JOUR?NAL OF BACTERiOLOGY, Aug. 1976, p. 739-746
Copyright © 1976 American Society for Microbiology
Vol. 127, No. 2
Printed in U.S.A.
Manganese Requirement of Phosphoglycerate Phosphomutase and Its Consequences for Growth and Sporulation of Bacillus subtilis YONG K. OH' AND ERNST FREESE* Laboratory of Molecular Biology, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland 20014 Received for publication 4 May 1976
In the absence of manganese, rapidly metabolizable carbohydrates such as glucose or glycerol are not completely metabolized by Bacillus subtilis growing in a nutrient sporulation medium; 3-phosphoglyceric acid (3PGA) accumulates inside the cells, growth stops at a low cell titer, and normal sporulation remains suppressed (no prespore septa). Upon the addition of manganese, 3PGA disappears, growth resumes, and normal sporulation takes place. These effects result from a specific manganese requirement of phosphoglycerate phosphomutase which catalyzes the interconversion of 3PGA and 2-phosphoglyceric acid (2PGA). Other metal ions cannot replace manganese, for which the enzyme has an apparent K. of 0.22 mM.
Manganese is an essential element for the sporulation of bacilli (4, 6, 28). It can replace Mg2+ as the cofactor of many enzymes, but some enzymes require Mn2+ specifically (21, 26, 27). In Bacillus licheniformis Mn2+ is apparently specifically needed to stabilize fructose1,6-bisphosphatase (23). In Bacillus subtilis, glutamine synthetase is more stable and behaves catalytically very differently in the presence of Mn2+ than in the presence of Mg2+ (10). After growth has stopped, the rate of potassium uptake in B. subtilis (W23) decreases if the medium contains no added manganese (12, 13); the rate of manganese uptake increases late in development (25). It is not known which of these Mn2+-specific functions are required for sporulation. In this paper, we demonstrate in B. subtilis Marburg a specific manganese requirement for phosphoglycerate phosphomutase (EC 5.4.2.1), one of the glycolytic enzymes. In the absence of manganese, growth stops at a reduced optical density, 3-phosphoglyceric acid (3PGA) accumulates inside the cells, and sporulation is arrested before prespore septation.
Media. NS medium contained 8.0 g of nutrient broth (Difco), 1.0 mM MgCl2, and 0.7 mM CaCl2 per liter. Its concentration of manganese was 150 to 200 nM, as was determined by atomic absorption at utilizing a graphite 403 nm furnace (Perkin-Elmer). Where stated, NS medium was supplemented with 50 juM MnCl2 (NS medium plus Mn2+). Growth and sporulation. The bacilli grown overnight on tryptose blood agar base (33 g/liter; Difco) plates were inoculated at an initial absorbancy at 600 nm (A6N) of about 0.05. For Fig. 1, the culture was grown to A6m = 0.5 and diluted 10-fold into the media (in Erlenmeyer flasks) to start the experiment. The cultures were shaken either in flasks (containing medium up to 0.1 of their volume) in a reciprocal water bath shaker at 37°C and 120 strokes/min (good aeration) or in 25-mm test tubes (containing 5 ml of medium) in a reciprocal shaker at 200 strokes/min (suboptimal aeration). The sporulation frequency (heat-resistant colony-forming units per viable cell) was determined at different times of growth by measuring the titers of heatresistant colony-forming units (heating for 15 min at 75°C) and of viable cells. Accumulation and assay of 3PGA. For 32P labeling, the standard strain 60015 was grown in NS medium containing 1 ,uCi of inorganic phosphate (PI) per ml; 20 mM glycerol was added when the culture reached an Am* = 0.6. At different times, 3MATERIALS AND METHODS ml samples of the culture were filtered through a Bacteria. Standard strain 60015 of transformable membrane filter (pore size, 0.45 jAm), and the filter B. subtilis Marburg (metC trpC) was used for most was extracted with 1 ml of ice-cold 0.46 M formic experiments. Strains 61310 (spt metC trpC) and acid. A 10-j,l portion of the extracts was chromato61402 (glpD hisA trpC) were described previously graphed on a thin-layer sheet containing poly(22). Strain 61540 (glpK ilvC pheA) was obtained ethyleneimine-impregnated cellulose MN 300 using 1 from L. Rutberg (19). Gene symbols and map loca- M LiCl as solvent (29), and an autoradiogram was tions have been described by Young and Wilson (30). produced. "4C labeling. For 14C labeling the strain was ' Present address: Microbiology L36, Smith, Kline, & grown as above, but 20 mM [U-_4C]glycerol (1 j,Ci/ French Laboratories, Philadelphia, Pa. 19101. 739
