Involvement of Phosphoenolpyruvate in the Catabolism of Caries-Conducive Disaccharides by Streptococcus mutans:

Transcription

1 INFECTION AND IMMUNITY, Mr. 1978, p /78/19-934$2./O Copyright X 1978 Americn Society for Microbiology Vol. 19, No. 3 Printed in U.S.A. Involvement of Phosphoenolpyruvte in the Ctbolism of Cries-Conducive Discchrides by Streptococcus mutns: Lctose Trnsport ROBERT CALMES Deprtment of Orl Biology, College of Dentistry, Albert B. Chndler Medicl Center, University of Kentucky, Lexington, Kentucky 456 Received for publiction 21 July 1977 The mechnisms for trnsport nd hydrolysis of lctose were investigted in five criogenic strins (HS6, AHT, FAl, NCTC 1449, nd SL1) representing the four serogenetic groups of Streptococcus mutns. The systems for trnsport nd hydrolysis of lctose hd the chrcteristics of phosphoenolpyruvte (PEP)- dependent lctose (Lc) phosphotrnsferse (PT) system nd phospho-,8-glctosidse (P-fl-gl), respectively, in ll strins tested, except strin HS6. Decryptified cells required PEP nd Mge for trnsport of the non-metbolizble model,8-glctosides o-nitrophenyl-,8-d-glctopyrnoside (ONPG) nd thiomethyl-,8-d-glctopyrnoside (TMG). Substitution of 2-phosphoglycerte (2- PG) for PEP lso stimulted the Lc PT system. Other potentil high-energy phosphte donors (denosine tri-, di-, nd monophosphtes nd gunosine triphosphte) did not stimulte the Lc PT system. Sodium fluoride hd no effect upon the PEP-dependent Lc PT system in decryptified cells with PEP s the energy source; however, when 2-PG ws used s the energy source, F- inhibited ONPG phosphoryltion. With intct cells which must generte PEP endogenously, the presence of F- in concentrtion o1 mm completely inhibited the Lc PT system, presumbly through inhibition of 2-PG hydrolyse (EC ; enolse). Both intct nd decryptified cells ccumulted phosphorylted derivtive of TMG tht behved chromtogrphiclly s TMG-phosphte. After lkline phosphtse tretment, the derivtive hd n Rf identicl to tht of TMG. No fb-glctosidse (fl-gl) ctivity ws detected with ONPG s the substrte; hydrolysis occurred only when ONPG-6-phosphte ws supplied s the substrte. Strin HS6 pprently trnsported lctose by n ctive trnsport-type system in which the ccumulted intrcellulr product ws the free discchride bsed on the following criteri: (i) ONPG trnsport nd hydrolysis in decryptified cells ws not stimulted by PEP; (ii) ONPG hydrolysis occurred in the bsence of PEP; nd (iii) ONPG-6-phosphte ws not hydrolyzed. These dt indicte tht, in ll strins tested except strin HS6, lctose trnsport ws medited by PEP-dependent Lc PT system, resulting in ccumultion of lctose-phosphte tht ws hydrolyzed by n enzyme similr to the P-,f-gl of group N streptococci nd Stphylococcus ureus; conversely, strin HS6 trnsported nd hydrolyzed lctose by PEP-independent trnsport system nd fl-gl, respectively. The utiliztion of sucrose by Streptococcus these crbohydrtes in humns becuse, except mutns nd other orl microorgnisms results for strch nd lctose, they re not usully considered to be mjor constituents of humn diets. in the ccumultion of dentl plque on teeth. The fermenttive metbolism of these plque Lctose, n cidogenic (13) discchride present bcteri yields end products, e.g, lctic cid, tht in bovine milk in high concentrtions, cn be ct directly nd indirectly upon the teeth nd considered s cries-conducive dietry crbohydrte of mjor import, especilly since milk is periodontium of humns, cusing cries nd periodontl disese (for reviews, see references 2 consumed in lrge quntities by humns in the nd 3). Studies with niml models hve shown so-clled "cries-prone" yers. In ddition, the tht cries will develop when the diet contins fermentbility of lctose by S. mutns nd other fermentble crbohydrtes other thn sucrose, plque microorgnisms my be of significnce in e.g., glucose, fructose, mltose, lctose, or strch "milk-bottle cries" of infnts. (12, 15). Little is known of the metbolism of Crbohydrte dissimiltion by S. mutns my 934 Downloded from on August 19, 218 by guest

