INFECTION AND IMMUNITY, June 1979, p. 821-828

Vol. 24, No. 3

0019-9567/79/06-0821/08$02.00/0

Phosphoenolpyruvate-Dependent Sucrose Phosphotransferase Activity in Streptococcus mutans NCTC 10449 ANDREW M. SLEE* AND JASON M. TANZER School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut 06032 Received for publication 29 March 1979

A phosphoenolpyruvate-dependent sucrose phosphotransferase system (PTS) has been demonstrated, by an enzyme-coupled reaction and product isolation, in decryptified cell suspensions of the cariogenic microorganism Streptococcus mutans NCTC 10449. The apparent sucrose PTS reaction for sucrose-adapted, sucrose-challenged cells displayed saturation kinetics with an apparent Km of 7.14 x 10- M, which was distinct from the Km of the glucose PTS activity of glucoseadapted, glucose-challenged cells. Both the sucrose and the glucose PTS activities appear to be inducible and under separate genetic control. The sucrose PTS reaction demonstrated in decryptified cells had an absolute requirement for phosphoenolpyruvate. Only 2-phosphoglycerate, the immediate glycolytic precursor of phosphoenolpyruvate, was found to substitute for phosphoenolpyruvate in this reaction in the absence of fluoride. The sucrose PTS activity of sucroseadapted cells was competitively inhibited by raffinose and lactose; these same sugars had no effect on the apparent glucose PTS activity. Fructose was the only carbohydrate tested other than sucrose which elicited an apparent PTS reaction in sucrose-adapted cells. The product of the sucrose PTS reaction was isolated and behaved chromatographically on a Dowex-l-X8 column like a monophosphate ester. Alkaline phosphatase treatment of the presumptive sucrose monophosphate liberated a component which behaved chromatographically like free sucrose. Subsequent acid hydrolysis of this component produced moieties which behaved chromatographically like glucose and fructose.

Dental caries has long been recognized as an infectious disease related to the presence of dense, adhesive, microbial deposits (dental plaque) on the surfaces of teeth (10, 14, 35). Demonstration of the etiological role of certain plaque-forming streptococci, namely Streptococcus mutans (of which there are at least five recognized serotypes [4]) as infectious agents in multisurface dental caries in rodents (11, 12, 21) and primates (3) has focused attention on a similar role of these microorganisms in humans. Their role in causation of dental caries in humans is now strongly established (10, 13, 22, 25, 26, 35), with S. mutans serotype c the most prevalent serotype (5, 35, 36, 41) associated with human disease. The direct correlation of dietary sucrose, specifically the frequency of consumption, with the incidence of dental caries associated with S. mutans in humans and in experimental animal model systems is striking and well established (12, 17, 28). The biochemistry and physiology of S. mutans with respect to sucrose metabolism is central to the causation and, therefore, the prevention of dental caries. Sucrose dissimilation by S. mutans involves (i) hexosyltransferase ac-

tivity by extracellular or cell-associated enzymes (6), or (ii) transport of the carbohydrate to intracellular sites for subsequent catabolism. Unfortunately, little is known about the transport of sucrose by these cariogenic microorganisms. It should be noted that a variety of sugars are transported by several species of facultative anaerobic and anaerobic bacteria by the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) (30-32). A glucose PTS has been reported in a number of oral streptococci (18, 33, 34). Similarly, PTSs have been reported for lactose (8) and for mannitol (29) in S. mutans, and preliminary reports have shown a sucrose PTS in S. sanguis [J. A. Mayo, T. W. Feary, and P. L. Doerr, J. Dent. Res. 57(Sp. Issue A):644, 1978] and S. mutans serotype d (E. J. St. Martin, Abstr. Annu. Meet. Am. Soc. Microbiol. 1978, K96, p. 142) and serotype c (A. M. Slee and J. M. Tanzer, J. Dent. Res. 58(Sp. Issue A):43, 1979, in press). However, except for lactose and the preliminary reports of sucrose PTSs, little is known of disaccharide transport in S. mutans. The following study indicates that sucrose is transported in S. mutans NCTC 10449 via an inducible PEP-dependent PTS which is distinct 821

822

SLEE AND TANZER

from a monosaccharide PTS of the same microorganism.