740
J. BACTERIOL.
OH AND FREESE
ml) was added when the culture reached an A,,, = 0.6. A 4-ml cell extract was prepared and chromatographed as above. In addition, both '4C-labeled and 32P-labeled extracts were chromatographed in a column of AG1-X8 (chloride form, 100 to 200 mesh, 0.8 by 12 cm) by elution with 0.02 M HCl at a flow rate of 1 ml/min and collection of 3-ml fractions. A 1.5-ml portion of each fraction was dried in a scintillation vial, and its radioactivity was determined in a TriCarb liquid scintillation counter. A reference sample of 3PGA (0.1 umol) was chromatographed in the same column, and 0.1 ml of each 3-ml fraction was assayed colorimetrically with chromotropic acid (1). The enzymatic identification of 3PGA (Table 1) was carried out by two different enzymatic coupling methods. In one method 3PGA was converted to glycerol-phosphate (glycerol-P) (8). In the other it was converted to lactate (7). In both cases the complete oxidation of reduced nicotinamide adenosine dinucleotide (NADH) (or the lack of it) were measured by the change in A 0. All enzymes were used at concentrations of 1 to 3 international units/ml. To determine the intracellular concentration of 3PGA, 30-ml samples of the culture were centrifuged at room temperature at 12,000 x g, and the cell pellets were extracted with 1.5 ml of ice-cold 0.46 M formic acid for 30 min. After neutralizing the extracts with 5 M K2C03, the amount of 3PGA was enzymatically determined by enzymatic coupling in the direction of glycerol-P formation. For the assay of 3PGA mutase (3) (Table 3; Fig. 6), cells were grown in NS medium with or without Mn2+ to an A6N of 1.0 to 1.2, centrifuged in the cold at 12,000 x g, washed twice with ice-cold 50 mM Tris-chloride buffer, pH 8.0, suspended at an A6N = 25 in this buffer, and lysed with lysozyme (150 ,ug/ ml) for 25 min at 37°C. After centrifugation of the lysate at 35,000 x g for 15 min, ammonium sulfate was added to the supernatant extract to give 60% ammonium sulfate in the cold. The precipitate was dissolved in 5 ml of Tris buffer and dialyzed against 100 ml of Tris buffer with continuous stirring at 4°C for 3 h; the buffer solution was once renewed. 3PGA mutase was assayed in the dialysate, the protein concentration of which was determined according to the method of Lowry et al. (20). The concentrations of 1-glucose (2) and L-malate (17) in the growth medium were enzymatically determined after cells had been removed from the culture by centrifugation.
TABLE 1. Enzymatic identification of 3PGA Enzymes sequentially added to the basal mixture
A. Assayed by enzyme coupling to produce glycerol-Pa None Glyceraldehyde 3-P dehydrogenase 3-P-Glycerate kinase 3-P-Glycerate mutase
NADH oxidation with different substrates
Ex-
3PGA
2PGA
tract
-
-
-
+
-
+
+
+ +
+
+ +
+
B. Assayed by enzyme coupling to produce L-lactateb
None Enolase 3-P-Glycerate mutase
a The basal mixture for PGA assayed by enzyme coupling to produce glycerol-P contained triose-P isomerase and glycerol-P dehydrogenase (see Materials and Methods). 3- and 2-PGA were used at 15 mM final concentrations. Symbols: +, signifies complete enzymatic oxidation of NADH; -, signifies no oxidation. b The basal mixture for PGA assayed by enzyme coupling to produce L-lactate contained pyruvate kinase and lactic dehydrogenase (see Materials and Methods). PGA concentrations and symbols are the same as above.
U, 0
n 0 a co 0
0.1
(D nnq-
c
n RESULTS :3 0 Manganese dependence of growth and sporulation. When our standard strain (60015) was shaken in NS-medium plus 10 mM glucose, growth stopped at a much lower A6. value than when the medium was supplemented with H O U RS 50 ,uM MnCl2 (Fig. 1). Whereas in the Mn2+1. FIG. and sporulation of strain 60015 in containing culture almost all cells produced NS mediumGrowth plus 10 mM glucose with and without 50 spores 7 to 8 h after the end of exponential MM MnCl2. Symbols: of culture with Mn2+ (0); growth, no sporulation was observed without A.00 without Mn2+ (0);A6. heat-resistant colony-forming added Mn2 . Inspection of thin sections under units per viable cell with Mn2+ (A); heat-resistant the electron microscope 5 h after growth had colony-forming units per viable cell without Mn2+ stopped showed that all (300 inspected) cells (A).
