STUDIES ON SPORE GERMINATION

Transcription

1 STUDIES ON SPORE GERMINATION I. EFFECr OF NITROGEN SOURCES ON SPoRE GERMNATION YOETSU HACHISUKA, NOBUO ASANO, NOBUO KATO, MITSUHARU OKAJIMA, MORIO KITAORI, AND TSUNEJI KUTNO Department of Bacteriology, School of Medicine, The University of Nagoya, Nagoya, Japan Little work has been done on the nitrogen sources of spore germination. Holzmuller (1909) reported spores of Bacillus mycoides did not germinate in distilled water and in physiological NaCl solution. Knaysi (1945) reported also that spores of strain C2 of B. mycoides did not germinate in solutions of potassium phosphate, potassium nitrate, lactose, potassium phosphate and nitrate, potassium phosphate and nitrate plus lactose, but that they did germinate in a solution of glucose without nitrogen sources. In recent papers Hills (1949, 1950) has shown that a mixture of adenosine, -tyrosine, and -alanine stimulated the germination of Bacillus anthraci8. Pulvertaft and Haynes (1951) reported that although 14 amino acids in an arbitrary mixture, DL-alanine, L-tyrosine, DL-cystine, DL-tryptophan, and methionine did not have stimulative effect on the germination of Bacilus subtilis, they did stimulate spore germination when adenosine was added. The results of the experiments reported here show the effects of various nitrogen sources on spore germination of B. subtilis. In the experiments a special method was adopted for measuring the rate of germination from the rate of decrease of optical density of culture media by electrophotometer. Received for publication September 24, 1954 MATERIALS AND METHODS Strain and spore suspenion. BacilU subtilis (PCI 219) was used. Spores were harvested from a five day culture on a meat extract agar medium at 37 C. It was confirmed by microscopy that no vegetative forms remained in the culture. The harvested spores were suspended in M/100 phosphate buffer (ph 7.2), heated at 80 C for 15 minutes, and washed repeatedly with phosphate buffer by centrifugation, and resupended in phosphate buffer. Medium. As shown in each table. Measurement of the rate of germination. Development of heat lability has been employed generally as a criterion of germination (Wynne, 1952). We have found that the optical density of liquid media inoculated with spores of B. subtilis in reasonable amounts decreases rapidly as spore germination proceeds. Powell (1950, 1951) has also used this method as a qualitative criterion of germination. We have already found that the rate of decrease of optical density of broth medium inoculated with spores, the number (per cent) of spores with translucent areas calculated on electron micrograph, and the number (per cent) of spores which have lost heat stability measured by the plating technique are almost consistent with one another. According to the observation with the electron microscope, the appearance of translucent areas in the spores appears to be the first and essential stage of spore germination (Knaysi, 1951). Therefore, it is possible to say that the decrease of optical density of a spore suspension is accompanied by spore germination; in fact, the method is excellent for quantitative studies and is easier and more exact than the usual method using the loss of heat stability as a criterion of germination. According to our method, the rate of germination may be calculated from the equation Ao-A Gt_= (Ao - a) (1) where Gt = rate of germination in some medium after t hours' incubation a = optical density of the medium Ao = optical density of the medium inoculated with the spores (before incubation) At = optical density of the medium inoculated with the spores (after incubation for t hours). This equation followed from the next explanation; as mentioned above the rate of spore germi- 399

