Vol. 135, lNo. 2
JoURNAL oF BACTEOLOGY, Aug. 1978, p. 408-414 0021-9193/78/0135-0408$02.00/0 CopyrightO 1978 American Society for Microbiology
Printed in U.,.S.A.
Pleiotropic Effect of a Rifampin-Resistant Mutation in Bacillus subtilis JUN-ICHI RYU Department of Biology, Tokyo Metropolitan University, Fukazawa, Setagaya-ku, Tokyo 158, Japan Received for publication 28 March 1978
Rifampin-reigstant (Rift) mutants were isolated spontaneously from Bacillus subtilis strain 168. A fraction of the mutants did not grow on a minimal medium. A high concentration of one of the L-amino acids (glutamic acid, glutamine, arginine, proline, aspartic acid, or asparagine) was required to restore their growth on the medium. Further analysis of one of the mutants (strain RF 161) suggested that the mutant is unable to use ammonia as a nitrogen source and requires amino acids instead. Activity of glutamate synthase was not detected in the crude extract of the mutant. The Rifr mutation was closely located to cysA and the drug resistance was cotransformed with the property of amino acid requirement at 100% frequency. All revertants to prototrophy tested showed the rifampinsensitive (Rif) property. The activity of the DNA-dependent RNA polymerase of the mutant was resistant to rifampin. It is concluded that some alteration of RNA polymerase may cause absence of the activity of an enzyme involved in the nitrogen metabolism.
Rifampin and its derivatives are known as specific antibiotics working on the , subunit of RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase; EC 2.7.7.6) (9, 15, 20, 26). Mutants resistant to these antibiotics have been useful as tools for genetic and biochemical studies of RNA polymerase (10, 26). Pleiotropic effects of Rift mutations have been observed in some bacteria. In Bacillus subtilis, some Rift mutations cause defects in spore formation (14, 23-25) and some lead to alterations in spore morphology (7, 13). In Escherichia coli, some Rifr mutants do not permit the growth of bacteriophages (22). There are Rifr mutations which reduce the frequency of lysogenization of phage P22 in Salmonella typhimurium (12) or lambda in E. coli (8). It has been observed both in E. coli and S. typhimurium that dnaA mutations are suppressed by Rif' mutations (2). It is thus assumed that these mutations in the ,B subunit of RNA polymerase are closely connected with the transcription of specific genes. In many cases so far reported, however, a particular enzyme whose production is affected specifically by the modification of RNA polymerase has not been identified. To study further the pleiotropic effects of Rif' mutations in B. subtilis, a search for a new class of mutants was carried out. Among the Rifr mutants examined, I found some mutants that required specific amino acids for growth in min-
results suggest that a modification of RNA polymerase causes a specific defect in the expression of the gene for glutamate synthase [glutamine (amide):2-oxoglutarate amino transferase oxidoreductase (NADP); EC 2.6.1.53], a key enzyme in nitrogen metabolism (3). MATERIALS AND METHODS Bacterial and phage strains. All bacterial strains used were derivatives of B. subtilis 168 (Marburg). B. subtilis 168 (thyA thyB hisB31), B. subtilis 168 (purA cysA14 metB4), and phages (4oe, SPOl, SP8, SPP1, 105C) were obtained from H. Saito. B. subtilis QB922 (gltA292 trpC2), a glutamate synthase-deficient mu-
tant, was obtained from H. Yoshikawa. Media. Nutrient broth agar (NB plate) contained the following in 1 liter of distilled water: meat extract, 10 g; Polypepton (Wako Chemical), 10 g; NaCl, 2 g; agar, 15 g (pH 7.0). Thymine (20 jg/ml) was always added to this medium. Modified Anagnostopoulos and Spizizen medium (1) was used as the minimal medium (MM). It contained the following in 1 liter of distilled water. KH2PO4, 14 g; K2HPO4, 6 g; (NH4)2SO4, 2 g; MgSO4* 7H20, 0.2 g, FeCl3 6H20, 2 mg, MnCl2 4H20, 2 mg; Na-citrate, 1 g; glucose, 2 g (pH 7.0). Required bases (20 ug/ml) and amino acids (40 pg/mi) were always added to this medium. In many experiments, MMCA was also used; it contained 0.1% Casamino Acids (Difco, vitamin free) in the MM. Agar (1.5%) was used for plates. Tryptose blood agar base (TBAB, Difco) was used for transformation. Reagents. Rifampin was a gift from Ciba Ltd. Calf thymus DNA was purchased from Worthington Bioimal medium. The characterization of such Rifr chemical Corp. Nucleotide triphosphates (ATP, GTP, mutants is described in the present paper. The UTP and CTP) were purchased from Sigma Chemical 408
VOL. 135, 1978
RIFAMPIN-RESISTANT MUTANTS OF B. SUBTILIS
