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Effect of temperature and inhibitors on sporulation and the calcium accumulation system in Bacillus megaterium
Camp. Biochem. Physiol. Vol. 77A, No. 3, pp. 525-531, 1984 Printed in Great Britain
0300-9629/84%3.00+ 0.00 Pergamon Press Ltd
EFFECT OF TEMPERATURE AND INHIBITORS ON SPORULATION AND THE CALCIUM ACCUMULATION SYSTEM IN BACILLUS MEGATERIUM AKWRO OTA* Department of Biochemical Sciences, Frick Chemical Laboratory, Princeton University, Princeton, NJ 08540, USA (Received 1 June 1983) Abstrad-1.
Incubation at 37°C seems to prevent the formation of the calcium accumulation system during sporulation in Bacillus megaterium. 2. The temperature sensitive period for the formation of the calcium accumulation system was approximately at the end of growth. 3. KCN inhibited not only the formation of the calcium accumulation system but also calcium uptake, whereas chloramphenicol inhibited the formation of the calcium accumulation system alone.
INTRODUCTION Most bacterial cells keep the intracellular calcium concentration lower than the extracellular calcium, as
a consequence of the extrusion of calcium by active transport (Silver and Kralovic, 1969; Silver et al., 1975; Devb and Brodie, 1981). The everted vesicles of the bacterial cells can accumulate calcium. Calcium accumulation has been shown in the everted vesicles of Escherichia coli cells (Rosen and McClees, 1974; Tsuchiya and Rosen, 1975, 1976), and in the vesicles of other bacterial cells (Golub and Bronner, 1974; Bhattacharyya and Barnes, 1976; Kobayashi et al., 1978; Belliveau and Lanyi, 1978; Barnes et al., 1978; Kumar et nl., 1979). Calcium accumulation in Bacilli is known to occur during sporulation (Young and Fitz-James, 1962; Pearce and Fitz-James, 1971; Bronner et al., 1971; Bronner and Freund, 1972;
Hogarth and Ellar, 1978). An active transport system for calcium existed in sporulating Bacillus megaterium (Hogarth and Ellar, 1979) and B. subtilis (Eisenstadt and Silver, 1972). Vesicles from sporulating cells of B. megaterium were supposed not to accumulate calcium. However, they could take up calcium as the vesicles of vegetative cells of this bacterium (Golub and Bronner, 1974) and those of other bacteria1 cells. This observation was explained by the assumption that both calcium uptake and efflux systems coexist in the sporulating cells of B. megaterium (Ota, 1980a). The assumption was supported by the fact that there was an exchange of calcium accumulation in the cells for external calcium in the medium (Ota, 1982a). Although B. megaterium mutant 3-12 cannot sporulate owing to a lack of dipicolinic acid synthesis, calcium uptake occurred during the period which corresponded to the calcium uptake by the wild-type strain in the sporulation sequence and the ability of calcium uptake decreased after attaining its maximum (Ota, 1980a, 1982a). The mutant could sporulate on the addition of dipicolinate (Fukuda and *Present address: Osaka University Medical Nakanoshima, Kita-ku, Osaka 530, Japan.
School, 525
Gilvarg, 1968), while dipicolinic acid-less mutants of B. cereus were reported to form heat sensitive spores
in the absence of added dipicolinate (Wise et al., 1967). A mutant capable of sporulation but unable to accumulate calcium could not be isolated, and the amount of the calcium accumulation correlated with the sporulation efficiency in the mutant when dipicolinate was added (Ota, 1980a). Heat-sensitive spores were formed in the absence of calcium in the wild-type strain of B. megaterium (Tamir and Gilvarg, 1966). From these results, it was suggested that calcium accumulation in the cells was not essential for sporulation but that the change in the cells which evoked calcium uptake was essential (Ota, 1980a). Several works have shown the property of calcium uptake during sporulation (Hogarth and Ellar, 1978, 1979; Eisenstadt and Silver, 1972; Ota, 1982a; SetoYoung and Ellar, 1981). This paper describes the influence of temperature and inhibitors on the formation of the calcium accumulation system and some properties of calcium accumulation in the process of sporulation in the wild-type strain of B. megaterium and its asporogenous mutant. In addition, other factors which affect sporulation and morphogenesis in bacteria are discussed. MATERIALS AND METHODS Organisms
A local strain (wild-type) and a mutant 3-12 of B. megaterium (Fukuda and Gilvarg, 1968) were used. The mutant 3-12 lacks dihydropicolinate synthase (EC 4.4.1.52) and needs diaminopimelate and lysine for growth. They were isolated from B. megaterium local strain after the treatment of N-methyl-N’-nitro-N-nitirosoguanidine (Fukuda and Gilvarg, 1968; Adelherg et al., 1965). Media The minimal medium (FCG) (Millett and Aubert, 1960), which was used for growth, excluded yeast extracts for the local strain, and then included diaminopimelic acid (0.005%) and L-lysine_HCl (0.01%) for the mutant strain (Fukuda and Gilvarg, 1968).
AKIHIROOTA
526 Growth and sporulation
condition
Cells were grown on a rotary shaker containing 25 ml at
32°C with vigorous aeration and at the end of growth, 2Spmol of calcium labelled with 4SCa (2.5 x lo3 counts/min nmol-‘) was added. In the measurement of calcium accumulation during 30 mitt, calcium was not added to the culture at the end of the growth. The vegetative growth of cells ceased on reaching 120-130Klett units at 660 nm in the turbidity, and pH was maintained at 6.5-6.8 throughout growth and sporulation. The temperature in the culture was shifted from 32 to 37°C in the experiment on the influence of temperature. -4
Purity of mutant 3-12
3-12 was examined by seeing whether or not the organisms could grow on a plate in the absence of mesodiaminopimelate and L-lysine-HCl. The wild-type strain which was diluted lOOtSfold with F media minus NH,Cl (pH 6.8) was grown on the other plate as a control. Contamination of the mutant 3-12 with the wildtype was estimated at less than 10e6. Measurement
of calcium accumulation
Each 0.1 ml of the medium was taken from the culture flask at a proper time and put into 5 ml of the solution (the culture medium minus glucose and CaCI,). The mixture was shaken vigorously and placed in duplicate on membrane filters, which were washed thoroughly with 30ml of the solution described above, and counted for radioactivity with a Packard 3003 Tri-Carb liquid scintillation counter. The scintillation fluid was prepared by Bray’s (1960) method. Measurement
of calcium accumulation
during 30 min
One millilitre of cells cultured without added calcium was taken from the culture flask at a proper time and put into a tube containing 100 mnol of the calcium labelled with 4SCa (2.5 x 10’ counts/min nmol-I). Next after 30min at 32°C with vigorous aeration, 0.1 ml was taken from the tube and the following procedure was the same as described in the measurement of calcium accumulation. Measurement
of sporulation
-2 Hours
The purity of mutant
0
2
after
4 End of
6
8
10
12
Growth
Fig. 1. Influence of higher temperature on sporulation and calcium accumulation in wild-type strain. Wild-type cells were grown at 32°C or 37°C with vigorous aeration. The calcium labelled with 4SCa were added to their culture at the end of growth. A 0.1 ml aliquot of the culture was taken at the indicated time and the calcium accumulation was measured. Detailed conditions and the procedure of the measurement are described in the Materials and Methods section. 0, Turbidity curve of culture in the wild-type at 32°C; A, turbidity curve of culture in the wild-type at 37°C; 0, turbidity curve of culture in the wild-type cultured in the medium containing 1 mM sodium dipicolinate at 37°C; 0, calcium accumulation in the wild-type cultured at 32°C; A, calcium accumulation in the wild-type cultured at 37°C; and n , calcium accumulation in the wild-type cultured in the medium containing 1 mM sodium dipicolinate at 37°C.