VOL. 127, 1976
Mn REQUIREMENT OF PGA MUTASE
741
grown without added Mn2+ were blocked at before 20 mM glycerol was added grew to the stage 0/I (i.e., before prespore septation), same low A . as when glycerol was added from whereas in the Mn2+-containing medium 85% of the beginning but experienced no lysis for sevthe cells had developed beyond stage II. In NS eral hours. This phenomenon was not further medium, without added glucose, the sporula- investigated because it was of no significance tion frequency was higher but still not normal; for this paper.) Glycerol did not reduce the maxit varied between 1 and 3% when the cultures imal A.,,, in mutants unable to metabolize glycwere grown in test tubes (presumably subopti- erol to Embden-Meyerhof path intermediates. mal aeration) and between 2 and 10% in Erlen- Such strains include a sugar-phosphoenolpyrumeyer flasks (good aeration) (see Materials and vate-phosphotranferase mutant (spt, 61310), Methods for conditions); the addition of Mn2+ to which cannot take up glycerol (15), a glycerol NS medium allowed 70% or better sporulation kinase mutant (glpK, 61540), and a glycerol-P in all cases. NS medium itself contained 150 to dehydrogenase (NAD-independent) mutant 200 nM manganese. (glpD, 61402) (22).
Prespore septation requires the normal functioning of so many biochemical reactions (14) that the biochemical reason for its absence would be difficult to analyze. But the difference in the maximal A,,) observed with and without Mn2+ should be accessible to a biochemical investigation. Figure 2 shows that this difference was much smaller, and the maximal A,;,., observed without Mn2+ was higher, when the NS medium contained no added carbohydrates than when it contained glucose, mannose, fructose, or glycerol. Apparently, the carbohydrates could be only inefficiently metabolized in the absence of Mn2+; possibly they caused the accumulation of some metabolic intermediate that prevented further growth or whose production effected the more rapid exhaustion of some other compound needed for growth. Addition of 50 uM MnCl2 restored growth in all cultures within 0.5 h (Fig. 2). (The lysis observed in the medium containing glycerol depended on the time of glycerol addition; a culture first grown in NS medium to an A611) of 0.6
Accumulation of 3PGA. Whereas glucose or glycerol were completely used up when cells were grown in NS medium plus Mn2+, they were still present in the medium at the end of growth in NS medium (Fig. 3). To identify the metabolic block, bacilli were grown in NS medium containing a trace amount of :2P. (1 ACiI ml), the concentration of Pi being that of the NS medium itself (1.3 mM Pi). When the A., reached 0.6, a sample was taken for extraction of the bacteria, and 20 mM glycerol was added to the rest of the culture. Forty minutes later, another sample was taken and 50 ,uM MnCl2 was added, which restored growth. After a further 20 min, a third sample was taken. Thinlayer chromatography of formic acid extracts showed the accumulation of a radioactive compound after glycerol addition; this material vanished again after manganese addition (Fig. 4). The accumulated compound was identified in crude cell extracts as 3PGA by the following three methods. (i) Enzymatic conversion of the compound in the direction of glyceraldehyde-3-
HOURS
FIG. 2. Growth of strain 60015 in NS medium with different carbohydrates with and without 50 MM MnCI2. (a) NS medium alone; (b) with 25 mM glucose; (c) with 25 mM mannose; (d) with 25 mM fructose; and (e) with 50 mM glycerol. Symbols: A6. with Mn2+ (O); A6N without Mn2+ (0); A600 after Mn2+ addition (A), in which case cells produced at least 70% spores 15 h later.
742
J. BACTERIOL.
OH AND FREESE 5.0
C) 1.0 0 0
0
0
cn
ate phosphomutase. The accumulation of 3PGA in the absence of Mn2+ suggested that Pglycerate phosphomutase (EC 5.4.2.1, D-phosphoglycerate 2,3-phosphosMutase, PGA mutase) required Mn2+ for its activity. This was verified in extracts of cells grown in NS medium; they exhibited no PGA mutase activity except when MnCl2 was added to the reaction mixture. To analyze the metal dependence, extracts of cells grown in NS medium and NS medium plus Mn2+ were precipitated with 60% ammonium
CD 5:
0.5
,
Copyright © 1976 American Society for Microbiology
Vol. 127, No. 2
Printed in U.S.A.
