INFECTION AND IMMUNITY, Feb. 1979, p. 224-231 0019-9567/79/02-0224/08$02.00/0
Vol. 23, No. 2
Effect of Growth Rate and Glucose Concentration on the Activity of the Phosphoenolpyruvate Phosphotransferase System in Streptococcus mutans Ingbritt Grown in Continuous Culture D. C. ELLWOOD, P. J. PHIPPS, AND I. R. HAMILTONt* Microbiological Research Establishment, Porton Down, Salisbury, England
Received for publication 10 October 1978
Streptococcus mutans Ingbritt was grown anaerobically in a chemostat with a glucose limitation, as well as with an excess of glucose (amino acid limitation) at dilution rates (D) between 0.05 and 0.4 h-1 (mean generation time = 12 to 1.5 h). The glucose-limited culture produced cells having 1.5- to 6.0-fold greater glycolytic activity than the cells from the glucose-excess culture. The preferred substrate for these cells was glucose, with the glycolytic rate for sucrose being only slightly lower; the rate for fructose was half that of glucose. The glycolytic rate of the glucose-limited cells was maximum at D = 0.1 h-', with a decline in rate as the growth rate approached D = 0.4 h-1. A comparison of the activity of phosphoenolpyruvate phosphotransferase system (PTS) in the two types of cells showed that the glucose-limited cells had 1.7- to 5.6-fold greater PTS activity for the three sugars than the glucose-excess-grown cells. Whereas little difference was seen between the three sugars with the latter cells, the glucose-PTS had the greatest activity with glucose-limited cells, with the maximum in cells grown at D = 0.1 h-1. Comparison of the rate of sugar uptake in the chemostat with the rate of PTS transport activity in the cells at each growth rate demonstrated that only under conditions of slow growth with a glucose limitation was the PTS system capable of supporting growth on glucose. Furthermore, PTS activity in cells grown with an excess of glucose was insignificant when compared with glucose uptake during growth in the chemostat. This evidence supports the observation that S. mutans possesses at least one other system, in addition to the PTS, for the transport of glucose into the cell. The organism was, however, devoid of glucose-proton symport transport activity. When Streptococcus mutans Ingbritt was grown in a complex medium under conditions of glucose limitation in a chemostat (J. R. Hunter, J. K. Baird, and D. C. Ellwood, Abstr. Annu. Meet. Br. Assoc. Dent. Res. 1973, 99, p. 954), the glucose-PTS activity decreased as the growth rate increased, suggesting the possible presence of at least one other transport system. Evidence for additional transport activity has also been obtained more recently (6) from studies with the same organism growing slowly at various pH values under conditions of glucose excess (amino acid limited). Cells growing at pH 5.5 exhibited two- to threefold greater glycolytic activity than cells growing at pH 6.5 but possessed only 11% of the glucose-PTS activity of the latter cells. As part of a more comprehensive study on the biochemical properties of various strains of S. t Permanent address: Department of Oral Biology, Faculty of Dentistry, University of Manitoba, Winnipeg, Manitoba, mutans grown under controlled conditions in the chemostat, we have examined the ability of R3E OW3 Canada.
Oral streptococci constitute a significant fraction of the microbial community on the tooth surface (1, 14). These bacteria are known to transport glucose into cells via the phosphoenolpyruvate (PEP) hexose phosphotransferase system (PTS) (5,12,17). In this system, a soluble enzyme (EI) catalyzes the phosphorylation of a heat-stable protein, employing PEP as the phosphoryl donor. Heat-stable protein is regenerated after the transport and phosphorylation of the appropriate sugar through a reaction catalyzed by a membrane-bound, sugar-specific enzyme (EII). Whereas studies with other bacteria, notably Escherichia coli, have shown that transport actually requires additional enzymic factors (see 16), little is known about the specific components of the PTS in the oral streptococci.