32°C. After the end of growth, their turbidity curve declined throughout the time course. Calcium uptake occurred 6 hr after the end of growth, which was delayed about 2 hr, and its amount was very small. These phenomena seem to be related to the result that sporulation did not occur during incubation at 37°C.
eficiency
The colony forming units of the sporulating culture determined after heat treatment for 15 min at 60°C were taken as a spore titre. The sporulation efficiency was also measured directly by counting cells containing spores in a Petroff Hauser chamber with the oil immersion lens of a phase contrast microscope. The specific pattern of the decline and rise of the culture turbidity during sporulation was used as a measure of sporulation efficiency (Fukuda and Gilvarg, 1968).
RESULTS Influence of temperature on calcium accumulation and sporulation in wild type
Influence of higher temperature on calcium accumulation during sporulation process in wild-type strain of B. megaterium is shown in Fig. 1. The wild-type strain of B. megaterium incubated at 32°C exhibited a secondary rise of its turbidity curve (Fig. 1). The specific pattern of this decline and rise of culture turbidity was conveniently used as a measure of sporulation efficiency (Fukuda and Gilvarg, 1968). A large amount of calcium was taken up, corresponding to the secondary rise, in the cells which had been incubated at 32°C as previously indicated (Ota, 1980a). Cells incubated at 37°C reached their end of growth about 2.5 hr earlier than cells incubated at
-2
0
2
Hours after
4
6 8 10 End of Growth
12
Fig. 2. Influence of timing in temperature shift up on sporulation and calcium accumulation in wild-type strain. Cells of the wild-type were grown at 32°C with vigorous aeration. At the indicated time, temperature was shifted from 32°C to 37°C following the same procedure as shown in Fig. I. A, Turbidity curve of culture in the wild-type at 37°C; 0, turbidity curve of culture temperature-shifted from 32°C to 37°C 1 hr before the end of growth in the wild-type; 0, turbidity curve of culture temperature-shifted from 32°C to 37°C at 0.5 hr after the end of growth; A, calcium accumulation by the wild-type culture at 37°C; m, calcium accumulation by the wild-type temperature-shifted from 32°C to 37°C 1 hr before the end of growth; 0, calcium accumulation by the wild-type temperature-shifted from 32°C to 37°C 0.5 hr after the end of growth.
Effect of
temperature on calcium accumulation in B. megareriwn
It was reported that temperature was strictly controlled because it was a critical factor in sporulation of the wild-type strain of B. megaterium (Fukuda and Gilvarg, 1968). Insufficient aeration during incubation lowered sporulation efficiency. An amount of calcium uptake was also decreased by the insufficient aeration during incubation. The calcium accumulation in the cells of the wild-type strain which had been incubated at 37°C increased to some degree in the presence of dipicolinate, but sporulation could not be observed. The amount of added dipicolinate allowed asporgenous mutant 3-12 to sporulate (Ota, 1980a; Fukuda and Gilvarg, 1968). The calcium accumulation of wild-type strain was different from that of mutant 3-12, for the former kept its level even after attaining its peak (Figs 1 and 2), whereas the latter decreased after its peak (Figs 3 and 4, Ota, 1980a). Influence of timing in temperature
and calcium accumulation
shift on sporuiation in wild-type
Influence of timing in temperature shift (32-37°C) on sporulation and calcium accumulation are shown in Fig. 2. When the temperature was raised to 37°C 1 hr before the end of growth, about 30% of the amount of calcium taken up at 32°C was observed. When the temperature was raised to 37°C 0.5 hr after end of growth, about 90% of the amount of calcium taken up at 32°C and some sporulation, was observed about 2 hr later than the control. Effect of inhibitors on calcium accumulation in mutant
3-12 Figure 3 indicates the effect of KCN on the formation of calcium accumulation system and on the decrease of calcium accumulation after attaining its peak in B. megaterium mutant 3-12. The calcium
” oL=& 4 Hours
6 after
12 End of
16 Growth
uptake was not observed when KCN was added to the culture which had been incubated at 32°C 2 hr after end of growth. The slope of the decrease curve of calcium accumulation becomes steeper on the addition of KCN. Since the calcium accumulation system contains both uptake and release systems (Ota, 1980a), the curve represents the amount of calcium remaining plus calcium taken up minus calcium released. A sharp decline of the curve after the addition of KCN is due to its inhibition of calcium uptake (Table 1). From this curve, it can be concluded that KCN does not inhibit the calcium release system. Effect of chloramphenicol on calcium accumulation system is shown in Fig. 4. The formation of calcium accumulation system was inhibited completely by the addition of chloramphenicol 2 hr after end of growth. The decrease curve of calcium accumulation became a little steeper on the addition of chloramphenicol. Effect of KCN and chloramphenicol on the calcium accumulation limited to a 30min period in B. megaterium mutant 3-12 is shown in Table 1. KCN at a concentration of 10m3M and of 10m4M inhibited 87% and 73% of the calcium accumulation of the control, respectively. About half the inhibition of the calcium accumulation in the control was shown by lOA M KCN. Chloramphenicol did not inhibit the calcium accumufation. Kumar et al. (1979) reported that KCN inhibited the respiration driven calcium uptake by the membrane vesicles of Mycobacterium phlei, while it had no effect on ATP-driven calcium uptake. Injuence of freezing on the formation accumulation system in mutant 3-12
of calcium
Figure 5 shows the influence of freezing on the formation of calcium accumulation system in B. megaterium mutant 3-12. The culture was taken at
20
Fig. 3. Effect of KCN on calcium accumulation in mutant 3-12. Cells of mutant 3-12 were cultivated at 32°C with vigorous aeration and 2.5 pmol calcium labelled with 45Ca were added at the end of growth. At the indicated time (arrows), 1ml KCN was added to the mutant 3-12 culture. A 0.1 ml aliquot of the culture was taken at the indicated time and then the calcium accumulation was measured as described in the Materials and Methods section. 0, Accumulation of calcium in mutant 3-12; A, amount of calcium in mutant 3-12 after 1 mM KCN was added at 2 hr before the end of growth; after 1mM KCN
527
0, amount of calcium in mutant 3-12 was added at 10 hr after the end of growth.
Hours
after
End of Growth
Fig. 4. Effect of chloramphenicol on calcium accumulation in mutant 3-12. Cells of mutant 3-12 were arown at 32°C with vigorous aeration, and 2.5 pmol calcium labelled with 45Ca were added at the end of growth. At the indicated time (arrows) chloramphenicol (50ng/ml) was added to the mutant 3-12 culture. A 0.1 ml aliquot of the culture was taken, and the calcium accumulation was measured by the procedure described in the Materials and Methods section. 0, Accumulation of calcium in mutant 3- 12; A, amount of calcium in mutant 3-12 after chloramphenicol50 pg/ml was added at 2 hr before end of growth; and 0, amount of calcium in mutant 3-12 after chloramphenicol50 pg/ml was added at 9 hr after the end of growth.