Manganese Requirement of Phosphoglycerate Phosphomutase and Its Consequences for Growth and Sporulation of Bacillus subtilis YONG K. OH' AND ERNST FREESE* Laboratory of Molecular Biology, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland 20014 Received for publication 4 May 1976
In the absence of manganese, rapidly metabolizable carbohydrates such as glucose or glycerol are not completely metabolized by Bacillus subtilis growing in a nutrient sporulation medium; 3-phosphoglyceric acid (3PGA) accumulates inside the cells, growth stops at a low cell titer, and normal sporulation remains suppressed (no prespore septa). Upon the addition of manganese, 3PGA disappears, growth resumes, and normal sporulation takes place. These effects result from a specific manganese requirement of phosphoglycerate phosphomutase which catalyzes the interconversion of 3PGA and 2-phosphoglyceric acid (2PGA). Other metal ions cannot replace manganese, for which the enzyme has an apparent K. of 0.22 mM.
Manganese is an essential element for the sporulation of bacilli (4, 6, 28). It can replace Mg2+ as the cofactor of many enzymes, but some enzymes require Mn2+ specifically (21, 26, 27). In Bacillus licheniformis Mn2+ is apparently specifically needed to stabilize fructose1,6-bisphosphatase (23). In Bacillus subtilis, glutamine synthetase is more stable and behaves catalytically very differently in the presence of Mn2+ than in the presence of Mg2+ (10). After growth has stopped, the rate of potassium uptake in B. subtilis (W23) decreases if the medium contains no added manganese (12, 13); the rate of manganese uptake increases late in development (25). It is not known which of these Mn2+-specific functions are required for sporulation. In this paper, we demonstrate in B. subtilis Marburg a specific manganese requirement for phosphoglycerate phosphomutase (EC 5.4.2.1), one of the glycolytic enzymes. In the absence of manganese, growth stops at a reduced optical density, 3-phosphoglyceric acid (3PGA) accumulates inside the cells, and sporulation is arrested before prespore septation.
Media. NS medium contained 8.0 g of nutrient broth (Difco), 1.0 mM MgCl2, and 0.7 mM CaCl2 per liter. Its concentration of manganese was 150 to 200 nM, as was determined by atomic absorption at utilizing a graphite 403 nm furnace (Perkin-Elmer). Where stated, NS medium was supplemented with 50 juM MnCl2 (NS medium plus Mn2+). Growth and sporulation. The bacilli grown overnight on tryptose blood agar base (33 g/liter; Difco) plates were inoculated at an initial absorbancy at 600 nm (A6N) of about 0.05. For Fig. 1, the culture was grown to A6m = 0.5 and diluted 10-fold into the media (in Erlenmeyer flasks) to start the experiment. The cultures were shaken either in flasks (containing medium up to 0.1 of their volume) in a reciprocal water bath shaker at 37°C and 120 strokes/min (good aeration) or in 25-mm test tubes (containing 5 ml of medium) in a reciprocal shaker at 200 strokes/min (suboptimal aeration). The sporulation frequency (heat-resistant colony-forming units per viable cell) was determined at different times of growth by measuring the titers of heatresistant colony-forming units (heating for 15 min at 75°C) and of viable cells. Accumulation and assay of 3PGA. For 32P labeling, the standard strain 60015 was grown in NS medium containing 1 ,uCi of inorganic phosphate (PI) per ml; 20 mM glycerol was added when the culture reached an Am* = 0.6. At different times, 3MATERIALS AND METHODS ml samples of the culture were filtered through a Bacteria. Standard strain 60015 of transformable membrane filter (pore size, 0.45 jAm), and the filter B. subtilis Marburg (metC trpC) was used for most was extracted with 1 ml of ice-cold 0.46 M formic experiments. Strains 61310 (spt metC trpC) and acid. A 10-j,l portion of the extracts was chromato61402 (glpD hisA trpC) were described previously graphed on a thin-layer sheet containing poly(22). Strain 61540 (glpK ilvC pheA) was obtained ethyleneimine-impregnated cellulose MN 300 using 1 from L. Rutberg (19). Gene symbols and map loca- M LiCl as solvent (29), and an autoradiogram was tions have been described by Young and Wilson (30). produced. "4C labeling. For 14C labeling the strain was ' Present address: Microbiology L36, Smith, Kline, & grown as above, but 20 mM [U-_4C]glycerol (1 j,Ci/ French Laboratories, Philadelphia, Pa. 19101. 739