Co. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from P-L Biochemicals Inc. [5-3H]UTP (1.0 Ci/mmol) was purchased from Amersham Products Inc. Isolation of the mutants. Rif' mutants were isolated spontaneously from B. 8ubti.s strain 168 (thyA thyB hisB31) (168TH) by streaking the cells on NB plates containing rifampin (20 pg/ml) and incubating them for 2 to 3 days at 370C. The average frequency of Rifr mutants was about 10-8. Growth experiments. Cells were usually incubated overnight on the MMCA plates. They were streaked on the plates or suspended in the liquid medium. Liquid cultures were incubated with shaking. Cell growth was monitored by measuring the turbidity of the cultures at 660 nm by a spectrophotometer (Hitachi EFO-3). All bacterial cultivations were done at 370C. Transformation. DNA was prepared by the pH 9 phenol method of Saito and Miura (21); cells were grown in MMCA medium. At late-log phase, cells in a 5-ml culture were collected by centrifgation. They were washed once with saline-ethylenediaminetetraacetic acid (EDTA) (0.1 M NaCl-0.01 M EDTA, pH 8.0) and suspended in 0.5 ml of lysozyme solution (400 ,ug of lysozyme in saline-EDTA per ml). After shaking gently for 15 min at 370C, 0.2 ml of sodium dodecyl sulfate (SDS) solution [0.1 M tris(hydroxymethyl)aminomethane (Tris)-1% SDS-0.1 M NaCl, pH 9.0] was added. Then an equal volume (0.7 ml) of a phenol solution (phenol-SDS solution, 2:1) was immediately added to the mixture. After the mixture was shaken gently for 10 min at room temperature, the aqueous layer was collected by centrifugation. The phenol treatment was repeated twice. The aqueous layer was dialyzed overnight against SSC (0.15 M NaCl-0.015 M sodium citrate) and used as the DNA source.
Competent cells were prepared essentially as described by Anagnostopoulos and Spizizen (1): B. subtilis 168 (purA cysA14 metB4) cells were grown overnight on TBAB and then transferred to C-1 medium (MM containing 0.2% Casamino Acids, 20 pg of required base and 50 pg of required amino acids per ml). Cell density was adjusted to 0.02 (optical density [OD] at 660 nm). After 5 h of incubation at 37°C, cells in a 4-ml culture were collected by centrifugation and transferred into twice the initial volume of C-2 medium (MM containing 0.1% Casamino Acids-2 mg of MgSO4 7H20 per ml). After 5 min, DNA solution (0.1 ml) was added to the 4-ml culture and incubated further for 90 min. Cells were then spread on appropriate selection media and incubated for 2 to 3 days at 370C. Preparation and assay of glutamate synthase. The method of Meers et al. (17) was used for assay of glutamate synthase: extracts were prepared by disrupting the cells (about 0.5 g [wet weight]) through sonic oscillation (1.5 min, Toyo 2N-100) in 2 ml of the Tris-buffer (50 mM Tris-hydrochloride pH 7.6-10 mM 2-mercaptoethanol). Samples were centrifuged at 10,000 rpm for 30 min, and the supernatant fluid was used as the enzyme source. These procedures were done at about 40C. The assay mixture (3 ml) contained the following: 5 mM 2-oxoglutarate, 5 mM glutamine,
409
0.25 mM NADPH, and extracts (10 to 300 P1). The enzyme activity was measured spectrophotometrically by recording the rate of oxidation of NADPH (indicated by the change in absorbance at 340 nm) at 370C by using a Hitachi-124 spectrophotometer. Preparation and assay of RNA polymerase. The modified method of Burgess (4) was used for assay of RNA polymerase: late-log celLs grown in MMCA medium were harvested by centrifugation and washed twice with buffer G (50 mM Tris-hydrochloride, pH 7.5-10 mM MgC12-200 mM KC1-0.1 mM EDTA). The cells (6 g [wet weight]) were suspended in 20 ml of the buffer (buffer G plus 1 mM dithiothreitol, 5% [vol/vol] glycerol) and disrupted by sonic oscillation (5 min). Unbroken cells and cell debris were removed by centrifugation at 15,000 rpm for 30 min. The supernatant fraction was centrifuged at 40,000 rpm for 2 h with a Hitachi 65P using an RP40 rotor. Crystalline ammonium sulfate was added to the supernatant fraction. The fraction that precipitated between 33 and 65% saturation was collected. The precipitate was dissolved in 0.5 to 1.0 ml of buffer A (Trishydrochloride, pH 7.9-10 mM MgCl2-100 mM KC1-0.1 mM EDTA-0.1 mM dithiothreitol-20% [vol/vol] glycerol) and was directly applied to the Sepharose-6B column equilibrated with Buffer A. The peak fraction of enzyme activity eluted from the column was used as the source of RNA polymerase. The assay mixture (0.25 ml) contained the following: 40 mM Tris-hydrochloride, pH 7.9, 10 mM MgCl2, 0.1 mM EDTA, 150 mM KCl, 0.15 mM each ATP, GTP, CTP, and UTP, 1 ,uCi of [3H]UTP (1.0 Ci/mmol), 20 pg of calf thymus DNA, and 20 p1 of the enzyme fraction. The reaction mixture was incubated for 20 min at 350C, and the reaction was stopped by adding 0.25 ml of cold 10% trichloroacetic acid. The mixture was allowed to stand for 1 h in an ice bath. The acid-insoluble fraction was collected on a Whatman glass-fiber filter (GF/C 2.4 cm), washed successively with 20 ml of cold 5% trichloroacetic acid and 10 ml of cold 5% acetic acid, and dried. The radioactivity was counted in a vial containing 10 ml of toluene-2,5-diphenyloxazole (0.4%) by a Beckman liquid scintillation counter (IS-IHI). Protein measurement. Protein was measured by the method of Lowry et al. (16).
