Vol. 21, No. 1
INFECTION AND IMMUNITY, July 1978, p. 206-212 0019-9567/78/0021-0206$02.00/0 Copyright (C 1978 American Society for Microbiology
Printed in U.S.A.
Lactate Dehydrogenase Mutants of Streptococcus mutans: Isolation and Preliminary Characterization JEFFREY D. HILLMAN Department of Microbiology, Forsyth Dental Center, Boston, Massachusetts 02115 Received for publication 7 February 1978
Mutants of Streptococcus mutans were isolated which lack the enzyme activity L(+)-lactate dehydrogenase. Reversion studies indicate that the genetic defects are in the structural gene for the enzyme. The mutants produce less titratable acid from glucose, adhere better to hydroxyapatite, and accumulate more plaque when grown in the presence of sucrose than does the parent strain.These findings suggest a possible use for the mutants as effector strains in the replacement therapy of dental caries. In recent years, considerable success has been achieved in preventing and controlling certain bacterial infections by purposefully colonizing susceptible host tissues with nonvirulent analogs of disease-causing microorganisms (6, 27). The basis of this phenomenon, termed bacterial interference, is in no single case completely understood, but in general terms appears to involve a competitive and/or antibiotic action of the nonvirulent so-called effector strain on its pathogenic counterpart (26). Bacterial infections in general should be amenable to control by this approach, the only prerequisites being that the particular pathogen involved be identified and that a competitive, nonvirulent analog be isolated. A large body of evidence has implicated Streptococcus mutans as a principal pathogen in dental caries of both rodents (9, 11, 15, 32) and humans (7, 16, 19). The characterisitc features of this organism which likely account for its cariogenic potential include its ability to accumulate on teeth (12, 22) and to produce, via fermentative processes, large amounts of lactic acid (8, 14, 28). In the course of our studies, we isolated certain mutant strains of S. mutans which appeared to make less acid than the normal, wild-type strain, and which should therefore be less virulent. The metabolic defect in these strains has been shown to be a deficiency in the enzyme activity, L(+)-lactate dehydrogenase (LDH), which is responsible for lactic acid production by this bacterium (2, 4). The present report describes the isolation and preliminary characterization of these mutants, emphasizing those phenotypic properties which may allow them to serve as effector strains in controlling the incidence and severity of dental caries.
MATERIALS AND METHODS Organisms and media. Strain BHT-2 is a spontaneous streptomycin-resistant derivative of S. mutans BHT; it was isolated by the methods of Lederberg (18), by using Trypticase soy agar containing 1 mg of streptomycin sulfate per ml as the selective medium. Glucose tetrazolium plates were prepared by the methods of Lederberg (17) and contained 25.5 g of antibiotic medium no. 2, 50 mg of 1,3,5-triphenyl tetrazoliunchloride, and 1% glucose (wt/vol) per liter of medium. Strains of bacteria were maintained in 50% glycerol stabs at -20'C and were streaked on glucose tetrazolium medium weekly to check contamination and reversion and to serve as a source of inocula. Mutagenesis. Exponentially growing cells in ToddHewitt broth containing 0.5% glucose were washed twice in 0.1 M phosphate-buffered saline (pH 7.0) and resuspended to a density of ca. 1.2 x 108 colony-forming units per ml. A 0.015-ml amount of ethyl methane sulfonate was added per ml of cell suspension and blended in a Vortex mixer into solution. After 60 min of incubation in a 370C water bath, mutagenesis was stopped by diluting the cell suspension with 9 volumes of phosphate-buffered saline. The cells were harvested by centrifugation and resuspended in 10 ml of ToddHewitt broth containing 0.5% glucose. The culture was then grown to stationary phase overnight. Enzyme assays. Crude cell extracts were prepared from 100 ml of overnight cultures grown in ToddHewitt broth containing 0.5% glucose. The