740
J. BACTERIOL.
OH AND FREESE
ml) was added when the culture reached an A,,, = 0.6. A 4-ml cell extract was prepared and chromatographed as above. In addition, both '4C-labeled and 32P-labeled extracts were chromatographed in a column of AG1-X8 (chloride form, 100 to 200 mesh, 0.8 by 12 cm) by elution with 0.02 M HCl at a flow rate of 1 ml/min and collection of 3-ml fractions. A 1.5-ml portion of each fraction was dried in a scintillation vial, and its radioactivity was determined in a TriCarb liquid scintillation counter. A reference sample of 3PGA (0.1 umol) was chromatographed in the same column, and 0.1 ml of each 3-ml fraction was assayed colorimetrically with chromotropic acid (1). The enzymatic identification of 3PGA (Table 1) was carried out by two different enzymatic coupling methods. In one method 3PGA was converted to glycerol-phosphate (glycerol-P) (8). In the other it was converted to lactate (7). In both cases the complete oxidation of reduced nicotinamide adenosine dinucleotide (NADH) (or the lack of it) were measured by the change in A 0. All enzymes were used at concentrations of 1 to 3 international units/ml. To determine the intracellular concentration of 3PGA, 30-ml samples of the culture were centrifuged at room temperature at 12,000 x g, and the cell pellets were extracted with 1.5 ml of ice-cold 0.46 M formic acid for 30 min. After neutralizing the extracts with 5 M K2C03, the amount of 3PGA was enzymatically determined by enzymatic coupling in the direction of glycerol-P formation. For the assay of 3PGA mutase (3) (Table 3; Fig. 6), cells were grown in NS medium with or without Mn2+ to an A6N of 1.0 to 1.2, centrifuged in the cold at 12,000 x g, washed twice with ice-cold 50 mM Tris-chloride buffer, pH 8.0, suspended at an A6N = 25 in this buffer, and lysed with lysozyme (150 ,ug/ ml) for 25 min at 37°C. After centrifugation of the lysate at 35,000 x g for 15 min, ammonium sulfate was added to the supernatant extract to give 60% ammonium sulfate in the cold. The precipitate was dissolved in 5 ml of Tris buffer and dialyzed against 100 ml of Tris buffer with continuous stirring at 4°C for 3 h; the buffer solution was once renewed. 3PGA mutase was assayed in the dialysate, the protein concentration of which was determined according to the method of Lowry et al. (20). The concentrations of 1-glucose (2) and L-malate (17) in the growth medium were enzymatically determined after cells had been removed from the culture by centrifugation.
TABLE 1. Enzymatic identification of 3PGA Enzymes sequentially added to the basal mixture
A. Assayed by enzyme coupling to produce glycerol-Pa None Glyceraldehyde 3-P dehydrogenase 3-P-Glycerate kinase 3-P-Glycerate mutase
NADH oxidation with different substrates
Ex-
3PGA
2PGA
tract
-
-
-
+
-
+
+
+ +
+
+ +
+
B. Assayed by enzyme coupling to produce L-lactateb
None Enolase 3-P-Glycerate mutase
a The basal mixture for PGA assayed by enzyme coupling to produce glycerol-P contained triose-P isomerase and glycerol-P dehydrogenase (see Materials and Methods). 3- and 2-PGA were used at 15 mM final concentrations. Symbols: +, signifies complete enzymatic oxidation of NADH; -, signifies no oxidation. b The basal mixture for PGA assayed by enzyme coupling to produce L-lactate contained pyruvate kinase and lactic dehydrogenase (see Materials and Methods). PGA concentrations and symbols are the same as above.
U, 0
n 0 a co 0
0.1
(D nnq-
c
n RESULTS :3 0 Manganese dependence of growth and sporulation. When our standard strain (60015) was shaken in NS-medium plus 10 mM glucose, growth stopped at a much lower A6. value than when the medium was supplemented with H O U RS 50 ,uM MnCl2 (Fig. 1). Whereas in the Mn2+1. FIG. and sporulation of strain 60015 in containing culture almost all cells produced NS mediumGrowth plus 10 mM glucose with and without 50 spores 7 to 8 h after the end of exponential MM MnCl2. Symbols: of culture with Mn2+ (0); growth, no sporulation was observed without A.00 without Mn2+ (0);A6. heat-resistant colony-forming added Mn2 . Inspection of thin sections under units per viable cell with Mn2+ (A); heat-resistant the electron microscope 5 h after growth had colony-forming units per viable cell without Mn2+ stopped showed that all (300 inspected) cells (A).