528
AKIHIRO OTA
Table 1. EBect of KCN and chloramphenicol 3-12
Inhibitors
Calcium acctmmlation (nmol/ml)
Inhibition (%)
10-j M 1O-4 M lo-‘M
19.5 2.5 5.3 9.9
0 87 73 49
50 pg/ml
19.4 19.8
0 0
Concentration
None KCN None Chloramphenicol
on calcium acctmmlation in mutant
Cells of mutant 3-12 were grown at 32°C with vigorous aeration. Calcium was not added to the culture at the end of growth. A 1 ml aliquot of the culture was taken from the culture flask when calcium accumulation would attain to its peak and put into a tube containing 100 nmol of calcium labelled with 45Ca plus KCN or chloramphenicol. After 30 mitt at 32°C with vigorous aeration, 0.1 ml was taken from the tube and the following procedure was the same as described in the Materials and Methods section.
6 hr after the end of growth, added to glycerol in order to make a 10% glycerol solution and stored at - 15°C for 64 hr. The stored cells were washed with medium to get rid of glycerol. The amount of calcium accumulation decreased on account of freezing, thawing and cell lysis due to the freeze-thaw, but maintained its original stage and then the same process proceeded in spite of the very low level of calcium accumulation. This indicates that the structure in the cells for calcium uptake system is not very labile to freezing and thawing under these conditions.
= 20 E
I
A
Hours
after
End
of
Growth
Fig. 5. Influence of freezing on calcium accumulation by mutant 3-12 in the medium containing glycerol. Mutant 3-12 cells were grown at 32°C with vigorous aeration. A 1 ml aliquot of the culture was taken from the culture flask at the proper time after end of growth, and the amount of calcium accumulated during 30 min was determined by the method of “Measurement of calcium accumulation during 30 min” as described in the Materials and Methods section. At the indicated time (arrows), 10 ml of the culture was taken and after the addition of glycerol in order to make a loo/, solution, was stored at - 15°C in a freezer. After 64 hr the stored culture cells collected by centrifuge were washed with the medium omitting glucose, yeast extract and calcium from medium FCG and supplementing with diaminopimelic acid (O.OOS~Jand L-lysine_HCl (O.Ol’/J in order to remove glycerol, then suspended in an original volume of the medium. The incubation was performed at 32°C. The accumulation of calcium was determined by the method of “Measurement of calcium accumulation during 30 mitt” as described in the Materials and Methods section. 0, Calcium accumulation in mutant 3-12 cells; and 0, calcium accumulation in mutant 3-12 cells taken at 6 hr after the end of growth and stored for 64 hr in a freezer.
DISCUSSION
Wild-type strains of B. megaterium could not sporulate at 37°C and the amount of their calcium uptake observed in the sporulation sequence was very small (Fig. 1). However, when the temperature was raised up to 37°C 30min after the end of growth, some sporulation and a considerable amount of calcium uptake were observed, although their period was delayed (Fig. 2). This result supports the previous conception (Ota, 1980a) that the change evoking calcium uptake in the cells is essential for sporulation in B. meguterium. The time required to reach the end of growth in the cells incubated at 37°C was shorter than that of the cells incubated at 32°C whereas calcium uptake at 37°C started later than that at 32”C, and the amount was very small (Figs 1 and 2). This suggests that the temperature-sensitive change near the end of growth affects the structural changes for the formation of the calcium accumulation system which corresponds to a rise of the secondary turbidity curve later. It is considered that dimorphism of fungal species is a good example of primitive morphogenesis (Haidle and Storck, 1966). Blastomyces dermatilidis can induce two morphological forms, a mycelial form and a yeast-like form. The organism grows as the mycelial form at 33°C and grows as the yeast-like form at 37°C (Levine and Ordal, 1946). The two morphogenetic patterns, M (mycelial) * Y (yeast-like) interconversion in Blastomyces dermatilidis, were called “thermal dimorphism” because that interconversion had been concluded to be solely a function of temperature (Nickerson, 1948). Structural changes in the cells of B. megaterium for the formation of the calcium accumulation system during sporulation seem to resemble this phenomenon in the influence of temperature. Some of fungi for the M e Y interconversion are influenced by - SH compound, glucose, citrate, amino acids, metals, CO*, 02, auxin, age, pH, X-ray irradiation etc. other than temperature (Haidle and Storck, 1966; Nickerson, 1948; Pine and Peacock, 1958; Scherr and Weaver, 1953; Bartnicki-Garcia and McMurrough, 1971; Takada et al., 1963; Johnson, 1954; Steele and Miller, 1974). Some bacteria can be induced to elongate into filaments by various treatments which are denoted B --+ F conversion (Nickerson, 1948).
Effect of temperature on calcium accumulation in B. meguterium
Even in the case of thermal dimorphism, oxygen consumption increased in the high temperature range (Nickerson and Edwards, 1949). In this case, not only the oxygen consumption but also the content of cytochrome oxidase and related enzyme etc. possibly increased. Thus, temperature alone is not considered to be a factor in this case. Oxygen consumption is important for the conversion (Nickerson, 1948). It was observed that oxygen consumption increased after transfer to sporulation medium (Croes, 1967; Hopper et al., 1974) and that the enzyme activities which may be related to the increase varied in yeast Succharomyces cerevisiae (Ota, 1979, 1982b). In addition, some enzymes (Ota, 1980b, 1982c) and the transport activity (Ota, 1982d, 1983a,b), which might be related indirectly to oxygen consumption, increased. The spore fo~ation was blctcked by ethanol whose sensitive period was between 40 and 90min after the end of growth in 3. subtilis, which was considered to be the result of a physical change upon the cell membrane (Bohin et al., 1976a). SpoO mutant of B. subtilis had higher membrane-bound nitrate reductase activities than their Spa+ parental strains. It was suggested that the increased nitrate reductase activity resulted from a mutational change in some membrane component other than the enzyme itself, that is, nitrate reductase in the modified membrane exhibited allotopic properties (Bohin er al., 1976b). The term allotopy was first described by Racker (1967). Lundzren and Beskid (1960) isolated ~em~rature-emotive mutants of 3: c&us ATCC 4342 in the minimal medium. and the snorulation interference was partially reversed when casein hydrolysate was added. Lundgren and Cooney (1962) reported that a temperature-sensitive asporogenous mutant of B. cereus took up about 30% as much of calcium as the parent during sporulation at 37°C. Calcium uptake increased somewhat when added to dipicolinate and cultured at 37°C (Fig. 1). This increase is probably because that calcium is transported as calcium dipicolinate. Furthermore, it might be possible that calcium dipicolinate has an influence on the cell structure leading to increase in calcium uptake. As the substances influencing M 5~$Y conversion are various in different cultural conditions of fungi, fungous species etc., substances influencing sporulation are various in those of bacteria. A dipicolinic acid-less mutant of B. cereus is able to form heatsensitive spores in the absence of dipicolinate (Wise et ai., 1967), but B. megaterium mutant 3-12 needs dipicolinate for its sporulation (Fukuda and Gilvarg, 1968). This indicates that dipicolinate participates not only in heat resistance of spores but also in the formation of spores in the case of the mutant 3-12. Temperature-sensitive mutants of B. subtilis could sporulate at 47°C by the infection of a sporeconverting bacteriophage PMBIZ, and the mutation to Spo (Ts) was suggested to be due to the mu~tion 1