2 400 YOETSU HACHISUKA ET AL. [VOL. 69 TABLE 1 Relation between the rate of germination and the percentage of spores with translucent areas in glucose-phosphate buffer media (ph 7.8, I/100) containing various nitrogen sources Per Cent of Rate of Germi- Incubation nation NitrgenSouces Timne Areas in Calculted 300 Spores from the Equation DL-Phenylalanine DL-Methionine nation proportionates with the decrease of optical density, so the rate of germination may be expressed mathematically by the equation tao-atx100 (2) Ao - Am where Am - optical density of the medium (optical density a) in which spore germination has occurred 100 per cent. By experimentation in broth medium which stimulates spore germination 100 per cent, the next equation may be established, and 0.3 was generally constant when Bo was within from 0.15 to 0.4, B,n b where b (3) b - optical density of broth medium Bo = optical density of broth medium inoculated with the spores (before incubation) Bm = optical density of broth medium in which spore germination has occurred 100 per cent. From preliminary experiments it has been established that in a broth medium incubated at 37 C the optical density of the medium reached the minimum value in 60 minutes, and, correspondingly, the numbers (per cent) of spores which have lost heat stability, as measured by the plating technique, and the numbers (per cent) of spores with translucent areas, as calculated on electron micrographs, reached 100 per cent. In a medium (optical density a), if spores germinate 100 per cent as in broth medium, equation (3) may be written as A._ Am-a = 0.3 Ao-a then Am = 0.3 Ao a (4) By using equations (4) and (2), equation (1) may be obtained as follows Ao - At Ao -At Gt ~ 100 m- 10 h - AAm Ao-(0.3Ao+0.7a)X 100 Ao - At 0 0.7(Ao - a) Ao, At, and "a" can be determined with an electrophotometer using the red filter. To evaluate if the rate of germination calculated from this equation has exactly traced the true ratio of the germination or not, the technique was compared with the number (per cent) of sporeshaving electron microscopically translucent areas. As shown in table 1, this equation may be useful. RESULTS Nitrogen sources which stimulate t germination. In broth almost all of the inoculated spores germinated, but in a synthetic medium (KNOs 10 g, Na,HP04* 12H20 6 g, KH2PO4 2 g, MgS0O4 6HiO 1 g, CaCl2.2H g, and FeSO4-7H mg in 1,000 ml of distilled water) only 30 per cent germinated. As this synthetic medium supported the growth of vegetative forms to a degree almost equal to that in broth medium, it is obvious that some essential substance or substances are needed for spore germination. Concerning these substances we have paid attention to nitrogen sources. For this purpose each nitrogen source tested was added as 0.15 per cent nitrogen content to m/100 phosphate buffer (ph 7.2) with or without 1 per cent glucose. The ph of each medium was adjusted to 7.2 with NaHCO, and then sterilized by autoclaving for 5 minutes at 120 C before spores were added. In phosphate buffer the spores did not germinate, but when glucose was added, 8 per cent of the spores germinated after 2 hours of incubation. The electron micrographs of spores in phosphate buffer before incubation or after 2 hours of incubation are

3 1955] STUDIES ON SPORE GERMINATION Q--- broth medium *_&ww casamino acid _=0400- glucose + casamino acid 6-<{-- glucose TABLE 2 The rate of spore germination of Bacillus subtilis in phosphate buffer medium containing various nitrogen sources respectively with or without glucose Kind of Nitrogen Sources Rate of Germination Without With Glucose Glucose I Tipne in houy ftfer inoculation. Figiere 1. Decrease of optical density of culture me(lia inoculated Nith spores of Bacillus subtilis. The basal medium was m/100 phosphate buffer (ph 7.2). Glucose was added in 1 per cent. Nitrogen sources were added in concentration of 0.15 Ier cent as nitrogen content. shown in figures 3 and 4. There are no spores with translucent areas. Inorganic salts. KNO3 and NH4Cl were examined. The results are shown in figure 1 and table 2. Although each substance added in phosplhate buffer had almost no effect on the germination, there was considerable stimulative effect in the presence of glucose. The electron iiicrograph of spores incubated for 2 hours in glucose-phosphate buffer medium containing KNO3, as shown in figure 5, indicates that some spores have translucent areas. Casamino acid. The addition of casamino acid to glucose-phosphate buffer medium stimulated the rate of spore germination, but without glucose such} stimulation did not occur. As shown in figure 1, the optical density of the glucose-phosphate buffer medium containing casamino acid Basal medium i,-cystine L-Leucine L-Tryptophan L-Histidine i,-lysine DL-Ornithine L-Arginine L-Isoleucine Di,-Alanine L-Glutamic acid 6 15 DL-Aspartic acid Adenosine Glycine KNO NH4CI i,-oxypro1ine L-Proline DL-Methionine DL-Tvrosine DL-Phenylalanine DL-Serine Dni,-Valine j,-asparagine DL-Isoleucine Casamino acid which was inoculated with spores decreased as rapidly as that of broth medium. As shown in table 2, the rate of germination after 2 hours of incubation was 100 per cent. From these results it appears that one or more of amino acids contained in casamino acid has a stimulative effect on spore germination and should be able to replace the "unknown" substances in broth. Therefore various amino acids were examined. Amino acids. The results in figure 2 and the rates of germination after 2 hours of incubation are shown in table 2. In the presence of glucose in phosphate buffer, the spore germination was stimulated when L-asparagine, DL-isoleucine, DLserine, or DL-valine was individually added. Germination in these media resembles that in broth or in the glucose-phosphate buffer mediium con-