2 VOL. 19, 1978 involve (i) hydrolysis by extrcellulr or cellssocited enzymes s for certin polyscchrides (1, 14, 4) nd sucrose (2), or (ii) trnsport of the crbohydrte to intrcellulr sites for subsequent ctbolism. Sugrs re trnsported in mny bcteri by the phosphoenolpyruvte (PEP)-dependent phosphotrnsferse (PT) system'(32), first described by Kundig et l. (23; for reviews, see references 33 nd 34). Glucose PT systems hve been reported in S. slivrius (2) nd S. mutns (36), nd PT systems for other monoscchrides (35, 36) nd hexitols (29, 35) hve lso been described. Except for previous report (I. R. Hmilton, J. Dent. Res. 55:B124, 1976), discchride trnsport mechnisms hve not been investigted in S. mutns, lthough other fculttive nerobes, e.g., Stphylococcus ureus (11, 17, 18) nd group N streptococci (27, 28), hve been shown to use PEP-dependent PT system for trnsport of lctose. Becuse of the reported criogenic potentil of lctose (13), systemtic investigtion of lctose ctbolism ws initited. In this communiction, I report the presence of PEP-dependent lctose (Lc) PT system nd 6-phospho-,f-glctosidse (P-,f-gl) in four strins of S. mutns nd describe some properties of the Lc PT system. Chrcteristics of P-,f-gl will be reported elsewhere. MATERIALS AND METHODS Bcteri. S. mutns strins NCTC 1449, FAl, SL1, HS6, nd AHT were generously provided by Albert T. Brown nd Aln L. Coykendll. These strins of S. mutns re representtive of ech of the four genetic groups bsed on gunosine-cytosine rtios nd deoxyribonucleic cid hydridiztion (9), kinetic ctivity of lctic dehydrogense (5), nd electrophoretic ptterns of hexitol phosphte dehydrogenses (4), ldolses (25), enzymes of extrcellulr polyscchride synthesis (7, 31), nd invertses (38). These chrcteristics ll correlte well with the serologicl groups described by Brtthll (1). All cultures were mintined by weekly trnsfer in complex medium consisting of 3% (wt/vol) fluid thioglycolte with 1.2% (wt/vol) beef extrct nd excess CCO3. Growth medi nd conditions of culture. Cells were grown in the complex medium of Jordn et l. (19) supplemented with.1% (wt/vol) discchrides or.2% (wt/vol) monoscchrides or hexitols. Sugrs nd hexitols were dded septiclly to the sterile bsl medium from filter-sterilized solutions. Before experimentl use, ll inocul were dpted to the pproprite crbon source by one trnsfer through the pproprite medium. Cultures were incubted t 37C in screw-cp Erlenmeyer flsks with no hed spce. Hrvesting of cells. Cells were hrvested from the mid-exponentil phse of growth by centrifugtion, wshed twice with equl volumes of ice-cold 15 mm KCl, nd resuspended in 5 mm potssium phosphte buffer (KPB) (ph 7). Preprtion of decryptified cells. The membrnes of wshed cells were perturbed by ddition of LACTOSE TRANSPORT IN S. MUTANS Il of toluene-cetone (1:4, vol/vol) per ml of cell suspension bsorbencyy t 75 nm ='2), followed by vigorous shking t room temperture for 5 min. The decryptified cell suspension ws chilled nd mintined in n ice bth.,8-gl ssy. Assy of f-glctosidse (fl-gl) ws performed by using the chromogenic substrte o-nitrophenyl-,l-d-glctopyrnoside (ONPG) (24). Unless otherwise specified, rection mixtures contined 1 mm ONPG nd 2 mm KPB in finl volume of 2.5 ml. The rection ws strted by dding intact or decryptified cells nd incubted t 37C. After 5 min, the rection ws terminted by ddition of n equl volume of ice-cold 1% N2CO3. The lklinized rection mixture ws chilled in ice for 5 min nd then centrifuged t 2, x g for 5 min t 4C to sediment cells from the ssy mixture. The bsorbnce of the superntnt fluid ws determined t 42 nm in Gilford Stsr I spectrophotometer set to zero with regent blnk. Under the prescribed conditions, solution contining uthentic o-nitrophenol (ONP) hd molr extinction coefficient of 1.87 x 1-3 M-l cm-', which ws used to convert bsorbnce dt to micromoles of ONP. The relese of 1 pmol of ONP from ONPG per min t 37C ws defined s unit of enzyme ctivity. Specific ctivity ws expressed s units per milligrm of protein. P-f8-gl ssy. A derivtive of ONPG phosphorylted t crbon 6 (ONPG-6-P) ws used s the substrte for phospho-fl-gl (P-,f-gl) ssys. The complete rection mixture contined 1 mm ONPG-6-P nd 2 mm KPB (ph 7) in finl volume of 2.5 ml. The rection ws strted by dding decryptified cells (intct cells were impermeble to ONPG-6-P) nd incubted t 37C for 5 min. Other spects of the ssy, including the definition of specific ctivity, re s for the fl-gl ssy described bove. Lc PT ssy. The stndrd ssy contined the following components in finl volume of 2.5 ml: 1 mm ONPG, 1 mm PEP, 1 mm MgCl2, 1 mm NF, nd 2 mm KPB (ph 7). The rection ws strted by dding intct cells or decryptified cells nd incubted by 37C for 2 min, unless otherwise specified. The reminder of the ssy, including the definition of the specific ctivity, ws s for the fl-gl nd P-,f-gl ssys described bove. EDTA wshing of decryptified cells. To deplete cells of Mg2e in order to show Mg2e dependence, decryptified cells were wshed with ethylenediminetetrcetic cid (EDTA) s follows: EDTA ws dded to 5-ml suspension of decryptified cells to finl concentrtion of 1 mm, nd the cells were incubted t room temperture with gentle gittion for 5 min. The suspension ws centrifuged t 2, x g for 5 min t 4C, nd the superntnt fluid ws discrded. The pellet ws resuspended in 5 ml of 5 mm KPB (ph 7) contining 1 mm EDTA nd 5 pj of toluenecetone (1:4, vol/vol) per ml. This procedure ws repeted five times; the finl pellet ws resuspended in 5 ml of solvent-contining buffer minus EDTA nd ssyed for Lc PT nd P-,f-gl ctivities. This tretment hd no effect upon the specific ctivities of the Lc PT system (with Mg2e dded bck) or P-fl-gl. [14CJTIMG trnsport. Another non-metbolizble nlog of lctose, ['4C]thiomethyl-fl-D-glctoside Downloded from on August 19, 218 by guest