Vegetative cell Undifferentiated
cell
529
in either the RNA polymerase subunit or the 30s ribosomal subunit. It was also suggested that PMBl2 might have at least three genes for spore conversion and that the products interact with a host cell pathway which is expressed in the earliest stage of sporulation (Kinney and Bramucci, 1981). Young (1976) characterized two temperature-sensitive sporulation mutants of B. subtilis; one was obstructed at stage II and the cells exhibited morphological abnormalities, and the other was obstructed at stage IV to V when formation of the spore cortex was almost complete but coat formation was arrested. The former had a short temperature sensitive period during the formation of spore septum, and the product of the defective gene was suggested to be a protein. The m-RNA which coded for this protein was short-lived. It was suggested that coat formation of the latter was arrested at an early stage and that synthesis of the product of the defective gene started long before it assumed its physiological function. Both of them failed to synthesize dipicolinic acid. A temperaturesensitive DNA synthesis-mutant of B. subtilis has been isolated in which the process of initiation of DNA repletion is inhibited at high temperature (Laurent and Vanier, 1973). Haidle and Storck (1966) showed that RNA precursors were discontinuously incorporated into RNA during Y -+ M conversion in Mucor rouxii. It might he possible that in wild type of B. megaterium the temperature shift up affected one or more of these RNA polymerase of r&some, the pattern of RNA synthesis, the DNA replication, the other enzymes or protein in the earliest stage of sporulation. Chloramphenicol inhibits the formation of the calcium accumulation system, but after the formation of the system it does not inhibit the calcium accumulation and the decrease of accumulation in the cells (Fig. 4 and Table 1). Chloramphe~col prevents protein breakdown as well as sporulation, but once the protein degradation is underway it does not diminish its rate, although protein synthesis and sporulation are completely blocked (Komberg et al., 1968). KCN inhibited both calcium uptake and the formation of the calcium uptake system (Table 1 and Fig. 3). This supports the previous suggestion that most of the calcium uptake is energy-dependent both in the mutant 3-12 (Ota, 1982a) and the cells of sporulating Bacilli (Hogarth and Ellar, 1979; Eisenstadt and Silver, 1972). Bronner et aL (1975) reported that energydependent calcium exclusion system was preserved until T6 stage in sporulation in 3. megaterium. It is not likely that KCN inhibits the decrease of the accumulated calcium (Fig. 3). This means that a large part of the calcium release of asporogenous cells in this stage is energy independent, and KCN does not inhibit the decrease or degradation of calcium accumulation system. I propose a tentative model of cell ~fferentiation as follows:
M factors I factors (change of membrane --*(change of gene+ components and membrane 4 expression etc.) structure etc.)
Spore Differentiated cell.
530
AKIHIRO OTA
I factors are the factors which affect the initiation
of differentiation, including the change of gene expression, environmental factors which affect genes etc. M factors are the factors that affect the cell membrane and change the membrane structure etc. In a case of sporulation in Bacilli, M factors contain the change in the cell which evoked calcium uptake. Additional factors essential for cell differentiation are supposed to exist. However, these two factors might be the primary factors at present. SUMMARY
Although a wild-type strain of B. megaterium cultivated at 37°C was incapable of sporulation, a very small amount of calcium accumulation was observed in the delayed period of sporulation sequence, and the calcium accumulation level increased somewhat in the presence of added sodium dipicolinate. The incubation at 37°C seems to prevent the system of calcium accumulation being formed. Influence of the temperature-shift timing from 32 to 37°C on calcium accumulation during sporulation was examined, and it was shown that the temperature sensitive period was approximately at the end of growth. KCN inhibited calcium uptake as well as the formation of calcium accumulation system in asporogenous mutant 3-12 under sporulation condition, and chloramphenicol inhibited the formation of calcium accumulation system alone. Neither KCN nor chloramphenicol inhibited the decrease or degradation of calcium accumulation system in the mutant. In spite of the decrease of the amount of calcium accumulation, the calcium accumulation system and the stage of sporulation in the mutant were maintained after 64 hr in the medium containing 10% glycerol at - 15°C. Factors necessary for sporulation and calcium accumulation were discussed. Acknowledgement-I wish to thank Dr Charles Gilvarg for his generous support and kind discussion. REFERENCES
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Lundgren D. G. and Beskid G. (1960) Isolation and investigation of induced asporogenic mutants. Can. J. Microbial. 6, 135-151.
Effect of temperature on calcium accumulation in 3. megaierium Lundgren D. G. and Cooney J. J. (1962) Chemical analysis of asporogenic mutants of Bacillus cereus. J. Bact. 83, 1287-1293.
Millet J. and Aubert J.-P. (1960) Le metabolisme de l’acide glutamique au cours de la sporulation chez Bacillus megaterium.
Annls Inst. Pasteur,
Paris 98, 282-290.
Nickerson W. J. (1948) Enzymatic control of cell division in micro-organisms. Nature, Lond. 162, 241-245. Nickerson W. J. and Edwards G. A. (1949) Studies on the physiological bases of morphogenesis in fungi. J. gen. Physiol. 33, 41-55.
Ota A. (1979) Biochemical changes in early phase of sporulation in Saccharomyces cerevisiae. XIth Int. Congr. Biochem., Toronto, Canada, Abstr., p, 512. Ota A. (198Oa) Calcium uptake by Bacillus megaterium mutant strains unable to sportdate. Znt. J. Biochem. 11, 363-367. Ota A. (1980b) Galactokinase activity of Saccharomyces cerevisiae during early ascosporulation. Microbios Lett. 14, 143-146.
Ota A. (1982a) Ca*+ uptake in an asporogenous mutant strain of BaciBus megaterium. Microbios 34, 185-196. Ota A. (1982b) Enzyme activities during early ascosporulation in Saccharomyces cerevisiae. bt. J. Biochem. 14, 11l-1 18. Ota A. (1982~) Some properties of catalase activity and its change during sporulation in the intact cells of yeast Saccharomyces
cerevisiae.
XIIth
Int. Congr.
Biochem.,
Perth, Australia, Abstr., p. 94. Ota A. (1982d) Flavin mononucleotide uptake during early a~ospo~lation in Sacc~romyces cerevisiae. M~crobios Left. 19, 17-20.
Ota A. (1983a) Sugar uptake during early ascosporulation in yeast Saccharomyces cerevisiae. Microbios, in press. Ota A. (1983b) Effect of inhibitors on glucose uptake in vegetative and sporulating cells of Saccharomyces cerevisiae. Microbios
Lett. 23, 135-138.