RESULTS Amino acid requiring mutants. A total of 203 Rifr mutants were isolated independently. They grew well on NB plates. When they were streaked on MM plates, however, some of them (26 strains) did not grow even after a prolonged incubation of more than 7 days. All the mutants of this type grew when Casamiino Acids (final concentration, 0.1%) were added to MM plates. Bases or several vitamins were not effective. Each of the 20 L-amino acids (final concentration, 1 mg/ml) was then added to MM plates. Growth of the mutants was observed after overnight incubation only on plates supplemented with glutamic acid, glutamine, arginine, proline, aspartic acid, or asparagine. Other amino acids were not effective even after 1 week of incuba-
410
J. BACTERIOL.
RYU
tion. Whemn serine was added, however, growth of the panent was also repressed. All the mutants tested (13 strains) showed the same requirement for amino acids. Further analyses were therefore done by 1using a typical strain (RF 161). This mutant siiowed no further abnonnal phenotype under u8 ual growth conditions; the doubling time of tIhe mutant in MMCA medium was 35 min and vwas similar to that of the parent. The mutant clells sporulated nornally in Schaeffer sporulatio)n medium. Some of the phages (se, SPOl, SI?8, SPP1, and 105C) formed plaques normally on the mutant cells. The grc)wth of the mutant was poor when any one of the six effective amino acids was added at concentraitions lower than 100 ug/ml. Figure 1 shows the) growth pattern of the mutant in MM medium isupplemented with glutamic acid at various ccDncentrations. When the nutrient supplement Nwas changed from Casamino Acids to glutamic acid, the growth of the mutant was restored a after a lag period (1 h). The growth yield of Ithe mutant was proportional to the concentraition of glutamic acid. The mutant required a bhigh concentration of amino acid (more
It 0
0.1
0 0
0
OD1 0
2
4
6
TIME IN HOURS FIG. 1. Growth of the RF 161 mutant ifl MMm dium supj lemented with an increased amount of glutamic I acid. Cells were grown exponentially in MMCA nuedium. At midlog phase, they were wahed three timeis on a membrane filter (Millipore A, 0.45 At zer eotime they weresuspended in MM medium jtm). supplenei tted with various concentrations of glutamic acu d. Symnbols: Micrograms of glutamic acid per ml, 0 ( U); 100 (0); 250 (0); 500 (A); 1,000 (A).
