JOURNAL OF BACTERIOLOGY, May 1979, p. 442-445 0021-9193/79/05-0442/04$02.00/0
Vol. 138, No. 2
Glucose-Triggered Germination of Bacillus megaterium Spores F. M. RACINE, S. S. DILLS,t AND J. C. VARY* Department of Biochemistry, University of Illinois Medical Center, Chicago, Illinois 60612 Received for publication 9 February 1979
Triggering of germination in Bacillus megaterium QM B1551 spores with Dglucose was studied. First, the interaction of glucose with spores for less than 1 min resulted in triggering almost 90% of the spores after the glucose was removed by dilution. Therefore only a brief time is needed for glucose to trigger germination, and then the continuous presence of glucose is not necessary. Detectable uptake of glucose began 2 to 3 min after absorbance loss started, and a nonmetabolizable glucose analog, methyl-a-D-glucopyranoside, triggered germination in the absence of detectable uptake. Several inhibitors that reduced or eliminated glucose uptake did not block triggering of germination. Therefore, glucose uptake may be a relatively late event and not a prerequisite for triggering of germination. In an attempt to understand how glucose can trigger germination of Bacillus megaterium spores (10), we have found that metabolism of the exogeneously added glucose is not necessary (9), nor is there an apparent requirement for functional respiratory chain-associated reactions (1). In the present study, the minimum times for exposure of spores to glucose that triggered germination were determined relative to the known sequence of events that occur during germination (3, 4). In addition, uptake of D-glucose was studied to assess the possible role of transport for triggering germination. MATERIALS AND METHODS The methods for preparing spores of B. megaterium QM B1551 and measuring triggering of germination were previously described (1, 9). The uptake of a-Dglucopyranose (glucose) during triggering and initiation of germination was measured by following the accumulation of D-[ U-'4C]glucose or methyl-a-D-[ U"4C]glucopyranoside (methyl-a-glucoside). Spores were preincubated at 30°C for 5 min, and then D-[ U'4C]glucose was added such that the final concentrations were 5 mM Tris-hydrochloride buffer (pH 8), 1 mg of spores per ml, lOO,g of chloramphenicol per ml, and 1 mM glucose (1 uCi/imol). Two methods of measuring glucose uptake were used. In method A, 0.1 ml of the reaction mixture was removed, and the spores were collected by vacuum filtration on a 0.45ym HAWP Millipore membrane filter (Millipore Corp., Bedford, Mass.) and washed three times with 2.0 ml of cold 5 mM Tris-hydrochloride (pH 8) containing 2 mM MgCl2. In method B, uptake of methyla-glucoside or glucose was measured under the same conditions, except that the monosaccharide concentra-
tion was 4 mM (1 ,uCi/,umol), 0.1 ml of reaction mixture was diluted with 2.0 ml of 5 mM Tris-hydrochloride (pH 8), 5 mM MgCl2, and either 20 mM methyl-aglucoside or 20 mM glucose prior to collection on the filter, and the filters were washed three times with 2.0 ml of the same buffer. Both methods gave comparable results, but method B was slightly more reproducible. The filters were dried and counted as previously described (9), and the results were plotted as nanomoles of methyl-a-glucoside or glucose per milligram of spores (dry weight). By these methods we could detect uptake of 0.1 nmol/mg of spores. Heat resistance loss, dipicolinic acid release, and absorbance loss were determined as previously described (3) with mixtures containing 10 mg of heatactivated spores per ml, 5 mM Tris-hydrochloride (pH 8), and 5 mM glucose at 30°C. The minimum times of exposure to glucose to trigger germination were measured by diluting samples of the same spore suspension 100-fold into 5 mM Tris (pH 8) at 30°C and then measuring the absorbance at 660 nm after 30 min. It should be noted that the dilution technique was sufficient to dilute the glucose to a final concentration of 50 ,uM. This concentration of glucose did not trigger germination, as demonstrated by the fact that the first sample in Fig. 1 did not lose absorbance. The details of this technique have been described and discussed
(6). Methyl-a-D-[ U-''C]glucopyranoside and D-[ U''C]glucopyranose were purchased from Amersham/ Searle, and other chemicals were reagent grade. Carbonyl cyanide m-chlorophenyl hydrazone and N,N'dicyclohexylcarbodiimide were dissolved in absolute ethanol and diluted 100-fold into the reaction mixtures.
t Present address: Department of Biology, University of California-San Diego, La Jolla, CA 92093.