cells were harvested by centrifugation at 40C and resuspended in 1/50 volume of 0.05 M potassium phosphate buffer (pH 6.2) containing 0.02 M fructose 1,6-diphosphate to stabilize LDH activity (2). Cells were broken by one passage through a French press at 20,000 lb/in2 and cell debris was removed by centrifugation at 15,000 x g for 30 min. The resulting supernatants served as the crude extracts. Extracts were kept on ice, and enzyme activities were measured within 3 h. The assay for L(+)-LDH activity was that described by Brown and Wittenberger (2). Heat lability studies used a modification of that assay: samples of extract containing 100 206
VOL. 21, 1978 to 300 Itg of protein were added to 1.1 ml of 0.05 M potassium phosphate buffer (pH 6.2), placed in a 550C water bath for various times, chilled in ice, and returned to 250C. Then 0.95-ml samples were assayed for remaining activity by the addition of 20 tl of 0.1 M reduced nicotinamide adenine dinucleotide, 20 pI of 1 M fructose 1,6-diphosphate, and 10 of 1 M sodium pyruvate; the rate of change in adsorbance at 340 nm was recorded. Other enzyme assays used in this study are cited in the text. Acid production by resting cells. One-liter batch cultures of BHT-2 and the mutants were grown aerobically standing in Todd-Hewitt broth containing 0.5% glucose at 370C overnight. Cells were harvested by centrifugation at 40C and washed twice with 1 mM potassium phosphate buffer (adjusted to the appropriate pH) containing 50 mM KCI. The pellets were resuspended to a final volume of 100 ml in buffer and were stored at 4"C until used (usually within 4 h). Fermentations were carried out with constant rapid stirring in a 150-ml closed reaction vessel maintained at 370C by a circulating water jacket. The pH was maintained at the desired level by a model 162 Automatic pH Controller (New Brunswick Scientific Co., New Brunswick, N.J.) with 1 N NaOH serving as the titrant. After 30 min of temperature and pH equilibration, a 1-ml sample of cell suspension was removed as a zero-time control. A 0.2-ml amount of 1.2 N H2SO4 was added to this sample and blended in a Vortex mixer before the addition of 0.01 ml of 20% (wt/vol) glucose. The fermentation was initiated by the addition of 1 ml of 20% glucose to the remaining 99 ml of cell suspension. The amount of titrant added was monitored at 1-min intervals, and at varying times 1ml samples of the cell suspension were removed and added to vials containing 0.2 ml of 1.2 N H2SO4. Samples were stored frozen at -20°C until glucose concentrations could be determined (glucose oxidase method, Sigma Chemical Co., St. Louis, Mo.). Dry weights of the cell suspensions were measured as described by Carlsson (3) and were between 5.4 and 6.5 mg per ml. Adsorption to hydroxyapatite. The affinity of strain BHT-2 and the mutants for untreated and saliva-treated hydroxyapatite was determined by the methods of Clark et al. (5). Cultures of the strains tested were grown overnight in Trypticase soy broth containing 0.2% glucose and 1 mCi of [3H]thymidine per ml. Cells were harvested by centrifugation, washed twice in 1 mM sodium phosphate buffer (pH 6.2) containing 50 mM KC1, 1 mM CaCl2, and 0.1 mM MgCl2, and resuspended in buffer to give 2.2 x 107 colony-forming units per ml. A 40-mg amount of washed spheroidal hydroxyapatite (BDH Biochemicals, Poole, England) was added to 1.5 ml of cell suspension and incubated for 1 h at 25°C with constant agitation. After 90 min, cells remaining unattached to hydroxyapatite were removed by repeated washings. The number of attached cells was determined by liquid scintillation counting of apatite samples, compared to that of controls. Saliva treatment entailed incubation of 40 mg of apatite with 1.5 ml of heat-inactivated, clarified whole saliva for 24 h at room temperature. Chemicals. Unless otherwise specified, all reagents
LDH MUTANTS OF S. MUTANS
207
used in this study were obtained from the Sigma Chemical Co. and were the highest grade available. Auxilliary enzymes for spectrophotometric assays were obtained from Boehringer Mannheim Corp., New York, N.Y.