than 500 ug/ml) to attain the growth yield of the parent in MM medium (optical density, 0.2). Thus, it was shown by experiments of the same kind that a similar high concentration was required for the normal growth of the mutant on any one of the six amino acids. It is noteworthy that each of these amino acids belongs to either the aspartate or glutamate family. Utilization of amino acid as nitrogen (N) sources. The phenotype of the mutant cells requiring a high concentration of amino acids suggested the possibility that these mutant cells might be defective in utilizing either glucose as a C source or ammonia sulfate as an N source in contrast to the parent, and consequently they might use amino acids instead. Thus, the growth of the mutant was tested in medium in which glucose or ammonium sulfate was replaced by one of the amino acids required by the mutant. As shown in Fig. 2, no growth of the mutant cells was observed when glucose was replaced by one of the six amino acids. By contrast, when ammonium sulfate was replaced by five amino acids (glutamic acid, glutamine, arginine, proline, and aspartic acid), growth of the mutant was restored after a lag period (about 1 h). These results show that glucose is indispensable to the mutant cells to resume their growth after a nutritional shift down; however, ammonium sulfate is not. When asparagine was added, growth of the cells was not observed during the period of observation. Figure 2 also shows that the presence of ammonium sulfate does not affect the growth pattern of the mutant cells. Slight promotion of growth was observed only when ammonium sulfate was added to the medium containing glutamic acid. These results suggest that ammonium sulfate is not utilized efficiently by the mutant cells, but instead, that amino acids required by the mutant are utilized as N sources. Further, the mutant did not utilize either of the two N sources, urea or potassium nitrate, which may be converted to ammonia and incorporated into amino acids; however, the parent utilized these N sources (Fig. 3). Absence of the activity of an enzyme. The above results suggest that the mutant has a defect in an enzyme that is necessary for the process of the assimilation of ammonia. It has been observed in B. subtilis that ammonia is incorporated into amino acids mainly through the glutamine synthetase-glutamate synthase enzyme activities of glutamate route (3). synthase of the parent (168TH) and the mutant (RF 161) are shown in Table 1. In this exerment, one of the revertants from RF 161 was also used. The revertant (161 R2) was obtained spontaneously on the MM plate, proved to be
The
RIFAMPIN-RESISTANT MUTANTS OF B. SUBTILIS
VOL. 135, 1978
2
(0 (0
411
/
0 OE
F
0.01 2
4
6
0 2
4
6
TIME IN HOURS FIG. 2. Growth of the RF 161 mutant after shifting down of the medium. Cells grown exponentially in MMCA medium were washed on a membrane filter. At zero time, they were suspended in MM medium supplemented with 500 pg each of the following amino acids: (A) glutamic acid; (B) glutamine; (C) arginine; (D) proline; (E) aspartic acid; (F) asparagine deprived ofglucose (U), ammonium sulfate (0), or none (0).
wild type with regard to the utilization of N sources, and showed Rif8 phenotype simultaneously. Sonic extracts were prepared respectively from cells at midlog phase in MMCA medium and those at 2.5 h after shifting down to MM medium. In the latter conditions, the parent and the revertant resumed growth, but the mutant did not. Neither crude extracts of the mutant obtained from cultures in MM medium (nongrowing conditions) nor those in MMCA medium (growing conditions) showed appreciable amounts of glutamate synthase activity, whereas those of the parent or the revertant showed a level of the enzyme activity similar to that reported previously (17). Therefore, it is highly probable that the mutant's requirement for amino acids is due to the absence of glutamate synthase activity. To confirm this point of view,
the character of a mutant defective in glutamate synthase (QB922 strain) was compared with the Rif' mutant. The growth of the glutamate synthase-deficient (gltA) mutant was reported to be restored by the addition of glutamate or aspartate (27). The test of amino acid requirements of this mutant showed, however, that one of the four amino acids (glutamine, argiline, proline, or asparagine) was also effective in the growth of the mutant. Moreover, it was pointed out by experiments similar to those of Fig. 1 that the mutant celLs required more than 500 jAg of one of those amino acids per ml, showing an identical phenotype to that of Rifr mutants examined in this paper. Revertant analysis. It may be assumed that a single mutation resulted in both Rifr and amino acid-requiring phenotype, since the fre-
412
J. BACTERIOL.
RYU
Mapping of the Rif' mutants. DNA extracted from RF 161 was transformed to the strain carrying the cysA14 mutation. Cys+ transformants were selected and their sensitivity to rifampin was tested on NB plates containing the drug (20 ,ug/ml). The Rifr phenotype was cotransformed with cysA at a frequency of 48% (74/154). Rifr mutations of two other strains (RF 183 and RF 271) were also transformed at a frequency of 36% (58/161) and 55% (87/159), respectively. It is thus suggested that these Rif' mutations are located at the rfn locus near cysA (10, 27). It was further shown that all the Rifr cotransformants acquired the property of amino
A t000/
0.11:
/ O/O 0
0
I~~~-~.l
002O .
o
I
0
.
acid requirement. Assay of RNA polymerase. The activity of RNA polymerase was measured in the presence
. .1 I
a
I
1I
B
aD1 a
I
o
2
I--
4
1
a
6
8
-i
TIME IN HOURS FIG. 3. Utilization of different N sources by 168TH (A) and RF 161 (B). Cells grown exponentially in MMCA medium were washed on a membrane filter. At zero time they were suspended in MM medium containing different N sources (0.4%). Symbols: (0) Urea; (0) potassium nitrate.
of various concentrations of rifampin after the partial purification of the enzyme. The activity of the parental enzyme as well as that of the revertant enzyme was completely inhibited in the presence of 0.1 ,ug of rifampin in the reaction mixture (0.25 ml), whereas the mutant enzyme retained 100% activity at the same concentration of the drug (Fig. 4). Partial inhibition of the activity of the mutant enzyme was observed when the drug at concentrations of 1 ,tg or more was added to the reaction mixture. Thus, com-
0~~~~~~
TABLE 1. Enzyme activity of glutamate synthase Glutamate synthase
Strain
Medium
168TH
MM
RF 161 161 R2
MMCA MM
MMCA MM
MMCA
activity' 148 24
JoURNAL oF BACTEOLOGY, Aug. 1978, p. 408-414 0021-9193/78/0135-0408$02.00/0 CopyrightO 1978 American Society for Microbiology
Printed in U.,.S.A.