4 402 YOETSU HACHISUKA ET AL. [VOL. 69 _0_ glucose t DL-inoleucine -. glucose +. DL*serine.ylu cose L-phenylalanine.mlj.. glucose * DL-vallne Figurse 4j. Spores incubated for 2 houirs in -m/100 phosphate b)uffelr mediuiii (pih 7.2) Time in hour after inoculation. Figuri e 2. Decrease of optical density of eulture media inoculated with spores of Bacilluls subtilis. The l)asal me(iiuim and concentration of glucose and Initrogeni sources, wi-ere the same as in figure 1. Figuire 3. The electroi inicrograph of resting sporess of Bacilluis suibtilis. taining casamino acid. On the contrary, without glucose the addition of these amino acids, respectively, showed no oir only a little effect. From these iesults it can be said that the stimulative effects of amino acids are dlisplayed only in the presence of glucose. The electron microgiaphs of spores incubated foir 2 hour s in glucose-phosphate buffer media Figure 5. Spores incubated for 2 houirs in glucose-phosphate buffer ine(litim (I per centt gluicose) containiing KNO:s. containing DL-iimethioniie, DL-plhenylalanine, DLvaline, an(l DL-serine ale showni in figures 6, 7, 8, and 9. All of the spores in the medlia containing DL-valine 01o DL-serine hlave tr ainslucent aireas. In the media containing DL-phenylalanine or DLmethionine, ther e remain a few intact spoires. These results agree with those shown in table 2. It is interesting to consider that although DLisoleucine showedl a remarkable stimulation in the presence of glucose on the spore germiination, L-isoleucine andl DL-leucine had no effect. Regaiding adlenosine, which has been observed bv Pulvertaft an(d Haynes (1951) to have an effect on the gerimination of B. subtilis even when added to washed spores in saline, we have failedi to demonstirate sutch a renmarkable effect. Is it lue to the (lifference of strain of B. subtilis? The difjerenice between tie nitrogen sources which are effective on the vegetative growth and those efective on the sporc germnination. In the prexious experiment the nitrogen sources which stimulate spore germination were exploredl. In this experi-

5 LS~~~~~~~~~~~~~~~~~..._ STUDIES ON SPORE GERMINATION 403.lo 4a..,w4p _9 lb so L Figuire 6 Figure 7 Fignure 8 Figure 9 Figutres 6-9. Spores incubated for 2 hours in glucose-phosphate buffer media containing DLmethionine (figure 6), DL-phenylalanine (figure 7), DL-valine (figure 8), and DL-serine (figure 9), respectively.... ment the nitrogen sources were studied to determine which were effective for spore germination and vegetative growth. Glucose-phosphate buffer medium was insufficient for this experiment. However, a medium sufficient for vegetative growth containing 0.6 per cent Na2HP04*12H20, 0.2 per cent KH2PO4, 0.1 per cent MgS , 0.01 per cent CaCl2*2H20, 25 mg per 100 ml FeSO4 7H20, and 1 per cent glucose was used as the basal medium. In this medium only 20 per cent of the inoculated spores germinated after 2 hours of incubation. Each nitrogen source was added in 1 per cent to basal medium. When an inorganic salt such as KNO3 or NH4Cl is added to this basal medium, vegetative forms grow profusely to a remarkable dense surface pellicle. The effect on germination was determined from the rate of germination after 5 hours of incubation, and the effect on vegetative growth was determined by the presence or absence of surface pellicles after 48 hours of incubation. From these results, nitrogen sources may be classified into four groups in accordance wn-ith their effect on growth and germination. Group I has no effect on growth nor germination. Group II has a stimulative effect on germination, but no effect on growth. Group III has no effect on germination, but has a stimulative effect on growth. Group IV has a stimulative effect on both growth and germination. The results are shown in table 3. According to the electron microgiaphs, spores develop to vegetative forms through fouir phases (Knaysi, 1951). The 1st is the phase of the appearance of translucent area in a spore. The 2nd is the phase of the formation of a germ pore. The 3rd is the phase of the emergence of a new bacillus (germ cell). The 4th is the phase of the development of new bacillus to a vegetative cell. Therefore, to examine to what phase the spores have developed in media in which the optical density decreased remarkably without forming