3 936 CALMES (TMG), ws used for n lterntive method to mesure lctose trnsport. The method of trnsport ssy ws similr to tht previously described (6). Briefly, the stndrd ssy contined 1 mm [14C]TMG (.1,tCi/jumol) nd 45 mm KPB (ph 7). When decryptified cells were used, the ssy included 1 mm PEP nd 1 mm MgCl2. Uptke ws initited by ddition of intct or decryptified cells, which were then incubted t 37C. Smples of 1,ul were removed t the indicted intervls nd pipetted onto the center of 25-mmdimeter membrne filter (type HA-6,.45-jm pore size; Millipore Corp.) where suction displced the lbeled medium from the cells, terminting uptke. The cells were immeditely wshed twice with 5-ml portions of isotherml 5 mm KPB (ph 7). Filtering nd wshing ws ccomplished in less thn 3 s. Zero-time smples were obtined by dding the pproprite mount of lbeled TMG fter 1,ul of cells-buffer mixture hd been diluted into 5 volumes of ice-cold 5 mm KPB; the entire volume ws then filtered nd wshed s bove. Filters were dried under n incndescent lmp, trnsferred to minicounting vils contining 3 ml of Aqusol, nd counted in Pckrd Tri- Crb liquid scintilltion spectrometer. Under these conditions no quenching occurred. Modifictions to this procedure re noted below. Extrction nd isoltion ofthe [14CJTMG trnsport product. The stndrd rection mixture for these experiments contined, in totl volume of 1 ml, 1 mm ["4C]TMG (.2 uci/iumol) nd 3 mm KPB (ph 7). The rection ws strted by dding intct or decryptified cells. When decryptified cells were used, the following dditions were mde to the stndrd rection mixture: 1 mm PEP, 1 mm MgCl2, nd 1 mm NF. After incubtion t 37 C for 3 min, the rection mixture ws chilled in ice bth for 5 min nd then centrifuged t 2, x g t 4 C for 15 min to sediment cells. The pellet ws extrcted for 1 min with boiling 8% (vol/vol) ethnol followed by chilling nd centrifugtion s bove. When decryptified cells were used, the superntnt fluid from the first centrifugtion ws combined with the ethnol extrct; for intct cells the superntnt fluid ws discrded. All extrcts were stored t -2 C. Chromtogrphy of the ['4C]TMG trnsport product. Portions of the extrcts were subjected to scending pper chromtogrphy on strips (2.5 by 3 cm) of Whtmn 1 pper. The solvent system contined ethyl cette-pyridine-wter (6:25:15, vol/vol/vol). In this solvent system, the stock ["C]- TMG ws chromtogrphiclly pure. When the solvent fronts reched 15 cm (c. 75 min), the chromtogrms were removed from the jr, ir dried, nd plced in vcuum oven t 4 C for 3 min to remove ll solvent. The strips were cut t 1-cm intervls; the pieces from one chromtogrm were counted s described bove to locte the position of the lbeled TMG nd derivtives. The corresponding rdioctive res from the other chromtogrms were removed for identifiction of the lbeled compounds. These methods resulted in t lest 9% recovery of pplied rdioctivity in ll cses. Identifiction of the phosphorylted derivtive of [14CJTMG trnsport. ['4C]TMG nd its derivtive were isolted from ech other by the method INFECT. IMMUN. described bove. The portions of the chromtogrms contining only the rdioctive derivtive were eluted with the smllest possible volume of distilled wter for 12 h. The elute ws Iyophilized nd redissolved in 2 IlI of distilled wter. A portion of the lbeled derivtive ws treted with.4 mg of highly purified lkline phosphtse in 5 mm tris(hydroxymethyl)- minomethne (Tris)-hydrochloride buffer (ph 8) in totl volume of 15 id for 6 min t 37 C. The entire 15 pd ws spotted on chromtogrm strip, developed, nd counted s described bove. Protein estimtions. The method of Lowry et l. (26) ws used to estimte the protein content of intct nd decryptified cells, using bovine serum lbumin s the stndrd. Chemicls. All biochemicls, substrtes, nd lkline phosphtse were purchsed from Sigm Chemicl Co. [14C]TMG nd Aqusol were obtined from New Englnd Nucler Corp. Fluid thioglycolte medium nd beef extrct were products of Difco. All other chemicls were of nlyticl grde nd were purchsed from Fisher Scientific Co. RESULTS Lc PT system chrcteristics. Cells of strin SLi decryptified with toluene-cetone redily phosphorylted