RESULTS
Isolation of mutants. S. mutans BHT-2 produces small white colonies when incubated in candle jars on glucose tetrazolium medium. After mutagenesis with ethyl methane sulfonate we were able to observe a small number (ca. 1% frequency) of red mutant colonies among the background of white, wild-type appearing colonies. Forty such colonies were isolated and purified by streaking. The majority of these strains proved likely to be defective in the early phases of glucose metabolism, since they yielded white colonies when streaked on tetrazolium medium containing carbon sources other than glucose. Five strains, however, appeared to be pleiotropically affected, producing red colonies when glucose, fructose, mannitol, or sorbitol served as the carbon source. On the basis of this phenotype, it seemed possible that the mutants possessed a defect in lactic acid production; indeed, cell extracts of two of these strains (JH137 and -140, Table 1) were found to have only ca. 1% of the parental levels of L(+)-LDH activity. Spectrophotometric assays of other glycolytic enzymes, including phosphoglucose isomerase, fructose diphosphate aldolase, and pyruvate kinase, revealed a two- to threefold higher level of specific activities in the mutants compared to the parent. In all cases, mixed wild-type and mutant extracts gave additive activities. In vitro assays using cell extracts of the other three strains, JH138, -139, and -141, gave similar results (data now shown). Growth properties. On the basis of certain properties, the five mutants could be divided TABLE 1. Specific activities of some enzymes in parent and mutant extracts Enzyme (8p act, U/mg of protein)' Strain
LDH 0.69 0.02 0.01 of cell
PGI FDA PK BHT-2 0.09 0.03 0.05 JH137 0.17 0.10 0.14 JH140 0.17 0.07 0.11 extracts and the assay for L(+)-LDH are described in the text. Other assays were phosphoglucose isomerase (PGI; 10), fructose diphosphate aldolase (FDA; 21), and pyruvate kinase (PK; 31). Specific activities are units (micromoles of product formed per minute) per milligram of protein. Protein was measured by the method of Lowry et al. (20) by using lyophilized bovine serum albumin as the standard.
"Preparation
208
HILLMAN
into two groups: the group 1 mutants, which consisted of three strains (JH137, -138, and -139), appeared less red on glucose tetrazolium plates than did the two representatives of group 2 (JH140 and -141). Both groups of mutants could easily be distinguished from the parent strain on this medium, not only by their color but also by their relatively larger colony size. A second parameter serving to differentiate the two classes of mutants was the terminal pH attained by growing and nongrowing cells of the various strains. The group 2 mutants, represented by JH140, were significantly less effective in reducing the pH of the culture liquor than were their parent, BHT-2, during 48 h of incubation in Todd-Hewitt broth containing 1% glucose (Table 2). Group 1 mutants, represented by JH137, produced intermediate values. The differences observed did not reflect differences in the amounts of glucose consumed. Cell yields for the various strains tested under these conditions also did not differ significantly, nor did apparent growth rates as had been previously reported (13). Similar differences in terminal pH attained were also observed using limiting (0.2%, wt/vol) glucose concentrations, or when washed, resting cell suspensions were incubated for 48 h in fermentation buffer (30) containing limiting or excess glucose. Third, the two classes of mutants could be distinguished on the basis of the amount of lactic acid they produced during fermentation of glucose (Table 2). Both growing and nongrowing cultures of group 1 strains yielded ca. 40 to 60% of the parental levels of lactic acid, whereas group 2 mutants produced no detectable lactic acid. On the basis of these results and the fact that all mutants were obtained from the same selection, it seems likely that the three strains of group 1 are sister strains, as are the two strains of group 2. Subsequent studies have therefore dealt with just one representative from each group. The likeliest explanation for the observed differences between the two groups is that group 1 strains possess a leaky LDH activity in situ which, because of its lability, has not been observed in vitro. Tending to support this notion was the isolation on glucose tetrazolium medium of a spontaneous LDH-deficient mutant (ca. i0' frequency), which appears phenotypically identical to the mutagen-induced mutants of group 2. Since, in general, spontaneous mutants tend to have single genetic lesions, multiple mutations in the group 2 strains seem an unlikely explanation for the phenotypic differences observed between groups. Acid production from glucose. Fermentation of glucose by washed cell suspensions of BHT-2 and the group 2 mutant, JH140, were
INFECT. IMMUN.
TABLE 2. Growth properties of BHT-2 and LDHdeficient mutants' Strain Terminal Strain pH
Cell yield
(ODmv)
Lactic acid produced
Glucose consumed
4.38 BHT-2 2.4 33.9 47.2 17.7 JH137 4.48 2.1 50.0 2.4
INFECTION AND IMMUNITY, July 1978, p. 206-212 0019-9567/78/0021-0206$02.00/0 Copyright (C 1978 American Society for Microbiology
Printed in U.S.A.