VOL. 127, 1976
Mn REQUIREMENT OF PGA MUTASE
741
grown without added Mn2+ were blocked at before 20 mM glycerol was added grew to the stage 0/I (i.e., before prespore septation), same low A . as when glycerol was added from whereas in the Mn2+-containing medium 85% of the beginning but experienced no lysis for sevthe cells had developed beyond stage II. In NS eral hours. This phenomenon was not further medium, without added glucose, the sporula- investigated because it was of no significance tion frequency was higher but still not normal; for this paper.) Glycerol did not reduce the maxit varied between 1 and 3% when the cultures imal A.,,, in mutants unable to metabolize glycwere grown in test tubes (presumably subopti- erol to Embden-Meyerhof path intermediates. mal aeration) and between 2 and 10% in Erlen- Such strains include a sugar-phosphoenolpyrumeyer flasks (good aeration) (see Materials and vate-phosphotranferase mutant (spt, 61310), Methods for conditions); the addition of Mn2+ to which cannot take up glycerol (15), a glycerol NS medium allowed 70% or better sporulation kinase mutant (glpK, 61540), and a glycerol-P in all cases. NS medium itself contained 150 to dehydrogenase (NAD-independent) mutant 200 nM manganese. (glpD, 61402) (22).
Prespore septation requires the normal functioning of so many biochemical reactions (14) that the biochemical reason for its absence would be difficult to analyze. But the difference in the maximal A,,) observed with and without Mn2+ should be accessible to a biochemical investigation. Figure 2 shows that this difference was much smaller, and the maximal A,;,., observed without Mn2+ was higher, when the NS medium contained no added carbohydrates than when it contained glucose, mannose, fructose, or glycerol. Apparently, the carbohydrates could be only inefficiently metabolized in the absence of Mn2+; possibly they caused the accumulation of some metabolic intermediate that prevented further growth or whose production effected the more rapid exhaustion of some other compound needed for growth. Addition of 50 uM MnCl2 restored growth in all cultures within 0.5 h (Fig. 2). (The lysis observed in the medium containing glycerol depended on the time of glycerol addition; a culture first grown in NS medium to an A611) of 0.6
Accumulation of 3PGA. Whereas glucose or glycerol were completely used up when cells were grown in NS medium plus Mn2+, they were still present in the medium at the end of growth in NS medium (Fig. 3). To identify the metabolic block, bacilli were grown in NS medium containing a trace amount of :2P. (1 ACiI ml), the concentration of Pi being that of the NS medium itself (1.3 mM Pi). When the A., reached 0.6, a sample was taken for extraction of the bacteria, and 20 mM glycerol was added to the rest of the culture. Forty minutes later, another sample was taken and 50 ,uM MnCl2 was added, which restored growth. After a further 20 min, a third sample was taken. Thinlayer chromatography of formic acid extracts showed the accumulation of a radioactive compound after glycerol addition; this material vanished again after manganese addition (Fig. 4). The accumulated compound was identified in crude cell extracts as 3PGA by the following three methods. (i) Enzymatic conversion of the compound in the direction of glyceraldehyde-3-
HOURS
FIG. 2. Growth of strain 60015 in NS medium with different carbohydrates with and without 50 MM MnCI2. (a) NS medium alone; (b) with 25 mM glucose; (c) with 25 mM mannose; (d) with 25 mM fructose; and (e) with 50 mM glycerol. Symbols: A6. with Mn2+ (O); A6N without Mn2+ (0); A600 after Mn2+ addition (A), in which case cells produced at least 70% spores 15 h later.
742
J. BACTERIOL.
OH AND FREESE 5.0
C) 1.0 0 0
0
0
cn
ate phosphomutase. The accumulation of 3PGA in the absence of Mn2+ suggested that Pglycerate phosphomutase (EC 5.4.2.1, D-phosphoglycerate 2,3-phosphosMutase, PGA mutase) required Mn2+ for its activity. This was verified in extracts of cells grown in NS medium; they exhibited no PGA mutase activity except when MnCl2 was added to the reaction mixture. To analyze the metal dependence, extracts of cells grown in NS medium and NS medium plus Mn2+ were precipitated with 60% ammonium
CD 5:
0.5
,
