INFECTION AND IMMUNITY, OCt. 1983, p. 19-26 Vol. 42, No. 10019-9567/83/100019-08$02.00/0Copyright © 1983, American Society for Microbiology

Carbohydrate Metabolism by Actinomyces viscosus Growingin Continuous Culture

I. R. HAMILTON'* AND D. C. ELLWOOD2Department of Oral Biology, Faculty of Dentistry, The University of Manitoba, Winnipeg, Canada' and

PHLS, Center for Applied Microbiology and Research, Porton Down, Salisbury, England2Received 28 February 1983/Accepted 18 July 1983

A human oral strain of Actinomyces viscosus, GN431/75, was grown anaerobi-cally in a defined medium in continuous culture with a glucose limitation atdilution rates (D) between 0.025 and 0.2 h-1 and with a nitrogen limitation at D =0.005 and 0.1 h-1. With 5 mg of glucose per ml, the culture was limited for carbonat D = 0.025 and 0.05 h-1, but became nitrogen limited (asparagine) at D = 0.1and 0.2 h-1. The molar growth yield (Ygiucose) decreased from 50.0 to 40.9 g ofcells per mol of glucose as the dilution rate was increased from 0.025 to 0.2 h-1reflecting the limitation of asparagine. With high glucose and low amino acidconcentrations (nitrogen limited), the cell yields at D = 0.05 and 0.1 h-' were 37to 33% lower than in the glucose-limited culture. The major products of metabo-lism were succinic and lactic acids with lesser amounts of acetic and formic acidsand ethanol. The rate of glucose fermentation by resting cells removed from theglucose-limited culture and assayed in a pH stat increased with the dilution rateand was always higher than that for the fermentation of sucrose (60%) andfructose (40%). Activity for the glucose-P-enolpyruvate phosphotransferase sys-tem was observed in whole homogenates, with the highest activity evident at D =0.1 h-1 with the glucose-limited culture. The observed activity was significantlylower than the rate of glucose metabolism at each dilution rate, suggesting thatglucose-P-enolpyruvate phosphotransferase system was underestimated or thatan additional transport system exists in the organism. The glucose-limited cultureshowed considerable ability to synthesize glycogen during the transition fromcarbon to nitrogen limitation, when 35% of the cell mass was present at thispolymer. The organism was shown to possess the glycogen synthetic enzymesADP glucose synthase and ADP glucose transferase, as well as the degradativeenzyme maltodextrin phosphorylase. Washed cells ofA. viscosus GN431/75 wereshown to be relatively insensitive to the inhibiting actions of NaF in pH-fall andconstant-pH experiments at all dilution rates. At pH 7.0, 25 mM NaF was requiredto completely inhibit glycolysis by glucose-limited cells at D = 0.05 h-1, whereasa concentration of only 11 mM NaF was required with cells of Streptococcusmutans grown and incubated under identical conditions. An interesting feature ofthe growth ofA. viscosus GN431/75 in the chemostat was the shift from individualnonadherent cells at the low dilution rates to the appearance at D = 0.2 h-1 oflarge cell aggregates forming tenacious adherent films reminiscent of its character-istics in the oral cavity.

Members of the genus Actinomyces are pre- inance ofActinomyces sp. in human root surfacedominant and unique constituents of dental lesions has indicated an etiological role for theseplaque microflora (2). Along with Streptococcus bacteria in human root surface caries (29, 30).sp., Actinomyces sp. can be isolated from a wide The exact biochemical niche for Actinomycesrange of animal species consuming a relatively sp. in the various plaque ecosystems is poorlyhigh carbohydrate diet and consequently has understood. Actinomyces species are less acido-come to be regarded as a component of the basic genic than are streptococci and lactobacilli; con-plaque community (3). Early animal models sug- sequently, they are generally considered to posegested a pathological role for Actinomyces sp. in less of a threat to enamel surfaces comparedperiodontal disease (19), and recent data have with cemental surfaces (7, 9). This appeared toimplicated members of the genus in human gingi- be substantiated in early studies where the infec-vitis (27). Furthermore, the numerical predom- tion of gnotobiotic rats with A. viscosus pro-

