JOURNAL OF BACTERiOLOGY, June 1982, p. 1024-10320021-9193/82/061024-09S02.00/0

Vol. 150, No. 3

Arginine Metabolism in Lactic StreptococciVAUGHAN L. CROW* AND TERENCE D. THOMAS

New Zealand Dairy Research Institute, Palmerston North, New Zealand

Received 26 October 1981/Accepted 13 January 1982

Streptococcus lactis metabolizes arginine via the arginine deiminase pathwayproducing ornithine, ammonia, carbon dioxide, and ATP. In the four strains of S.lactis examined, the specific activities of arginine deiminase and ornithinetranscarbamylase were 5- to 10-fold higher in galactose-grown cells comparedwith glucose- or lactose-grown cells. The addition of arginine increased thespecific activities of these two enzymes with all growth sugars. The specificactivity of the third enzyme involved in arginine metabolism (carbamate kinase)was not altered by the composition of the growth medium. In continuous culturesarginine deiminase was not induced, and arginine was not metabolized, untilglucose limitation occurred. In batch cultures the metabolism of glucose andarginine was sequential, whereas galactose and arginine were metabolized concur-rently, and the energy derived from arginine metabolism was efficiently coupled togrowth. No arginine deiminase activity was detected in the nine Streptococcuscremoris strains examined, thus accounting for their inability to metabolizearginine. All nine strains of S. cremoris had specific activities of carbamate kinasesimilar to those found in S. lactis, but only five S. cremoris strains had ornithinetranscarbamylase activity.

The arginine deiminase pathway (formerly re-ferred to as the arginine dihydrolase pathway)occurs in many bacteria, including some speciesof streptococci (1). This pathway involves thethree enzymes arginine deiminase (EC 3.5.3.6),ornithine transcarbamylase (EC 2.1.3.3), andcarbamate kinase (EC 2.7.2.2) which catalyzethe following reactions, respectively

L-arginine + H20 L-citrulline + NH3L-citrulline + Pi = L-ormithine +

carbamyl phosphatecarbamyl phosphate + ADP = ATP +

CO2 + NH3

Although there is considerable detail availableconcerning the properties of all three enzymesfrom Streptococcus faecalis (8, 9, 13-16, 22, 23)and to a lesser extent concerning ornithine trans-carbamylase and carbamate kinase from Strep-tococcus lactis (10, 24, 25), the factors affectingenzyme synthesis in bacterial cells are ill de-fined. Although various reports indicate thatsome streptococci are induced for arginine me-tabolism when grown on galactose (2, 32), onlow concentrations of glucose (20), or in thepresence of high arginine concentrations (8, 23),there appear to be no specific activity dataavailable for the enzymes concerned.

Lactic streptococci (Streptococcus cremoris,S. lactis, and Streptococcus diacetylactis) havea vital role as starters in commercial milk fer-

mentations, where their essential function is thehomolactic fermentation of lactose. S. cremorisstrains are generally preferred to S. lactis ascheddar cheese starters (12). Lactic streptococcipossess only limited catabolic and biosyntheticabilities, and their complex growth requirementsinclude a fermentable sugar for carbon and ener-gy supply. Orla-Jensen (21) in 1919 establishedS. cremoris as a type distinct from S. lactis onthe basis of a number of characteristics includingthe tendency to form longer chains, lower maxi-mum growth temperature, and less fermentativepower. Sherman (27) described these differencesbetween the two organisms as relative ratherthan definitive.A definitive difference between the two "spe-

cies" was established by a number of workers(5, 7, 20, 35) who showed that some streptococcisuch as S. lactis hydrolyzed peptone (and specif-ically arginine) with the production of ammonia,whereas S. cremoris strains lacked this ability.Agar media were subsequently developed (26,28, 34) to differentiate S. lactis from S. cremorison the basis of ability to produce ammonia fromarginine; such media are important in selectingstrains for use in milk fermentations. It is there-fore surprising that arginine metabolism has notbeen more extensively investigated in lacticstreptococci.The present study was undertaken to examine

the enzymes involved in arginine metabolism inlactic streptococci and to investigate why S.

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ARGININE METABOLISM IN LACTIC STREPTOCOCCI 1025

cremoris strains are unable to metabolize argi-nine.

MATERIAIS AND METHODS

Organisms and culture conditions. All strains werefrom the collection held at the New Zealand DairyResearch Institute. Arginine-negative mutants of S.lactis ML% and H1 were kindly supplied by H. A. Heapand A. W. Jarvis, and the arginine-positive mutant ofS. cremoris 166 was kindly supplied by L. E. Pearce.

