Molec. gen. Genet. 175, 113-120 (1979) © by Springer-Verlag 1979
RNA Synthesis During Spore Germination in Bacillus subtilis* A. Sloma and I. Smith Department of Microbiology, The Public Health Research Institute of The City of New York, Inc., New York, New York 10016, USA
Summary. We have analyzed the R N A synthesized during spore germination in Bacillus subtilis. Early in germination there is little incorporation of [3H]uridine into R N A . A large increase in incorporation into R N A was found at 45 60 rain into germination which was in part due to increases in the specific activity of the U T P pool. When corrected for specific activity changes, the instantaneous rate of R N A synthesis showed a seven to tenfold increase between 30 and 45 rain of germination. Polyacrylamide gel electrophoresis studies showed that the R N A synthesized during germination appeared very similar to the R N A made during vegetative growth. D N A - R N A hybridization studies indicated that m R N A and r R N A were synthesized throughout germination. Their relative proportions remained constant and were very similar to the composition of R N A synthesized during vegetative growth.
Introduction
When Bacillus subtilis spores are incubated in a suitable medium, they undergo a series of changes that lead to the outgrowth of vegetative cells. Very little is known about how these changes are regulated. A r m s t r o n g and Sueoka (1968) reported that R N A synthesis is biphasic during germination. In the early phase (2-10 rain) they found that only r R N A is made, while both r R N A and m R N A are synthesized in the second phase which begins at 15 rain. Protein synthesis started at the beginning of the second phase in their experiments. Similar results in B. subtilis were * In partial fulfillment of the requirements for the doctoral degree by A.S. in the Department of Microbiology at the New York University School of Medicine For offprints contact." I. Smith
described by Woese and Bleyman (1969). On the other hand, Balassa and Contesse (1965) have also reported that both r R N A and m R N A are synthesized from the beginning of germination in B. subtilis. In B. cereus, an immediate synthesis of all classes of R N A has been reported by Spiegelman et al. (1969) and Torriani et al. (1969). If there is a biphasic pattern of R N A synthesis during germination, this would be evidence for a unique type of regulation in which only r R N A was made at a specific stage of growth. If, however, the R N A synthesized throughout the germination process is similar or identical to the R N A made during vegetative growth, it would indicate that there is no unusual transcriptional regulation during outgrowth. This study was undertaken to compare the R N A made during germination with N a t made during vegetative growth in B. subtilis, to 'resolve the published contradictions appearing in the literature regarding the nature of R N A synthesis during B. subtilis germination and outgrowth. We have found that there was no transition from a stage where only r R N A is made to one where both r R N A and m R N A are transcribed. Proportions o f r R N A and m R N A remained relatively constant throughout the germination process, and these proportions were the same as those of vegetative growth.
Materials and Methods Strains. B. subtilis strain BD68 (pyrA, argC4, leu-2), obtained from
Dr. D. Dubnau, was used in all experiments. Media. Sporulation media (SM) used contained per liter, nutrient broth (Difco), 8 gm; KC1, 1 gin; MgSO4-7 H20, 0.25 gin; 1 nM FeSO4; 1 mM Ca(NO3)2; 0.1 nM MnC12 (Schaeffer et al., 1965). Germination media (GM) contained per liter, NaCI, 3 gm ; KCI, 0.5 gin; (NH~)SO4, 2.0 gin; Na citrate-2 H20, 1.0 gin: MgSO4.7H20, 0.2gm; L-alanine, 0.5gin; casein hydrolysate, 0.2 gin; yeast extract, 0.2 gm; glucose, 5 gin; 25 mM Tris, pH 7.5;
0026-8925/79/0175/0113/$01.60
114
A. Sloma and I. Smith: R N A Synthesis During Spore Germination in B, subtilis
NaHzPO4, 5 m M ; uridine, 100 gg/ml; arginine 100 gg/ml; leucine 100 gg/ml. W h e n labeling with [3H]uridine (Moravek), 20 gg/ml uridine was used; when labeling with [32p]o4, 0.5 m M NaH2PO4 was used. Other media (VY, 1 x SS, etc.) have been described (Morell et al., 1967).