442
RESULTS AND DISCUSSION In the known sequence of events that occur during initiation of germination, the times for triggering germination were determined. This
GLUCOSE-TRIGGERED B. MEGATERIUM GERMINATION
VOL. 138, 1979
was done by brief exposures of spores to glucose, followed by dilution. Similar techniques have been used with L-alanine (2, 7) to show that less than 1 min of exposure to L-alanine was sufficient to trigger germination and that the continued presence of the L-alanine was not necessary. As shown in Fig. 1, a fraction of the spores (-50%) were triggered for germination at 45 s, when only 11% loss in heat resistance, 4% dipicolinic acid loss, and no absorbance loss had occurred. These results indicate that a transient exposure to glucose is sufficient to trigger germination, and that triggering of germination in the population of spores occurs before loss in heat resistance. The kinetics of glucose uptake relative to absorbance loss was determined (Fig. 2). Loss in absorbance began at 3 min, whereas glucose uptake was first detected at 6 min, suggesting that glucose uptake is a late event. Used as a control, dormant spores neither accumulated glucose nor lost absorbance. Not shown is the fact that the rate of absorbance loss could be altered by changing the concentration of spores and/or glucose. However, in those experiments glucose uptake always occurred later than absorbance loss. It should also be noted that this experiment was run in the presence of chloramphenicol, but the same results were found if chloramphenicol was omitted (data not shown). Since glucose uptake was not inhibited by chlor-
0
443
amphenicol, a glucose transport system presumably preexisted in the spore and became activated during initiation of germination. This phenomenon of activating preexisting enzyme systems during initiation of germination has been previously suggested (1, 11) based on similar evidence. Previous results (1) showed that a variety of inhibitors of electron transport and energy production did not inhibit triggering of germination but did block ATP synthesis or decreases in pH in the medium. It was of interest to test some of these inhibitors for their effect on glucose uptake. A number of metabolic inhibitors and protonmotive force uncouplers were tested (Table 1) and found to reduce but not eliminate the amount of glucose uptake. However, both HgC12 and p-hydroxymercuribenzoate completely blocked glucose uptake for 20 min without stopping triggering of germination, as evidenced by the partial losses in absorbance. It was previously shown (1, 5) that the partial losses in absorbance, such as shown in Table 1, represent triggering of germination by the whole population of spores, but that complete initiation of germination is then stopped by 1 mM HgCl2. Although the characterization of the glucose uptake mechanism was not the aim of this study, it is worthwhile noting our observations for any further investigations. From a survey of the types of inhibitors and combinations of inhibi-
0
2 40
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60
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0
0
100 1 0 3
5
10~~t
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3
TIME (min) FIG. 1. Minimum time for triggering germination. Heat-activated spores (10 mg/ml) were incubated in 5 mM Tris-hydrochloride (pH 8) and 5 mM glucose at 30°C. Minimum times for triggering (0), and heatresistance (0), dipicolinic acid (x), and absorbance (A) losses were measured as described in the text, and the results were normalized to percent completion. The total losses in heat resistance, dipicolinic acid, and absorbance were 98%, 90 pg/mg of spores, and 44%, respectively, within 30 min.