6 404 YOETSU HACHISUKA ET AL. [VOL. 69 TABLE 3 Effect of nitrogen sources on vegetative growth and spore germination of Bacillus subtilis Kind of Nitrogen Sources Effection Effect on L A Figure 10. All of spores have translucent areas, and some of them have germ pore. Basal medium - - L-Alanine - - L-Cystine - - L-Tryptophan - - L-Isoleucine - Adenosine + - L-Leucine i - Glvcine 4i _ DL-Serine DL-Valine DL-Phenylalanine DL-Methionine +++ L-Glutamic acid DL-Aspartic acid i +++ L-Histidine KNO3 ±4+ + L-Arginine - ++ DL-Ornithine _ + Figure 11. Spores with translucent areas, new bacilli, and a shell of spore are seen. r Figure 12. Almost same as figure 11. Figures Spores incubated for 48 hours in the synthetic medium containing DL-serine (figure 10), DL-phenylalanine (figure 11), and DLmethionine (figure 12), respectively, which belong to the second group described in table 2. surface pellicle, electron micrographs were taken of the spores which were incubated for 48 hours in the media containing one of the amino acids belonging to the second group as a nitrogen DL-Isoleucine L-Asparagine L-Oxyproline DL-Alanine X -Proline + ++ NH4Cl The effect on the spore germination was determined from the rate of germination in 5 hour culture per cent, per cent, per cent, per cent, - less than 20 per cent. (The rate of germination in the basal medium is 20 per cent.) The effect on the vegetative growth was determined from whether surface pellicles were formed or not in 48 hour culture and the degree of their thickness when pellicles were formed. +++ thick pellicle (as thick as will not sink by shaking), ++ fairly thick pellicle, + thin pellicle (easily sunk by shaking), ±t pellicle like scurf, - not formed. source. These are shown in figures 10 to 12. In the case of DL-serine, all spores have translucent areas and some have a germ pore, while in the case of DL-phenylalanine and DL-methionine the vegetative cells seem to be new bacilli and shells of spores appear. From these findings it may be considered that in the case of DL-serine, the development of germination stopped at the sec-

