+ Ger+

30 50

thr+ Ger+ cys+ thr+ GerPAB cys+ thr Ger+ cys+ thr GerPAB cys+

0 25 20 34

37.5

thr-5-GerPA'_3-cysB3

thr-5, Two-factor transduction; thr-5 cysB3, three-factor cross. "The Ger+ and Ger PAO phenotypes were scored as in the experiments of Tables 4 and 5.

a

TABLE 7. Mapping of GerALA-97 ts and smo by four-factor transduction cross Determinants

Recipienta thr-5 cysB3

No. tested for Gerb 5 of 14 1 of 1

5 of 25 4 of 20

Donor

Selection

GerALA_97 ts smo

cys+

Recombinant classes

thr cys+ smo+ thr+ cys+ smo+ thr cys+ smo thr+ cys+ smo thr+ Ger+ cys+ smo+ thr+ GerALA CyS+ smo+ thr+ Ger+ cys+ smo thr+ GerALAcys+ smo thr Ger + cys + smo + thr GerAL ACys+ smo+ thr Ger+ cys+ smo thr GerAUA cys+ smo

No.

Order suggested

25 14 20 1

thr-5-cysB3-smo

0 5

thr-5-GerALA_97 tscysB3-smo

0 1

4 1

3 1

a After scoring for thr and smo, spores were collected from 15 transductants representing all four combinations of the thr and smo alleles. b Spores were tested for germination in ALA buffer to determine Ger phenotype.

grown on TBAB at 42 C, in which conditions designation, Wrd) was mapped, to aid location they produced colonies of various sizes. This of the GerA'--ts mutation. Even though it may character (also given a temporary phenotypic have been due to mutation at another locus, this

91

GERMINATION MUTANTS OF B. SUBTILIS

VOL. 123, 1975

could be closely linked to GerALA-ts due to mutagenesis with nitrosoguanidine. Transduction crosses were first done using donor lysates of strain 1488 and strains with auxotrophic reference markers as recipients (Table 1). In this way Wrd was linked to Iys-i (Table 9). The GerALA-88 ts phenotype was found to have been simultaneously inherited in the Wrd transductants, at least in the small sample scored (Table 9). Similarly, the Wrd and GerAIA-58 ts markers in strain 1558 were also linked to lys-1 (Table

10). This mutant was obtained from the same selection experiment as mutant 1488 and may have resulted from division of the same mutated bacterium. Reliable data for the order of trpC2, Iys-1, and GerAIA-88 ts or -58 ts could not be obtained. The Wrd mutation may have fiad adverse physiological effects on transduction as this took place at low frequency when strains with this phenotype were used as recipients. The two remaining markers, GerALA-84 and GerPAB -85, were not linked to thr-5 or cysB3.

TABLE 8. Mapping of GerALA-86 ts by a three-factor transduction cross Determinants

Donor

Recipienta thr-5 cysB3

GerALA_86 ts

Selection Recombinant classes

cys+

No. tested for GerP 5 of 19

55

19

thr+ Ger+ cys+ thr+ Ger ALA cys + thr Ger+ cys+ thr GerALA cys+

9 of 55 a

cys+ thr cys+ thr+

Order suggested

No.

0 5 1 8

thr-5-GerALA 86 ts-cysB3

After scoring for thr, spores were collected from 14 transductants (thr+ and thr).

Spores were tested for germination in ALA buffer to determine Ger phenotype. TABLE 9. Mapping of GerALA-88 ts (Wrd) by two-factor transduction Determinants

Recipienta Iys-l

Donor

Selection

GerALA-88 ts Wrd

Iys+

No. tested for Gerb 5 of 44

No.