224
VOL. 23, 1979
GROWTH RATE AND GLUCOSE EFFECTS ON PTS
225
sucrose by washed cells suspensions incubated anaerobically at a constant pH in a pH stat (6). Cells were collected via the overflow from each chemostat into a container cooled in ice for various periods, usually overnight. They were then harvested by centrifugation (8,000 x g for 15 min) and washed once in potassium phosphate buffer (20 mM, pH 7.0) by centrifugation. The cells were suspended in saline at a concentration of =30 mg/ml and stored in ice until used. For each experiment, washed cells (7 to 10 mg) were suspended in a volume of 2.0 ml of 20 mM potassium phosphate buffer. The sugar (100 pI of 30mg/ml solution in water) was added, and the pH was kept constant at the required value by the addition of standardized 0.1 M NaOH with the radiometer pHstat system (model 26 pH meter, type SBR2c Titregraph Titrator II and Autoburette ABU 12). The cell suspension was mixed by a magnetic stirrer, and the MATERIALS AND METHODS suspension was kept anaerobic by the passage of a Bacterial rain. The S. mutans, strain Ingbritt, slow stream of nitrogen gas through the mixture. The used in this study was kindly supplied by J. Sandham, rate of alkali addition was recorded for sufficient time Toronto, Ontario, Canada. The purity of the chemo- (usually for 4 to 8 min) to permit the establishment of stat culture was checked daily by slide examination a linear rate. Replicate samples done sequentially were and by streaking on nutrient agar plates incubated reproducible within 5%. Samples taken after storage aerobically and anaerobically in an atmosphere of of the cell suspension in ice for 18 h were within 10%, nitrogen + 5% CO2. In addition, the purity of the test as were samples prepared from cells taken directly organism was determined initially, and at intervals from the chemostat while the culture was in steady throughout the run, for its ability to agglutinate with state. Units of glycolytic activity are expressed as type c antisera kindly supplied by G. Bowden, London nanomoles of metabolic acid neutralized x milligrams Hospital, England. Because cells obtained directly (dry weight) of cells-' x minute-. from the chemostat frequently show variation in agAssay for PEP phosphotransferase activity. glutination, the tests were always performed on cells Sugar transport via the PEP PTS was assayed by the growing on the nutrient agar plates used for routine method of Kornberg and Reeves (13) with washed purity checks. cells made permeable with toluene. Cell samples (50 Growth conditions. Cultures were grown in a ml) were removed directly from the chemostat, cenPorton-type chemostat (10) with a 500-ml working trifuged at 10,000 x g (15 min), washed by centrifucapacity; pH was maintained at pH 6.5, and the gas gation, and suspended in phosphate buffer (50 mM, phase was nitrogen + 5% CO2. The dilution rate was pH 7.0) at a concentration of 4 to 10 mg of cells (dry maintained at values between 0.05 and 0.4 h-1, and the weight) per ml and mixed vigorously on a Vortex mixer cultures were allowed to reach equilibrium for at least with 0.01 volume of toluene for 60 a. As shown in 10 mean generations at each dilution rate before har- Table 1, this toluene treatment gave the optimal revesting. sults for cells derived from all growth rates with gluTo compare the properties of cells grown under cose-limited cultures; 10 pl toluene was also shown to conditions of glucose limitation and glucose excess be optimal for glucose-excess cultures. The conditions (nitrogen limitation), the test organism was inoculated simultaneously into two chemostats, each containing TABLE 1. Effect of toluene concentration on PEP a defined medium slightly modified to that of Carlsson phosphotransferase activity in cells of S. mutans inaltered by (M3) (2). The basic M3 medium was Ingbritt grown in complex medium at different creasing the concentration of aspartic acid and cysdilution rates with a glucose limitation teine to 400 mg/liter and that of leucine, isoleucine, and lysine to 200 mg/liter while removing glucose and PTS activity at D (h-Q) of: Toluenea acetate. In addition, MgS04.7H20 was increased to 0.05 0.1 0.5 0.3 0.4 0.2 400 mg/liter while KH2PO4 and K2HPO4 were decreased to 1.33 and 2.66 g/liter, respectively. For the 0 10.2b 11.4 11.1 5.6 2.8 1.2
cells grown at various growth rates under conditions of glucose limitation and glucose excess (nitrogen limitation) to transport and metabolize various sugars. The results show that PTS activity is repressed at all growth rates tested (mean generation time = 1.5 to 12 h) under conditions of glucose excess compared with activity in cells grown with a glucose limitation. A comparison of the rate of sugar utilization by S. mutans Ingbritt growing in the chemostat with the rate of transport via the phosphotransferase system demonstrated that only under conditions of slow growth (mean generation time >4 to 7 h) was the PTS adequate to account for all of the glucose transported by cells of the organism.