Lactate Dehydrogenase Mutants of Streptococcus mutans: Isolation and Preliminary Characterization JEFFREY D. HILLMAN Department of Microbiology, Forsyth Dental Center, Boston, Massachusetts 02115 Received for publication 7 February 1978
Mutants of Streptococcus mutans were isolated which lack the enzyme activity L(+)-lactate dehydrogenase. Reversion studies indicate that the genetic defects are in the structural gene for the enzyme. The mutants produce less titratable acid from glucose, adhere better to hydroxyapatite, and accumulate more plaque when grown in the presence of sucrose than does the parent strain.These findings suggest a possible use for the mutants as effector strains in the replacement therapy of dental caries. In recent years, considerable success has been achieved in preventing and controlling certain bacterial infections by purposefully colonizing susceptible host tissues with nonvirulent analogs of disease-causing microorganisms (6, 27). The basis of this phenomenon, termed bacterial interference, is in no single case completely understood, but in general terms appears to involve a competitive and/or antibiotic action of the nonvirulent so-called effector strain on its pathogenic counterpart (26). Bacterial infections in general should be amenable to control by this approach, the only prerequisites being that the particular pathogen involved be identified and that a competitive, nonvirulent analog be isolated. A large body of evidence has implicated Streptococcus mutans as a principal pathogen in dental caries of both rodents (9, 11, 15, 32) and humans (7, 16, 19). The characterisitc features of this organism which likely account for its cariogenic potential include its ability to accumulate on teeth (12, 22) and to produce, via fermentative processes, large amounts of lactic acid (8, 14, 28). In the course of our studies, we isolated certain mutant strains of S. mutans which appeared to make less acid than the normal, wild-type strain, and which should therefore be less virulent. The metabolic defect in these strains has been shown to be a deficiency in the enzyme activity, L(+)-lactate dehydrogenase (LDH), which is responsible for lactic acid production by this bacterium (2, 4). The present report describes the isolation and preliminary characterization of these mutants, emphasizing those phenotypic properties which may allow them to serve as effector strains in controlling the incidence and severity of dental caries.
MATERIALS AND METHODS Organisms and media. Strain BHT-2 is a spontaneous streptomycin-resistant derivative of S. mutans BHT; it was isolated by the methods of Lederberg (18), by using Trypticase soy agar containing 1 mg of streptomycin sulfate per ml as the selective medium. Glucose tetrazolium plates were prepared by the methods of Lederberg (17) and contained 25.5 g of antibiotic medium no. 2, 50 mg of 1,3,5-triphenyl tetrazoliunchloride, and 1% glucose (wt/vol) per liter of medium. Strains of bacteria were maintained in 50% glycerol stabs at -20'C and were streaked on glucose tetrazolium medium weekly to check contamination and reversion and to serve as a source of inocula. Mutagenesis. Exponentially growing cells in ToddHewitt broth containing 0.5% glucose were washed twice in 0.1 M phosphate-buffered saline (pH 7.0) and resuspended to a density of ca. 1.2 x 108 colony-forming units per ml. A 0.015-ml amount of ethyl methane sulfonate was added per ml of cell suspension and blended in a Vortex mixer into solution. After 60 min of incubation in a 370C water bath, mutagenesis was stopped by diluting the cell suspension with 9 volumes of phosphate-buffered saline. The cells were harvested by centrifugation and resuspended in 10 ml of ToddHewitt broth containing 0.5% glucose. The culture was then grown to stationary phase overnight. Enzyme assays. Crude cell extracts were prepared from 100 ml of overnight cultures grown in ToddHewitt broth containing 0.5% glucose. The cells were harvested by centrifugation at 40C and resuspended in 1/50 volume of 0.05 M potassium phosphate buffer (pH 6.2) containing 0.02 M fructose 1,6-diphosphate to stabilize LDH activity (2). Cells were broken by one passage through a French press at 20,000 lb/in2 and cell debris was removed by centrifugation at 15,000 x g for 30 min. The resulting supernatants served as the crude extracts. Extracts were kept on ice, and enzyme activities were measured within 3 h. The assay for L(+)-LDH activity was that described by Brown and Wittenberger (2). Heat lability studies used a modification of that assay: samples of extract containing 100 206