Cotransduc-

44 36

55

lys+ Wrd lys+ Wrd+ Iys+ Wrd GerAL A-88 ts lys+ Wrd Ger+ 1ys+ Wrd+ GerALA88 ts lys+ Wrd+ Ger+

5 of 36 a

Recombinant classes

5 0 0 5

After scoring Wrd, spores were collected from 10 transductants (Wrd+ and Wrd). Spores were tested for Ger phenotype. TABLE 10. Mapping of GerALA-58 ts (Wrd) by two-factor transduction Determinants

Recipienta lys-I No. tested for Gerb 4 of 52 2 of 28 a

b

Donor

Selection

Ger ALA_ 58 ts Wrd

Iys +

Recombinant classes

lys+ Wrd lys+ Wrd lys+ Wrd GerAL A-58 ts Iys+ Wrd Ger+ Iys+Wrd+ Ger LA-58 ts Iys+ Wrd+ Ger+

After scoring Wrd, spores were collected from six transductants (Wrd+ and Wrd). Spores were tested for Ger phenotype.

No.

Cotransduc-

52 28

65

4 0 0 2

92

TROWSDALE AND SMITH

This was established in an extensive analysis of recombinants in crosses similar to those on Tables 4 to 8 and using strains 1484 and 1485 as donors. Further crosses were done using lysates of strain 1485 and reference auxotrophs (Table 1) as recipients. In each case 80 transductants were analyzed using tetrazolium (see later). Low linkage of GerA"A-85 and hisAl was established in this way. Three (out of 80 hisA+) clones that were pink-orange colored, in comparison to the other deep-red colonies, were confirmed as Gerr^B by germination studies. However no linkage of GerP"^-85 to argC4 or cysB3 could be demonstrated. This is not surprising in view of the newly published data on the B. subtilis genetic map, showing that hisAl is not linked to argC4 (20). No evidence of linkage of GerAIA-84 to hisAl was obtained. Scoring Ger recombinants using tetrazolium. The basis of the tetrazolium reaction and its relationship to germination is not understood, but it appeared to have potential for scoring Ger recombinants. It was used, as indicated above, to locate GerP -85. Also, strains 1524, 1549, 1554, and 1559 (GerP-'-98, GerALJA97 ts, Ger'"-3, and GerP"3-87, respectively, in the genetic background of strain BD111 transduced to cysB+) were all readily distinguishable from strain 1562 (BD111 transduced to cysB+ using strain 168) by the tetrazolium reaction. Colonies of the latter were deep red, in contrast to the pink-orange coloration of those of the former after 12 h of incubation in the test. Curiously strain 168 was not so easily distinguishable from the Ger mutants. Only after 72 h of incubation did the 168 colonies become colored, in comparison to the Ger colonies which had no red coloration after this time. In other words, in the genetic background of the strain 168 that we used there was a difference between Ger+ and Ger clones, but the overall level of coloration was decreased. This suggested that the strain of B. subtilis 168 (the parental strain of all the Ger mutants) was not isogenic with the other reference strains used. Indeed, when examining possible linkage of Germ-84 and GerP"-85 to lys-i, clones of two tetrazolium phenotypes (i.e., deep red and pink-orange after 12 h of incubation) were obtained, but the latter phenotype was not due to inheritance of the Ger markers-all the transductants were Ger+ when tested for germination by OD changes. In control crosses lys+ transductants of the two phenotypes were also obtained when using strain 168, but not strain BD111 as donor. Strain 168 must have a mutation linked to lys-i which lowers the degree of