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20 HAMILTON AND ELLWOOD

duced only a limited amount offissure caries andno smooth surface caries (31). However, consid-erable caries formation was observed when theorganism was associated with the indigenousflora of specific-pathogen-free rats fed glucoseand sucrose diets. This specific interactive effectof A. viscosus was attributed, in part, to theability of the organism to produce large quanti-ties of plaque, as well as to acid formationresulting from carbohydrate metabolism.The anaerobic metabolism of glucose by A.

naeslundii results in the formation of acetate,formate, and succinate in the presence of C02,but only lactate in its absence (5). Althoughearly work showed A. viscosus to produce lac-tate and succinate from glucose under anaerobicconditions (18), more recent work has shownthat strains of A. naeslundii and A. viscosusfermented glucose with the production of lac-tate, succinate, acetate, formate, and ethanol(8). Succinate formation in A. viscosus is depen-dent upon the presence of carbon dioxide sinceoxaloacetate is generated from P-enolpyruvate(PEP) by PEP carboxylase (4). Oxaloacetate isfurther converted to succinate by the sequentialactions of malic dehydrogenase, fumarase, andsuccinic dehydrogenase.

In spite of the obvious importance of Actino-myces species in the various oral ecosystems,little is know about their biochemistry at theslow growth rates typical of human dentalplaque (24, 28). Consequently, we employedcontinuous culture to grow a human strain of A.viscosus at mean generation times between 3.5and 28 h in a defined medium with a carbon or anitrogen limitation. The growth yields and meta-bolic end products were analyzed at each dilu-tion rate, and washed cells were tested forglycolytic activity (in the presence and absenceof fluoride), for the presence of the PEP phos-photransferase hexose transport system, and forthe ability to synthesize glycogen.

MATERIALS AND METHODSBacteriological methods. A. viscosus GN431/75, a

human isolate, was kindly supplied by G. H. W.Bowden, formerly of the London Hospital DentalCollege, London, England. The organism was main-tained on blood agar, was catalase variable, and react-ed in whole cell agglutination with A. viscosusWVU627 antiserum. Consequently, the organism fitsinto cluster 1 of the numerical taxonomic classificationof Fillery et al. (11).The Streptococcus mutans Ingbritt strain used in

this study was kindly supplied by H. J. Sandham,University of Toronto. The methods of maintenanceand purity control have been described previously(14).Growth conditions. The culture was grown in a

Porton-type chemostat (16) with a 500-ml workingcapacity; the pH was maintained at pH 6.5, and the gasphase was 5% CO2 in nitrogen. The dilution rate (D)

was maintained at values between 0.025 and 0.2 h-1(mean generation time, 28 to 3.5 h), and the culturewas allowed to reach equilibrium for at least 10 meangenerations at each dilution rate before harvesting.The medium for growth in the chemostat was the

defined medium (M3) of Carlsson (6), modified aspreviously described (10), to obtain conditions ofglucose and nitrogen limitation (glucose excess). Theglucose-limited (glucose concentration, 5 mglml) cul-ture was grown in the modified M3 medium at dilutionrates of 0.025, 0.05, 0.1, and 0.2 h-'. For the nitrogen-limited culture, the modified M3 medium was furtheraltered to contain one-fifth of the concentration ofamino acids, whereas glucose was added at 55 mg/mlsuch that arginine and asparagine limited growth. Thisculture was grown at dilution rates of0.05 and 0.1 h-1.

S. mutans Ingbritt was grown with a glucose limita-tion in the same medium as that for the A. viscosusculture by methods previously described (10).