glucose excess (nitrogen limited) culture, the basic medium was modified to contain 1/5 the concentration of amino acids, while glucose was added at 55 mg/ml such that during growth asparagine and arginine were limiting growth. The glucose-limited culture was grown in the basal M3 medium modified to contain 5 mg of glucose per ml. Washed cell experiments. The glycolytic activity of the chemostat-grown cells was studied by measuring the rate of acid production from glucose, fructose, or
5 10 15 20 25
40.0 47.4 46.4 45.7 21.7
52.2
45.1
26.5
13.2
52.0
44.2
21.0
9.4
5.3 7.6 7.0 5.7 5.0
' Volume of toluene per milliliter of cell suspension (30 mg/ml). b Nanomoles x milligram (dry weight) of cells-' x minutes.
226
ELLWOOD, PHIPPS, AND HAMILTON
of assay were as previously described (6). Net PITS activity is expressed as nanomoles of pyruvate fornned x milligram (dry weight) of cells- x minute'. Assay for glucose transport via proton 8ynmport. Cells of S. mutans Ingbritt were assayed for glucose - Ho symport activity under the conditi ons described by Henderson et al. (8). Cells, growing in the chemostat under conditions of glucose limitation sat a dilution rate of D = 0.4 h-1, were collected in ice foi h, harvested, and washed as previously described (6) One-half of the cells were suspended in saline (43 mg/ml), whereas the remainder were suspended in 420 mM phosphate buffer (pH 7.0) (36 mg/ml). A portion of the latter cells was incubated at 370C for 60 miu i to deplete any glycogen present, washed, and suspendded in anoxic 150 mM KCI-2 mM glycylglycine buffer ((pH 6.8) (8). Proton uptake was measured at 250C with118 mg (dry weight) of cells suspended in 6 ml of the lai r buffer. Rate of glucose transport in the chemositat. Glucose uptake by cells growing in the chemostat i determined by the equation of Herbert and Kornb
INFECTr. IMMUN.
TABLE 2. Effect of dilution rate on the growth parameters of S. mutans Ingbritt growing under conditions ofglucose limitation and glucose excess (nitrogen limited)
Condition' D - 0.05 h-1
irM
Ex
Organiim
Residual glucose
Ya.e (mg of
ml)
(mg/ml)
cells/mg of glucose)
0.81 0.63
0 14.5
0.172 0.016
0.93 0.46
0 14.2
0.186 0.011
1.02 0.59
0 15.6
0.204 0.015
yield (mg
0.1 h-1
D
IAM Ex D - 0.2h-1 Lim
Ex
D = 0.4 h-1 Lim 0.88 0 0.176 Ex 0.58 16.0 0.015 'Lim, Glucose limitation; Ex, glucose excess.