Pleiotropic Effect of a Rifampin-Resistant Mutation in Bacillus subtilis JUN-ICHI RYU Department of Biology, Tokyo Metropolitan University, Fukazawa, Setagaya-ku, Tokyo 158, Japan Received for publication 28 March 1978
Rifampin-reigstant (Rift) mutants were isolated spontaneously from Bacillus subtilis strain 168. A fraction of the mutants did not grow on a minimal medium. A high concentration of one of the L-amino acids (glutamic acid, glutamine, arginine, proline, aspartic acid, or asparagine) was required to restore their growth on the medium. Further analysis of one of the mutants (strain RF 161) suggested that the mutant is unable to use ammonia as a nitrogen source and requires amino acids instead. Activity of glutamate synthase was not detected in the crude extract of the mutant. The Rifr mutation was closely located to cysA and the drug resistance was cotransformed with the property of amino acid requirement at 100% frequency. All revertants to prototrophy tested showed the rifampinsensitive (Rif) property. The activity of the DNA-dependent RNA polymerase of the mutant was resistant to rifampin. It is concluded that some alteration of RNA polymerase may cause absence of the activity of an enzyme involved in the nitrogen metabolism.
Rifampin and its derivatives are known as specific antibiotics working on the , subunit of RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase; EC 2.7.7.6) (9, 15, 20, 26). Mutants resistant to these antibiotics have been useful as tools for genetic and biochemical studies of RNA polymerase (10, 26). Pleiotropic effects of Rift mutations have been observed in some bacteria. In Bacillus subtilis, some Rift mutations cause defects in spore formation (14, 23-25) and some lead to alterations in spore morphology (7, 13). In Escherichia coli, some Rifr mutants do not permit the growth of bacteriophages (22). There are Rifr mutations which reduce the frequency of lysogenization of phage P22 in Salmonella typhimurium (12) or lambda in E. coli (8). It has been observed both in E. coli and S. typhimurium that dnaA mutations are suppressed by Rif' mutations (2). It is thus assumed that these mutations in the ,B subunit of RNA polymerase are closely connected with the transcription of specific genes. In many cases so far reported, however, a particular enzyme whose production is affected specifically by the modification of RNA polymerase has not been identified. To study further the pleiotropic effects of Rif' mutations in B. subtilis, a search for a new class of mutants was carried out. Among the Rifr mutants examined, I found some mutants that required specific amino acids for growth in min-
results suggest that a modification of RNA polymerase causes a specific defect in the expression of the gene for glutamate synthase [glutamine (amide):2-oxoglutarate amino transferase oxidoreductase (NADP); EC 2.6.1.53], a key enzyme in nitrogen metabolism (3). MATERIALS AND METHODS Bacterial and phage strains. All bacterial strains used were derivatives of B. subtilis 168 (Marburg). B. subtilis 168 (thyA thyB hisB31), B. subtilis 168 (purA cysA14 metB4), and phages (4oe, SPOl, SP8, SPP1, 105C) were obtained from H. Saito. B. subtilis QB922 (gltA292 trpC2), a glutamate synthase-deficient mu-
tant, was obtained from H. Yoshikawa. Media. Nutrient broth agar (NB plate) contained the following in 1 liter of distilled water: meat extract, 10 g; Polypepton (Wako Chemical), 10 g; NaCl, 2 g; agar, 15 g (pH 7.0). Thymine (20 jg/ml) was always added to this medium. Modified Anagnostopoulos and Spizizen medium (1) was used as the minimal medium (MM). It contained the following in 1 liter of distilled water. KH2PO4, 14 g; K2HPO4, 6 g; (NH4)2SO4, 2 g; MgSO4* 7H20, 0.2 g, FeCl3 6H20, 2 mg, MnCl2 4H20, 2 mg; Na-citrate, 1 g; glucose, 2 g (pH 7.0). Required bases (20 ug/ml) and amino acids (40 pg/mi) were always added to this medium. In many experiments, MMCA was also used; it contained 0.1% Casamino Acids (Difco, vitamin free) in the MM. Agar (1.5%) was used for plates. Tryptose blood agar base (TBAB, Difco) was used for transformation. Reagents. Rifampin was a gift from Ciba Ltd. Calf thymus DNA was purchased from Worthington Bioimal medium. The characterization of such Rifr chemical Corp. Nucleotide triphosphates (ATP, GTP, mutants is described in the present paper. The UTP and CTP) were purchased from Sigma Chemical 408
VOL. 135, 1978
RIFAMPIN-RESISTANT MUTANTS OF B. SUBTILIS