MATERIALS AND METHODS Microorganisms, growth medium, and conditions of culture. S. mutans serotype c representative strain NCTC 10449 was used throughout these studies. Stock cultures were maintained by monthly transfer in complex medium consisting of fluid thioglycolate (Difco) with 20% (vol/vol) beef extract and excess CaCO3. Cells for experimental use were grown in the defined chemical medium (FMC) of Terleckyj et al. (40) supplemented with 5 mM carbohydrate. Carbohydrates were added aseptically to the filter-sterilized basal medium. Before experimental use, all cultures were adapted to the appropriate carbohydrate source by three transfers through defined medium supplemented with the appropriate carbohydrate. Cultures were incubated at 370C in screw-capped Erlenmeyer flasks with no head space. Harvesting of cells. Cells were harvested from the late exponential to the early stationary phase of growth (usually an 18-h incubation) by centrifugation, washed twice with the same volume of ice-cold 50 mM phosphate buffer (pH 7.0), and resuspended in the same buffer to an optical density at 600 nm of 1.95. Samples (10 ml) of these resuspended cells were stored at -20'C until used. No loss of PTS activity was noted for up to 3 weeks when such procedures were employed. Preparation of decryptified cells. The membrane integrity of washed cells was perturbed by the addition of 0.01 volume of toluene-acetone (1:4), followed by vigorous agitation at room temperature for 90 s with a Vortex mixer. The resultant decryptified cells were maintained at 0C until used. Sucrose PTS assay. A modification of the scheme of Kornberg and Reeves (20) was used to detect sucrose PTS activity in decryptified cells. The standard assay contained the following components in a final volume of 2.0 ml: 1.0 mM PEP, 0.1 mM reduced nicotinamide adenine dinucleotide (NADH), 2.0 U of lactate dehydrogenase, 1 mM MgCl2, 10 mM NaF, and 25 mM potasium phosphate buffer, pH 7.0. Decryptified cells at a concentration equivalent to 0.2 mg of bacterial protein were added, and the reaction mixture was preincubated at 37°C for 5 min. After the preincubation the carbohydrate of interest was added, and the utilization of NADH was monitored at 340 nm with a Gilford 240 spectrophotometer at 37°C. Extraction of "C-labeled sucrose transport product. The standard reaction mixture for these experiments contained, in a total volume of 3.0 ml: 0.5 mM [U-14C]sucrose (0.2 MCi/4Lmol), 2.0 mM PEP, 1 mM MgCl2, 20 mM NaF, and 25 mM phosphate buffer, pH 7.0. The reaction was started by adding decryptifled cells. After incubation for 15 min at 37°C, the reaction mixture was chilled in an ice bath for 10 min and centrifuged at 20,000 x g and 4°C for 15 min to sediment cells The pellet was extracted for 10 min with boiling 80% ethanol, which was followed by chilling and centrifugation. The supernatant fluids were

INFECT. IMMUN. combined, and the ethanol was evaporated. The extract was resuspended in water and stored at -20°C until used. Chromatography of the ['4C]sucrose transport product. Samples of the water-dissolved ethanolic extracts were placed onto a Dowex-1-formate (AG-1X8; 200 to 400 mesh; Bio-Rad Laboratories, Richmond, Calif.) column (12 by 0.5 cm) and eluted by methods which separate uncharged or positively charged solutes from monophosphates and diphosphates (2, 7, 39). Specifically, fractions were collected upon successive elution with water, a convex exponential gradient of water to 4.0 N formic acid, and 3.5 N formic acid containing 0.5 N ammonium formate. The column was regenerated by extensive washing with 5 N ammonium formate, followed by washing with water until the effluent was formate free. These procedures have been detailed previously (39). Each fraction was assayed for 14C radioactivity by using Aquasol (New England Nuclear Corp., Boston, Mass.) and liquid scintillation counting (model S-150; Beckman Instruments, Inc., Irvine, Calif.). A plot of the effluent profile of radioactivity revealed peaks whose effluent volumes were consistent with their identity as known monoand diphosphate enters of carbohydrates. These peaks were pooled, freeze-dried to remove formic acid and ammonium formate, and redissolved in small volumes of water to facilitate further study. All such samples were maintained at -20°C until used. Isolation and identification of the transport product. Each Dowex-1 pooled peak, after evaporation and redissolution in water, was studied by descending chromatography on Whatman no. 1 paper, using the solvent system of Damonte et al. (9) and techniques previously described (37). Chromatograms were run for 44 h. Authentic standards of carbohydrates and glycolytic intermediates were run on the same paper chromatogram. However, it should be noted that authentic standards of the possible phosphate esters of sucrose are not available for cochromatography. Thus, radioactive peaks were identified by cutting and counting 0.25- or 0.5-cm strips of the paper chromatogram. Those radioactive peaks which did not correspond to the positions of cochromatographed standards were further studied because they may represent sucrose phosphate ester(s). The location of the standards was established by their colorimetric reactions upon spraying with the reagent of Koch et al. (19) for free sugars or the reagent of Hanes and Isherwood (16) for phosphate esters, using techniques previously described (39). The Dowex-1 peak which thus contained a phosphate ester of an unidentifiable solute was sampled, treated with alkaline phosphatase [0.4 mg of alkaline phosphatase in 0.1 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 8.5, for 90 min at 37°C], and rechromatographed on Dowex-1. The pooled water-eluted peak (representing uncharged or positively charged material) was divided into two aliquots. One aliquot was treated with 0.1 N HCl at 100°C for 10 min, and the other was not treated. Both were then rechromatographed on Whatman no. 1 paper, as detailed above. Protein determination. The method of Lowry et

VOL. 24, 1979 al. (27) was used to estimate the protein content of decryptified cells, using bovine serum albumin as the standard. Chemicals. Unless otherwise indicated, all biochemicals, substrates, and alkaline phosphatase (derived from Escherichia coli) were purchased from Sigma Chemical Co. [U-'4C]sucrose and Aquasol were obtained from New England Nuclear Corp.