Uerg
(9): D(SD- S)
(1) (x) x 60 where q, = the rate of glucose utilization (nanomoles x milligram [dry weight] of cells-l x minute-), D = the dilution rate (hours-'), So = glucose concentration in the inflowing medium (nanomoles per milliliter), S = glucose concentration in the outflowing culture (nanomoles per milliliter), and x = dry weight of cells (milligrams per milliliter). Rate of acid production in the chemostat. Because S. mutans Ingbritt ferments glucose during growth with the production of acid end products and because the chemostat cultures were maintained at pH 6.5 by the addition of NaOH, it was possible to determine the rate of acid production in the chemostat from the following equation, which is analogous to equation 1: Das x A q
(x) X 60
(2)
where qm = the rate of alkali addition (nanomoles x milligram [dry weight] of cells-1 X minute'), A = concentration of alkali (nanomoles per milliliter), x = dry weight of cells (milligrams per milliliter), and Dam = dilution rate for alkali (volume of alkali added, hour-' per volume of culture vessel). Analytical procedures. Glucose and metabolic end products present in the culture fluid were assayed after rapid filtration (
Vol. 23, No. 2
Effect of Growth Rate and Glucose Concentration on the Activity of the Phosphoenolpyruvate Phosphotransferase System in Streptococcus mutans Ingbritt Grown in Continuous Culture D. C. ELLWOOD, P. J. PHIPPS, AND I. R. HAMILTONt* Microbiological Research Establishment, Porton Down, Salisbury, England
Received for publication 10 October 1978
Streptococcus mutans Ingbritt was grown anaerobically in a chemostat with a glucose limitation, as well as with an excess of glucose (amino acid limitation) at dilution rates (D) between 0.05 and 0.4 h-1 (mean generation time = 12 to 1.5 h). The glucose-limited culture produced cells having 1.5- to 6.0-fold greater glycolytic activity than the cells from the glucose-excess culture. The preferred substrate for these cells was glucose, with the glycolytic rate for sucrose being only slightly lower; the rate for fructose was half that of glucose. The glycolytic rate of the glucose-limited cells was maximum at D = 0.1 h-', with a decline in rate as the growth rate approached D = 0.4 h-1. A comparison of the activity of phosphoenolpyruvate phosphotransferase system (PTS) in the two types of cells showed that the glucose-limited cells had 1.7- to 5.6-fold greater PTS activity for the three sugars than the glucose-excess-grown cells. Whereas little difference was seen between the three sugars with the latter cells, the glucose-PTS had the greatest activity with glucose-limited cells, with the maximum in cells grown at D = 0.1 h-1. Comparison of the rate of sugar uptake in the chemostat with the rate of PTS transport activity in the cells at each growth rate demonstrated that only under conditions of slow growth with a glucose limitation was the PTS system capable of supporting growth on glucose. Furthermore, PTS activity in cells grown with an excess of glucose was insignificant when compared with glucose uptake during growth in the chemostat. This evidence supports the observation that S. mutans possesses at least one other system, in addition to the PTS, for the transport of glucose into the cell. The organism was, however, devoid of glucose-proton symport transport activity. When Streptococcus mutans Ingbritt was grown in a complex medium under conditions of glucose limitation in a chemostat (J. R. Hunter, J. K. Baird, and D. C. Ellwood, Abstr. Annu. Meet. Br. Assoc. Dent. Res. 1973, 99, p. 954), the glucose-PTS activity decreased as the growth rate increased, suggesting the possible presence of at least one other transport system. Evidence for additional transport activity has also been obtained more recently (6) from studies with the same organism growing slowly at various pH values under conditions of glucose excess (amino acid limited). Cells growing at pH 5.5 exhibited two- to threefold greater glycolytic activity than cells growing at pH 6.5 but possessed only 11% of the glucose-PTS activity of the latter cells. As part of a more comprehensive study on the biochemical properties of various strains of S. t Permanent address: Department of Oral Biology, Faculty of Dentistry, University of Manitoba, Winnipeg, Manitoba, mutans grown under controlled conditions in the chemostat, we have examined the ability of R3E OW3 Canada.
Oral streptococci constitute a significant fraction of the microbial community on the tooth surface (1, 14). These bacteria are known to transport glucose into cells via the phosphoenolpyruvate (PEP) hexose phosphotransferase system (PTS) (5,12,17). In this system, a soluble enzyme (EI) catalyzes the phosphorylation of a heat-stable protein, employing PEP as the phosphoryl donor. Heat-stable protein is regenerated after the transport and phosphorylation of the appropriate sugar through a reaction catalyzed by a membrane-bound, sugar-specific enzyme (EII). Whereas studies with other bacteria, notably Escherichia coli, have shown that transport actually requires additional enzymic factors (see 16), little is known about the specific components of the PTS in the oral streptococci.
