Vol. 19, No. 3
INFECTION AND IMMUNITY, Mar. 1978, p. 934-942 0019-9567/78/0019-0934$02.00/O Copyright X 1978 American Society for Microbiology
Printed in U.S.A.
Involvement of Phosphoenolpyruvate in the Catabolism of Caries-Conducive Disaccharides by Streptococcus mutans: Lactose Transport ROBERT CALMES Department of Oral Biology, College of Dentistry, Albert B. Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40506 Received for publication 21 July 1977
The mechanisms for transport and hydrolysis of lactose were investigated in five cariogenic strains (HS6, AHT, FAl, NCTC 10449, and SL1) representing the four serogenetic groups of Streptococcus mutans. The systems for transport and hydrolysis of lactose had the characteristics of a phosphoenolpyruvate (PEP)dependent lactose (Lac) phosphotransferase (PT) system and phospho-,8-galactosidase (P-fl-gal), respectively, in all strains tested, except strain HS6. Decryptified cells required PEP and Mge for transport of the non-metabolizable model ,8-galactosides o-nitrophenyl-,8-D-galactopyranoside (ONPG) and thiomethyl-,8-D-galactopyranoside (TMG). Substitution of 2-phosphoglycerate (2PG) for PEP also stimulated the Lac PT system. Other potential high-energy phosphate donors (adenosine tri-, di-, and monophosphates and guanosine triphosphate) did not stimulate the Lac PT system. Sodium fluoride had no effect upon the PEP-dependent Lac PT system in decryptified cells with PEP as the energy source; however, when 2-PG was used as the energy source, F- inhibited ONPG phosphorylation. With intact cells which must generate PEP endogenously, the presence of F- in concentration o10 mM completely inhibited the Lac PT system, presumably through inhibition of 2-PG hydrolyase (EC 4.2.1.11; enolase). Both intact and decryptified cells accumulated a phosphorylated derivative of TMG that behaved chromatographically as TMG-phosphate. After alkaline phosphatase treatment, the derivative had an Rf identical to that of TMG. No fB-galactosidase (fl-gal) activity was detected with ONPG as the substrate; hydrolysis occurred only when ONPG-6-phosphate was supplied as the substrate. Strain HS6 apparently transported lactose by an active transport-type system in which the accumulated intracellular product was the free disaccharide based on the following criteria: (i) ONPG transport and hydrolysis in decryptified cells was not stimulated by PEP; (ii) ONPG hydrolysis occurred in the absence of PEP; and (iii) ONPG-6-phosphate was not hydrolyzed. These data indicate that, in all strains tested except strain HS6, lactose transport was mediated by a PEP-dependent Lac PT system, resulting in accumulation of lactose-phosphate that was hydrolyzed by an enzyme similar to the P-,f-gal of group N streptococci and Staphylococcus aureus; conversely, strain HS6 transported and hydrolyzed lactose by a PEP-independent transport system and fl-gal, respectively. The utilization of sucrose by Streptococcus mutans and other oral microorganisms results in the accumulation of dental plaque on teeth. The fermentative metabolism of these plaque bacteria yields end products, e.g, lactic acid, that act directly and indirectly upon the teeth and periodontium of humans, causing caries and periodontal disease (for reviews, see references 2 and 3). Studies with animal models have shown that caries will develop when the diet contains fermentable carbohydrates other than sucrose, e.g., glucose, fructose, maltose, lactose, or starch (12, 15). Little is known of the metabolism of
these carbohydrates in humans because, except for starch and lactose, they are not usually considered to be major constituents of human diets. Lactose, an acidogenic (13) disaccharide present in bovine milk in high concentrations, can be considered as a caries-conducive dietary carbohydrate of major import, especially since milk is consumed in large quantities by humans in the so-called "caries-prone" years. In addition, the fermentability of lactose by S. mutans and other plaque microorganisms may be of significance in "milk-bottle caries" of infants. Carbohydrate dissimilation by S. mutans may 934
VOL. 19, 1978
involve (i) hydrolysis by extracellular or cellassociated enzymes as for certain polysaccharides (10, 14, 40) and sucrose (2), or (ii) transport of the carbohydrate to intracellular sites for subsequent catabolism. Sugars are transported in many bacteria by the phosphoenolpyruvate (PEP)-dependent phosphotransferase (PT) system'(32), first described by Kundig et al. (23; for reviews, see references 33 and 34). Glucose PT systems have been reported in S. salivarius (20) and S. mutans (36), and PT systems for other monosaccharides (35, 36) and hexitols (29, 35) have also been described. Except for a previous report (I. R. Hamilton, J. Dent. Res. 55:B124, 1976), disaccharide transport mechanisms have not been investigated in S. mutans, although other facultative anaerobes, e.g., Staphylococcus aureus (11, 17, 18) and group N streptococci (27, 28), have been shown to use a PEP-dependent PT system for transport of lactose. Because of the reported cariogenic potential of lactose (13), a systematic investigation of lactose catabolism