Co. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from P-L Biochemicals Inc. [5-3H]UTP (1.0 Ci/mmol) was purchased from Amersham Products Inc. Isolation of the mutants. Rif' mutants were isolated spontaneously from B. 8ubti.s strain 168 (thyA thyB hisB31) (168TH) by streaking the cells on NB plates containing rifampin (20 pg/ml) and incubating them for 2 to 3 days at 370C. The average frequency of Rifr mutants was about 10-8. Growth experiments. Cells were usually incubated overnight on the MMCA plates. They were streaked on the plates or suspended in the liquid medium. Liquid cultures were incubated with shaking. Cell growth was monitored by measuring the turbidity of the cultures at 660 nm by a spectrophotometer (Hitachi EFO-3). All bacterial cultivations were done at 370C. Transformation. DNA was prepared by the pH 9 phenol method of Saito and Miura (21); cells were grown in MMCA medium. At late-log phase, cells in a 5-ml culture were collected by centrifgation. They were washed once with saline-ethylenediaminetetraacetic acid (EDTA) (0.1 M NaCl-0.01 M EDTA, pH 8.0) and suspended in 0.5 ml of lysozyme solution (400 ,ug of lysozyme in saline-EDTA per ml). After shaking gently for 15 min at 370C, 0.2 ml of sodium dodecyl sulfate (SDS) solution [0.1 M tris(hydroxymethyl)aminomethane (Tris)-1% SDS-0.1 M NaCl, pH 9.0] was added. Then an equal volume (0.7 ml) of a phenol solution (phenol-SDS solution, 2:1) was immediately added to the mixture. After the mixture was shaken gently for 10 min at room temperature, the aqueous layer was collected by centrifugation. The phenol treatment was repeated twice. The aqueous layer was dialyzed overnight against SSC (0.15 M NaCl-0.015 M sodium citrate) and used as the DNA source.
Competent cells were prepared essentially as described by Anagnostopoulos and Spizizen (1): B. subtilis 168 (purA cysA14 metB4) cells were grown overnight on TBAB and then transferred to C-1 medium (MM containing 0.2% Casamino Acids, 20 pg of required base and 50 pg of required amino acids per ml). Cell density was adjusted to 0.02 (optical density [OD] at 660 nm). After 5 h of incubation at 37°C, cells in a 4-ml culture were collected by centrifugation and transferred into twice the initial volume of C-2 medium (MM containing 0.1% Casamino Acids-2 mg of MgSO4 7H20 per ml). After 5 min, DNA solution (0.1 ml) was added to the 4-ml culture and incubated further for 90 min. Cells were then spread on appropriate selection media and incubated for 2 to 3 days at 370C. Preparation and assay of glutamate synthase. The method of Meers et al. (17) was used for assay of glutamate synthase: extracts were prepared by disrupting the cells (about 0.5 g [wet weight]) through sonic oscillation (1.5 min, Toyo 2N-100) in 2 ml of the Tris-buffer (50 mM Tris-hydrochloride pH 7.6-10 mM 2-mercaptoethanol). Samples were centrifuged at 10,000 rpm for 30 min, and the supernatant fluid was used as the enzyme source. These procedures were done at about 40C. The assay mixture (3 ml) contained the following: 5 mM 2-oxoglutarate, 5 mM glutamine,
409
0.25 mM NADPH, and extracts (10 to 300 P1). The enzyme activity was measured spectrophotometrically by recording the rate of oxidation of NADPH (indicated by the change in absorbance at 340 nm) at 370C by using a Hitachi-124 spectrophotometer. Preparation and assay of RNA polymerase. The modified method of Burgess (4) was used for assay of RNA polymerase: late-log celLs grown in MMCA medium were harvested by centrifugation and washed twice with buffer G (50 mM Tris-hydrochloride, pH 7.5-10 mM MgC12-200 mM KC1-0.1 mM EDTA). The cells (6 g [wet weight]) were suspended in 20 ml of the buffer (buffer G plus 1 mM dithiothreitol, 5% [vol/vol] glycerol) and disrupted by sonic oscillation (5 min). Unbroken cells and cell debris were removed by centrifugation at 15,000 rpm for 30 min. The supernatant fraction was centrifuged at 40,000 rpm for 2 h with a Hitachi 65P using an RP40 rotor. Crystalline ammonium sulfate was added to the supernatant fraction. The fraction that precipitated between 33 and 65% saturation was collected. The precipitate was dissolved in 0.5 to 1.0 ml of buffer A (Trishydrochloride, pH 7.9-10 mM MgCl2-100 mM KC1-0.1 mM EDTA-0.1 mM dithiothreitol-20% [vol/vol] glycerol) and was directly applied to the Sepharose-6B column equilibrated with Buffer A. The peak fraction of enzyme activity eluted from the column was used as the source of RNA polymerase. The assay mixture (0.25 ml) contained the following: 40 mM Tris-hydrochloride, pH 7.9, 10 mM MgCl2, 0.1 mM EDTA, 150 mM KCl, 0.15 mM each ATP, GTP, CTP, and UTP, 1 ,uCi of [3H]UTP (1.0 Ci/mmol), 20 pg of calf thymus DNA, and 20 p1 of the enzyme fraction. The reaction mixture was incubated for 20 min at 350C, and the reaction was stopped by adding 0.25 ml of cold 10% trichloroacetic acid. The mixture was allowed to stand for 1 h in an ice bath. The acid-insoluble fraction was collected on a Whatman glass-fiber filter (GF/C 2.4 cm), washed successively with 20 ml of cold 5% trichloroacetic acid and 10 ml of cold 5% acetic acid, and dried. The radioactivity was counted in a vial containing 10 ml of toluene-2,5-diphenyloxazole (0.4%) by a Beckman liquid scintillation counter (IS-IHI). Protein measurement. Protein was measured by the method of Lowry et al. (16).