224
VOL. 23, 1979
GROWTH RATE AND GLUCOSE EFFECTS ON PTS
225
sucrose by washed cells suspensions incubated anaerobically at a constant pH in a pH stat (6). Cells were collected via the overflow from each chemostat into a container cooled in ice for various periods, usually overnight. They were then harvested by centrifugation (8,000 x g for 15 min) and washed once in potassium phosphate buffer (20 mM, pH 7.0) by centrifugation. The cells were suspended in saline at a concentration of =30 mg/ml and stored in ice until used. For each experiment, washed cells (7 to 10 mg) were suspended in a volume of 2.0 ml of 20 mM potassium phosphate buffer. The sugar (100 pI of 30mg/ml solution in water) was added, and the pH was kept constant at the required value by the addition of standardized 0.1 M NaOH with the radiometer pHstat system (model 26 pH meter, type SBR2c Titregraph Titrator II and Autoburette ABU 12). The cell suspension was mixed by a magnetic stirrer, and the MATERIALS AND METHODS suspension was kept anaerobic by the passage of a Bacterial rain. The S. mutans, strain Ingbritt, slow stream of nitrogen gas through the mixture. The used in this study was kindly supplied by J. Sandham, rate of alkali addition was recorded for sufficient time Toronto, Ontario, Canada. The purity of the chemo- (usually for 4 to 8 min) to permit the establishment of stat culture was checked daily by slide examination a linear rate. Replicate samples done sequentially were and by streaking on nutrient agar plates incubated reproducible within 5%. Samples taken after storage aerobically and anaerobically in an atmosphere of of the cell suspension in ice for 18 h were within 10%, nitrogen + 5% CO2. In addition, the purity of the test as were samples prepared from cells taken directly organism was determined initially, and at intervals from the chemostat while the culture was in steady throughout the run, for its ability to agglutinate with state. Units of glycolytic activity are expressed as type c antisera kindly supplied by G. Bowden, London nanomoles of metabolic acid neutralized x milligrams Hospital, England. Because cells obtained directly (dry weight) of cells-' x minute-. from the chemostat frequently show variation in agAssay for PEP phosphotransferase activity. glutination, the tests were always performed on cells Sugar transport via the PEP PTS was assayed by the growing on the nutrient agar plates used for routine method of Kornberg and Reeves (13) with washed purity checks. cells made permeable with toluene. Cell samples (50 Growth conditions. Cultures were grown in a ml) were removed directly from the chemostat, cenPorton-type chemostat (10) with a 500-ml working trifuged at 10,000 x g (15 min), washed by centrifucapacity; pH was maintained at pH 6.5, and the gas gation, and suspended in phosphate buffer (50 mM, phase was nitrogen + 5% CO2. The dilution rate was pH 7.0) at a concentration of 4 to 10 mg of cells (dry maintained at values between 0.05 and 0.4 h-1, and the weight) per ml and mixed vigorously on a Vortex mixer cultures were allowed to reach equilibrium for at least with 0.01 volume of toluene for 60 a. As shown in 10 mean generations at each dilution rate before har- Table 1, this toluene treatment gave the optimal revesting. sults for cells derived from all growth rates with gluTo compare the properties of cells grown under cose-limited cultures; 10 pl toluene was also shown to conditions of glucose limitation and glucose excess be optimal for glucose-excess cultures. The conditions (nitrogen limitation), the test organism was inoculated simultaneously into two chemostats, each containing TABLE 1. Effect of toluene concentration on PEP a defined medium slightly modified to that of Carlsson phosphotransferase activity in cells of S. mutans inaltered by (M3) (2). The basic M3 medium was Ingbritt grown in complex medium at different creasing the concentration of aspartic acid and cysdilution rates with a glucose limitation teine to 400 mg/liter and that of leucine, isoleucine, and lysine to 200 mg/liter while removing glucose and PTS activity at D (h-Q) of: Toluenea acetate. In addition, MgS04.7H20 was increased to 0.05 0.1 0.5 0.3 0.4 0.2 400 mg/liter while KH2PO4 and K2HPO4 were decreased to 1.33 and 2.66 g/liter, respectively. For the 0 10.2b 11.4 11.1 5.6 2.8 1.2
cells grown at various growth rates under conditions of glucose limitation and glucose excess (nitrogen limitation) to transport and metabolize various sugars. The results show that PTS activity is repressed at all growth rates tested (mean generation time = 1.5 to 12 h) under conditions of glucose excess compared with activity in cells grown with a glucose limitation. A comparison of the rate of sugar utilization by S. mutans Ingbritt growing in the chemostat with the rate of transport via the phosphotransferase system demonstrated that only under conditions of slow growth (mean generation time >4 to 7 h) was the PTS adequate to account for all of the glucose transported by cells of the organism.