VOL. 21, 1978 to 300 Itg of protein were added to 1.1 ml of 0.05 M potassium phosphate buffer (pH 6.2), placed in a 550C water bath for various times, chilled in ice, and returned to 250C. Then 0.95-ml samples were assayed for remaining activity by the addition of 20 tl of 0.1 M reduced nicotinamide adenine dinucleotide, 20 pI of 1 M fructose 1,6-diphosphate, and 10 of 1 M sodium pyruvate; the rate of change in adsorbance at 340 nm was recorded. Other enzyme assays used in this study are cited in the text. Acid production by resting cells. One-liter batch cultures of BHT-2 and the mutants were grown aerobically standing in Todd-Hewitt broth containing 0.5% glucose at 370C overnight. Cells were harvested by centrifugation at 40C and washed twice with 1 mM potassium phosphate buffer (adjusted to the appropriate pH) containing 50 mM KCI. The pellets were resuspended to a final volume of 100 ml in buffer and were stored at 4"C until used (usually within 4 h). Fermentations were carried out with constant rapid stirring in a 150-ml closed reaction vessel maintained at 370C by a circulating water jacket. The pH was maintained at the desired level by a model 162 Automatic pH Controller (New Brunswick Scientific Co., New Brunswick, N.J.) with 1 N NaOH serving as the titrant. After 30 min of temperature and pH equilibration, a 1-ml sample of cell suspension was removed as a zero-time control. A 0.2-ml amount of 1.2 N H2SO4 was added to this sample and blended in a Vortex mixer before the addition of 0.01 ml of 20% (wt/vol) glucose. The fermentation was initiated by the addition of 1 ml of 20% glucose to the remaining 99 ml of cell suspension. The amount of titrant added was monitored at 1-min intervals, and at varying times 1ml samples of the cell suspension were removed and added to vials containing 0.2 ml of 1.2 N H2SO4. Samples were stored frozen at -20°C until glucose concentrations could be determined (glucose oxidase method, Sigma Chemical Co., St. Louis, Mo.). Dry weights of the cell suspensions were measured as described by Carlsson (3) and were between 5.4 and 6.5 mg per ml. Adsorption to hydroxyapatite. The affinity of strain BHT-2 and the mutants for untreated and saliva-treated hydroxyapatite was determined by the methods of Clark et al. (5). Cultures of the strains tested were grown overnight in Trypticase soy broth containing 0.2% glucose and 1 mCi of [3H]thymidine per ml. Cells were harvested by centrifugation, washed twice in 1 mM sodium phosphate buffer (pH 6.2) containing 50 mM KC1, 1 mM CaCl2, and 0.1 mM MgCl2, and resuspended in buffer to give 2.2 x 107 colony-forming units per ml. A 40-mg amount of washed spheroidal hydroxyapatite (BDH Biochemicals, Poole, England) was added to 1.5 ml of cell suspension and incubated for 1 h at 25°C with constant agitation. After 90 min, cells remaining unattached to hydroxyapatite were removed by repeated washings. The number of attached cells was determined by liquid scintillation counting of apatite samples, compared to that of controls. Saliva treatment entailed incubation of 40 mg of apatite with 1.5 ml of heat-inactivated, clarified whole saliva for 24 h at room temperature. Chemicals. Unless otherwise specified, all reagents
LDH MUTANTS OF S. MUTANS
207
used in this study were obtained from the Sigma Chemical Co. and were the highest grade available. Auxilliary enzymes for spectrophotometric assays were obtained from Boehringer Mannheim Corp., New York, N.Y.