was initiated. In this communication, I report the presence of a PEP-dependent lactose (Lac) PT system and 6-phospho-,f-galactosidase (P,f-gal) in four strains of S. mutans and describe some properties of the Lac PT system. Characteristics of P-,f-gal will be reported elsewhere. MATERIALS AND METHODS Bacteria. S. mutans strains NCTC 10449, FAl, SL1, HS6, and AHT were generously provided by Albert T. Brown and Alan L. Coykendall. These strains of S. mutans are representative of each of the four genetic groups based on guanosine-cytosine ratios and deoxyribonucleic acid hydridization (9), kinetic activity of lactic dehydrogenase (5), and electrophoretic patterns of hexitol phosphate dehydrogenases (4), aldolases (25), enzymes of extracellular polysaccharide synthesis (7, 31), and invertases (38). These characteristics all correlate well with the serological groups described by Bratthall (1). All cultures were maintained by weekly transfer in complex medium consisting of 3% (wt/vol) fluid thioglycolate with 1.2% (wt/vol) beef extract and excess CaCO3. Growth media and conditions of culture. Cells were grown in the complex medium of Jordan et al. (19) supplemented with 0.1% (wt/vol) disaccharides or 0.2% (wt/vol) monosaccharides or hexitols. Sugars and hexitols were added aseptically to the sterile basal medium from filter-sterilized solutions. Before experimental use, all inocula were adapted to the appropriate carbon source by one transfer through the appropriate medium. Cultures were incubated at 370C in screw-cap Erlenmeyer flasks with no head space. Harvesting of cells. Cells were harvested from the mid-exponential phase of growth by centrifugation, washed twice with equal volumes of ice-cold 15 mM KCl, and resuspended in 50 mM potassium phosphate buffer (KPB) (pH 7). Preparation of decryptified cells. The membranes of washed cells were perturbed by addition of
LACTOSE TRANSPORT IN S. MUTANS
935
50 Il of toluene-acetone (1:4, vol/vol) per ml of cell suspension absorbencyy at 750 nm ='20), followed by vigorous shaking at room temperature for 5 min. The decryptified cell suspension was chilled and maintained in an ice bath. ,8-Gal assay. Assay of f-galactosidase (fl-gal) was performed by using the chromogenic substrate o-nitrophenyl-,l-D-galactopyranoside (ONPG) (24). Unless otherwise specified, reaction mixtures contained 1 mM ONPG and 20 mM KPB in a final volume of 2.5 ml. The reaction was started by adding intAct or decryptified cells and incubated at 370C. After 5 min, the reaction was terminated by addition of an equal volume of ice-cold 10% Na2CO3. The alkalinized reaction mixture was chilled in ice for 5 min and then centrifuged at 20,000 x g for 5 min at 40C to sediment cells from the assay mixture. The absorbance of the supernatant fluid was determined at 420 nm in a Gilford Stasar I spectrophotometer set to zero with a reagent blank. Under the prescribed conditions, a solution containing authentic o-nitrophenol (ONP) had a molar extinction coefficient of 1.87 x 10-3 M-l cm-', which was used to convert absorbance data to micromoles of ONP. The release of 1 pmol of ONP from ONPG per min at 370C was defined as a unit of enzyme activity. Specific activity was expressed as units per milligram
of protein.
P-f8-gal assay. A derivative of ONPG phosphorylated at carbon 6 (ONPG-6-P) was used as the substrate for phospho-fl-gal (P-,f-gal) assays. The complete reaction mixture contained 1 mM ONPG-6-P and 20 mM KPB (pH 7) in a final volume of 2.5 ml. The reaction was started by adding decryptified cells (intact cells were impermeable to ONPG-6-P) and incubated at 370C for 5 min. Other aspects of the assay, including the definition of specific activity, are as for the fl-gal assay described above. Lac PT assay. The standard assay contained the following components in a final volume of 2.5 ml: 10 mM ONPG, 1 mM PEP, 1 mM MgCl2, 10 mM NaF, and 20 mM KPB (pH 7). The reaction was started by adding intact cells or decryptified cells and incubated by 370C for 20 min, unless otherwise specified. The remainder of the assay, including the definition of the specific activity, was as for the fl-gal and P-,f-gal assays described above. EDTA washing of decryptified cells. To deplete cells of Mg2e in order to show Mg2e dependence, decryptified cells were washed with ethylenediaminetetraacetic acid (EDTA) as follows: EDTA was added to a 5-ml suspension of decryptified cells to a final concentration of 10 mM, and the cells were incubated at room temperature with gentle agitation for 5 min. The suspension was centrifuged at 20,000 x g for 5
min at 40C, and the supernatant fluid was discarded. The pellet was resuspended in 5 ml of 50 mM KPB (pH 7) containing 10 mM EDTA and 50 pJ of tolueneacetone (1:4, vol/vol) per ml. This procedure was repeated five times; the final pellet was resuspended in 5 ml of solvent-containing buffer minus EDTA and assayed for Lac PT and P-,f-gal activities. This treatment had no effect upon the specific activities of the Lac PT system (with Mg2e added back) or P-fl-gal. [14CJTIMG transport. Another non-metabolizable analog of lactose, ['4C]thiomethyl-fl-D-galactoside
936
INFECT. IMMUN.