the lctose nlog ONPG in the presence of PEP nd Mg2e (Tble 1). In the bsence of PEP, or fter het tretment, little ONPG-6-P ws formed. Some ctivity (18%) ws observed in the bsence of dded Mg2e. Presumbly this ws due to bound Mg2e not removed by repeted EDTA tretment. When this tretment ws omitted, the mount of Lc PT system ctivity present without dded Mg2e incresed to 74% of the complete rection. Attempts to determine the ction specificity using the chloride slts of Mn2", C2+, Sn2+, Co2+, Zn2+, Sr+, nd Cu2" were not successful; these ctions ll interfered to some extent with the coupled indictor enzyme, P-fl-gl. The ddition TABLE 1. Lc PT system in S. mutns SLY Reltive mt of ONPG-6-P Assy condition formed/min per mg of protein (%)b Complete... 1 Heted cells PEP Mg2. 18 Decryptified cell suspensions were prepred from mid-exponentil-phse lctose-grown cells. Experimentl conditions were s described in Mterils nd Methods. Ech rection contined 4.85 mg of bcteril protein (specific ctivity of the complete rection ws 3.51 X 1-3). b ONPG-6-P formed in the complete rection mixture ws rbitrrily set t 1%. c Contins cells heted t 1C for 1 min. Downloded from on August 19, 218 by guest

4 VOL. 19, 1978 LACTOSE TRANSPORT IN S. MUTANS 937 of lctose t ten times the concentrtion of ONPG present in the rection reduced phosphoryltion of ONPG by 94%, indicting tht both lctose nd its nlog compete for the sme trnsport system. The Lc PT system ws inctive in Tris-hydrochloride buffer. Inclusion of 2 mm potssium phosphte to the Tris-hydrochloride buffer did not prevent this inctivtion. The synthesis of ONPG-6-P by decryptified cells ws exmined in the presence of severl potentil high-energy phosphte-contining metbolites to determine the most effective phosphte donor for lctose (Tble 2). No stimultion of ONPG-6-P synthesis ws observed when denosine tri-, di-, or monophosphte or gunosine triphosphte ws supplied s the energy source. PEP nd its immedite glycolytic precursor, 2-phosphoglycerte (2-PG), were the only phosphte donors tested which were cpble of stimulting ONPG-6-P formtion. Inclusion of 1 mm NF in the rection prevented ONPG-6-P synthesis when 2-PG ws used s the energy source, but NF hd no effect when PEP ws the phosphte donor. Tble 3 illustrtes the effect of fluoride on the Lc PT system in intct nd decryptified cells. Intct cells (impermeble to exogenous PEP nd 2-PG) exposed to fluoride t concentrtions 21 mm exhibited little Lc PT system ctivity. However, when decryptified cells (permeble to exogenous PEP nd 2-PG) were exposed to concentrtions of fluoride s high s 1 mm, in the presence of exogenous TABLE 2. Effect of vrious energy sources upon the Lc PT system in S. mutns SLP Reltive mt of ONPG-6-P Energy source (1 MM)u formed/min per mg of protein PEP (complete rection)... 1 PEP, minus 1 mm NF PG 6 2-PG, minus 1 mm NF... ATP ADP 5 AMP... 6 GTP... 4 None... 5 Decryptified cell suspensions were prepred from mid-exponentil-phse lctose-grown cells. Experimentl conditions were s described in Mterils nd Methods, except for the indicted substitutions for PEP. Ech rection contined 2.5 mg of bcteril protein (specific ctivity of the complete rection ws 5.2 x 1-3). b ATP, Adenosine triphosphte; ADP, denosine diphosphte; AMP, denosine monophosphte; GTP, gunosine triphosphte. c ONPG-6-P formed in the complete rection ws rbitrrily set t 1%. TABLE 3. Effect offluoride on the Lc PT system in S. mutns SLj Reltive mt of ONPG-6-P formed/min per mg of protein NF (%)b (mm) Intct cells Decryptified cells Intct or decryptified cell suspensions were prepred from mid-exponentil-phse lctose-grown cells. Experimentl conditions were s described in Mterils nd Methods, except tht the NF concentrtion ws vried s indicted. Ech rection contined either 3.8 mg of bcteril protein (intct cells; specific ctivity for the zero rection ws 12.5 x 1-') or 1.37 mg of bcteril protein (decryptified cells; specific ctivity for the zero rection ws 16.7 x 1-3). b ONPG-6-P formed in the zero rection ws rbitrrily set t 1%. PEP, Lc PT system ctivity ws not significntly ffected. The bsolute requirement for PEP, the bility of 2-PG to substitute for PEP, nd the differentil effect of fluoride upon intct nd decryptified cells is consistent with the hypothesis tht lctose trnsport in S. mutns is group trnsloction process medited by the PEP-dependent Lc PT system. This system for lctose trnsport is similr to those previously described in Stphylococcus ureus (11, 17, 18) nd group N streptococci (27, 28). ONPG-6-P synthesis ws liner with respect to time for t lest 2 min in the presence of PEP (Fig. 1) nd ws proportionl to the protein concentrtion up to t lest 