Washed-cell experiments. Cells were collected forvarying periods (usually less than 16 h) via the over-flow from each chemostat into a container cooled to0°C. They were then harvested by centrifugation(8,000 x g, 15 min) and washed once in potassiumphosphate buffer (20 mM, pH 7.0). The cells weresuspended in saline (0.98% NaCl in water) at a concen-tration of 30 mg (dry weight) of cells per ml and storedin ice until used.The rate of acid production from the anaerobic

fermentation of glucose, sucrose, and fructose and theinhibitory effect of fluoride on washed cells of A.viscosus GN431/75 were studied under conditions of(i) constant pH in a pH-stat or (ii) where the pH fallwas monitored (15). Glucose and fructose were used ata concentration of 10 mM, and sucrose was used at aconcentration of 5 mM. The units of glycolytic activitywere expressed as nanomoles of metabolic acid neu-tralized per milligram (dry weight) of cells per minute.In all cases, at least two samples were removed foranalysis at each dilution rate; steady-state conditionswere established between samples.

Cell breakage. Whole homogenates and cell-freeextracts of A. viscosus GN431/75 were obtained fromwashed-cell suspensions (30 mg/ml) prepared in phos-phate buffer (50 mM, pH 7.0). Cell disruption wasachieved by sonication for 5 min in a MSE sonicator(MSE, London, England) at full power. Microscopicexamination indicated few intact cells in the prepara-tion, which for our purposes will be considered thewhole homogenate. Intact cells and cell wall fragmentswere removed from portions of the whole homogenateby centrifugation at 15,000 x g for 25 min; the super-natant was considered the cell-free extract.Enzyme assays. Activity for the PEP phosphotrans-

ferase system (PTS) was assayed with whole homoge-nates by measuring the rate of the glucose-dependentconversion of PEP to pyruvate by a modification of themethod of Kornberg and Reeves (22). Attempts topermeabilize the cells by the usual toluene treatment(20), with toluene-ethanol (1:9), or by freeze-thawingwere without success; consequently, the whole-ho-mogenate preparation was used as the source of activi-ty (23). Cell samples were usually removed directlyfrom the growth chamber of the chemostat, and thewhole homogenate was prepared as described above.The conditions of the assay have been describedpreviously (14), except that each assay contained 20 to

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TABLE 1. Effect of dilution rate on growth parameters and metabolic end products of A. viscosus GN431M5grown in chemically defined medium with a glucose and nitrogen limitation (glucose excess)

Yield of Residual Concn (mM) of metabolic end productsDilution rate (h-1) cells glucose Y9ucOSCa Lactic Succinic Acetic Formic Ethanol

(mg/ml) (mM) acid acid acid acid

Glucose-limited'0.025 1.40 0 50.0 33.3 55.1 15.5 8.7 15.20.05 1.15 0 41.7 33.3 50.8 17.2 8.7 15.20.1 1.11 1.7 42.5 26.7 36.4 13.7 8.7 17.30.2 0.87 6.1 40.9 24.4 22.0 17.2 13.0 17.3

Nitrogen-limitedc0.05 0.44 141 2.68 12.2 12.7 3.5 4.4 00.1 0.37 144 2.36 13.3 13.6 3.5 4.4 0a Grams of cells per mole of glucose.bInitial glucose concentration, 28 mM.Initial glucose concentration, 306 mM.

200 ,ug of whole homogenate on a dry weight basis.Controls contained no glucose, and net PTS activity isexpressed as nanomoles of pyruvate formed per milli-gram (dry weight) of cells per minute.ADP glucose synthase, ADP glucose transferase,

and phosphorylase were assayed as previously de-scribed (12).

Analytical procedures. Metabolic products, residualglucose, and glucoamylase-specific glycogen in cellswere assayed as previously described (14).