CALMES
(TMG), was used for an alternative method to measure lactose transport. The method of transport assay was similar to that previously described (6). Briefly, the standard assay contained 1 mM [14C]TMG (0.1 ,tCi/jumol) and 45 mM KPB (pH 7). When decryptified cells were used, the assay included 1 mM PEP and 1 mM MgCl2. Uptake was initiated by addition of intact or decryptified cells, which were then incubated at 370C. Samples of 100 ,ul were removed at the indicated intervals and pipetted onto the center of a 25-mmdiameter membrane filter (type HA-6, 0.45-jm pore size; Millipore Corp.) where suction displaced the labeled medium from the cells, terminating uptake. The cells were immediately washed twice with 5-ml portions of isothermal 50 mM KPB (pH 7). Filtering and washing was accomplished in less than 30 s. Zero-time samples were obtained by adding the appropriate amount of labeled TMG after 100 ,ul of cells-buffer mixture had been diluted into 50 volumes of ice-cold 50 mM KPB; the entire volume was then filtered and washed as above. Filters were dried under an incandescent lamp, transferred to minicounting vials containing 3 ml of Aquasol, and counted in a Packard TriCarb liquid scintillation spectrometer. Under these conditions no quenching occurred. Modifications to this procedure are noted below. Extraction and isolation ofthe [14CJTMG transport product. The standard reaction mixture for these experiments contained, in a total volume of 1 ml, 1 mM ["4C]TMG (0.2 uCi/iumol) and 30 mM KPB (pH 7). The reaction was started by adding intact or decryptified cells. When decryptified cells were used, the following additions were made to the standard reaction mixture: 1 mM PEP, 1 mM MgCl2, and 10 mM NaF. After incubation at 37°C for 30 min, the reaction mixture was chilled in a ice bath for 5 min and then centrifuged at 20,000 x g at 4°C for 15 min to sediment cells. The pellet was extracted for 10 min with boiling 80% (vol/vol) ethanol followed by chilling and centrifugation as above. When decryptified cells were used, the supernatant fluid from the first centrifugation was combined with the ethanol extract; for intact cells the supernatant fluid was discarded. All extracts were stored at -20°C. Chromatography of the ['4C]TMG transport product. Portions of the extracts were subjected to ascending paper chromatography on strips (2.5 by 30 cm) of Whatman 1 paper. The solvent system contained ethyl acetate-pyridine-water (60:25:15, vol/vol/vol). In this solvent system, the stock ["C]TMG was chromatographically pure. When the solvent fronts reached 15 cm (ca. 75 min), the chromatograms were removed from the jar, air dried, and placed in a vacuum oven at 40°C for 30 min to remove all solvent. The strips were cut at 1-cm intervals; the pieces from one chromatogram were counted as described above to locate the position of the labeled TMG and derivatives. The corresponding radioactive areas from the other chromatograms were removed for identification of the labeled compounds. These methods resulted in at least 90% recovery of applied radioactivity in all cases. Identification of the phosphorylated derivative of [14CJTMG transport. ['4C]TMG and its derivative were isolated from each other by the method
described above. The portions of the chromatograms containing only the radioactive derivative were eluted with the smallest possible volume of distilled water for 12 h. The eluate was Iyophilized and redissolved in 200 IlI of distilled water. A portion of the labeled derivative was treated with 0.4 mg of highly purified alkaline phosphatase in 50 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 8) in a total volume of 150 id for 60 min at 37°C. The entire 150 pd was spotted on a chromatogram strip, developed, and counted as described above. Protein estimations. The method of Lowry et al. (26) was used to estimate the protein content of intact and decryptified cells, using bovine serum albumin as the standard. Chemicals. All biochemicals, substrates, and alkaline phosphatase were purchased from Sigma Chemical Co. [14C]TMG and Aquasol were obtained from New England Nuclear Corp. Fluid thioglycolate medium and beef extract were products of Difco. All other chemicals were of analytical grade and were purchased from Fisher Scientific Co. RESULTS
Lac PT system characteristics. Cells of strain SLi decryptified with toluene-acetone readily phosphorylated the lactose analog ONPG in the presence of PEP and Mg2e (Table 1). In the absence of PEP, or after heat treatment, little ONPG-6-P was formed. Some activity (18%) was observed in the absence of added Mg2e. Presumably this was due to bound Mg2e not removed by repeated EDTA treatment. When this treatment was omitted, the amount of Lac PT system activity present without added Mg2e increased to 74% of the complete reaction. Attempts to determine the cation specificity using the chloride salts of Mn2", Ca2+, Sn2+, Co2+, Zn2+, Sr+, and Cu2" were not successful; these cations all interfered to some extent with the coupled indicator enzyme, P-fl-gal. The addition TABLE 1. Lac PT system in S. mutans SLYa Assay condition
Relative amt of ONPG-6-P formed/min per mg of protein (%)b
Complete .....................