J. BACTEIOT..

coloration with the tetrazolium test. The marker may not be connected with germination. However there were other indications that strain 168 was slightly defective in germination. This became apparent when the Ger markers were introduced into other strains. For example, when germination of strain 1498 (GerPa -98 smo trpC2) was compared with Ger".-98 smo trpC2 (thr-5+ cysB3+) transductants in the BD111 genetic background (Table 5), the relative OD,., after 60 min germination in PAB medium was 1.0 for 1498, and from 0.86 to 0.95 for the latter strains. All the spore preparations for these tests were made in the same conditions, at the same time. A similar effect was noted for the other Ger markers when introduced into BD111; germination was always slightly increased. Spores of strain 168 itself germinated slightly less well than those of strain BD111 (measured by the fall in OD,,,), although it has not been ruled out that this is due to differences in the nutritional requirements of the two strains. Other markers did affect germination slightly. For example, in the cross shown in Table 7, the fall in relative OD,,, after 60 min in ALA plus GLC plus FRU buffer at 37 C in spores of the transductants was enhanced by about 5% by the presence of either the smo or the thr allele. DISCUSSION The scheme proposed by Prasad et al. (31) provides an explanation of the germination of B. subtilis spores by ALA, or by ASN plus GLC plus FRU (plus KCl), and the dependence of certain mutants on the mixture of compounds. The properties of some of our Ger mutants may also be explained by this scheme. Our mutants (and strain 168 in ASN plus GLC plus FRU buffer) did not have an obligatory requirement for KCl and this may mean that traces of K+ ions were already present in the spores. Insufficient washing, or the different method of preparing the spores, could account for this. Mutants 1486 and 1497 required both GLC and FRU, in addition to ALA or ASN, to germinate at 42 C, suggesting that they were blocked in a reaction in fructoneogenesis necessary for both NADH (or NADPH) and fructose6-phosphate production. A previously described mutant lacking phosphoglycerate kinase had a similar phenotype (31). Spores of B. subtilis were shown to respond more rapidly to the addition of GLC (plus KCl) after preincubation with ASN plus FRU at 44 C (39). The stimulatory effect of preincubation of 1486 and 1497 spores at 42 C, on subsequent germination at

VOL. 123, 1975

GERMINATION MUTANTS OF B. SUBTILIS

30 C, is probably a manifestation of the same reaction. It takes place in these two mutants even in ALA plus FRU buffer, because in these conditions germination does not occur at 42 C. Strain 1484 was different than previously described mutants as it required GLC but not FRU for germination in ALA buffer. It is unlikely that GLC provided the FRU as it could be replaced by 2-deoxyglucose which is not metabolized to fructose-6-phosphate (31). That the ALA itself was providing the FRU was suggested by the inhibitory effect of D-alanine in the absence of FRU (i.e., in ALA plus GLC buffer) but not in its presence (in ALA [or ASN ] plus GLC plus FRU buffer). Presumably the GLC is necessary for this mutant to form enough NADH (or NADPH). It is possible that one of the enzymes normally providing reducing power is missing, so that all the NADH made by the metabolism of ALA is reutilized for fructose6-phosphate production. Alternatively the level of NADH oxidase may be high in this mutant. The ts germination of 1488 spores with L-aamino-n-butyrate, L-valine, or L-alanine indicates that there is a step (the ts step) necessary for germination affected by all three amino acids. It is improbable that these compounds are all metabolized to pyruvate. Perhaps they act as triggers (9) and stimulate metabolism of endogenous ALA. In fact there is little evidence of uptake or metabolism of ALA when a radioactive label is used (Dring, personnal communication; 35). There is a large pool of free ALA within the dormant spore (24) which could be activated by triggers. D-Alanine inhibited germination by any of the three amino acids, again suggesting that they initiated germination by the same mechanism. The GerP" mutants may be defective in major germination functions or may have structural defects in their spores, as they germinated poorly under all the conditions used. A summary of the mapping experiments is given in Fig. 4. Location of five Ger markers between thr-5 and cysB3 is of interest. They were all taken from the same mutagenesis and selection experiment and could have arisen by division of the same initial mutated cell. There are two pieces of evidence against this. First, some of the mutants carried different additional lesions (e.g., smo, thi). Second, the Ger mutants were of at least three different phenotypes, two GerA and three GerPAB, one of which had slightly different properties (Table 2). Few mutations, and no sporulation markers, have so far been located in this region (42). The two GerALAts markers linked to lys-1 were