RESULTSGrowth parameters and end products. The

growth of A. viscosus GN431/75 in the chemo-stat in a medium containing 5 mg of glucose perml was limited by glucose at D = 0.025 and 0.05h-1 (Table 1). Obviously, at the higher dilutionrates, growth was limited by some other nutri-ent. Analysis of the amino acid content of themedium at each dilution rate (Table 2) revealedthat the organism utilized all of the availableasparagine and, thus, that the culture was limit-ed by glucose and asparagine atD = 0.25 and 0.5h-1 and by asparagine alone at D = 0.1 and 0.2h-1. It is also of interest to note that, with theexception of methionine and leucine, the degreeof amino acids utilization by the culture de-creased as the dilution rate was increased,whereas ornithine was actually synthesized andexcreted during growth.Table 1 indicates that for the glucose-limited

culture, the cell yield fell from 1.40 to 0.87 mg/mlof medium as the dilution rate was increasedfrom 0.025 to 0.2 h-1, whereas the molar growthyield (Ygiucose) fell from 50.0 to 40.9 mM over thesame range. The major products of metabolismwere succinic and lactic acids with minoramounts of acetic and formic acids and ethanol.The concentration of the two major end prod-ucts fell as the dilution rate was increased. Asthe dilution rate increased from D = 0.025 to 0.2h-1, the morphology of the culture changed. At

the lower dilution rates, the culture formed ahomogenous suspension of discrete cells withonly minor branches and aggregates. At D = 0.2h-, however, the cells became highlybranched, and the culture formed tenaciousfilms on the walls of the chemostat. Consequent-ly, it was necessary to increase the agitatorspeed to maintain a homogenous liquid suspen-sion. The cell yield under nitrogen-limited condi-tions at D = 0.05 and 0.1 h-1 was only 37 and33% of that at the same dilution rates with theglucose-limited culture, whereas the Yglucosewere only 16 and 13%, respectively. Again,

TABLE 2. Amino acid utilization by A. viscosusGN431/75 grown at pH 6.5 in a chemostat with a

glucose limitation at D = 0.025 to 0.2 h-1

ATSAGPGAV

LT

LACCT

Amt (~Lmol/ml)Amino acid present in

original medium

Lspartate 1.0'hreonine 1.06erine 1.78Lsparagine 2.65'lutamine 3.2roline 0.8'lycine 2.5Llanine 1.1'aline 0.7lethionine 1.2soleucine 2.4,eucine 0.2'yrosine 0.5'henylalanine 0.5listidine 0.4,ysine 1.0Lrginine 0.4'ysteine 0.3)rnithine 0.05'ryptophan 0.5a Values preceded by

ornithine.

Utilization (%) at D of:

0.025 0.05 0.1 0.2

33 19 0 029 16 9 867 43 35 26100 100 100 10012 7 10 419 13 11 046 37 34 2077 65 41 289 12 0 026 27 27 2729 22 23 90 0 0 040 42 32 2840 34 20 625 33 25 2052 41 35 2695 90 80 7517 0 20 10

+140a +160 +160 +16036 26 26 24

+ indicate the formation of

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22 HAMILTON AND ELLWOOD

A-:stat. The highest activity was seen with glucosewhen the endpoint in the pH stat was at valuesbetween 7.0 and 5.5. The rates for sucrose andfructose were 60 and 40%, respectively, of thatfor glucose, and as would be expected, all of therates decreased as the pH was lowered in the pHstat.

Glucose transport activity. Many anaerobicbacteria are known to transport sugars via thePTS, which utilizes the energy of PEP to trans-

, , , port and phosphorylate the sugar in question0.1 0.2 (26). Experiments were undertaken to determine

DILUTION RATE whether glucose-PTS activity was present incells of A. viscosus GN431/75 growing in contin-uous culture with glucose. Low activity for the

B. glucose-PTS was observed with whole homoge-nates prepared by the sonication of washed cellsfor 5 min and assayed by the method of Korn-berg and Reeves (22). Glucose-PTS activity wasmaximal at pH 7.0, and the assay of wholehomogenates prepared from cells growing with aglucose limitation at D = 0.025 to 0.2 h-1

, , " indicated maximum activity at D = 0.1 h-7.0 6.5 6.0 55 (Table 3).