531
Racker E. (1967) Resolution and reconstitution of the inner mitochondrial membrane. Fe& Proc. Fe&t Am. Sots. exp. Biol. 26, 1335-1340. Rosen B. P. and McClees J. S. (1974) Active transport of calcium in inverted membrane vesicles of Escherichia coli. Proc. natn. Acad. Sci. U.S.A. 71, 5042-5046.
Scherr G. H. and Weaver R. H. (1953) The dimorphism phenomenon in yeasts. Bact. Rev. 17, 51-92. Seto-Young D. L. T. and Ellar D. J. (1981) Studies on calcium transport during growth and sport&ion. Microbios 30, 191-208. Silver S. and Kralovic M. L. (1969) Manganese accumulation by Escherichia cob: Evidence for a specific transport system. Biochem. biophys. Res. Commun. 34, 64@645.
Silver S., Toth K. and Scribner H. (1975) Facilitated transport of calcium by cells and subcelluiar membranes of Bacillus subtilis and Escherichia coli. J. Bact. 122, 88&885.
Steele S. D. and Miller J. J. (1974) Pseudomycehum development and sporulation in Saccharomycesfragilis. Can. J. Microbial.
28, 265-267.
Takada H., Morisbita H. and Ya8i T. (1963) Evidence for dimorphic character by Endomycopsis fdbuliger. 7iiuns. Mycoi.
Sot. Japan 4, 126130.
Tamir H. and Gilvarg C. (1966) Density gradient centrifugation for the separation of sporulating forms of Bacteria. J. biol. Chem. 241, 1085-1090. Tsuchiya T. and Rosen 3. P. (1975) Characterization of an active transport system for calcium in inverted membrane vesicles of ~scber~=hia coli. J. biol. Chem. Uo, 7687-7692. Tsuchiva T. and Rosen B. P. (1976) Calcium transnort driven by a proton gradient in inverted membrane ve&les of Escherichia coli. J. biol. Chem. 251, 962-967. Wise J., Swanson A. and Halvorson H. 0. (1967) Dipicolinic acid-less mutants of Bacillus cereus. J. Bact. 94, 2075-2076. Young I. E. and Fitz-James P. C. (1962) Chemical and rno~holo~~l studies of bacterial spore formation-IV. The development of spore refractility. J. cell Biol. 12,
Pearce S. M. and Fitz-James P. C. (19’71)Spore refractility in variants of Baci~i~ cereus treated with actinomycin D. J. Bact. 107, 337-344. Pine L. and Peacock C. L. (1958) Studies on the growth of 115-133. Histoplasma capsulatum--IV. Factors influencing conYoung M. (1976) Use of temperature-sensitive version of the mycehal phase to the yeast phase. J. Bact. _ study gene expression during sporulation 75, 167-174. subtilis. J. Bact. 126, 928-936.
mutants to in Bacillus
0300-9629/84%3.00+ 0.00 Pergamon Press Ltd
EFFECT OF TEMPERATURE AND INHIBITORS ON SPORULATION AND THE CALCIUM ACCUMULATION SYSTEM IN BACILLUS MEGATERIUM AKWRO OTA* Department of Biochemical Sciences, Frick Chemical Laboratory, Princeton University, Princeton, NJ 08540, USA (Received 1 June 1983) Abstrad-1.
Incubation at 37°C seems to prevent the formation of the calcium accumulation system during sporulation in Bacillus megaterium. 2. The temperature sensitive period for the formation of the calcium accumulation system was approximately at the end of growth. 3. KCN inhibited not only the formation of the calcium accumulation system but also calcium uptake, whereas chloramphenicol inhibited the formation of the calcium accumulation system alone.
INTRODUCTION Most bacterial cells keep the intracellular calcium concentration lower than the extracellular calcium, as
a consequence of the extrusion of calcium by active transport (Silver and Kralovic, 1969; Silver et al., 1975; Devb and Brodie, 1981). The everted vesicles of the bacterial cells can accumulate calcium. Calcium accumulation has been shown in the everted vesicles of Escherichia coli cells (Rosen and McClees, 1974; Tsuchiya and Rosen, 1975, 1976), and in the vesicles of other bacterial cells (Golub and Bronner, 1974; Bhattacharyya and Barnes, 1976; Kobayashi et al., 1978; Belliveau and Lanyi, 1978; Barnes et al., 1978; Kumar et nl., 1979). Calcium accumulation in Bacilli is known to occur during sporulation (Young and Fitz-James, 1962; Pearce and Fitz-James, 1971; Bronner et al., 1971; Bronner and Freund, 1972;
Hogarth and Ellar, 1978). An active transport system for calcium existed in sporulating Bacillus megaterium (Hogarth and Ellar, 1979) and B. subtilis (Eisenstadt and Silver, 1972). Vesicles from sporulating cells of B. megaterium were supposed not to accumulate calcium. However, they could take up calcium as the vesicles of vegetative cells of this bacterium (Golub and Bronner, 1974) and those of other bacteria1 cells. This observation was explained by the assumption that both calcium uptake and efflux systems coexist in the sporulating cells of B. megaterium (Ota, 1980a). The assumption was supported by the fact that there was an exchange of calcium accumulation in the cells for external calcium in the medium (Ota, 1982a). Although B. megaterium mutant 3-12 cannot sporulate owing to a lack of dipicolinic acid synthesis, calcium uptake occurred during the period which corresponded to the calcium uptake by the wild-type strain in the sporulation sequence and the ability of calcium uptake decreased after attaining its maximum (Ota, 1980a, 1982a). The mutant could sporulate on the addition of dipicolinate (Fukuda and *Present address: Osaka University Medical Nakanoshima, Kita-ku, Osaka 530, Japan.
School, 525
Gilvarg, 1968), while dipicolinic acid-less mutants of B. cereus were reported to form heat sensitive spores
in the absence of added dipicolinate (Wise et al., 1967). A mutant capable of sporulation but unable to accumulate calcium could not be isolated, and the amount of the calcium accumulation correlated with the sporulation efficiency in the mutant when dipicolinate was added (Ota, 1980a). Heat-sensitive spores were formed in the absence of calcium in the wild-type strain of B. megaterium (Tamir and Gilvarg, 1966). From these results, it was suggested that calcium accumulation in the cells was not essential for sporulation but that the change in the cells which evoked calcium uptake was essential (Ota, 1980a). Several works have shown the property of calcium uptake during sporulation (Hogarth and Ellar, 1978, 1979; Eisenstadt and Silver, 1972; Ota, 1982a; SetoYoung and Ellar, 1981). This paper describes the influence of temperature and inhibitors on the formation of the calcium accumulation system and some properties of calcium accumulation in the process of sporulation in the wild-type strain of B. megaterium and its asporogenous mutant. In addition, other factors which affect sporulation and morphogenesis in bacteria are discussed. MATERIALS AND METHODS Organisms
A local strain (wild-type) and a mutant 3-12 of B. megaterium (Fukuda and Gilvarg, 1968) were used. The mutant 3-12 lacks dihydropicolinate synthase (EC 4.4.1.52) and needs diaminopimelate and lysine for growth. They were isolated from B. megaterium local strain after the treatment of N-methyl-N’-nitro-N-nitirosoguanidine (Fukuda and Gilvarg, 1968; Adelherg et al., 1965). Media The minimal medium (FCG) (Millett and Aubert, 1960), which was used for growth, excluded yeast extracts for the local strain, and then included diaminopimelic acid (0.005%) and L-lysine_HCl (0.01%) for the mutant strain (Fukuda and Gilvarg, 1968).