Spore Preparation. BD68 cultures were grown in SM at 35°C for 3 days with vigorous shaking. The cultures were then centrifuged, washed with cold distilled H 2 0 three times a n d resuspended into 100 m M Na E D T A , 50 m M NaC1, p i t 6.9 (LM), using 20 ml/per liter of culture. Lysozyme (500 l~g/ml) was added to the resuspended cell pellet at 37 ° C for 1 h. The crude spore preparation was washed with distilled H 2 0 three times and resuspended in i x SS (20 ml per liter). Pancreatic D N a s e (5 gg/ml) was added and the mixture was incubated for 30 min at 37 ° C. The preparation was then washed three times in distilled H 2 0 and lyophilized. The resulting spores were completely free of vegetative cells and were 100% refractile as viewed by phase contrast microscopy.
Spore Germination. Lyophilized spores were resuspended in distilled H 2 0 at a concentration of 5 m g / m l and heat-shocked for 30 rain at 70 ° C. Spores were germinated in G M at a concentration of 0.5 m g dry wt. spores/ml GM. This corresponds to 1 to 2 x l0 s colony forming units/ml. The culture was vigorously shaken at 37°C.
Labeling of RNA. Pulse-labeled R N A was obtained by incubating 5 ml germinating spores or 5 ml vegetative cells grown to mid-log phase, both at a cell concentration of 1 x 10S/ml, with 50 gCi/ml [5-3H]uridine (Moravek) or 200 gCi/ml [32p]NazPO4 (Amersham). Uptake was stopped by pouring the samples over crushed ice containing 10 m M NAN3. Samples were centrifuged at 4 ° C a n d washed in 100 m M NaC1, 10 m M Na acetate, p H 5 ( R N A buffer). To fully label spore R N A , 0.1 gCi/ml [2-1~C]uracil (New England Nuclear) was added to a freshly inoculated culture of BD68 in SM. After 3 days of growth at 35 ° C, spores were prepared as above. To prepare R N A , radioactive cells were resuspended in 5 ml of cold R N A buffer, after washing as described in the preceding section. To each radioactive sample, 1 ml of nonradioactive carrier cells was added, if the radioactive samples were to be analyzed by gel electrophoresis (the carrier cells were prepared as follows: BD68 cells grown in VY [50 ml] to a cell concentration of 2 × 10S/ml were centrifuged and washed in 10 m l / R N A buffer. The cells were resuspended in 10 ml cold R N A buffer). If the samples were to be used to prepare R N A for R N A : D N A hybridization, 5 r a g of yeast R N A in R N A buffer were added to each radioactive sample instead of carrier cells. To each 5 ml sample, 4 ml of 0.17 m m glass beads (VWR Scientific) were added, and each sample was broken for 2 min in a Braun homogenizer under constant passage of CO2 to keep the samples cold. After breakage, the sample was quickly put into 1% SDS and centrifuged 5000 r p m for 5 min to remove cell debris. To the supernatant an equal volume of hot redistilled phenol saturated with R N A buffer was added. Samples were vortexed two times and centrifuged at 10,000 r p m for 15 rain. The aqueous phase containing the R N A was removed, and to it was added 2 volumes of cold ethanol. The R N A was precipitated overnight at - 2 0 ° C. The R N A pellet was resuspended in 5 ml R N A buffer containing 0.5% SDS, and centrifuged 15,000 rpm for 30 rain. If R N A was used for hybridization, the R N A pellet was resuspended in 5 ml 10 m M Tris, pH 7.5; 10 m M MgC12; and R N a s e free pancratic D N a s e (Worthington) (5 gg/ml) was added at 0 ° C for 30 min. The R N A was reextracted with R N A buffer-saturated phenol, re-precipitated with ethanol, and resuspended in 5 ml R N A buffer containing 0.5% SDS. The R N A was then precipitated a second time with 2 volumes of cold ethanol. After 2 h at - 2 0 ° C the precipitated R N A was centrifuged 15,000 r p m for 30 rain and resuspended
in 0.5 ml R N A buffer containing 0.5% SDS. Recovery of R N A was always greater than 70%.