93

FIG. 4. Abbreviated map of the B. subtilis chromosome (after Harford [13] and Lepesant-Kejzlarova et al. [19]). The markers mapped in the present work are shown in boxes; the orientation of those in the same box is not known. A linkage which has been demonstrated only by two-factor crosses is indicated by a dotted line. GerALA-84 was not mapped. 0, Origin; T, terminus of replication.

also obtained in one selection experiment. They were probably of common origin as they were similar in both germination and vegetative growth. Insufficient genetic analysis has been done to determine clearly whether the unstable growth of these mutants on TBAB agar at 42 C was a consequence of the GerA"A_ts mutation. The choice of nitrosoguanidine as a mutagen was unfortunate, as it has been reported to cause multiple lesions at high frequency (11), and indeed most of the Ger mutants had additional mutations. For further studies it would be desirable to introduce the Ger markers

into isogenic strains. By nature of their phenotypes more sophisticated genetic analysis of the Ger mutants will be difficult, although the tetrazolium reaction is promising as a rapid method for screening recombinants. The basis of the reaction in relation to germination is not understood. A similar technique (without preheating the plates) was used to distinguish between some sporulation mutants (2), and tetrazolium was also used in tests to identify respiratory mutants in yeast (29) and sugar metabolism mutants in Escherichia coli (17). Balassa (2) suggested that the ability of spores (as old colonies)

94

TROWSDALE AND SMITH

to reduce tetrazolium was related to the appearance of NADH oxidase, glucose dehydrogenases, or other dehydrogenases in sporulation. Such dehydrogenases may be important for germination (31). The strain of B. subtilis 168 differed from strains we obtained from other laboratories in the tetrazolium test although it was from the same initial source (32). Presumably this is due to an additional spontaneous mutation (near lys-1) in our strain. All the auxotrophs used for mapping germinated in ALA buffer although there were small differences in the extent of fall in OD due to certain markers (thr-5 or smo). None of the Ger markers, except possibly GerALA-88 ts and -58 ts, resulted in abnormal vegetative growth, hence the defective products were probably specific to germination. Vary and Kornberg (37) described some ts germination mutants of B. megaterium QM B1551 which were also ts for vegetative growth, showing that some gene products may be required for both stages. Similar mutants have been isolated for the outgrowth stage (3, 26; also see 7). This type of mutant may have been lost in our selection experiments. We have not been able to identify the specific defects in the Ger mutants. There were no visible differences in thin sections of the mutant spores when examined by electron microscopy, and levels of alanine dehydrogenase and spore dipicolinic acid were normal (unpublished data). It would be interesting to identify these defects. The GerPAB mutants in particular are probably defective in important components of the germination mechanism, for example lytic activity. ACKNOWLEDGMENTS We are grateful to G. W. Gould, P. Piggot, and J. Dring for helpful discussions. The work was supported by a Science Research Council Cooperative award in Pure Science.

LITERATURE CITED 1. Anagnostopoulos, C., and J. Spizizen. 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81:741-746. 2. Balassa, G. 1969. Biochemical genetics of bacterial sporulation. I. Unidirectional pleiotropic interactions among genes controlling sporulation in Bacillus subtilis. Mol. Gen. Genet. 104:73-103. 3. Dawes, I. W., and H. 0. Halvorson. 1974. Temperaturesensitive mutants of Bacillus subtilis defective in spore outgrowth. Mol. Gen. Genet. 131:147-157. 4. Dring, G. J., and G. W. Gould. 1971. Movement of potassium during L-alanine-initiated germination of Bacillus subtilis spores, p. 133-142. In A. N. Barker, G. W. Gould, and J. Wolf (ed.), Spore research 1971. Academic Press Inc., London. 5. Freese, E., and M. Cashel. 1965. Initial stages of germina-