100 4 6 18 g2. a Decryptified cell suspensions were prepared from mid-exponential-phase lactose-grown cells. Experimental conditions were as described in Materials and Methods. Each reaction contained 4.85 mg of bacterial protein (specific activity of the complete reaction was 3.51 X 10-3). b ONPG-6-P formed in the complete reaction mixture was arbitrarily set at 100%. c Contains cells heated at 1000C for 10 min. Heated cells .................. -PEP .......................
-M
VOL. 19, 1978
LACTOSE TRANSPORT IN S. MUTANS
of lactose at ten times the concentration of ONPG present in the reaction reduced phosphorylation of ONPG by 94%, indicating that both lactose and its analog compete for the same transport system. The Lac PT system was inactive in Tris-hydrochloride buffer. Inclusion of 20 mM potassium phosphate to the Tris-hydrochloride buffer did not prevent this inactivation. The synthesis of ONPG-6-P by decryptified cells was examined in the presence of several potential high-energy phosphate-containing metabolites to determine the most effective phosphate donor for lactose (Table 2). No stimulation of ONPG-6-P synthesis was observed when adenosine tri-, di-, or monophosphate or guanosine triphosphate was supplied as the energy source. PEP and its immediate glycolytic precursor, 2-phosphoglycerate (2-PG), were the only phosphate donors tested which were capable of stimulating ONPG-6-P formation. Inclusion of 10 mM NaF in the reaction prevented ONPG-6-P synthesis when 2-PG was used as the energy source, but NaF had no effect when PEP was the phosphate donor. Table 3 illustrates the effect of fluoride on the Lac PT system in intact and decryptified cells. Intact cells (impermeable to exogenous PEP and 2-PG) exposed to fluoride at concentrations 210 mM exhibited little Lac PT system activity. However, when decryptified cells (permeable to exogenous PEP and 2-PG) were exposed to concentrations of fluoride as high as 100 mM, in the presence of exogenous TABLE 2. Effect of various energy sources upon the Lac PT system in S. mutans SLPa Relative amt of ONPG-6-P
Energy source (1 MM)u PEP (complete reaction) PEP, minus 10 mM NaF
formed/min per mg of protein
....... .......
2-PG
6
2-PG, minus 10 mM NaF ....... ATP ........................ ADP
AMP GTP None
100 110
44 0.4 5
........................ ........................ ........................
6 4 5
Decryptified cell suspensions were prepared from mid-exponential-phase lactose-grown cells. Experia
mental conditions were as described in Materials and Methods, except for the indicated substitutions for PEP. Each reaction contained 2.5 mg of bacterial protein (specific activity of the complete reaction was 5.02 x 10-3). b ATP, Adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; GTP, guanosine triphosphate. c ONPG-6-P formed in the complete reaction was arbitrarily set at 100%.
937
TABLE 3. Effect of fluoride on the Lac PT system in S. mutans SLja NaF
Relative amt of ONPG-6-P formed/min per mg of protein (%)b
(mM) Intact cells
Decryptified cells
100 88 90 92 90 a Intact or decryptified cell suspensions were pre0 0.1 1.0 10 100
100 103 100 6 2
pared from mid-exponential-phase lactose-grown cells. Experimental conditions were as described in Materials and Methods, except that the NaF concentration was varied as indicated. Each reaction contained either 3.8 mg of bacterial protein (intact cells; specific activity for the zero reaction was 12.5 x 10-') or 1.37 mg of bacterial protein (decryptified cells; specific activity for the zero reaction was 16.7 x 10-3). b ONPG-6-P formed in the zero reaction was arbitrarily set at 100%.
PEP, Lac PT system activity was not significantly affected. The absolute requirement for PEP, the ability of 2-PG to substitute for PEP, and the differential effect of fluoride upon intact and decryptified cells is consistent with the hypothesis that lactose transport in S. mutans is a group translocation process mediated by the PEP-dependent Lac PT system. This system for lactose transport is similar to those previously described in Staphylococcus aureus (11, 17, 18) and group N streptococci (27, 28). ONPG-6-P synthesis was linear with respect to time for at least 20 min in the presence of PEP (Fig. 1) and was proportional to the protein concentration up to at least 5 mg of cell protein (Fig. 2); in the absence of PEP, little ONPG-6-P synthesis occurred regardless of the length of incubation or the amount of protein present in the reaction. A pH optimum of 8.5 to 9 was observed for lactose transport (Fig. 3). Others have reported a pH optimum of 7 to 7.5 in group N streptococci using a similar assay system (8). Isolation and identification of the product of lactose transport. Evidence that lactose was transported in S. mutans by vectorial phosphorylation was obtained by isolation and identification of the transport product from intact or decryptified cells. To prevent catabolism of the accumulated transport product, a non-metabolizable analog of lactose, TMG, was used. Using intact cells, the rate of ['4C]TMG accumulation was linear for about 5 min, reaching a maximum by 20 min (Fig. 4). In a similar experiment using decryptified cells, at 20 min an ethanolic extract of the cells was chromatographed (Fig. 5). In the
938
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I-_
0
a
400 .I
z
-J
INFECTION AND IMMUNITY, Mar. 1978, p. 934-942 0019-9567/78/0019-0934$02.00/O Copyright X 1978 American Society for Microbiology
Printed in U.S.A.