UTP Pool Measurements. Pulse-labeled U T P was obtained by incubating 5 ml of germinating spores with 100 gCi/ml [5-3H]uridine for 2 rain. The samples were centrifuged for 2 rain at 5,000 r p m at 0 ° C. The cell pellets were resuspended in 0.2 ml 2 M formic acid, pH 3.4 at 0 ° C and kept on ice for 15 rain. The samples were then centrifuged in the cold in an Eppendorf microcentrifuge. The supernatant solution containing the extracted nucleotides was quickly frozen, and they were thawed immediately before applying to PEI-cullulose F thin layer plates (E. Merck). The radioactive nucleotides, with 50 nmoles of cold U T P as carrier, were resolved on the PEI plates with 0.85 M KH2PO4, p H 3.4 (Cashel et al., 1969). After the chromatography plates were developed and dried, the U T P spots were visualized under shortwave UV light (254 nM). The spots were then cut from the plate and resuspended in 0.7 M MgC12, 0.02 M Tris, p H 7.4 (Randerath and Randerath, 1967) After 30 rain at r o o m temperature the sample was counted after suspension in Triton-toluene-PPO. To measure the preexisting [~4C]UTP pool, an equal a m o u n t of [14C]uracil-labeled spores (see above) was incubated in G M (0.5 mg spores/m1) and shaken vigorously in a separate flask. At each time point the pulse-labeled spores and prelabeled spores were removed at the same time and prepared as above. The absolute a m o u n t of U T P in each radioactive spot was too low to detect. Therefore, the [3H]UTP pool specific activity was measured by applying 10 gl of the 3H labeling media (20 ~tg/ml cold uridine, 100 gCi/ml [5-3H]uridine) to a PEI-cellulose T L C plate, and eluting as above. The recovered U T P was measured in a Gilford Spectrophotometer and the concentration calculated using the extinction coefficient of uridine. The radioactivity of the eluted nucleotide was the then counted as above, and the specific activity was calculated. The specific activity of the [I~C]UTP pool was determined by disrupting in a Braun homogenizer 15 m g of the same [14C]uridine labeled spores used in the above experiments in 1.0 ml of 0.3 N K O H . The extract was kept overnight at 37 ° C to hydrolyze cellular R N A and then neutralized with HC1. To this extract, 0.2 ml of acid-washed Norite (5 mg/ml) and 0.2 ml of concentrated HCI were added. The micture was vortexed a n d centrifuged at 5,000 r p m for 5 rain. The Norite pellet was separated a n d the supernatant extract again treated with Norite and centrifuged. The pellets were pooled, washed three times with distilled water, and nucleotides were eluted from the Norite by the addition of 1.0 ml of e t h a n o l : H z O : c o n c . N H 4 O H in a proportion of 100:100:8. After 30 min, the extract was centrifuged at 5,000 r p m for 5 min to remove the Norite. The nucleotides were concentrated under v a c u u m to a final volume of 0.3 ml. 40 gl of extract was applied to a PEI-cellulose T L C plate, which was developed by chromatography using 0.5 N Na-formate p H 3.4 as the solvent. This system separated U M P from all other nucleotides (Randerath and Randerath, 1967). The U M P spot was visualized under UV, eluted, O.D. measured, and the radioactivity counted as above. Since the prelabeled spores were fully labeled, the specific activity of the U M P spot was assumed to be equivalent to the specific activity of the [14C]UTP pool. It would have been preferable to prelabel spores with radioactive R N A precursor and then to germinate these spores in the presence of the same specific activity of the precursor, thus insuring the same specific activity of the triphosphate pool during germination. This would have obviated the laborious, indirect technique employed in the U T P pool determinations. Unfortunately, spores were prepared in complex medium, while germination was performed in a m u c h more defined medium to increase uridine uptake. This made it impossible to maintain the same specific activity of the U T P pool throughout sporulation and germination. Data from double labeling experiments in which the same samples
A. Sloma and I. Smith: R N A Synthesis During Spore Germination in B. subtilis of [~4C]uracil-labeled spores were pulsed with [~H]uridine during germination were not usable because the almost 100-fold range in uptake into the U T P pool during germination completely masked the low radioactivity of the [14C]UTP pool, especially at the 45 and 60 rain stages, even when appropriate spillover corrections were attempted.