444
RACINE, DILLS, AND VARY
J. BACTERIOL.
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previously shown that methyl-a-glucoside triggers germination, yet is not metabolized by germinating spores (9). As shown in Fig. 3, no uptake was detected during 40 min. Based on inhibitor studies and on measurement of glucose or methyl-a-glucoside uptake, triggering of germination occurred in the absence of detectable uptake. It appears that only a brief exposure to glucose is necessary to trigger germination. It should be noted that in the above studies with either radioactive glucose or radioactive methyl-a-glucoside, uptake (or binding) of
Vol. 138, No. 2
Glucose-Triggered Germination of Bacillus megaterium Spores F. M. RACINE, S. S. DILLS,t AND J. C. VARY* Department of Biochemistry, University of Illinois Medical Center, Chicago, Illinois 60612 Received for publication 9 February 1979
Triggering of germination in Bacillus megaterium QM B1551 spores with Dglucose was studied. First, the interaction of glucose with spores for less than 1 min resulted in triggering almost 90% of the spores after the glucose was removed by dilution. Therefore only a brief time is needed for glucose to trigger germination, and then the continuous presence of glucose is not necessary. Detectable uptake of glucose began 2 to 3 min after absorbance loss started, and a nonmetabolizable glucose analog, methyl-a-D-glucopyranoside, triggered germination in the absence of detectable uptake. Several inhibitors that reduced or eliminated glucose uptake did not block triggering of germination. Therefore, glucose uptake may be a relatively late event and not a prerequisite for triggering of germination. In an attempt to understand how glucose can trigger germination of Bacillus megaterium spores (10), we have found that metabolism of the exogeneously added glucose is not necessary (9), nor is there an apparent requirement for functional respiratory chain-associated reactions (1). In the present study, the minimum times for exposure of spores to glucose that triggered germination were determined relative to the known sequence of events that occur during germination (3, 4). In addition, uptake of D-glucose was studied to assess the possible role of transport for triggering germination. MATERIALS AND METHODS The methods for preparing spores of B. megaterium QM B1551 and measuring triggering of germination were previously described (1, 9). The uptake of a-Dglucopyranose (glucose) during triggering and initiation of germination was measured by following the accumulation of D-[ U-'4C]glucose or methyl-a-D-[ U"4C]glucopyranoside (methyl-a-glucoside). Spores were preincubated at 30°C for 5 min, and then D-[ U'4C]glucose was added such that the final concentrations were 5 mM Tris-hydrochloride buffer (pH 8), 1 mg of spores per ml, lOO,g of chloramphenicol per ml, and 1 mM glucose (1 uCi/imol). Two methods of measuring glucose uptake were used. In method A, 0.1 ml of the reaction mixture was removed, and the spores were collected by vacuum filtration on a 0.45ym HAWP Millipore membrane filter (Millipore Corp., Bedford, Mass.) and washed three times with 2.0 ml of cold 5 mM Tris-hydrochloride (pH 8) containing 2 mM MgCl2. In method B, uptake of methyla-glucoside or glucose was measured under the same conditions, except that the monosaccharide concentra-
tion was 4 mM (1 ,uCi/,umol), 0.1 ml of reaction mixture was diluted with 2.0 ml of 5 mM Tris-hydrochloride (pH 8), 5 mM MgCl2, and either 20 mM methyl-aglucoside or 20 mM glucose prior to collection on the filter, and the filters were washed three times with 2.0 ml of the same buffer. Both methods gave comparable results, but method B was slightly more reproducible. The filters were dried and counted as previously described (9), and the results were plotted as nanomoles of methyl-a-glucoside or glucose per milligram of spores (dry weight). By these methods we could detect uptake of 0.1 nmol/mg of spores. Heat resistance loss, dipicolinic acid release, and absorbance loss were determined as previously described (3) with mixtures containing 10 mg of heatactivated spores per ml, 5 mM Tris-hydrochloride (pH 8), and 5 mM glucose at 30°C. The minimum times of exposure to glucose to trigger germination were measured by diluting samples of the same spore suspension 100-fold into 5 mM Tris (pH 8) at 30°C and then measuring the absorbance at 660 nm after 30 min. It should be noted that the dilution technique was sufficient to dilute the glucose to a final concentration of 50 ,uM. This concentration of glucose did not trigger germination, as demonstrated by the fact that the first sample in Fig. 1 did not lose absorbance. The details of this technique have been described and discussed
(6). Methyl-a-D-[ U-''C]glucopyranoside and D-[ U''C]glucopyranose were purchased from Amersham/ Searle, and other chemicals were reagent grade. Carbonyl cyanide m-chlorophenyl hydrazone and N,N'dicyclohexylcarbodiimide were dissolved in absolute ethanol and diluted 100-fold into the reaction mixtures.
t Present address: Department of Biology, University of California-San Diego, La Jolla, CA 92093.