Involvement of Phosphoenolpyruvate in the Catabolism of Caries-Conducive Disaccharides by Streptococcus mutans: Lactose Transport ROBERT CALMES Department of Oral Biology, College of Dentistry, Albert B. Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40506 Received for publication 21 July 1977
The mechanisms for transport and hydrolysis of lactose were investigated in five cariogenic strains (HS6, AHT, FAl, NCTC 10449, and SL1) representing the four serogenetic groups of Streptococcus mutans. The systems for transport and hydrolysis of lactose had the characteristics of a phosphoenolpyruvate (PEP)dependent lactose (Lac) phosphotransferase (PT) system and phospho-,8-galactosidase (P-fl-gal), respectively, in all strains tested, except strain HS6. Decryptified cells required PEP and Mge for transport of the non-metabolizable model ,8-galactosides o-nitrophenyl-,8-D-galactopyranoside (ONPG) and thiomethyl-,8-D-galactopyranoside (TMG). Substitution of 2-phosphoglycerate (2PG) for PEP also stimulated the Lac PT system. Other potential high-energy phosphate donors (adenosine tri-, di-, and monophosphates and guanosine triphosphate) did not stimulate the Lac PT system. Sodium fluoride had no effect upon the PEP-dependent Lac PT system in decryptified cells with PEP as the energy source; however, when 2-PG was used as the energy source, F- inhibited ONPG phosphorylation. With intact cells which must generate PEP endogenously, the presence of F- in concentration o10 mM completely inhibited the Lac PT system, presumably through inhibition of 2-PG hydrolyase (EC 4.2.1.11; enolase). Both intact and decryptified cells accumulated a phosphorylated derivative of TMG that behaved chromatographically as TMG-phosphate. After alkaline phosphatase treatment, the derivative had an Rf identical to that of TMG. No fB-galactosidase (fl-gal) activity was detected with ONPG as the substrate; hydrolysis occurred only when ONPG-6-phosphate was supplied as the substrate. Strain HS6 apparently transported lactose by an active transport-type system in which the accumulated intracellular product was the free disaccharide based on the following criteria: (i) ONPG transport and hydrolysis in decryptified cells was not stimulated by PEP; (ii) ONPG hydrolysis occurred in the absence of PEP; and (iii) ONPG-6-phosphate was not hydrolyzed. These data indicate that, in all strains tested except strain HS6, lactose transport was mediated by a PEP-dependent Lac PT system, resulting in accumulation of lactose-phosphate that was hydrolyzed by an enzyme similar to the P-,f-gal of group N streptococci and Staphylococcus aureus; conversely, strain HS6 transported and hydrolyzed lactose by a PEP-independent transport system and fl-gal, respectively. The utilization of sucrose by Streptococcus mutans and other oral microorganisms results in the accumulation of dental plaque on teeth. The fermentative metabolism of these plaque bacteria yields end products, e.g, lactic acid, that act directly and indirectly upon the teeth and periodontium of humans, causing caries and periodontal disease (for reviews, see references 2 and 3). Studies with animal models have shown that caries will develop when the diet contains fermentable carbohydrates other than sucrose, e.g., glucose, fructose, maltose, lactose, or starch (12, 15). Little is known of the metabolism of
these carbohydrates in humans because, except for starch and lactose, they are not usually considered to be major constituents of human diets. Lactose, an acidogenic (13) disaccharide present in bovine milk in high concentrations, can be considered as a caries-conducive dietary carbohydrate of major import, especially since milk is consumed in large quantities by humans in the so-called "caries-prone" years. In addition, the fermentability of lactose by S. mutans and other plaque microorganisms may be of significance in "milk-bottle caries" of infants. Carbohydrate dissimilation by S. mutans may 934
VOL. 19, 1978
involve (i) hydrolysis by extracellular or cellassociated enzymes as for certain polysaccharides (10, 14, 40) and sucrose (2), or (ii) transport of the carbohydrate to intracellular sites for subsequent catabolism. Sugars are transported in many bacteria by the phosphoenolpyruvate (PEP)-dependent phosphotransferase (PT) system'(32), first described by Kundig et al. (23; for reviews, see references 33 and 34). Glucose PT systems have been reported in S. salivarius (20) and S. mutans (36), and PT systems for other monosaccharides (35, 36) and hexitols (29, 35) have also been described. Except for a previous report (I. R. Hamilton, J. Dent. Res. 55:B124, 1976), disaccharide transport mechanisms have not been investigated in S. mutans, although other facultative anaerobes, e.g., Staphylococcus aureus (11, 17, 18) and group N streptococci (27, 28), have been shown to use a PEP-dependent PT system for transport of lactose. Because of the reported cariogenic potential of lactose (13), a systematic investigation of lactose catabolism was initiated. In this communication, I report the presence of a PEP-dependent lactose (Lac) PT system and 6-phospho-,f-galactosidase (P,f-gal) in four strains of S. mutans and describe some properties of the Lac PT system. Characteristics of P-,f-gal will be reported elsewhere. MATERIALS AND METHODS Bacteria. S. mutans strains NCTC 10449, FAl, SL1, HS6, and AHT were generously provided by Albert T. Brown and Alan L. Coykendall. These strains of S. mutans are representative of each of the four genetic groups based on guanosine-cytosine ratios and deoxyribonucleic acid hydridization (9), kinetic activity of lactic dehydrogenase (5), and electrophoretic patterns of hexitol phosphate dehydrogenases (4), aldolases (25), enzymes of extracellular polysaccharide synthesis (7, 31), and invertases (38). These characteristics all correlate well with the serological groups described by Bratthall (1). All cultures were maintained by weekly transfer in complex medium consisting of 3% (wt/vol) fluid thioglycolate with 1.2% (wt/vol) beef extract and excess CaCO3. Growth media and conditions of culture. Cells were grown in the complex medium of Jordan et al. (19) supplemented with 0.1% (wt/vol) disaccharides or 0.2% (wt/vol) monosaccharides or hexitols. Sugars and hexitols were added aseptically to the sterile basal medium from filter-sterilized solutions. Before experimental use, all inocula were adapted to the appropriate carbon source by one transfer through the appropriate medium. Cultures were incubated at 370C in screw-cap Erlenmeyer flasks with no head space. Harvesting of cells. Cells were harvested from the mid-exponential phase of growth by centrifugation, washed twice with equal volumes of ice-cold 15 mM KCl, and resuspended in 50 mM potassium phosphate buffer (KPB) (pH 7). Preparation of decryptified cells. The membranes of washed cells were perturbed by addition of
LACTOSE TRANSPORT IN S. MUTANS
935
50 Il of toluene-acetone (1:4, vol/vol) per ml of cell suspension absorbencyy at 750 nm ='20), followed by vigorous shaking at room temperature for 5 min. The decryptified cell suspension was chilled and maintained in an ice bath. ,8-Gal assay. Assay of f-galactosidase (fl-gal) was performed by using the chromogenic substrate o-nitrophenyl-,l-D-galactopyranoside (ONPG) (24). Unless otherwise specified, reaction mixtures contained 1 mM ONPG and 20 mM KPB in a final volume of 2.5 ml. The reaction was started by adding intAct or decryptified cells and incubated at 370C. After 5 min, the reaction was terminated by addition of an equal volume of ice-cold 10% Na2CO3. The alkalinized reaction mixture was chilled in ice for 5 min and then centrifuged at 20,000 x g for 5 min at 40C to sediment cells from the assay mixture. The absorbance of the supernatant fluid was determined at 420 nm in a Gilford Stasar I spectrophotometer set to zero with a reagent blank. Under the prescribed conditions, a solution containing authentic o-nitrophenol (ONP) had a molar extinction coefficient of 1.87 x 10-3 M-l cm-', which was used to convert absorbance data to micromoles of ONP. The release of 1 pmol of ONP from ONPG per min at 370C was defined as a unit of enzyme activity. Specific activity was expressed as units per milligram
of protein.
P-f8-gal assay. A derivative of ONPG phosphorylated at carbon 6 (ONPG-6-P) was used as the substrate for phospho-fl-gal (P-,f-gal) assays. The complete reaction mixture contained 1 mM ONPG-6-P and 20 mM KPB (pH 7) in a final volume of 2.5 ml. The reaction was started by adding decryptified cells (intact cells were impermeable to ONPG-6-P) and incubated at 370C for 5 min. Other aspects of the assay, including the definition of specific activity, are as for the fl-gal assay described above. Lac PT assay. The standard assay contained the following components in a final volume of 2.5 ml: 10 mM ONPG, 1 mM PEP, 1 mM MgCl2, 10 mM NaF, and 20 mM KPB (pH 7). The reaction was started by adding intact cells or decryptified cells and incubated by 370C for 20 min, unless otherwise specified. The remainder of the assay, including the definition of the specific activity, was as for the fl-gal and P-,f-gal assays described above. EDTA washing of decryptified cells. To deplete cells of Mg2e in order to show Mg2e dependence, decryptified cells were washed with ethylenediaminetetraacetic acid (EDTA) as follows: EDTA was added to a 5-ml suspension of decryptified cells to a final concentration of 10 mM, and the cells were incubated at room temperature with gentle agitation for 5 min. The suspension was centrifuged at 20,000 x g for 5
min at 40C, and the supernatant fluid was discarded. The pellet was resuspended in 5 ml of 50 mM KPB (pH 7) containing 10 mM EDTA and 50 pJ of tolueneacetone (1:4, vol/vol) per ml. This procedure was repeated five times; the final pellet was resuspended in 5 ml of solvent-containing buffer minus EDTA and assayed for Lac PT and P-,f-gal activities. This treatment had no effect upon the specific activities of the Lac PT system (with Mg2e added back) or P-fl-gal. [14CJTIMG transport. Another non-metabolizable analog of lactose, ['4C]thiomethyl-fl-D-galactoside
936