The PEP-dependent transport of sucrose andpH fructose was also observed, with a specific ac-

lycolytic activity by washed cells of glu- tivity similar to that found for glucose. ForA. viscosus GN431/75 cultures. (A) Rate example, at D = 0.2 h-1, activities of 2.18 andetabolism at D = 0.05 to 0.2 h- l. (B) Cells 1.94 nmol/mg of cells per min were observed for= 0.2 h-1 and incubated with glucose (0), sucrose and fructose, respectively. In addition,and fructose (A), in a pH stat at pH 7.0 to the glucose-PTS activity in the nitrogen-limitedis expressed as nanomoles per milligram culture was lower than that for the glucose-of cells per minute. limited culture at D = 0.05 h-1, which is similar

to that seen with S. mutans (10).When estimated with intact cells, the satura-

id lactic acids were the major end tion constant (Ks) for glucose at pH 7.0 was 2 xvhereas acetic and formic acids were 10-4 M with a Vmax of 1,052 nmollmin, indicat-minor amounts. ing that the observed PTS activity representedc activity. Since previous research only a fraction of the maximum transport activi-

has indicated that Actinomyces species are lessglycolytic than other members of the plaquemicroflora, such as the streptococci or lactoba-cilli (9), we undertook to test the glycolyticactivity of cells of A. viscosus GN431/75 grownat rates typical of dental plaque. Therefore,glucose-limited cells in steady state at eachdilution rate were collected and assayed anaero-bically for glycolytic activity in a pH stat withglucose, fructose, or sucrose as the carbonsource. The glycolytic rate with glucose in-creased from 12 to 40 nmol/mg of cells per minas the dilution rate was increased from D =0.025 to 0.2 h-V (Fig. 1A). Although the rates forsucrose and fructose showed a similar increasewith increasing dilution rates, the actual specificactivity was less than that for glucose. This canbe seen in Fig. 1B, which plots the glycolyticrate for cells collected from the chemostat at D= 0.2 h-1 and incubated with glucose, sucrose,and fructose at different pH values in the pH

ty.Effects of fluoride. Fluoride is known to have a

TABLE 3. Glucose-PTS transport activity in wholehomogenatesa

Glucose-PTS activity' inDilution rate cultures that were:

(h'l)Glucose limitedc Nitrogen limited

0.025 0.84 d0.05 1.64 1.180.1 2.190.2 1.85

a The homogenates were prepared from cells of A.viscosus GN431/75 grown in continuous culture withglucose as the carbon source at D = 0.025 to 0.2 h-1.

b Nanomoles of pyruvate formed per milligram ofcells per minute.

c Growth at D = 0.1 and 0.2 h-1 was actually limitedby nitrogen.

d -, Not done.

40 L-

HL120

Do

401

wH-

FIG. 1. Gcose-limitedof glucose megrown at D =sucrose (0),5.5. The rate(dry weight)

succinic anproducts, Mpresent in r

Glycolyti4

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significant inhibitory effect on carbohydrate me-tabolism by the acidogenic oral bacteria presentin dental plaque (2, 13). Consequently, the ef-fects of NaF were determined in both pH-falland pH-stat experiments with washed cellsgrown at each of the four dilution rates em-ployed with a glucose limitation. The compara-tive effects of fluoride at concentrations of 0, 5,and 9 mM in pH-fall experiments can be see inFig. 2 for cells grown at D = 0.025, 0.1, and 0.2h-1 and incubated in buffer with glucose. In theabsence of fluoride, the rate of acid formation(i.e., pH fall) increased as the dilution rate wasincreased; however, the more active cells werealso shown to be more insensitive to fluorideunder the same conditions. For example, the pHfalls were 0.5, 1.2, and 1.4 pH U for the cells atD = 0.025, 0.1, and 0.2 h-1, respectively, over a20-min incubation period in the presence of 9mM NaF.