442
RESULTS AND DISCUSSION In the known sequence of events that occur during initiation of germination, the times for triggering germination were determined. This
GLUCOSE-TRIGGERED B. MEGATERIUM GERMINATION
VOL. 138, 1979
was done by brief exposures of spores to glucose, followed by dilution. Similar techniques have been used with L-alanine (2, 7) to show that less than 1 min of exposure to L-alanine was sufficient to trigger germination and that the continued presence of the L-alanine was not necessary. As shown in Fig. 1, a fraction of the spores (-50%) were triggered for germination at 45 s, when only 11% loss in heat resistance, 4% dipicolinic acid loss, and no absorbance loss had occurred. These results indicate that a transient exposure to glucose is sufficient to trigger germination, and that triggering of germination in the population of spores occurs before loss in heat resistance. The kinetics of glucose uptake relative to absorbance loss was determined (Fig. 2). Loss in absorbance began at 3 min, whereas glucose uptake was first detected at 6 min, suggesting that glucose uptake is a late event. Used as a control, dormant spores neither accumulated glucose nor lost absorbance. Not shown is the fact that the rate of absorbance loss could be altered by changing the concentration of spores and/or glucose. However, in those experiments glucose uptake always occurred later than absorbance loss. It should also be noted that this experiment was run in the presence of chloramphenicol, but the same results were found if chloramphenicol was omitted (data not shown). Since glucose uptake was not inhibited by chlor-
0
443
amphenicol, a glucose transport system presumably preexisted in the spore and became activated during initiation of germination. This phenomenon of activating preexisting enzyme systems during initiation of germination has been previously suggested (1, 11) based on similar evidence. Previous results (1) showed that a variety of inhibitors of electron transport and energy production did not inhibit triggering of germination but did block ATP synthesis or decreases in pH in the medium. It was of interest to test some of these inhibitors for their effect on glucose uptake. A number of metabolic inhibitors and protonmotive force uncouplers were tested (Table 1) and found to reduce but not eliminate the amount of glucose uptake. However, both HgC12 and p-hydroxymercuribenzoate completely blocked glucose uptake for 20 min without stopping triggering of germination, as evidenced by the partial losses in absorbance. It was previously shown (1, 5) that the partial losses in absorbance, such as shown in Table 1, represent triggering of germination by the whole population of spores, but that complete initiation of germination is then stopped by 1 mM HgCl2. Although the characterization of the glucose uptake mechanism was not the aim of this study, it is worthwhile noting our observations for any further investigations. From a survey of the types of inhibitors and combinations of inhibi-
0
2 40
0
z w
60
-
000
0
0
100 1 0 3
5
10~~t
IS
3
TIME (min) FIG. 1. Minimum time for triggering germination. Heat-activated spores (10 mg/ml) were incubated in 5 mM Tris-hydrochloride (pH 8) and 5 mM glucose at 30°C. Minimum times for triggering (0), and heatresistance (0), dipicolinic acid (x), and absorbance (A) losses were measured as described in the text, and the results were normalized to percent completion. The total losses in heat resistance, dipicolinic acid, and absorbance were 98%, 90 pg/mg of spores, and 44%, respectively, within 30 min.
444
RACINE, DILLS, AND VARY
J. BACTERIOL.
0
r
c 0
0
(a m
C
-.4
m
2. wI z
0 1.2
-5
.
0
(0f
0
AA
.d
5
-A _.,
A
_A
previously shown that methyl-a-glucoside triggers germination, yet is not metabolized by germinating spores (9). As shown in Fig. 3, no uptake was detected during 40 min. Based on inhibitor studies and on measurement of glucose or methyl-a-glucoside uptake, triggering of germination occurred in the absence of detectable uptake. It appears that only a brief exposure to glucose is necessary to trigger germination. It should be noted that in the above studies with either radioactive glucose or radioactive methyl-a-glucoside, uptake (or binding) of