7 1955] STUDIES ON SPORE GERMINATION 405 ond phase, while in the case of DL-phenylalanine and DL-methionine, germination proceeded to the 4th phase. These results may show that the effects of amino acids of the second group on the development of the germination are not necessarily identical. DISCUSSION For quantitative studies of spore germination it is most important to know the percentage of spores germinated. Up to date, investigators have used the loss of heat stability as a criterion of the germination. In practice this method has many troublesome procedures which sometimes lead to erroneous conclusions; for example, the second heating, dilution, plating, and counting, etc Ṫhe change accompanying the beginning of the germination of Bacillus species is, besides the loss of heat stability, electron microscopically, the appearance of translucent area in the spore (Knaysi, 1951), phase-contrast microscopically, the darkening of the spore (Pulvertaft and Haynes, 1951), and nephelometrically, the decrease of turbidity of spore suspension (Powell, 1950, 1951). Our finding that the optical density of a spore suspension of B. subtili8 decreases rapidly as spore germination proceeds is the phenomenon corresponding to the appearance of translucent areas in spores observed by electron microscope. The decrease of optical density of a spore suspension accompanied by germination has proved to be an excellent criterion for the quantitative studies of the germination. Using this criterion, we have established an equation from which the rate of germination can easily be calculated. From the present investigation it is clear that L-asparagine, DL-isoleucine, DL-valine, and DLserine have a stimulative effect on spore germination of B. subtilis (PCI 219) in glucose solution buffered with inorganic phosphates, or in a nitrogen-free synthetic medium composed of inorganic salts and glucose. Defect of any factor, i.e., effective amino acid and glucose, repeals the stimulative effect for the spores to develop to the 1st phase of germination. The mechanism of the effect of these factors on the germination has not yet been decided, but it may be probable that these factors are required as the "sparking factor" for changing the resting spores to active spores. The reasons for this deduction are as follows: (1) Hardwick and Foster (1953) have reported that in spores active enzymes are not demonstrated and enzymes may be present in inactive forms such as protein precursors more or les closely related to the specific enzymes. According to our work, the spores in the lst phase have a considerable respiratory activity which is not demonstrable in resting spores (Hachisuka et al., unpublished data). (2) According to the unpublished experiment (Hachisuka et al.) DL-isoleucine is effective on the germination in glucose-phosphate buffer even at the concentration of 107 mol. From this fact it may be considered that the amino acids effective on the germination are not used as materials for the synthesis of cell substances. In any case, in order to clear the mechanism of the germination of B. subtils, it is most important to decide the action of the amino acids which are stimulative only with glucose in phosphate buffer. It is interesting that some amino acids such as L-glutamic acid and DL-alanine, which have a remarkable effect on the vegetative growth of B. ssubtilis and many other species, have little effect on the germination of B. subtilis. On the contrary, the effective amino acids on the germination are not necessarily effective on growth. This fact may support the above mentioned supposition that the amino acids effective on germination are used in other ways than as materials for the synthesis of cell substances. ACKNOWLEDGMENT The authors wish to express their appreciation to Professor Dr. Kazuo Ogasawara, Department of Bacteriology, School of Medicine, Nagoya University, and to Assistant Professor Dr. Masahiko Yoneda, Department of Bacteriology, School of Medicine, Nagoya City University, for helpful encouragement throughout the work. SUMMARY The rate of spore germination of Bacillus subtilis (PCI 219) can be easily determined by measuring the decrease of optical density of culture media in which spores germinate. By the method using the decrease of optical density of culture media as a criterion of the germination, the effects of various nitrogen sources on the germination have been studied. From the present work it is clear that some amino acids stimulate germination in the presence of glucose and inorganic

8 4:06 YOETSU HACHISUKA ET AL. [vol. 69 phosphate. There is no identity between the nitrogen requirements of the germination and those of the vegetative growth. Possible explanations of these phenomena are discussed. REFERENCES HARDwIcK, W. A., AND FOSTER, J. W Enzymatic changes during sporogenesis in some aerobic bacteria. J. Bacteriol., 65, HILLS, G. M Chemical factors in the germination of spore-bearing aerobes. The effects of amino acids on the germination of Bacillu anthracis, with some observation on the relation of optical form to biological activity. Biochem. J. (London), 45, HILLS, G. M Chemical factors in the germination of spore-bearing aerobes. Observations on the influence of species, strain and conditions of growth. J. Gen. Microbiol., 4, HoLzmtJLLER, K Die Gruppe des Bacillus mycoidee Flfgge. Ein Beitrag zur Morphologie und Physiologie der Spaltpilze. Centr. Bakteriol. Parasitenk., Abt. II, 2X, KNAYSI, G Investigation of the existence and nature of reserve material in the endospore of a strain of Bacillus mycoidea by an indirect method. J. Bacteriol., 49, KNAYSI, G Elements of bacterial cytology. Second edition, p Comstock Publishing Co., Inc., Ithaca, N. Y. POWELL, J. F Factors affecting the germination of thick suspensions of Bacillus subtilis spores in L-alanine solution. J. Gen. Microbiol., 4, POWELL, J. F The sporulation and germination of a strain of Bacillw megatherium. J. Gen. Microbiol., 6, PULVERTAFT, J. V., AND HAYNES, J; A Adenosine and spore germination; Phase-contrast studies. J. Gen. Microbiol., 5, WYNNE, E. S Some physiological aspects of bacterial spore formation and spore germination. Bacteriol. Revs., 2,