Surprisingly, washed cells of A. viscosusgrowing on limiting glucose in the chemostat atD = 0.1 h-1 possessed significant endogenousactivity when the cells were incubated without acarbon source with various amounts of NaF(Fig. 3A). By comparison with cells receivingglucose (Fig. 3B), the cell metabolized the en-dogenous reserves in the absence of glucose atrates almost equivalent to those of cells receiv-ing glucose. In addition, those without exoge-nous glucose were somewhat more insensitive toNaF concentrations up to 9 mM.

In the pH-stat experiments (Fig. 4), relativelylittle difference could be seen between the cellsfrom two different dilution rates (D = 0.05 and0.2 h-1) with respect to the level of NaF re-quired to bring about complete inhibition ofglucose metabolism. Concentrations of NaF be-tween 22 and 25 mM were required to inhibitcompletely glucose fermentation by the washed

A. D=0.025 B. D=0.1 C. D=0.2

7

pH

6-

5-

0 10 20 0 10 20 0 10 20

MINFIG. 2. Effect of NaF on the pH fall by washed

cells of the glucose-limited culture of A. viscosusGN431/5 grown in a chemostat at D = 0.025, 0.1,and 0.2 h-l and incubated with 10 mM glucose.Symbols: 0, no NaF; 0, 5 mM NaF; A, 9 mM NaF.

pH

A. endogenous B. glucose

MINFIG. 3. Comparison of effect of NaF on the pH fall

by washed cells of the glucose-limited culture of A.viscosus GN431/75 grown at D = 0.1 h-V and incubat-ed with and without 10 mM glucose. Symbols: 0, noNaF; 0, 5 mM NaF; A, 9 mM NaF; A, 13 mM NaF.

40 A. D=0.2

20

<l 40B. D=O.05

20

00 10 20

NaF - mM

FIG. 4. Effect of NaF on the glycolytic rate bywashed cells of A. viscosus GN431/75 grown at D =0.05 and 0.2 h-1 and incubated with glucose in a pHstat at endpoints 7.0 to 5.5. The rate is expressed asnanomoles per milligram (dry weight) of cells perminute. Endpoint pHs were 7.0 (0), 6.5 (0), 6.0 (A)and 5.5 (A).

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24 HAMILTON AND ELLWOOD

TABLE 4. Glycogen content of cells of A. viscosusGN431/75 growing in continuous culture with glucose

as the carbon source

Dilution rate (h-') Glycogen content'

0.025 0.0410.05 0.1030.1 0.3640.2 0.359

a Milligrams of glucose per milligram of cells.

cells with the endpoint set at pH 7.0. When thecells were incubated with glucose and fluoride atprogressively lower pH endpoints, lower con-centrations of NaF were required to bring aboutinhibition, confirming the well-known pH effectof fluoride (13, 15).Formation of glycogen. Cells of A. viscosus

GN431/75 grown at D = 0.1 h-1 with limitingglucose exhibited significant acid productionupon incubation in buffer in the absence of anexogenous carbon source (Fig. 3). This suggest-ed that the cells might be capable of synthesizingan intracellular storage compound. An assay forglucoamylase-specific carbohydrate material inKOH-hydrolyzed cells grown with a glucoselimitation at each dilution rate revealed a signifi-cant increase in the cellular concentration of thispolysaccharide at D = 0.1 h-1 when the culturebecame nitrogen limited (Table 4). The levelremained at this high value at D = 0.2 h-1. Ananalysis of the iodine spectrum of the isolatedmaterial showed a wavelength maximum at 457nm, and authentic glycogen gave a value of 464nm, indicating that the cellular material wasglycogen.The assay of cell-free extracts of A. viscosus

GN431/75 for the glycogen synthetic enzymesADP glucose synthase and ADP glucose trans-ferase (25) revealed significant differences be-tween glucose- and nitrogen-limited cells grownat the same dilution rate (D = 0.05 h- 1; Table 5).ADP glucose synthase was not detected in theglucose-limited culture, and the transferase ac-

tivity was threefold lower than that in the nitro-gen-limited cells. The glycogen-degrading en-

zyme glycogen phosphorylase was also present

in higher levels in the latter cells and was more

active with dextrin as the primer, suggesting thatthe enzyme is actually a maltodextrin phosphor-ylase.