115
IOC
d
RNA Synthesis During Spore Germination in Bacillus subtilis* A. Sloma and I. Smith Department of Microbiology, The Public Health Research Institute of The City of New York, Inc., New York, New York 10016, USA
Summary. We have analyzed the R N A synthesized during spore germination in Bacillus subtilis. Early in germination there is little incorporation of [3H]uridine into R N A . A large increase in incorporation into R N A was found at 45 60 rain into germination which was in part due to increases in the specific activity of the U T P pool. When corrected for specific activity changes, the instantaneous rate of R N A synthesis showed a seven to tenfold increase between 30 and 45 rain of germination. Polyacrylamide gel electrophoresis studies showed that the R N A synthesized during germination appeared very similar to the R N A made during vegetative growth. D N A - R N A hybridization studies indicated that m R N A and r R N A were synthesized throughout germination. Their relative proportions remained constant and were very similar to the composition of R N A synthesized during vegetative growth.
Introduction
When Bacillus subtilis spores are incubated in a suitable medium, they undergo a series of changes that lead to the outgrowth of vegetative cells. Very little is known about how these changes are regulated. A r m s t r o n g and Sueoka (1968) reported that R N A synthesis is biphasic during germination. In the early phase (2-10 rain) they found that only r R N A is made, while both r R N A and m R N A are synthesized in the second phase which begins at 15 rain. Protein synthesis started at the beginning of the second phase in their experiments. Similar results in B. subtilis were * In partial fulfillment of the requirements for the doctoral degree by A.S. in the Department of Microbiology at the New York University School of Medicine For offprints contact." I. Smith
described by Woese and Bleyman (1969). On the other hand, Balassa and Contesse (1965) have also reported that both r R N A and m R N A are synthesized from the beginning of germination in B. subtilis. In B. cereus, an immediate synthesis of all classes of R N A has been reported by Spiegelman et al. (1969) and Torriani et al. (1969). If there is a biphasic pattern of R N A synthesis during germination, this would be evidence for a unique type of regulation in which only r R N A was made at a specific stage of growth. If, however, the R N A synthesized throughout the germination process is similar or identical to the R N A made during vegetative growth, it would indicate that there is no unusual transcriptional regulation during outgrowth. This study was undertaken to compare the R N A made during germination with N a t made during vegetative growth in B. subtilis, to 'resolve the published contradictions appearing in the literature regarding the nature of R N A synthesis during B. subtilis germination and outgrowth. We have found that there was no transition from a stage where only r R N A is made to one where both r R N A and m R N A are transcribed. Proportions o f r R N A and m R N A remained relatively constant throughout the germination process, and these proportions were the same as those of vegetative growth.
Materials and Methods Strains. B. subtilis strain BD68 (pyrA, argC4, leu-2), obtained from
Dr. D. Dubnau, was used in all experiments. Media. Sporulation media (SM) used contained per liter, nutrient broth (Difco), 8 gm; KC1, 1 gin; MgSO4-7 H20, 0.25 gin; 1 nM FeSO4; 1 mM Ca(NO3)2; 0.1 nM MnC12 (Schaeffer et al., 1965). Germination media (GM) contained per liter, NaCI, 3 gm ; KCI, 0.5 gin; (NH~)SO4, 2.0 gin; Na citrate-2 H20, 1.0 gin: MgSO4.7H20, 0.2gm; L-alanine, 0.5gin; casein hydrolysate, 0.2 gin; yeast extract, 0.2 gm; glucose, 5 gin; 25 mM Tris, pH 7.5;
0026-8925/79/0175/0113/$01.60
114
A. Sloma and I. Smith: R N A Synthesis During Spore Germination in B, subtilis
NaHzPO4, 5 m M ; uridine, 100 gg/ml; arginine 100 gg/ml; leucine 100 gg/ml. W h e n labeling with [3H]uridine (Moravek), 20 gg/ml uridine was used; when labeling with [32p]o4, 0.5 m M NaH2PO4 was used. Other media (VY, 1 x SS, etc.) have been described (Morell et al., 1967).