5 mg of cell protein (Fig. 2); in the bsence of PEP, little ONPG-6-P synthesis occurred regrdless of the length of incubtion or the mount of protein present in the rection. A ph optimum of 8.5 to 9 ws observed for lctose trnsport (Fig. 3). Others hve reported ph optimum of 7 to 7.5 in group N streptococci using similr ssy system (8). Isoltion nd identifiction of the product oflctose trnsport. Evidence tht lctose ws trnsported in S. mutns by vectoril phosphoryltion ws obtined by isoltion nd identifiction of the trnsport product from intct or decryptified cells. To prevent ctbolism of the ccumulted trnsport product, non-metbolizble nlog of lctose, TMG, ws used. Using intct cells, the rte of ['4C]TMG ccumultion ws liner for bout 5 min, reching mximum by 2 min (Fig. 4). In similr experiment using decryptified cells, t 2 min n ethnolic extrct of the cells ws chromtogrphed (Fig. 5). In the Downloded from on August 19, 218 by guest

5 938 CALMES INFECT. IMMUN. 4 -J<. z I-_.I z -5 Ci CL 2 I. 1_ / MINUTES FIG. 1. Formtion of ONPG-6-P s function of time. Decryptified cell suspensions were prepred from mid-exponentil-phse lctose-grown cells of strin SLL. Experimentl conditions were s described in Mterils nd Methods. Ech rection contined 4.85 mg of bcterilprotein (specific ctivity ofthe Lc PT system ws 3.89 X 1r`). The mount ofonpg-6-p formed ws determined t the indicted intervls. Rections were performed with () nd without () PEP. 15 z!i cc 1 I- so 5. pw mg CELL PROTEIN FIG. 2. Formtion of ONPG-6-P s function of protein concentrtion. Decryptified cell suspensions were prepred from mid-exponentil-phse lctosegrown cells of strin SLI. Experimentl conditions were s described in Mterils nd Methods. Ech rection contined the indicted mount of bcteril protein (specific ctivity of the Lc PT system ws 3.16 x 1r-). The mount of ONPG-6-P formed ws determined t 2 min. Rections were performed with () nd PEP () without. IL V- 2 ( ph FIG. 3. Effect of ph upon ONPG-6-P formtion. Decryptified cell suspensions were prepred from mid-exponentil-phse lctose-grown cells of strin SLI. Experimentl conditions were es described in Mterils nd Methods, except tht the ph of the phosphte buffer system ws vried s indicted. Ech rection contined 1.37 mg of bcteril protein (specific ctivity of the Lc PT system ws 5.2 x 13 t ph 7). The mount of ONPG-6-P formed ws determined t 2 min. D::! Z C I MINUTES Time course for [14CJTMG trnsport. In- FIG. 4. tct cell suspensions were prepred from mid-exponentil-phse lctose-grown cells of strin SL1. Experimentl conditions for TMG trnsport were s described in Mterils nd Methods. [14CJTMG ccumultion ws determined t the indicted intervls. The rection contined 4.5 mg of bcteril protein in totl volume of 1 ml. solvent system used, uthentic ['4C]TMG migrted t n Rf of.46 (Fig. 5A), wheres the ionic trnsport product remined t the origin (Fig. 5D). In the bsence of exogenous PEP, Downloded from on August 19, 218 by guest

6 VOL. 19, e 3 F 2 1 c 2 1 I~- -J 5 c ORIGIN X S m. _ -A _ SOLVENT FRONTt I~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~ I... s A MIGRATION IN cm FIG. 5. Chromtogrphy of '4C]TMG derivtive. Decryptified cell suspensions were prepred from mid-exponentil-phse lctose-grown cells of strin SLI. Experimentl conditions for TMG trnsport were s described in Mterils nd Methods in the presence or bsence ofpep. Ech rection contined 4.5 mg of bcteril protein in totl volume of I ml. After 2 min, the r4c]tmg nd ny derivtives were extrcted nd chromtogrphed s described in Mterils nd Methods. (A) Authentic stndrd /'4C1- TMG; (B) smple extrcted from the "minus PEP" rection; (C) smple extrcted from the 'plus PEP" rection, 1 mmnf; (D) rechromtogrphy ofthe r4c]tmg derivtive fter eltion from the chromtogrm depicted in (C); (E) distribution of lbel fter tretment of the [14C]TMG derivtive with lkline phosphtse. smll mount of the ["4C]TMG ws derivtized (Fig. 5B) due to endogenously generted PEP. Addition of 1 mm PEP to the rection stimulted conversion of pproximtely 5% of the [I4C]TMG to its derivtive within the 2-min time period (Fig. 5C). Addition of 1 mm NF hd no effect on the rte of conversion in the A B C L D E LACTOSE TRANSPORT IN S. MUTANS 939 presence of PEP. The lbeled derivtive ws cut from the pproprite re of the chromtogrm. Elution nd rechromtogrphy of the lbeled derivtive indicted tht it ws essentilly free of contminting ["4C]TMG or other lbeled derivtives (Fig. 5D). The derivtive ws concentrted nd then treted with highly purified lkline phosphtse. Rechromtogrphy of the lkline phosphtse-treted derivtive yielded compound tht behved chromtogrphiclly s [14C]TMG (Fig. 5E). Similr results