J. BACTERIOL. tion, p. 144-151. In L. L. Campbell and H. 0. Halvorson (ed.), Spores III. American Society for Microbiology, Ann Arbor, Mich. 6. Freese, E., S. W. Park, and M. Cashel. 1964. The developmental significance of alanine dehydrogenase in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 51:1164-1172. 7. Galizzi, A., F. Gorrini, A. Rollier, and M. Polsinelli. 1973. Mutants of Bacillus subtilis temperature sensitive in the outgrowth phase of spore germination. J. Bacteriol. 113:1482-1490. 8. Gould, G. W. 1966. Stimulation of L-alanine-induced germination of Bacills cereus spores by D-cycloserine and O-carbamyl-D-serine. J. Bacteriol. 92:1261-1262. 9. Gould, G. W. 1969. Germination, p. 397-444. In G. W. Gould and A. Hurst (ed.), The bacterial spore. Academic Press Inc., London. 10. Grant, G. F., and M. I. Simon. 1969. Synthesis of bacterial flagella. II. PBS1 transduction of flagellaspecific markers in Bacillus subtilis. J. Bacteriol. "9:116-124. 11. Guerola, N., J. L. Ingraham, and E. Cerda-Olmedo. 1971. Induction of closely linked multiple mutations by nitrosoguanidine. Nature (London) New Biol. 230:122-125. 12 Halvorson, H. O., J. C. Vary, and W. Steinberg. 1966. Developmental changes during the formation and breaking of the dormant state in bacteria. Annu. Rev. Microbiol. 20:169-188. 13. Harford, N. 1975. Bidirectional chromosome replication in Bacillus subtilis 168. J. Bacteriol. 121:835-847. 14. Karamata, D., and J. D. Gross. 1970. Isolation and genetic analysis of temperature-sensitive mutants of B. subtilis defective in DNA synthesis. Mol. Gen. Genet. 108:277-287. 15. Keynan, A., and H. Halvorson. 1965. Transformation of a dormant spore into a vegetative cell, p. 174-179. In L. L. Campbell and H. 0. Halvorson (ed.), Spores HI. American Society for Microbiology, Ann Arbor, Mich. 16. Lechowich, R. V., and Z. J. Ordal. 1962. The influence of the sporulation temperature on the heat resistance and chemical composition of bacterial spores. Can. J. Microbiol. 8:287-295. 17. Lederberg, J. 1948. Detection of fermentative variants with tetrazolium. J. Bacteriol. 56:695. 18. Lepesant, J.-A., F. Kunst, J. Lepesant-Kejzlarova, and R. Dedonder. 1972. Chromosomal location of mutations affecting sucrose metabolism in Bacillus subtilis Marburg. Mol. Gen. Genet. 118:135-160. 19. Lepesant-Kejzlarova, J., J.-A. Lepesant, J. Walle, A. Billault, and R. Dedonder. 1975. Revision of the linkage-map of Bacillus subtilis 168: indications for circularity of the chromosome. J. Bacteriol. 121:823-834. 20. Lepesant-Kejzlarovh, J., J. Walle, A. Billault, F. Kunst, J.-A. Lepesant, and R. Dedonder. 1974. Etablissement de la carte genetique de Bacillus subtilis: r6-examen de la localisation du segment chromosomique compris entre les marqueurs sac Q36 et gtaA12. C. R. Acad. Sci. Paris. Ser. D 278:1911-1914. 21. Levinson, H. S., and M. T. Hyatt. 1964. Effect of sporulation medium on heat resistance, chemical composition, and germination of Bacillus megaterium spores. J. Bacteriol. 87:876-886. 22. McCormick, N. G., F. Freeherry, and H. S. Levinson. 1972. Germination properties and proteins of spores of Bacillus megaterium QM B1551 mutants, p. 421-429. In H. 0. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores V. American Society for Microbiology, Washington, D.C. 23. McCormick, N. G., and H. 0. Halvorson. 1963. The production and properties of spores with varying levels