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(TMG), was used for an alternative method to measure lactose transport. The method of transport assay was similar to that previously described (6). Briefly, the standard assay contained 1 mM [14C]TMG (0.1 ,tCi/jumol) and 45 mM KPB (pH 7). When decryptified cells were used, the assay included 1 mM PEP and 1 mM MgCl2. Uptake was initiated by addition of intact or decryptified cells, which were then incubated at 370C. Samples of 100 ,ul were removed at the indicated intervals and pipetted onto the center of a 25-mmdiameter membrane filter (type HA-6, 0.45-jm pore size; Millipore Corp.) where suction displaced the labeled medium from the cells, terminating uptake. The cells were immediately washed twice with 5-ml portions of isothermal 50 mM KPB (pH 7). Filtering and washing was accomplished in less than 30 s. Zero-time samples were obtained by adding the appropriate amount of labeled TMG after 100 ,ul of cells-buffer mixture had been diluted into 50 volumes of ice-cold 50 mM KPB; the entire volume was then filtered and washed as above. Filters were dried under an incandescent lamp, transferred to minicounting vials containing 3 ml of Aquasol, and counted in a Packard TriCarb liquid scintillation spectrometer. Under these conditions no quenching occurred. Modifications to this procedure are noted below. Extraction and isolation ofthe [14CJTMG transport product. The standard reaction mixture for these experiments contained, in a total volume of 1 ml, 1 mM ["4C]TMG (0.2 uCi/iumol) and 30 mM KPB (pH 7). The reaction was started by adding intact or decryptified cells. When decryptified cells were used, the following additions were made to the standard reaction mixture: 1 mM PEP, 1 mM MgCl2, and 10 mM NaF. After incubation at 37°C for 30 min, the reaction mixture was chilled in a ice bath for 5 min and then centrifuged at 20,000 x g at 4°C for 15 min to sediment cells. The pellet was extracted for 10 min with boiling 80% (vol/vol) ethanol followed by chilling and centrifugation as above. When decryptified cells were used, the supernatant fluid from the first centrifugation was combined with the ethanol extract; for intact cells the supernatant fluid was discarded. All extracts were stored at -20°C. Chromatography of the ['4C]TMG transport product. Portions of the extracts were subjected to ascending paper chromatography on strips (2.5 by 30 cm) of Whatman 1 paper. The solvent system contained ethyl acetate-pyridine-water (60:25:15, vol/vol/vol). In this solvent system, the stock ["C]TMG was chromatographically pure. When the solvent fronts reached 15 cm (ca. 75 min), the chromatograms were removed from the jar, air dried, and placed in a vacuum oven at 40°C for 30 min to remove all solvent. The strips were cut at 1-cm intervals; the pieces from one chromatogram were counted as described above to locate the position of the labeled TMG and derivatives. The corresponding radioactive areas from the other chromatograms were removed for identification of the labeled compounds. These methods resulted in at least 90% recovery of applied radioactivity in all cases. Identification of the phosphorylated derivative of [14CJTMG transport. ['4C]TMG and its derivative were isolated from each other by the method
described above. The portions of the chromatograms containing only the radioactive derivative were eluted with the smallest possible volume of distilled water for 12 h. The eluate was Iyophilized and redissolved in 200 IlI of distilled water. A portion of the labeled derivative was treated with 0.4 mg of highly purified alkaline phosphatase in 50 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 8) in a total volume of 150 id for 60 min at 37°C. The entire 150 pd was spotted on a chromatogram strip, developed, and counted as described above. Protein estimations. The method of Lowry et al. (26) was used to estimate the protein content of intact and decryptified cells, using bovine serum albumin as the standard. Chemicals. All biochemicals, substrates, and alkaline phosphatase were purchased from Sigma Chemical Co. [14C]TMG and Aquasol were obtained from New England Nuclear Corp. Fluid thioglycolate medium and beef extract were products of Difco. All other chemicals were of analytical grade and were purchased from Fisher Scientific Co. RESULTS
Lac PT system characteristics. Cells of strain SLi decryptified with toluene-acetone readily phosphorylated the lactose analog ONPG in the presence of PEP and Mg2e (Table 1). In the absence of PEP, or after heat treatment, little ONPG-6-P was formed. Some activity (18%) was observed in the absence of added Mg2e. Presumably this was due to bound Mg2e not removed by repeated EDTA treatment. When this treatment was omitted, the amount of Lac PT system activity present without added Mg2e increased to 74% of the complete reaction. Attempts to determine the cation specificity using the chloride salts of Mn2", Ca2+, Sn2+, Co2+, Zn2+, Sr+, and Cu2" were not successful; these cations all interfered to some extent with the coupled indicator enzyme, P-fl-gal. The addition TABLE 1. Lac PT system in S. mutans SLYa Assay condition
Relative amt of ONPG-6-P formed/min per mg of protein (%)b
Complete .....................