AKIHIROOTA
526 Growth and sporulation
condition
Cells were grown on a rotary shaker containing 25 ml at
32°C with vigorous aeration and at the end of growth, 2Spmol of calcium labelled with 4SCa (2.5 x lo3 counts/min nmol-‘) was added. In the measurement of calcium accumulation during 30 mitt, calcium was not added to the culture at the end of the growth. The vegetative growth of cells ceased on reaching 120-130Klett units at 660 nm in the turbidity, and pH was maintained at 6.5-6.8 throughout growth and sporulation. The temperature in the culture was shifted from 32 to 37°C in the experiment on the influence of temperature. -4
Purity of mutant 3-12
3-12 was examined by seeing whether or not the organisms could grow on a plate in the absence of mesodiaminopimelate and L-lysine-HCl. The wild-type strain which was diluted lOOtSfold with F media minus NH,Cl (pH 6.8) was grown on the other plate as a control. Contamination of the mutant 3-12 with the wildtype was estimated at less than 10e6. Measurement
of calcium accumulation
Each 0.1 ml of the medium was taken from the culture flask at a proper time and put into 5 ml of the solution (the culture medium minus glucose and CaCI,). The mixture was shaken vigorously and placed in duplicate on membrane filters, which were washed thoroughly with 30ml of the solution described above, and counted for radioactivity with a Packard 3003 Tri-Carb liquid scintillation counter. The scintillation fluid was prepared by Bray’s (1960) method. Measurement
of calcium accumulation
during 30 min
One millilitre of cells cultured without added calcium was taken from the culture flask at a proper time and put into a tube containing 100 mnol of the calcium labelled with 4SCa (2.5 x 10’ counts/min nmol-I). Next after 30min at 32°C with vigorous aeration, 0.1 ml was taken from the tube and the following procedure was the same as described in the measurement of calcium accumulation. Measurement
of sporulation
-2 Hours
The purity of mutant
0
2
after
4 End of
6
8
10
12
Growth
Fig. 1. Influence of higher temperature on sporulation and calcium accumulation in wild-type strain. Wild-type cells were grown at 32°C or 37°C with vigorous aeration. The calcium labelled with 4SCa were added to their culture at the end of growth. A 0.1 ml aliquot of the culture was taken at the indicated time and the calcium accumulation was measured. Detailed conditions and the procedure of the measurement are described in the Materials and Methods section. 0, Turbidity curve of culture in the wild-type at 32°C; A, turbidity curve of culture in the wild-type at 37°C; 0, turbidity curve of culture in the wild-type cultured in the medium containing 1 mM sodium dipicolinate at 37°C; 0, calcium accumulation in the wild-type cultured at 32°C; A, calcium accumulation in the wild-type cultured at 37°C; and n , calcium accumulation in the wild-type cultured in the medium containing 1 mM sodium dipicolinate at 37°C.
32°C. After the end of growth, their turbidity curve declined throughout the time course. Calcium uptake occurred 6 hr after the end of growth, which was delayed about 2 hr, and its amount was very small. These phenomena seem to be related to the result that sporulation did not occur during incubation at 37°C.
eficiency
The colony forming units of the sporulating culture determined after heat treatment for 15 min at 60°C were taken as a spore titre. The sporulation efficiency was also measured directly by counting cells containing spores in a Petroff Hauser chamber with the oil immersion lens of a phase contrast microscope. The specific pattern of the decline and rise of the culture turbidity during sporulation was used as a measure of sporulation efficiency (Fukuda and Gilvarg, 1968).
RESULTS Influence of temperature on calcium accumulation and sporulation in wild type
Influence of higher temperature on calcium accumulation during sporulation process in wild-type strain of B. megaterium is shown in Fig. 1. The wild-type strain of B. megaterium incubated at 32°C exhibited a secondary rise of its turbidity curve (Fig. 1). The specific pattern of this decline and rise of culture turbidity was conveniently used as a measure of sporulation efficiency (Fukuda and Gilvarg, 1968). A large amount of calcium was taken up, corresponding to the secondary rise, in the cells which had been incubated at 32°C as previously indicated (Ota, 1980a). Cells incubated at 37°C reached their end of growth about 2.5 hr earlier than cells incubated at
-2
0
2
Hours after
4
6 8 10 End of Growth
12
Fig. 2. Influence of timing in temperature shift up on sporulation and calcium accumulation in wild-type strain. Cells of the wild-type were grown at 32°C with vigorous aeration. At the indicated time, temperature was shifted from 32°C to 37°C following the same procedure as shown in Fig. I. A, Turbidity curve of culture in the wild-type at 37°C; 0, turbidity curve of culture temperature-shifted from 32°C to 37°C 1 hr before the end of growth in the wild-type; 0, turbidity curve of culture temperature-shifted from 32°C to 37°C at 0.5 hr after the end of growth; A, calcium accumulation by the wild-type culture at 37°C; m, calcium accumulation by the wild-type temperature-shifted from 32°C to 37°C 1 hr before the end of growth; 0, calcium accumulation by the wild-type temperature-shifted from 32°C to 37°C 0.5 hr after the end of growth.