glucose excess (nitrogen limited) culture, the basic medium was modified to contain 1/5 the concentration of amino acids, while glucose was added at 55 mg/ml such that during growth asparagine and arginine were limiting growth. The glucose-limited culture was grown in the basal M3 medium modified to contain 5 mg of glucose per ml. Washed cell experiments. The glycolytic activity of the chemostat-grown cells was studied by measuring the rate of acid production from glucose, fructose, or
5 10 15 20 25
40.0 47.4 46.4 45.7 21.7
52.2
45.1
26.5
13.2
52.0
44.2
21.0
9.4
5.3 7.6 7.0 5.7 5.0
' Volume of toluene per milliliter of cell suspension (30 mg/ml). b Nanomoles x milligram (dry weight) of cells-' x minutes.
226
ELLWOOD, PHIPPS, AND HAMILTON
of assay were as previously described (6). Net PITS activity is expressed as nanomoles of pyruvate fornned x milligram (dry weight) of cells- x minute'. Assay for glucose transport via proton 8ynmport. Cells of S. mutans Ingbritt were assayed for glucose - Ho symport activity under the conditi ons described by Henderson et al. (8). Cells, growing in the chemostat under conditions of glucose limitation sat a dilution rate of D = 0.4 h-1, were collected in ice foi h, harvested, and washed as previously described (6) One-half of the cells were suspended in saline (43 mg/ml), whereas the remainder were suspended in 420 mM phosphate buffer (pH 7.0) (36 mg/ml). A portion of the latter cells was incubated at 370C for 60 miu i to deplete any glycogen present, washed, and suspendded in anoxic 150 mM KCI-2 mM glycylglycine buffer ((pH 6.8) (8). Proton uptake was measured at 250C with118 mg (dry weight) of cells suspended in 6 ml of the lai r buffer. Rate of glucose transport in the chemositat. Glucose uptake by cells growing in the chemostat i determined by the equation of Herbert and Kornb
INFECTr. IMMUN.
TABLE 2. Effect of dilution rate on the growth parameters of S. mutans Ingbritt growing under conditions ofglucose limitation and glucose excess (nitrogen limited)
Condition' D - 0.05 h-1
irM
Ex
Organiim
Residual glucose
Ya.e (mg of
ml)
(mg/ml)
cells/mg of glucose)
0.81 0.63
0 14.5
0.172 0.016
0.93 0.46
0 14.2
0.186 0.011
1.02 0.59
0 15.6
0.204 0.015
yield (mg
0.1 h-1
D
IAM Ex D - 0.2h-1 Lim
Ex
D = 0.4 h-1 Lim 0.88 0 0.176 Ex 0.58 16.0 0.015 'Lim, Glucose limitation; Ex, glucose excess.
Uerg
(9): D(SD- S)
(1) (x) x 60 where q, = the rate of glucose utilization (nanomoles x milligram [dry weight] of cells-l x minute-), D = the dilution rate (hours-'), So = glucose concentration in the inflowing medium (nanomoles per milliliter), S = glucose concentration in the outflowing culture (nanomoles per milliliter), and x = dry weight of cells (milligrams per milliliter). Rate of acid production in the chemostat. Because S. mutans Ingbritt ferments glucose during growth with the production of acid end products and because the chemostat cultures were maintained at pH 6.5 by the addition of NaOH, it was possible to determine the rate of acid production in the chemostat from the following equation, which is analogous to equation 1: Das x A q
(x) X 60
(2)
where qm = the rate of alkali addition (nanomoles x milligram [dry weight] of cells-1 X minute'), A = concentration of alkali (nanomoles per milliliter), x = dry weight of cells (milligrams per milliliter), and Dam = dilution rate for alkali (volume of alkali added, hour-' per volume of culture vessel). Analytical procedures. Glucose and metabolic end products present in the culture fluid were assayed after rapid filtration (