RESULTS Amino acid requiring mutants. A total of 203 Rifr mutants were isolated independently. They grew well on NB plates. When they were streaked on MM plates, however, some of them (26 strains) did not grow even after a prolonged incubation of more than 7 days. All the mutants of this type grew when Casamiino Acids (final concentration, 0.1%) were added to MM plates. Bases or several vitamins were not effective. Each of the 20 L-amino acids (final concentration, 1 mg/ml) was then added to MM plates. Growth of the mutants was observed after overnight incubation only on plates supplemented with glutamic acid, glutamine, arginine, proline, aspartic acid, or asparagine. Other amino acids were not effective even after 1 week of incuba-
410
J. BACTERIOL.
RYU
tion. Whemn serine was added, however, growth of the panent was also repressed. All the mutants tested (13 strains) showed the same requirement for amino acids. Further analyses were therefore done by 1using a typical strain (RF 161). This mutant siiowed no further abnonnal phenotype under u8 ual growth conditions; the doubling time of tIhe mutant in MMCA medium was 35 min and vwas similar to that of the parent. The mutant clells sporulated nornally in Schaeffer sporulatio)n medium. Some of the phages (se, SPOl, SI?8, SPP1, and 105C) formed plaques normally on the mutant cells. The grc)wth of the mutant was poor when any one of the six effective amino acids was added at concentraitions lower than 100 ug/ml. Figure 1 shows the) growth pattern of the mutant in MM medium isupplemented with glutamic acid at various ccDncentrations. When the nutrient supplement Nwas changed from Casamino Acids to glutamic acid, the growth of the mutant was restored a after a lag period (1 h). The growth yield of Ithe mutant was proportional to the concentraition of glutamic acid. The mutant required a bhigh concentration of amino acid (more
It 0
0.1
0 0
0
OD1 0
2
4
6
TIME IN HOURS FIG. 1. Growth of the RF 161 mutant ifl MMm dium supj lemented with an increased amount of glutamic I acid. Cells were grown exponentially in MMCA nuedium. At midlog phase, they were wahed three timeis on a membrane filter (Millipore A, 0.45 At zer eotime they weresuspended in MM medium jtm). supplenei tted with various concentrations of glutamic acu d. Symnbols: Micrograms of glutamic acid per ml, 0 ( U); 100 (0); 250 (0); 500 (A); 1,000 (A).