Effect of
temperature on calcium accumulation in B. megareriwn
It was reported that temperature was strictly controlled because it was a critical factor in sporulation of the wild-type strain of B. megaterium (Fukuda and Gilvarg, 1968). Insufficient aeration during incubation lowered sporulation efficiency. An amount of calcium uptake was also decreased by the insufficient aeration during incubation. The calcium accumulation in the cells of the wild-type strain which had been incubated at 37°C increased to some degree in the presence of dipicolinate, but sporulation could not be observed. The amount of added dipicolinate allowed asporgenous mutant 3-12 to sporulate (Ota, 1980a; Fukuda and Gilvarg, 1968). The calcium accumulation of wild-type strain was different from that of mutant 3-12, for the former kept its level even after attaining its peak (Figs 1 and 2), whereas the latter decreased after its peak (Figs 3 and 4, Ota, 1980a). Influence of timing in temperature
and calcium accumulation
shift on sporuiation in wild-type
Influence of timing in temperature shift (32-37°C) on sporulation and calcium accumulation are shown in Fig. 2. When the temperature was raised to 37°C 1 hr before the end of growth, about 30% of the amount of calcium taken up at 32°C was observed. When the temperature was raised to 37°C 0.5 hr after end of growth, about 90% of the amount of calcium taken up at 32°C and some sporulation, was observed about 2 hr later than the control. Effect of inhibitors on calcium accumulation in mutant
3-12 Figure 3 indicates the effect of KCN on the formation of calcium accumulation system and on the decrease of calcium accumulation after attaining its peak in B. megaterium mutant 3-12. The calcium
” oL=& 4 Hours
6 after
12 End of
16 Growth
uptake was not observed when KCN was added to the culture which had been incubated at 32°C 2 hr after end of growth. The slope of the decrease curve of calcium accumulation becomes steeper on the addition of KCN. Since the calcium accumulation system contains both uptake and release systems (Ota, 1980a), the curve represents the amount of calcium remaining plus calcium taken up minus calcium released. A sharp decline of the curve after the addition of KCN is due to its inhibition of calcium uptake (Table 1). From this curve, it can be concluded that KCN does not inhibit the calcium release system. Effect of chloramphenicol on calcium accumulation system is shown in Fig. 4. The formation of calcium accumulation system was inhibited completely by the addition of chloramphenicol 2 hr after end of growth. The decrease curve of calcium accumulation became a little steeper on the addition of chloramphenicol. Effect of KCN and chloramphenicol on the calcium accumulation limited to a 30min period in B. megaterium mutant 3-12 is shown in Table 1. KCN at a concentration of 10m3M and of 10m4M inhibited 87% and 73% of the calcium accumulation of the control, respectively. About half the inhibition of the calcium accumulation in the control was shown by lOA M KCN. Chloramphenicol did not inhibit the calcium accumufation. Kumar et al. (1979) reported that KCN inhibited the respiration driven calcium uptake by the membrane vesicles of Mycobacterium phlei, while it had no effect on ATP-driven calcium uptake. Injuence of freezing on the formation accumulation system in mutant 3-12
of calcium
Figure 5 shows the influence of freezing on the formation of calcium accumulation system in B. megaterium mutant 3-12. The culture was taken at
20
Fig. 3. Effect of KCN on calcium accumulation in mutant 3-12. Cells of mutant 3-12 were cultivated at 32°C with vigorous aeration and 2.5 pmol calcium labelled with 45Ca were added at the end of growth. At the indicated time (arrows), 1ml KCN was added to the mutant 3-12 culture. A 0.1 ml aliquot of the culture was taken at the indicated time and then the calcium accumulation was measured as described in the Materials and Methods section. 0, Accumulation of calcium in mutant 3-12; A, amount of calcium in mutant 3-12 after 1 mM KCN was added at 2 hr before the end of growth; after 1mM KCN
527
0, amount of calcium in mutant 3-12 was added at 10 hr after the end of growth.
Hours
after
End of Growth
Fig. 4. Effect of chloramphenicol on calcium accumulation in mutant 3-12. Cells of mutant 3-12 were arown at 32°C with vigorous aeration, and 2.5 pmol calcium labelled with 45Ca were added at the end of growth. At the indicated time (arrows) chloramphenicol (50ng/ml) was added to the mutant 3-12 culture. A 0.1 ml aliquot of the culture was taken, and the calcium accumulation was measured by the procedure described in the Materials and Methods section. 0, Accumulation of calcium in mutant 3- 12; A, amount of calcium in mutant 3-12 after chloramphenicol50 pg/ml was added at 2 hr before end of growth; and 0, amount of calcium in mutant 3-12 after chloramphenicol50 pg/ml was added at 9 hr after the end of growth.
528
AKIHIRO OTA
Table 1. EBect of KCN and chloramphenicol 3-12
Inhibitors
Calcium acctmmlation (nmol/ml)
Inhibition (%)
10-j M 1O-4 M lo-‘M
19.5 2.5 5.3 9.9
0 87 73 49
50 pg/ml
19.4 19.8
0 0
Concentration
None KCN None Chloramphenicol
on calcium acctmmlation in mutant
Cells of mutant 3-12 were grown at 32°C with vigorous aeration. Calcium was not added to the culture at the end of growth. A 1 ml aliquot of the culture was taken from the culture flask when calcium accumulation would attain to its peak and put into a tube containing 100 nmol of calcium labelled with 45Ca plus KCN or chloramphenicol. After 30 mitt at 32°C with vigorous aeration, 0.1 ml was taken from the tube and the following procedure was the same as described in the Materials and Methods section.
6 hr after the end of growth, added to glycerol in order to make a 10% glycerol solution and stored at - 15°C for 64 hr. The stored cells were washed with medium to get rid of glycerol. The amount of calcium accumulation decreased on account of freezing, thawing and cell lysis due to the freeze-thaw, but maintained its original stage and then the same process proceeded in spite of the very low level of calcium accumulation. This indicates that the structure in the cells for calcium uptake system is not very labile to freezing and thawing under these conditions.
= 20 E
I
A
Hours
after
End
of
Growth
Fig. 5. Influence of freezing on calcium accumulation by mutant 3-12 in the medium containing glycerol. Mutant 3-12 cells were grown at 32°C with vigorous aeration. A 1 ml aliquot of the culture was taken from the culture flask at the proper time after end of growth, and the amount of calcium accumulated during 30 min was determined by the method of “Measurement of calcium accumulation during 30 min” as described in the Materials and Methods section. At the indicated time (arrows), 10 ml of the culture was taken and after the addition of glycerol in order to make a loo/, solution, was stored at - 15°C in a freezer. After 64 hr the stored culture cells collected by centrifuge were washed with the medium omitting glucose, yeast extract and calcium from medium FCG and supplementing with diaminopimelic acid (O.OOS~Jand L-lysine_HCl (O.Ol’/J in order to remove glycerol, then suspended in an original volume of the medium. The incubation was performed at 32°C. The accumulation of calcium was determined by the method of “Measurement of calcium accumulation during 30 mitt” as described in the Materials and Methods section. 0, Calcium accumulation in mutant 3-12 cells; and 0, calcium accumulation in mutant 3-12 cells taken at 6 hr after the end of growth and stored for 64 hr in a freezer.
DISCUSSION
Wild-type strains of B. megaterium could not sporulate at 37°C and the amount of their calcium uptake observed in the sporulation sequence was very small (Fig. 1). However, when the temperature was raised up to 37°C 30min after the end of growth, some sporulation and a considerable amount of calcium uptake were observed, although their period was delayed (Fig. 2). This result supports the previous conception (Ota, 1980a) that the change evoking calcium uptake in the cells is essential for sporulation in B. meguterium. The time required to reach the end of growth in the cells incubated at 37°C was shorter than that of the cells incubated at 32°C whereas calcium uptake at 37°C started later than that at 32”C, and the amount was very small (Figs 1 and 2). This suggests that the temperature-sensitive change near the end of growth affects the structural changes for the formation of the calcium accumulation system which corresponds to a rise of the secondary turbidity curve later. It is considered that dimorphism of fungal species is a good example of primitive morphogenesis (Haidle and Storck, 1966). Blastomyces dermatilidis can induce two morphological forms, a mycelial form and a yeast-like form. The organism grows as the mycelial form at 33°C and grows as the yeast-like form at 37°C (Levine and Ordal, 1946). The two morphogenetic patterns, M (mycelial) * Y (yeast-like) interconversion in Blastomyces dermatilidis, were called “thermal dimorphism” because that interconversion had been concluded to be solely a function of temperature (Nickerson, 1948). Structural changes in the cells of B. megaterium for the formation of the calcium accumulation system during sporulation seem to resemble this phenomenon in the influence of temperature. Some of fungi for the M e Y interconversion are influenced by - SH compound, glucose, citrate, amino acids, metals, CO*, 02, auxin, age, pH, X-ray irradiation etc. other than temperature (Haidle and Storck, 1966; Nickerson, 1948; Pine and Peacock, 1958; Scherr and Weaver, 1953; Bartnicki-Garcia and McMurrough, 1971; Takada et al., 1963; Johnson, 1954; Steele and Miller, 1974). Some bacteria can be induced to elongate into filaments by various treatments which are denoted B --+ F conversion (Nickerson, 1948).