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of L-alanine dehydrogenase. Ann. N.Y. Acad. Sci. 102:763-772. 24. Murrell, W. G. 1969. Chemical composition of spores and spore structures, p. 215-273. In G. W. Gould and A. Hurst (ed.), The bacterial spore. Academic Press Inc., New York. 25. Nitta, Y., Y. Yashuda, K. Tochikubo, and Y. Hachisuka, 1974. L-Amino acid dehydrogenases in Bacillus subtilis spores. J. Bacteriol. 117:588-592. 26. Nukushina, J.-I., and Y. Ikeda. 1969. Genetic analysis of the developmental processes during germination and outgrowth of Bacillus subtilis spores with temperaturesensitive mutants. Genetics 63:63-74. 27. O'Connor, R., and H. Halvorson. 1959. Intermediate metabolism of aerobic spores. IV. Alanine deamination during the germination of spores of Bacillus cereus. J. Bacteriol. 78:844-851. 28. O'Connor, R. J., and H. 0. Halvorson. 1961. L-Alanine dehydrogenase: a mechanism controlling the specificity of amino acid-induced germination of Bacillus cereus spores. J. Bacteriol. 82:706-713. 29. Ogur, M., R. St. John, and S. Nagai. 1957. Tetrazolium overlay technique for population studies of respiratory deficiency in yeast. Science 125:928-929. 30. Prasad, C. 1974. Initiation of spore germination in Bacillus subtilis: relationship to inhibition of L-alanine metabolism. J. Bacteriol. 119:805-810. 31. Prasad, C., M. Diesterhaft, and E. Freese. 1972. Initiation of spore germination in glycolytic mutants of Bacillus subtilis. J. Bacteriol. 110:321-328. 32. Takahashi, I. 1961. Genetic transduction in Bacillus subtilis. Biochem. Biophys. Res. Commun. 5:171-175. 33. Takahashi, I. 1963. Transducing phages of Bacillus subtilis. J. Gen. Microbiol. 31:211-217.

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34. Trowsdale, J., D. A. Smith, and G. W. Gould. 1973. Mapping of germination mutants of Bacillus subtilis 168, p. 103-117. In A. N. Barker, G. W. Gould, and J. Wolf (ed.), Spore research, 1973. Academic Press Inc., London. 35. Uchiyama, H., K. Tanaka, and T. Yanagita. 1965. Role of alanine in the spore germination of Bacillus subtilis. J. Gen. Appl. Microbiol. 11:233-242. 36. Vary, J. C. 1972. Spore germination of Bacillus megaterium QM B1551 mutants. J. Bacteriol. 112:640-642. 37. Vary, J. C., and A. Kornberg. 1970. Biochemical studies of bacterial sporulation and germination. XXI. Temperature-sensitive mutants for initiation of germination. J. Bacteriol. 101:327-329. 38. Warren, S. C. 1969. Spore germination mutants of Bacillus cereus. J. Gen. Microbiol. 55:xviii-xix. 39. Wax, R., and E. Freese. 1968. Initiation of the germination of Bacillus subtilis spores by a combination of compounds in place of L-alanine. J. Bacteriol. 95:433-438.

40. Wax, R., E. Freese, and M. Cashel. 1967. Separation of two functional roles of L-alanine in the initiation of Bacillus subtilis spore germination. J. Bacteriol. 94:522-529. 41. Young, F. E., C. Smith, and B. C. Reilly. 1969. Chromosomal location of genes regulating resistance to bacteriophage in Bacillus subtilis. J. Bacteriol. 98:1087-1097. 42. 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 Micro-

biology, Washington, D.C.

Isolation, characterization, and mapping of Bacillus subtilis 168 germination mutants.

Vol. 123, No. 1 Printed in U.S.A. JOURNAL OF BACrERIOLOGY, JUlY 1975, p. 83-95 Copyright 0 1975 American Society for Microbiology Isolation, Charact...
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