than 500 ug/ml) to attain the growth yield of the parent in MM medium (optical density, 0.2). Thus, it was shown by experiments of the same kind that a similar high concentration was required for the normal growth of the mutant on any one of the six amino acids. It is noteworthy that each of these amino acids belongs to either the aspartate or glutamate family. Utilization of amino acid as nitrogen (N) sources. The phenotype of the mutant cells requiring a high concentration of amino acids suggested the possibility that these mutant cells might be defective in utilizing either glucose as a C source or ammonia sulfate as an N source in contrast to the parent, and consequently they might use amino acids instead. Thus, the growth of the mutant was tested in medium in which glucose or ammonium sulfate was replaced by one of the amino acids required by the mutant. As shown in Fig. 2, no growth of the mutant cells was observed when glucose was replaced by one of the six amino acids. By contrast, when ammonium sulfate was replaced by five amino acids (glutamic acid, glutamine, arginine, proline, and aspartic acid), growth of the mutant was restored after a lag period (about 1 h). These results show that glucose is indispensable to the mutant cells to resume their growth after a nutritional shift down; however, ammonium sulfate is not. When asparagine was added, growth of the cells was not observed during the period of observation. Figure 2 also shows that the presence of ammonium sulfate does not affect the growth pattern of the mutant cells. Slight promotion of growth was observed only when ammonium sulfate was added to the medium containing glutamic acid. These results suggest that ammonium sulfate is not utilized efficiently by the mutant cells, but instead, that amino acids required by the mutant are utilized as N sources. Further, the mutant did not utilize either of the two N sources, urea or potassium nitrate, which may be converted to ammonia and incorporated into amino acids; however, the parent utilized these N sources (Fig. 3). Absence of the activity of an enzyme. The above results suggest that the mutant has a defect in an enzyme that is necessary for the process of the assimilation of ammonia. It has been observed in B. subtilis that ammonia is incorporated into amino acids mainly through the glutamine synthetase-glutamate synthase enzyme activities of glutamate route (3). synthase of the parent (168TH) and the mutant (RF 161) are shown in Table 1. In this exerment, one of the revertants from RF 161 was also used. The revertant (161 R2) was obtained spontaneously on the MM plate, proved to be
The
RIFAMPIN-RESISTANT MUTANTS OF B. SUBTILIS
VOL. 135, 1978
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TIME IN HOURS FIG. 2. Growth of the RF 161 mutant after shifting down of the medium. Cells grown exponentially in MMCA medium were washed on a membrane filter. At zero time, they were suspended in MM medium supplemented with 500 pg each of the following amino acids: (A) glutamic acid; (B) glutamine; (C) arginine; (D) proline; (E) aspartic acid; (F) asparagine deprived ofglucose (U), ammonium sulfate (0), or none (0).
wild type with regard to the utilization of N sources, and showed Rif8 phenotype simultaneously. Sonic extracts were prepared respectively from cells at midlog phase in MMCA medium and those at 2.5 h after shifting down to MM medium. In the latter conditions, the parent and the revertant resumed growth, but the mutant did not. Neither crude extracts of the mutant obtained from cultures in MM medium (nongrowing conditions) nor those in MMCA medium (growing conditions) showed appreciable amounts of glutamate synthase activity, whereas those of the parent or the revertant showed a level of the enzyme activity similar to that reported previously (17). Therefore, it is highly probable that the mutant's requirement for amino acids is due to the absence of glutamate synthase activity. To confirm this point of view,
the character of a mutant defective in glutamate synthase (QB922 strain) was compared with the Rif' mutant. The growth of the glutamate synthase-deficient (gltA) mutant was reported to be restored by the addition of glutamate or aspartate (27). The test of amino acid requirements of this mutant showed, however, that one of the four amino acids (glutamine, argiline, proline, or asparagine) was also effective in the growth of the mutant. Moreover, it was pointed out by experiments similar to those of Fig. 1 that the mutant celLs required more than 500 jAg of one of those amino acids per ml, showing an identical phenotype to that of Rifr mutants examined in this paper. Revertant analysis. It may be assumed that a single mutation resulted in both Rifr and amino acid-requiring phenotype, since the fre-
412
J. BACTERIOL.
RYU
Mapping of the Rif' mutants. DNA extracted from RF 161 was transformed to the strain carrying the cysA14 mutation. Cys+ transformants were selected and their sensitivity to rifampin was tested on NB plates containing the drug (20 ,ug/ml). The Rifr phenotype was cotransformed with cysA at a frequency of 48% (74/154). Rifr mutations of two other strains (RF 183 and RF 271) were also transformed at a frequency of 36% (58/161) and 55% (87/159), respectively. It is thus suggested that these Rif' mutations are located at the rfn locus near cysA (10, 27). It was further shown that all the Rifr cotransformants acquired the property of amino
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acid requirement. Assay of RNA polymerase. The activity of RNA polymerase was measured in the presence
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TIME IN HOURS FIG. 3. Utilization of different N sources by 168TH (A) and RF 161 (B). Cells grown exponentially in MMCA medium were washed on a membrane filter. At zero time they were suspended in MM medium containing different N sources (0.4%). Symbols: (0) Urea; (0) potassium nitrate.
of various concentrations of rifampin after the partial purification of the enzyme. The activity of the parental enzyme as well as that of the revertant enzyme was completely inhibited in the presence of 0.1 ,ug of rifampin in the reaction mixture (0.25 ml), whereas the mutant enzyme retained 100% activity at the same concentration of the drug (Fig. 4). Partial inhibition of the activity of the mutant enzyme was observed when the drug at concentrations of 1 ,tg or more was added to the reaction mixture. Thus, com-
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TABLE 1. Enzyme activity of glutamate synthase Glutamate synthase
Strain
Medium
168TH
MM
RF 161 161 R2
MMCA MM
MMCA MM
MMCA
activity' 148 24