Effect of temperature on calcium accumulation in B. meguterium
Even in the case of thermal dimorphism, oxygen consumption increased in the high temperature range (Nickerson and Edwards, 1949). In this case, not only the oxygen consumption but also the content of cytochrome oxidase and related enzyme etc. possibly increased. Thus, temperature alone is not considered to be a factor in this case. Oxygen consumption is important for the conversion (Nickerson, 1948). It was observed that oxygen consumption increased after transfer to sporulation medium (Croes, 1967; Hopper et al., 1974) and that the enzyme activities which may be related to the increase varied in yeast Succharomyces cerevisiae (Ota, 1979, 1982b). In addition, some enzymes (Ota, 1980b, 1982c) and the transport activity (Ota, 1982d, 1983a,b), which might be related indirectly to oxygen consumption, increased. The spore fo~ation was blctcked by ethanol whose sensitive period was between 40 and 90min after the end of growth in 3. subtilis, which was considered to be the result of a physical change upon the cell membrane (Bohin et al., 1976a). SpoO mutant of B. subtilis had higher membrane-bound nitrate reductase activities than their Spa+ parental strains. It was suggested that the increased nitrate reductase activity resulted from a mutational change in some membrane component other than the enzyme itself, that is, nitrate reductase in the modified membrane exhibited allotopic properties (Bohin er al., 1976b). The term allotopy was first described by Racker (1967). Lundzren and Beskid (1960) isolated ~em~rature-emotive mutants of 3: c&us ATCC 4342 in the minimal medium. and the snorulation interference was partially reversed when casein hydrolysate was added. Lundgren and Cooney (1962) reported that a temperature-sensitive asporogenous mutant of B. cereus took up about 30% as much of calcium as the parent during sporulation at 37°C. Calcium uptake increased somewhat when added to dipicolinate and cultured at 37°C (Fig. 1). This increase is probably because that calcium is transported as calcium dipicolinate. Furthermore, it might be possible that calcium dipicolinate has an influence on the cell structure leading to increase in calcium uptake. As the substances influencing M 5~$Y conversion are various in different cultural conditions of fungi, fungous species etc., substances influencing sporulation are various in those of bacteria. A dipicolinic acid-less mutant of B. cereus is able to form heatsensitive spores in the absence of dipicolinate (Wise et ai., 1967), but B. megaterium mutant 3-12 needs dipicolinate for its sporulation (Fukuda and Gilvarg, 1968). This indicates that dipicolinate participates not only in heat resistance of spores but also in the formation of spores in the case of the mutant 3-12. Temperature-sensitive mutants of B. subtilis could sporulate at 47°C by the infection of a sporeconverting bacteriophage PMBIZ, and the mutation to Spo (Ts) was suggested to be due to the mu~tion 1
Vegetative cell Undifferentiated
cell
529
in either the RNA polymerase subunit or the 30s ribosomal subunit. It was also suggested that PMBl2 might have at least three genes for spore conversion and that the products interact with a host cell pathway which is expressed in the earliest stage of sporulation (Kinney and Bramucci, 1981). Young (1976) characterized two temperature-sensitive sporulation mutants of B. subtilis; one was obstructed at stage II and the cells exhibited morphological abnormalities, and the other was obstructed at stage IV to V when formation of the spore cortex was almost complete but coat formation was arrested. The former had a short temperature sensitive period during the formation of spore septum, and the product of the defective gene was suggested to be a protein. The m-RNA which coded for this protein was short-lived. It was suggested that coat formation of the latter was arrested at an early stage and that synthesis of the product of the defective gene started long before it assumed its physiological function. Both of them failed to synthesize dipicolinic acid. A temperaturesensitive DNA synthesis-mutant of B. subtilis has been isolated in which the process of initiation of DNA repletion is inhibited at high temperature (Laurent and Vanier, 1973). Haidle and Storck (1966) showed that RNA precursors were discontinuously incorporated into RNA during Y -+ M conversion in Mucor rouxii. It might he possible that in wild type of B. megaterium the temperature shift up affected one or more of these RNA polymerase of r&some, the pattern of RNA synthesis, the DNA replication, the other enzymes or protein in the earliest stage of sporulation. Chloramphenicol inhibits the formation of the calcium accumulation system, but after the formation of the system it does not inhibit the calcium accumulation and the decrease of accumulation in the cells (Fig. 4 and Table 1). Chloramphe~col prevents protein breakdown as well as sporulation, but once the protein degradation is underway it does not diminish its rate, although protein synthesis and sporulation are completely blocked (Komberg et al., 1968). KCN inhibited both calcium uptake and the formation of the calcium uptake system (Table 1 and Fig. 3). This supports the previous suggestion that most of the calcium uptake is energy-dependent both in the mutant 3-12 (Ota, 1982a) and the cells of sporulating Bacilli (Hogarth and Ellar, 1979; Eisenstadt and Silver, 1972). Bronner et aL (1975) reported that energydependent calcium exclusion system was preserved until T6 stage in sporulation in 3. megaterium. It is not likely that KCN inhibits the decrease of the accumulated calcium (Fig. 3). This means that a large part of the calcium release of asporogenous cells in this stage is energy independent, and KCN does not inhibit the decrease or degradation of calcium accumulation system. I propose a tentative model of cell ~fferentiation as follows:
M factors I factors (change of membrane --*(change of gene+ components and membrane 4 expression etc.) structure etc.)
Spore Differentiated cell.
530
AKIHIRO OTA
I factors are the factors which affect the initiation
of differentiation, including the change of gene expression, environmental factors which affect genes etc. M factors are the factors that affect the cell membrane and change the membrane structure etc. In a case of sporulation in Bacilli, M factors contain the change in the cell which evoked calcium uptake. Additional factors essential for cell differentiation are supposed to exist. However, these two factors might be the primary factors at present. SUMMARY
Although a wild-type strain of B. megaterium cultivated at 37°C was incapable of sporulation, a very small amount of calcium accumulation was observed in the delayed period of sporulation sequence, and the calcium accumulation level increased somewhat in the presence of added sodium dipicolinate. The incubation at 37°C seems to prevent the system of calcium accumulation being formed. Influence of the temperature-shift timing from 32 to 37°C on calcium accumulation during sporulation was examined, and it was shown that the temperature sensitive period was approximately at the end of growth. KCN inhibited calcium uptake as well as the formation of calcium accumulation system in asporogenous mutant 3-12 under sporulation condition, and chloramphenicol inhibited the formation of calcium accumulation system alone. Neither KCN nor chloramphenicol inhibited the decrease or degradation of calcium accumulation system in the mutant. In spite of the decrease of the amount of calcium accumulation, the calcium accumulation system and the stage of sporulation in the mutant were maintained after 64 hr in the medium containing 10% glycerol at - 15°C. Factors necessary for sporulation and calcium accumulation were discussed. Acknowledgement-I wish to thank Dr Charles Gilvarg for his generous support and kind discussion. REFERENCES
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