DISCUSSIONThe change in dilution rate of the glucose-

limited culture resulted in a significant decreasein the yield as the growth rate increased (Table1). This was undoubtedly due to the limitation ofasparagine and a possible rate-limiting reactionin glycolysis. The calculation of the distributionof carbon in the metabolic end products (Table1) in relation to the exogenous glucose sourceindicates that substantial end product carbonoriginated in the amino acid pool. Increasing thedilution rate (i.e., the growth rate) to D = 0.2h-1 resulted in a notable reduction in the forma-tion of succinate and, to a lesser extent, lactate.At the same time, considerable glucose carbonwas being diverted to glycogen (Table 4).One possible explanation for these observa-

tions is that a rate-limiting reaction exists in theglycolytic pathway at some point after the for-mation of glucose-6-P. The normal metabolicsequence in Actinomyces sp. would have a splitof the triose phosphate pool at PEP, with a

portion of the carbon diverted to succinate viaoxaloacetate, whereas the remainder is convert-ed to pyruvate, resulting in the formation oflactate, formate, acetate, and ethanol. The for-mation of acetate and formate obviously takesprecedence under these conditions (Table 1),probably because of the ATP-forming reactioncatalyzed by acetate kinase.The relationship of amino acid metabolism to

the energy pathway is somewhat obscure. Sur-prisingly, at D = 0.2 h-', the utilization ofaspartate, a possible precursor of oxaloacetate,was reduced to zero, suggesting that the aspar-tate requirement for protein synthesis was beingsupplied by asparagine. One would assume thatin this situation asparagine carbon is not contrib-uting to succinate formation.As mentioned earlier, the shift from a glucose

plus asparagine limitation to one where onlyasparagine was limiting (i.e., at D = 0.1 and 0.2h-1) resulted in a significant increase in glyco-

TABLE 5. Activity of the enzymes of glycogen metabolism in cell-free extracts of A. viscosus GN431/75grown in continuous culture at D = 0.05 h-'

GrowthSynthetic enzyme activitya Degradative enzyme activity'

limitation ADP glucose ADP glucose Phosphorylase Phosphorylasesynthase transferase (glycogen as primer) (dextrin as primer)

Glucose NDb 0.63 2.4 4.6Nitrogen 7.3 1.9 7.2 15.0

a Nanomoles per milligram of protein per minute.b ND, Not detected.

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gen synthesis. The phenomenon of increasedglycogen synthesis in a nitrogen-deficient medi-um is not new (17); however, the results in thisstudy indicate that glycogen synthesis in A.viscosus must be closely regulated since theappearance of only 1.7 mM of residual glucose atD = 0.1 h-1 (Table 1) resulted in an almostfourfold increase in the glycogen content of thecells (Table 2). Although glycogen has beenisolated from A. viscosus and characterized (21),little had been know previously about the meta-bolic systems involved. The appearance ofADPglucose synthase and ADP glucose transferasein extracts of A. viscosus GN431/5 indicatesthat glycogen synthesis follows the pathway thathas been observed in other bacteria (25).A comparison of the glycolytic activity for A.

viscosus GN431/75 in Fig. 1A under glucose-limiting conditions with the activity for the glu-cose-PTS in Table 3 reveals that the latter activi-ty does not contribute significantly to the overalltransport activity observed in the cells. By cal-culating the glycolytic rate in terms of glucoseconcentration, assuming a molar ratio of 2, onecan see that the glucose uptake rate (neglectingthose glucose molecules involved in glycogensynthesis) increased from 6 to 20 nmol/mg ofcells per min, whereas the glucose-PTS activityvaried from a low of 0.8 (D = 0.025 h-1) to ahigh of 2.2 nmol/mg per min at D = 0.1 h-1.Clearly, either the glucose-PTS activity wasgrossly underestimated, or the cell must possessan additional transport system to account for therate of transport activity. In fact, it is possiblethat both explanations may be tenable, andmuch more work must be done before the truenature of the transport process can be elucidat-ed.