were obtined when intct cells were used (dt not shown) except tht inclusion of 1 mm NF in the rection mixture blocked formtion of the phosphorylted TMG derivtive. Comprison of Lc PT ctivity in vrious strins. Strins representing ech of the serogenetic groups (1, 9) ofs. mutns were exmined for Lc PT system ctivity. Growth in lctosecontining medium induced the synthesis of the enzyme systems for lctose ctbolism in ll strins tested (Tble 4). In ddition, PEP stimultion of ONPG-6-P synthesis ws observed in ll strins, except strin HS6 of genetic group IV (serologicl group ). Although strin HS6 grew well on lctose, no stimultion of lctose trnsport by PEP ws observed. A second strin, AHT, of the sme serogenetic group s strin HS6 ws tested to determine whether the biochemicl nomly observed in strin HS6 ws chrcteristic of tht serogenetic group. It ws not; strin AHT possessed Lc PT system similr to tht of strins FAL, NCTC 1449, nd SLL. Other physiologicl differences were observed between strins HS6 nd SLL. Strin SL1 ws unble to hydrolyze ONPG in the bsence TABLE 4. Effect of PEP nd the growth substrte upon lctose ctbolism in vrious strins of S. mutns Sp ct of the Lc PT system' Seroge- Strin netic Lctose Glucose group +PEP -PEP +PEP -PEP HS6 IV AHT FA1 b, II NCTC c, I SL1 d, III Decryptified cell suspensions were prepred from mid-exponentil-phse cells grown in either lctose or glucose medium. Experimentl conditions were s described in Mterils nd Methods. Ech rection contined 2 to 4 mg of bcteril protein. bserologicl groups of Brtthll (1) (, b, c, nd d) nd the corresponding genetic groups of Coykendll (9) (IV, II, I, III). c Micromoles of ONP formed per minute per milligrm of protein x 1-3. Downloded from on August 19, 218 by guest

7 94 CALMES of PEP, wheres strin HS6 hydrolyzed ONPG eqully well in the presence or bsence of PEP, lbeit t n overll lower rte thn strin SLi (Tble 5) (these vritions in rte re discussed below). Furthermore, strin SL1, but not HS6, hydrolyzed ONPG-6-P. These results suggest tht strin HS6 hs different enzyme systems for dissimiltion of lctose thn the other strins tested; i.e., strin HS6 my use n ctive trnsport system followed by hydrolysis with /8-gl s in Streptococcus lctis 7692 (21, 22, 27, 28). In the present study, vritions in the mounts of stimultion by PEP nd the specific ctivities of the trnsport system were observed. Addition of PEP stimulted phosphoryltion of ONPG from 13- to 22-fold in strins AHT, FAl, NCTC 1449, nd SL1, wheres the specific ctivity of the induced systems vried over n pproximte sixfold rnge (Tble 4). Similr vritions in the specific ctivities of invertse were observed mong strins of S. mutns by Tnzer et l. (37, 38) Ėffect of the growth substrte on the Lc PT system. The inducible nture of the Lc PT system ws the first observed in strin SLi grown t the expense of vriety of discchrides, monoscchrides, nd hexitols. Incresed levels of Lc PT ctivity were noted when lctose nd/or glctose were used s the crbon source (Tble 6). The bility of glctose to induce the Lc PT system hs been observed in Stphylococcus ureus (3), nd lctic streptococci (28), where glctose is the preferentil inducer of these enzymes. The dt shown here indicte tht lctose, rther thn glctose, is the preferentil inducer of the enzymes of lctose metbolism in S. mutns, nd tht glctose, lthough ble to prtilly induce the Lc PT system- when present s the sole crbon source, inhibits full induction of the system when simultneously present with lctose in the medium. TABLE 5. Comprison of ONPG nd ONPG-6-P hydrolysis in two strins of S. mutns Sp ctb Substrte Strin HS6 Strin SL1 ONPG ONPG + PEP ONPG-6-P Decryptified cell suspensions were prepred from mid-exponentil-phse lctose-grown cells of both strins. Experimentl conditions for ech ssy were s described in Mterils nd Methods. Rections contined either 1.5 mg (strin SL1) or 4.6 mg (strin HS6) of bcteril protein. b See Tble 4, footnote c. INIFECT. IMMUN. TABLE 6. Effect ofgrowth substrte on Lc PT system ctivity in S. mutns SLj Reltive mt of ONPG-6-P Growth substrte formed/min per mg of protein (%)b Lctose... 1 Lctose/glctose Glctose Mltose... 5 Sucrose... 4 Glucose... 4 Mnnitol... 3 Sorbitol... 4 Decryptified cell suspensions were prepred from mid-exponentil-phse cells grown on the indicted substrte. Experimentl conditions were s described in Mterils nd Methods. Ech rection contined 2 to 5 mg of bcteril protein. b ONPG-6-P formed by the lctose-grown cells ws rbitrrily set t 1%. DISCUSSION The Lc PT system of Streptococcus mutns described here displyed chrcteristics similr to those systems reported for the trnsport of monoscchrides nd hexitols by this bcterium (29, 35, 36) nd for lctose trnsport by group N streptococci (27, 28, 39) nd Stphylococcus ureus (11, 17, 18). The system phosphorylted /3- glctosides only when PEP ws included s the energy source nd phosphte donor. 