100 4 6 18 g2. a Decryptified cell suspensions were prepared from mid-exponential-phase lactose-grown cells. Experimental conditions were as described in Materials and Methods. Each reaction contained 4.85 mg of bacterial protein (specific activity of the complete reaction was 3.51 X 10-3). b ONPG-6-P formed in the complete reaction mixture was arbitrarily set at 100%. c Contains cells heated at 1000C for 10 min. Heated cells .................. -PEP .......................
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LACTOSE TRANSPORT IN S. MUTANS
of lactose at ten times the concentration of ONPG present in the reaction reduced phosphorylation of ONPG by 94%, indicating that both lactose and its analog compete for the same transport system. The Lac PT system was inactive in Tris-hydrochloride buffer. Inclusion of 20 mM potassium phosphate to the Tris-hydrochloride buffer did not prevent this inactivation. The synthesis of ONPG-6-P by decryptified cells was examined in the presence of several potential high-energy phosphate-containing metabolites to determine the most effective phosphate donor for lactose (Table 2). No stimulation of ONPG-6-P synthesis was observed when adenosine tri-, di-, or monophosphate or guanosine triphosphate was supplied as the energy source. PEP and its immediate glycolytic precursor, 2-phosphoglycerate (2-PG), were the only phosphate donors tested which were capable of stimulating ONPG-6-P formation. Inclusion of 10 mM NaF in the reaction prevented ONPG-6-P synthesis when 2-PG was used as the energy source, but NaF had no effect when PEP was the phosphate donor. Table 3 illustrates the effect of fluoride on the Lac PT system in intact and decryptified cells. Intact cells (impermeable to exogenous PEP and 2-PG) exposed to fluoride at concentrations 210 mM exhibited little Lac PT system activity. However, when decryptified cells (permeable to exogenous PEP and 2-PG) were exposed to concentrations of fluoride as high as 100 mM, in the presence of exogenous TABLE 2. Effect of various energy sources upon the Lac PT system in S. mutans SLPa Relative amt of ONPG-6-P
Energy source (1 MM)u PEP (complete reaction) PEP, minus 10 mM NaF
formed/min per mg of protein
....... .......
2-PG
6
2-PG, minus 10 mM NaF ....... ATP ........................ ADP
AMP GTP None
100 110
44 0.4 5
........................ ........................ ........................
6 4 5
Decryptified cell suspensions were prepared from mid-exponential-phase lactose-grown cells. Experia
mental conditions were as described in Materials and Methods, except for the indicated substitutions for PEP. Each reaction contained 2.5 mg of bacterial protein (specific activity of the complete reaction was 5.02 x 10-3). b ATP, Adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; GTP, guanosine triphosphate. c ONPG-6-P formed in the complete reaction was arbitrarily set at 100%.
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TABLE 3. Effect of fluoride on the Lac PT system in S. mutans SLja NaF
Relative amt of ONPG-6-P formed/min per mg of protein (%)b
(mM) Intact cells
Decryptified cells
100 88 90 92 90 a Intact or decryptified cell suspensions were pre0 0.1 1.0 10 100
100 103 100 6 2
pared from mid-exponential-phase lactose-grown cells. Experimental conditions were as described in Materials and Methods, except that the NaF concentration was varied as indicated. Each reaction contained either 3.8 mg of bacterial protein (intact cells; specific activity for the zero reaction was 12.5 x 10-') or 1.37 mg of bacterial protein (decryptified cells; specific activity for the zero reaction was 16.7 x 10-3). b ONPG-6-P formed in the zero reaction was arbitrarily set at 100%.
PEP, Lac PT system activity was not significantly affected. The absolute requirement for PEP, the ability of 2-PG to substitute for PEP, and the differential effect of fluoride upon intact and decryptified cells is consistent with the hypothesis that lactose transport in S. mutans is a group translocation process mediated by the PEP-dependent Lac PT system. This system for lactose transport is similar to those previously described in Staphylococcus aureus (11, 17, 18) and group N streptococci (27, 28). ONPG-6-P synthesis was linear with respect to time for at least 20 min in the presence of PEP (Fig. 1) and was proportional to the protein concentration up to at least 5 mg of cell protein (Fig. 2); in the absence of PEP, little ONPG-6-P synthesis occurred regardless of the length of incubation or the amount of protein present in the reaction. A pH optimum of 8.5 to 9 was observed for lactose transport (Fig. 3). Others have reported a pH optimum of 7 to 7.5 in group N streptococci using a similar assay system (8). Isolation and identification of the product of lactose transport. Evidence that lactose was transported in S. mutans by vectorial phosphorylation was obtained by isolation and identification of the transport product from intact or decryptified cells. To prevent catabolism of the accumulated transport product, a non-metabolizable analog of lactose, TMG, was used. Using intact cells, the rate of ['4C]TMG accumulation was linear for about 5 min, reaching a maximum by 20 min (Fig. 4). In a similar experiment using decryptified cells, at 20 min an ethanolic extract of the cells was chromatographed (Fig. 5). In the
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