Observations of the data in Fig. 3 and 4suggest that A. viscosus GN431/75 is relativelyinsensitive to the inhibitory action of fluoride.The relative insensitivity of Actinomyces sp. tofluoride has been noted by other workers (1).The significance of the fluoride insensitivity ofA. viscosus GN431/75 can be readily seen bycomparing it with the fluoride inhibition of S.mutans, grown in a separate chemostat in thesame medium at D = 0.05 h-1Washed cells of S. mutans possessed higl.

glycolytic activity and reduced the pH of themedium to near pH 4.0 within 10 min (Fig. 5).Cells of A. viscosus GN431M5 at the sameconcentration reduced the pH of the mediumonly to 5.3 within a 20-min period, indicating asignificant difference in the rate of glucose me-tabolism between cells of the two organisms.Interestingly, the addition of NaF had a muchgreater inhibitory effect on the faster-metaboliz-ing S. mutans cells than on the slower-metabo-lizing A. viscosus cells. The quantitative inhibi-

7

6

pH

5

4

r A. S. mutons K B. A. viscosus

0 10 20 0 10 20

MINFIG. 5. Comparison of effect of NaF on the pH fall

by washed cells of glucose-limited cultures of S.mutans Ingbritt and A. viscosus GN431/74 grown withglucose at D = 0.05 h-1 under identical conditions andincubated with 10 mM glucose. Symbols: 0, no NaF;0,0.23 mM NaF; A, 0.47 mM NaF; A, 2.3 mM NaF;*, 4.6 NaF.

tory etiect of NaF on these two cell types wasestimated in pH-stat experiments (data notshown), where cells metabolized glucose in thepresence of increasing concentrations of NaF.Glycolytic activity by S. mutans cells in theabsence of NaF was more than fourfold higherthan that for the A. viscosus cells (e.g., 76 versus18 nmol/mg of cells per min). On the other hand,metabolism by S. mutans was completely inhib-ited by 11 mM NaF, whereas A. viscosus wascompletely inhibited only when the concentra-tion reached 25 mM.The difference in fluoride sensitivity is proba-

bly associated with the relative rates at whichthey degrade glucose, since we have shownpreviously (13, 15, 20) that streptococcal cellscan possess a degree of immunity to fluorideunder conditions where growth or metabolismhas been reduced. In other words, slow-metabo-lizing cells are more fluoride insensitive than arecells metabolizing sugar rapidly. This conditionwould fit the case of A. viscosus since it metabo-lizes glucose relatively slowly.Another feature of fluoride insensitivity re-

lates to the involvement of the PTS in glucosetransport. Studies with S. mutans Ingbritt in

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26 HAMILTON AND ELLWOOD

continuous culture have demonstrated that cellswith low PTS activity are more fluoride insensi-tive than cells with high levels of this transportsystem (14). S. mutans Ingbritt possesses rela-tively high levels of the glucose-PTS under theconditions of glucose limitation at D = 0.05 h-1(11), whereas A. viscosus possesses low levelsof this system at this dilution rate (Table 3). Thefluoride susceptibility of this transport processresides not in the enzymes of the PTS, which areimmune to the effects of fluoride (20), but in theenzyme enolase, which generates PEP from 2-P-glycerate and which is known to be inhibited bya low concentration of fluoride.

ACKNOWLEDGMENTS

We thank M. Longyear, J. Hunter, and W. Harris for theirexcellent technical assistance.A portion of this research was supported by a research grant

(MT-3546) to one of us (I.R.H.) from the Medical ResearchCouncil of Canada.

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