Spore Preparation. BD68 cultures were grown in SM at 35°C for 3 days with vigorous shaking. The cultures were then centrifuged, washed with cold distilled H 2 0 three times a n d resuspended into 100 m M Na E D T A , 50 m M NaC1, p i t 6.9 (LM), using 20 ml/per liter of culture. Lysozyme (500 l~g/ml) was added to the resuspended cell pellet at 37 ° C for 1 h. The crude spore preparation was washed with distilled H 2 0 three times and resuspended in i x SS (20 ml per liter). Pancreatic D N a s e (5 gg/ml) was added and the mixture was incubated for 30 min at 37 ° C. The preparation was then washed three times in distilled H 2 0 and lyophilized. The resulting spores were completely free of vegetative cells and were 100% refractile as viewed by phase contrast microscopy.
Spore Germination. Lyophilized spores were resuspended in distilled H 2 0 at a concentration of 5 m g / m l and heat-shocked for 30 rain at 70 ° C. Spores were germinated in G M at a concentration of 0.5 m g dry wt. spores/ml GM. This corresponds to 1 to 2 x l0 s colony forming units/ml. The culture was vigorously shaken at 37°C.
Labeling of RNA. Pulse-labeled R N A was obtained by incubating 5 ml germinating spores or 5 ml vegetative cells grown to mid-log phase, both at a cell concentration of 1 x 10S/ml, with 50 gCi/ml [5-3H]uridine (Moravek) or 200 gCi/ml [32p]NazPO4 (Amersham). Uptake was stopped by pouring the samples over crushed ice containing 10 m M NAN3. Samples were centrifuged at 4 ° C a n d washed in 100 m M NaC1, 10 m M Na acetate, p H 5 ( R N A buffer). To fully label spore R N A , 0.1 gCi/ml [2-1~C]uracil (New England Nuclear) was added to a freshly inoculated culture of BD68 in SM. After 3 days of growth at 35 ° C, spores were prepared as above. To prepare R N A , radioactive cells were resuspended in 5 ml of cold R N A buffer, after washing as described in the preceding section. To each radioactive sample, 1 ml of nonradioactive carrier cells was added, if the radioactive samples were to be analyzed by gel electrophoresis (the carrier cells were prepared as follows: BD68 cells grown in VY [50 ml] to a cell concentration of 2 × 10S/ml were centrifuged and washed in 10 m l / R N A buffer. The cells were resuspended in 10 ml cold R N A buffer). If the samples were to be used to prepare R N A for R N A : D N A hybridization, 5 r a g of yeast R N A in R N A buffer were added to each radioactive sample instead of carrier cells. To each 5 ml sample, 4 ml of 0.17 m m glass beads (VWR Scientific) were added, and each sample was broken for 2 min in a Braun homogenizer under constant passage of CO2 to keep the samples cold. After breakage, the sample was quickly put into 1% SDS and centrifuged 5000 r p m for 5 min to remove cell debris. To the supernatant an equal volume of hot redistilled phenol saturated with R N A buffer was added. Samples were vortexed two times and centrifuged at 10,000 r p m for 15 rain. The aqueous phase containing the R N A was removed, and to it was added 2 volumes of cold ethanol. The R N A was precipitated overnight at - 2 0 ° C. The R N A pellet was resuspended in 5 ml R N A buffer containing 0.5% SDS, and centrifuged 15,000 rpm for 30 rain. If R N A was used for hybridization, the R N A pellet was resuspended in 5 ml 10 m M Tris, pH 7.5; 10 m M MgC12; and R N a s e free pancratic D N a s e (Worthington) (5 gg/ml) was added at 0 ° C for 30 min. The R N A was reextracted with R N A buffer-saturated phenol, re-precipitated with ethanol, and resuspended in 5 ml R N A buffer containing 0.5% SDS. The R N A was then precipitated a second time with 2 volumes of cold ethanol. After 2 h at - 2 0 ° C the precipitated R N A was centrifuged 15,000 r p m for 30 rain and resuspended
in 0.5 ml R N A buffer containing 0.5% SDS. Recovery of R N A was always greater than 70%.