RESULTS
Isolation of mutants. S. mutans BHT-2 produces small white colonies when incubated in candle jars on glucose tetrazolium medium. After mutagenesis with ethyl methane sulfonate we were able to observe a small number (ca. 1% frequency) of red mutant colonies among the background of white, wild-type appearing colonies. Forty such colonies were isolated and purified by streaking. The majority of these strains proved likely to be defective in the early phases of glucose metabolism, since they yielded white colonies when streaked on tetrazolium medium containing carbon sources other than glucose. Five strains, however, appeared to be pleiotropically affected, producing red colonies when glucose, fructose, mannitol, or sorbitol served as the carbon source. On the basis of this phenotype, it seemed possible that the mutants possessed a defect in lactic acid production; indeed, cell extracts of two of these strains (JH137 and -140, Table 1) were found to have only ca. 1% of the parental levels of L(+)-LDH activity. Spectrophotometric assays of other glycolytic enzymes, including phosphoglucose isomerase, fructose diphosphate aldolase, and pyruvate kinase, revealed a two- to threefold higher level of specific activities in the mutants compared to the parent. In all cases, mixed wild-type and mutant extracts gave additive activities. In vitro assays using cell extracts of the other three strains, JH138, -139, and -141, gave similar results (data now shown). Growth properties. On the basis of certain properties, the five mutants could be divided TABLE 1. Specific activities of some enzymes in parent and mutant extracts Enzyme (8p act, U/mg of protein)' Strain
LDH 0.69 0.02 0.01 of cell
PGI FDA PK BHT-2 0.09 0.03 0.05 JH137 0.17 0.10 0.14 JH140 0.17 0.07 0.11 extracts and the assay for L(+)-LDH are described in the text. Other assays were phosphoglucose isomerase (PGI; 10), fructose diphosphate aldolase (FDA; 21), and pyruvate kinase (PK; 31). Specific activities are units (micromoles of product formed per minute) per milligram of protein. Protein was measured by the method of Lowry et al. (20) by using lyophilized bovine serum albumin as the standard.
"Preparation
208
HILLMAN
into two groups: the group 1 mutants, which consisted of three strains (JH137, -138, and -139), appeared less red on glucose tetrazolium plates than did the two representatives of group 2 (JH140 and -141). Both groups of mutants could easily be distinguished from the parent strain on this medium, not only by their color but also by their relatively larger colony size. A second parameter serving to differentiate the two classes of mutants was the terminal pH attained by growing and nongrowing cells of the various strains. The group 2 mutants, represented by JH140, were significantly less effective in reducing the pH of the culture liquor than were their parent, BHT-2, during 48 h of incubation in Todd-Hewitt broth containing 1% glucose (Table 2). Group 1 mutants, represented by JH137, produced intermediate values. The differences observed did not reflect differences in the amounts of glucose consumed. Cell yields for the various strains tested under these conditions also did not differ significantly, nor did apparent growth rates as had been previously reported (13). Similar differences in terminal pH attained were also observed using limiting (0.2%, wt/vol) glucose concentrations, or when washed, resting cell suspensions were incubated for 48 h in fermentation buffer (30) containing limiting or excess glucose. Third, the two classes of mutants could be distinguished on the basis of the amount of lactic acid they produced during fermentation of glucose (Table 2). Both growing and nongrowing cultures of group 1 strains yielded ca. 40 to 60% of the parental levels of lactic acid, whereas group 2 mutants produced no detectable lactic acid. On the basis of these results and the fact that all mutants were obtained from the same selection, it seems likely that the three strains of group 1 are sister strains, as are the two strains of group 2. Subsequent studies have therefore dealt with just one representative from each group. The likeliest explanation for the observed differences between the two groups is that group 1 strains possess a leaky LDH activity in situ which, because of its lability, has not been observed in vitro. Tending to support this notion was the isolation on glucose tetrazolium medium of a spontaneous LDH-deficient mutant (ca. i0' frequency), which appears phenotypically identical to the mutagen-induced mutants of group 2. Since, in general, spontaneous mutants tend to have single genetic lesions, multiple mutations in the group 2 strains seem an unlikely explanation for the phenotypic differences observed between groups. Acid production from glucose. Fermentation of glucose by washed cell suspensions of BHT-2 and the group 2 mutant, JH140, were
INFECT. IMMUN.
TABLE 2. Growth properties of BHT-2 and LDHdeficient mutants' Strain Terminal Strain pH
Cell yield
(ODmv)
Lactic acid produced
Glucose consumed
4.38 BHT-2 2.4 33.9 47.2 17.7 JH137 4.48 2.1 50.0 2.4