RESULTS Detection of sucrose PTS. Cells of S. mutans NCTC 10449, decryptified with tolueneacetone and incubated in the presence of exogenous PEP and sucrose, exhibited a decrease in optical density at 340 mm which was linear with time. In the absence of exogenous PEP or sucrose or after heat treatment (80'C for 15 min) of the cells, no decrease in optical density at 340 nm was observed. Such a decrease thus reflected the generation of pyruvate from PEP and the consequent utilization of NADH in the conversion of pyruvate to lactate mediated by lactate dehydrogenase. Thus, the amount of NADH consumed was reflective of the amount of PEP utilized and, concurrently, with the amount of presumptive sucrose phosphate formed. The primary energy source for this presumptive sucrose PTS reaction was found to be PEP. Neither 3-phosphoglyceric acid, 2,3-diphosphoglyceric acid, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, cyclic adenosine monophosphate, guanosine triphosphate, or guanosine diphosphate could substitute as the apparent phosphate donor, in the presence or absence of NaF, in the reaction (Table 1). PEP and its immediate glycolytic precursor 2-phosphoglyceric acid were the only potential phosphate donors tested which stimulated apparent sucrose phosphate formation. The latter phosphate donor only stimulated PTS activity in the absence of NaF. Such a result is to be expected because of the wellknown inhibition of S. mutans enolase by NaF (15). Up to 50 mM NaF had no significant effect on the observed sucrose PTS activity in the presence of PEP. The absolute requirement for PEP, the ability of 2-phosphoglyceric acid to substitute for PEP in the absence of NaF, and the absence of an F- effect on the decrease in optical density at 340 nm in the presence of PEP are all consistent with the hypothesis that at least one mode of sucrose permeation by S. mutans NCTC 10449 is a group translocation process mediated by a PEP-dependent PTS. Distinction between sucrose and glucose PTSs. Table 2 shows that sucrose-adapted cells exhibited strong sucrose PTS activity but no glucose PTS activity. In contrast, glucoseadapted cells exhibited glucose PTS activity but

SUCROSE PTS ACTIVITY IN S. MUTANS

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TABLE 1. Relative sucrose PTS activity of

decryptified cells of S. mutans NCTC 10449a Relative activity Experimental system' Complete reaction system

100 minus PEP 2 minus PEP plus 2PG 5 minus PEP plus 2PG minus NaF 45 minus PEP plus 3PG 2 minus PEP plus 2,3-diPG 3 minus PEP plus ATP 3 minus PEP plus ADP 2 minus PEP plus AMP 1 minus PEP plus cAMP 0 minus PEP plus GTP 3 minus PEP plus GDP 1 Complete system plus heat treatment 2 (80'C for 15 min) a Decryptified cell suspensions were prepared from lateexponential-phase sucrose-grown cells. Experimental conditions were as described in the text, except for the indicated substitutions for PEP. Each reaction contained 0.2 mg of bacterial protein. b Abbreviations: 2PG, 2-phosphoglyceric acid; 3PG, 3-phosphoglyceric acid; 2,3-diPG, 2,3-diphosphoglyceric acid; ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; cAMP, cyclic adenosine monophosphate; GTP, guanosine triphosphate; GDP, guanosine diphosphate. All potential energy sources were employed at a final concentration of 0.1 mM. The relative activity (micromoles ofoxidized nicotinamide adenine dinucleotide produced per minute per milligram of protein) in the complete reaction system was arbitrarily set at 100%. c

TABLE 2. Sucrose PTS and glucose PTS activities for sucrose-grown and glucose-grown S. mutans NCTC 10449a Sucrose PTS Glucose PTS Growth conditions

activity (jmol activity (umol Carbohydrate of NAD /min of NAD+/min challenge per mg of per mg of protein) b 78.9

protein) SucroseSucrose

Phosphoenolpyruvate-dependent sucrose phosphotransferase activity in Streptococcus mutans NCTC 10449.

INFECTION AND IMMUNITY, June 1979, p. 821-828 Vol. 24, No. 3 0019-9567/79/06-0821/08$02.00/0 Phosphoenolpyruvate-Dependent Sucrose Phosphotransfera...
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