2-PG, the immedite precursor of PEP, ws unble to replce PEP s the energy source in decryptified cells when sodium fluoride ws present. Furthermore, no phosphorylted product of TMG ws synthesized by intct cells in the presence of sodium fluoride (R. Clmes, unpublished observtion). These results indicte tht t lest one site of fluoride ction is the inhibition of enolse, s hs been suggested for S. mutns (36), other orl streptococci (2, 36), nd S. lctis ML3 (39). Enolse nd other possible sites of fluoride inhibition hve been reviewed by Hmilton (16). The bility of S. mutns nd other fculttive nerobes to persist in the orl environment is t lest prtly due to their bility to extrct energy for biosynthesis from wide vriety of crbohydrtes present in the diet of the host. As Rosemn (33) pointed out, the PEP-dependent system offers severl importnt physiologicl dvntges to such microorgnisms, which live in ecologicl niches where environmentl conditions re such tht the energy source for biosynthesis nd growth is widely vried nd fluctutes rpidly. (i) By tightly coupling sugr trnsport with its subsequent metbolism, the rtes of Downloded from on August 19, 218 by guest

8 VOL. 19, 1978 both processes become interdependent, enbling fine control nd rpid dpttion to chnging environmentl conditions. (ii) If conditions chnge so tht the energy source becomes limited (s in the orl cvity during fsting), the energy-generting system llows for conservtion of denosine triphosphte becuse the intrcellulr trnsport product is sugr phosphte tht cn enter metbolic pthwys without further expenditure of energy. (iii) For microorgnisms tht synthesize nd store intrcellulr polyscchride (IPS), PEP prticiptes in three metbolic systems: trnsport, glycolysis, nd IPS synthesis. By linking ctbolism of sugr with biosynthesis of IPS, PEP cts s n interlocking metbolic key tht enbles the cell to tune its overll physiology to chnging environment. The trnsport of lctose in S. mutns by n inducible PEP-dependent Lc PT system nd the subsequent hydrolysis of the ccumulted lctose-phosphte by P-f8-gl reported in this communiction re consistent with the physiologicl dvntges discussed bove (33). Any crbohydrte consumed in quntity over lifetime nd/or by specific ge groups (such s lctose), which cn serve s substrte for bcteril glycolysis (with concomitnt production of cids [13] nd other products), is potentilly dngerous to the orl helth of the host. If the virulence of S. mutns is ssocited with its metbolic potentil (2, 3), it follows tht n understnding of the dissimiltory mechnisms for ll mjor dietry crbohydrtes is necessry if methods re to be devised which will eventully nullify the pthogenic potentil of S. mutns nd reduce or eliminte the occurrence of dentl cries in humns. ACKNOWLEDGMENTS I thnk Thoms T. Lillich for his criticl review nd Albert Brown for his mny helpful discussions. I m lso indebted to Debi Wlton nd Lois McCrcken for their expert ssistnce in the preprtion of this mnuscript. This investigtion ws supported by grnt from the University of Kentucky College of Dentistry Reserch Committee. LITERATURE CITED 1. Brtthll, D Demonstrtion of five serologicl groups of streptococcl strins resembling Streptococcus mutns. Odontol. Revy 21: Brown, A. T Crbohydrte metbolism in criesconducive, orl streptococci, p In H. L. Sipple nd K. W. McNutt (ed.), Sugrs in nutrition, vol. 33. Acdemic Press Inc., New York. 3. Brown, A. T The role of dietry crbohydrtes in plque formtion nd orl disese. Nutr. Rev. 33: Brown, A. T., nd C. E. Ptterson Heterogeneity of Streptococcus mutns strins bsed on their mnnitol-l-phosphte dehydrogenses: criterion for rpid clssifiction. Infect. Immun. 6: Brown, A. T., nd C. L. Wittenberger Fructose- LACTOSE TRANSPORT IN S. MUTANS 941 1,6-uipnospnte-ependent lctte enydrogense from criogenic streptococcus: purifiction nd regultory properties. J. Bcteriol. 11: Clmes, R., nd S. J. Del Ftty cid trnsport by the lipophilic bcterium, Nocrdi steroides. J. Bcteriol. 126: Cirdi, J. E., G. J. Hgege, nd C. L Wittenberger Multi-component nture of the glucosyltrnsferse system of Streptococcus mutns. J. Dent. Res. 55:C87-C Cords, B. R., nd L L McKy Chrcteriztion of lctose-fermenting revertnts from lctose-negtive Streptococcus lctis C2 mutnts. J. Bcteriol. 119: Coykendll, A. L Four types of Streptococcus mutns bsed on their genetic, ntigenic nd biochemicl chrcteristics. J. Gen. Microbiol. 