UTP Pool Measurements. Pulse-labeled U T P was obtained by incubating 5 ml of germinating spores with 100 gCi/ml [5-3H]uridine for 2 rain. The samples were centrifuged for 2 rain at 5,000 r p m at 0 ° C. The cell pellets were resuspended in 0.2 ml 2 M formic acid, pH 3.4 at 0 ° C and kept on ice for 15 rain. The samples were then centrifuged in the cold in an Eppendorf microcentrifuge. The supernatant solution containing the extracted nucleotides was quickly frozen, and they were thawed immediately before applying to PEI-cullulose F thin layer plates (E. Merck). The radioactive nucleotides, with 50 nmoles of cold U T P as carrier, were resolved on the PEI plates with 0.85 M KH2PO4, p H 3.4 (Cashel et al., 1969). After the chromatography plates were developed and dried, the U T P spots were visualized under shortwave UV light (254 nM). The spots were then cut from the plate and resuspended in 0.7 M MgC12, 0.02 M Tris, p H 7.4 (Randerath and Randerath, 1967) After 30 rain at r o o m temperature the sample was counted after suspension in Triton-toluene-PPO. To measure the preexisting [~4C]UTP pool, an equal a m o u n t of [14C]uracil-labeled spores (see above) was incubated in G M (0.5 mg spores/m1) and shaken vigorously in a separate flask. At each time point the pulse-labeled spores and prelabeled spores were removed at the same time and prepared as above. The absolute a m o u n t of U T P in each radioactive spot was too low to detect. Therefore, the [3H]UTP pool specific activity was measured by applying 10 gl of the 3H labeling media (20 ~tg/ml cold uridine, 100 gCi/ml [5-3H]uridine) to a PEI-cellulose T L C plate, and eluting as above. The recovered U T P was measured in a Gilford Spectrophotometer and the concentration calculated using the extinction coefficient of uridine. The radioactivity of the eluted nucleotide was the then counted as above, and the specific activity was calculated. The specific activity of the [I~C]UTP pool was determined by disrupting in a Braun homogenizer 15 m g of the same [14C]uridine labeled spores used in the above experiments in 1.0 ml of 0.3 N K O H . The extract was kept overnight at 37 ° C to hydrolyze cellular R N A and then neutralized with HC1. To this extract, 0.2 ml of acid-washed Norite (5 mg/ml) and 0.2 ml of concentrated HCI were added. The micture was vortexed a n d centrifuged at 5,000 r p m for 5 rain. The Norite pellet was separated a n d the supernatant extract again treated with Norite and centrifuged. The pellets were pooled, washed three times with distilled water, and nucleotides were eluted from the Norite by the addition of 1.0 ml of e t h a n o l : H z O : c o n c . N H 4 O H in a proportion of 100:100:8. After 30 min, the extract was centrifuged at 5,000 r p m for 5 min to remove the Norite. The nucleotides were concentrated under v a c u u m to a final volume of 0.3 ml. 40 gl of extract was applied to a PEI-cellulose T L C plate, which was developed by chromatography using 0.5 N Na-formate p H 3.4 as the solvent. This system separated U M P from all other nucleotides (Randerath and Randerath, 1967). The U M P spot was visualized under UV, eluted, O.D. measured, and the radioactivity counted as above. Since the prelabeled spores were fully labeled, the specific activity of the U M P spot was assumed to be equivalent to the specific activity of the [14C]UTP pool. It would have been preferable to prelabel spores with radioactive R N A precursor and then to germinate these spores in the presence of the same specific activity of the precursor, thus insuring the same specific activity of the triphosphate pool during germination. This would have obviated the laborious, indirect technique employed in the U T P pool determinations. Unfortunately, spores were prepared in complex medium, while germination was performed in a m u c h more defined medium to increase uridine uptake. This made it impossible to maintain the same specific activity of the U T P pool throughout sporulation and germination. Data from double labeling experiments in which the same samples
A. Sloma and I. Smith: R N A Synthesis During Spore Germination in B. subtilis of [~4C]uracil-labeled spores were pulsed with [~H]uridine during germination were not usable because the almost 100-fold range in uptake into the U T P pool during germination completely masked the low radioactivity of the [14C]UTP pool, especially at the 45 and 60 rain stages, even when appropriate spillover corrections were attempted.
115
IOC
d