83: dcost, T., nd R. J. Gibbons Hydrolysis of levn by humn plque streptococci. Arch. Orl Biol. 13: Egn, J. B., nd M. L Morse Crbohydrte trnsport in Stphylococcus ureus. Ill. Studies of the trnsport process. Biochim. Biophys. Act 112: Frostell, G., P. H. Keyes, nd R. HI Lrson Effect of vrious sugrs nd sugr substitutes on dentl cries in hmsters nd rts. J. Nutr. 93: Gibbons, R. J., nd J. vnhoute Dentl cries. Annu. Rev. Med. 26: Guggenheim, B., nd J. J. Burckhrdt Isoltion nd properties of dextrnse from Streptococcus mutns OMZ 176. Helv. OdontoL Act 18: Guggenheim, B., K. G. Konig, E. Herzog, nd H. R. Mihlemnn The criogenicity of different dietry crbohydrtes tested on rts in reltive gnotobiosis with streptococcus producing extrcellulr polyscchride. Helv. Odontol. Act 1: Hmilton, L. R Effects of fluoride on enzymtic regultion of bcteril crbohydrte metbolism. Cries Res. 11(Suppl. 1): Hengstenberg, W., J. B. Egn, nd M. L. Morse Crbohydrte trnsport in Stphylococcus ureus. V. The ccumultion of phosphorylted crbohydrte derivtives, nd evidence for new enzyme-splitting lctose phosphte. Proc. Ntl. Acd. Sci. U.S.A. 58: Hengstenberg, W., J. B. Egn, nd M. L Morse Crbohydrte trnsport in Stphylococcus ureus: the nture of the derivtives ccumulted. J. Biol. Chem. 243: Jordn, HI V., R. J. Fitzgerld, nd A. E. Bowler Inhibition of experimentl cries by sodium metbisulfite nd its effect on the growth nd metbolism of selected bcteri. J. Dent. Res. 39: Knpk, J. A., nd I. RI Hmilton Fluoride inhibition of enolse ctivity in vivo nd its reltionship to the inhibition of glucose-6-phosphte formtion in Streptococcus slivrius. Arch. Biochem. Biophys. 146: Kshket, E. R., nd T. H. Wilson Role of metbolic energy in the trnsport ofb-glctosides by Streptococcus lctis. J. Bcteriol. 19: Kshket, E. R., nd T. H. Wilson Protonmotive force in fermenting Streptococcus lctis 7962 in reltion to sugr ccumultion. Biochem. Biophys. Res. Commun. 59: Kundig, W., S. Ghosh, nd S. Rosemn Phosphte bound to histidine s n intermedite in novel phosphotrnsferse system. Proc. Nil. Acd. Sci. U.S.A. 52: Lederberg, J The,f-glctosidse of Escherichi coli, strin K-12. J. Bcteriol. 6: London, J., N. M. Chce, nd K. Kline Aldolse of lctic cid bcteri: immunologicl reltionships mong ldolses of streptococci nd grm-positive non- Downloded from on August 19, 218 by guest

9 942 CALMES sporeforming nerobes. Int. J. Syst. Bcteriol. 26: Lowry,. H., N. J. Rosebrough, A. L. Frr, nd R. J. Rndll Protein mesurement with the Folin phenol regent. J. Biol. Chem. 193: McKy, L. L., A. Miller HI, W. E. Sndine, nd P. R. Elliker Mechnisms of lctose utiliztion by lctic cid streptococci: enzymtic nd genetic nlysis. J. Bcteriol. 12: McKy, L. L., L. A. Wlter, W. E. Sndine, nd P. R. Elliker Involvement of phosphoenolpyruvte in lctose utiliztion by group N streptococci. J. Bcteriol. 99: Mrynski, J. H., nd C. L. Wittenberger Mnnitol trnsport in Streptococcus mutns. J. Bcteriol. 124: Morse, J. L., K. L. Hill, J. B. Egn, nd W. Hengstenberg Metbolism of lctose in Stphylococcus ureus nd its genetic bsis. J. Bcteriol. 95: Nlbndin, J., J. L. Freedmn, J. M. Tnzer, nd S. M. Lovelce Ultrstructure of mutnts of Streptococcus mutns with reference to gglutintion, dhesion, nd extrcellulr polyscchride. Infect. Immun. 1: Romno, A. H., S. J. Eberhrd, S. L. Dingle, nd T. D. McDowell Distribution of phosphoenolpyruvte: glucose phosphotrnsferse system in bcteri. J. Bcteriol. 14: Rosemn, S The trnsport of crbohydrtes by INFECT. IMMUN. bcteril phosphotrnsferse system. J. Gen. Physiol. 54: 138s-184s. 34. Rosemn, S The bcteril phosphoenoenolpyruvte: sugr phosphotrnsferse system, p In G. E. W. Wolstenhome nd D. W. Fitzsimons (ed.), Energy trnsformtion in biologicl systems. Cib Foundtion Symposium 31 (new series). Americn Elsevier, New York. 35. Schchtele, C. F Glucose trnsport in Streptococcus mutns: preprtion of cytoplsmic membrnes nd chrcteristics of phosphotrnsferse ctivity. J. Dent. Res. 54: Schchtele, C. F., nd J. A. Myo Phosphoenolpyruvte dependent glucose trnsport in orl streptococci. J. Dent. Res. 52: Tnzer, J. M., A. T. Brown, nd M. F. McInerney Identifiction, preliminry chrcteriztion, nd evidence for regultion of invertse in Streptococcus mutns. J. Bcteriol. 116: Tnzer, J. M., A. T. Brown, M. F. Mclnerney, nd F. M. Woodiel Comprison of invertses of Streptococcus mutns. Infect. Immun. 16: Thompson, J., nd T. D. Thoms Phosphoenolpyruvte nd 2-phosphoglycerte: endogenous energy source(s) for sugr ccumultion by strved cells of Streptococcus lctis. J. Bcteriol. 13: vnhoute, J., nd H. M. Jnsen Levn degrdtion by streptococci isolted from humn dentl plque. Arch. Orl Biol. 13: Downloded from on August 19, 218 by guest