Unless otherwise specified, static batch cultureswere grown at 30°C in T5 complex broth (30) whichcontained 28 mM galactose (or glucose) or 14 mMlactose with various amounts of arginine added (up toa final concentration of 48 mM). The basal concentra-tion offree arginine in the complex broth was 0.6 mM,and the initial pH was 7.2. Standardized carbohydrateand arginine solutions were ifiter sterilized before theywere added to autoclaved broth. Strains were trans-ferred at least three times on the appropriate carbohy-drate-arginine medium before cells were used in anyexperiments. Where indicated, S. lactis strains werealso maintained and grown in a chemically definedmedium (29). Continuous culture experiments in-volved anaerobic growth of S. lactis strains in definedmedium containing 28 mM glucose and 5.5 mM argi-nine; the detailed culture conditions have been report-ed elsewhere (29).Enzyme assays. Unless indicated otherwise, expo-

nentially growing cells were harvested from batchcultures when the residual carbohydrate concentrationwas about half the initial value. Cells from 100 ml ofmedium were washed and suspended in 5 ml of 20 mMphosphate buffer (pH 6.5) containing 50 mM NaCl and10 mM MgCl2. Cell disruption was achieved by shak-ing with glass beads for 2 min at 0 to 5°C in a Mickledisintegrator, and debris was removed by centrifuga-tion at 35,000 x g for 5 min. Arginine deiminase wasassayed in a reaction mixture (1 ml) containing 50 mMphosphate buffer (pH 6.7), 50 mM L-argmne, and theappropriate amount of cell-free extract. At suitabletime intervals, the reaction was stopped by the addi-tion oft ml of 10%o trichloroacetic acid, and precipitatedprotein was removed by centrifugation at 20,000 x g

for 5 min. The citrulline formed was measured by amodification of the Archibald method (4). To 1 ml ofthe centrifuged trichloroacetic acid-treated sample, 1.4ml of acid mixture (18 M H2SO4-14 M H3P04; 1:3,vol/vol) and 0.5 ml of 3% diacetyl monoxime wereadded, mixed together, and boiled in the dark for 15min. After cooling in the dark for 10 min, the absor-bance of samples and citnrlline standards (made inreaction mixture and treated in the same way) wasmeasured at 490 nm. Ornithine transcarbamylase as-say mixture (1 ml) contained 200 mM Tris-hydrochlo-ride (pH 8.5), 10 mM carbamyl phosphate, 5 mMornithine, and diluted cell-free extract. The reactionwas stopped, and citrulline was measured as describedabove for arginine deiminase. Carbamate kinase wasassayed by coupling ATP production from carbamylphosphate and ADP to NADP+ reduction via hexoki-nase and glucose 6-phosphate dehydrogenase. Thereaction mixture (1 ml) contained 100 mM Tris-hydro-chloride buffer (pH 8.5), 5 mM MgCl2, 10 mM glucose,0.5 mM NADP+, 5 mM ADP, S U of glucose 6-

phosphate dehydrogenase, 12.5 U of hexokinase, 10

mM carbamyl phosphate, and diluted cell-free extract.Controls lacking carbamyl phosphate were routinelyincluded and deducted from values obtained for com-plete reaction mixtures. The amount of carbamylphosphate used, as estimated with Escherichia colicarbamate kinase, was equal to the amount ofNADPHproduced when corrected for myokinase activity. Allthree enzymes were assayed at 25°C, and reactionrates were linear with time and extract concentrationunder the conditions used. Protein was determined bya modification (6) of the Lowry method, with bovineserum albumin as the standard.

Experiments with nongrowing cels. Cells from 100-ml exponential cultures were washed in 100 ml of 20mM phosphate buffer (pH 6.5) containing 50mM NaCland 10 mM MgCl2 and suspended in 10 ml of the samebuffer at 3 to 6 mg (dry weight) of bacteria per ml.Portions of this suspension, appropriately diluted,were placed in a Radiometer pH-stat apparatus (TTA31 titration assembly linked to an ABU 12 Autobur-ette, pH meter 26, Titrator 11, and Titrigraph SBR2C). The stirred suspension (2.5 ml) was adjusted topH 6.50 and maintained at 30°C. Arginine (50 ,ul, 0.5M, pH 6.5) was added, and the rate of acid (0.1 M HCI)addition required for pH control at 6.50 was recordedfor 5 min (see Table 3). In these experiments theactivity is expressed as micromoles of arginine utilizedper milligram (dry weight) of bacteria per minute. Thearginine concentration was measured in some sam-ples, and it was confirmed that 2 mol ofH+ was addedper mol of arginine utilized. In other experiments (seeTable 4), the cell suspension (10 ml) was maintained atpH 6.50 by the addition of either 1 M NaOH or 1 MHCI as appropriate. Samples (0.4 ml) were taken atintervals and added to an equal volume of 10%o tri-chloroacetic acid. After spinning at 20,000 x g for 5min, samples were stored at -20°C before assay ofsubstrate and products. The rate of arginine utilizedper milligam (dry weight) of bacteria per minute wasdetermined from enzymatic analysis of NH4( produc-tion. From a number of samples arginine was mea-sured both enzymatically and by the amino acid ana-lyzer. These analyses confirmed that 2 mol of NH4+and 1 mol of ornithine were produced per mol ofarginine utilized.

Experiments with growing cells. Cells previouslyadapted to grow on the appropriate sugar and arginineconcentration were inoculated into 100 ml of mediumand incubated at 30°C. During growth, 5-ml sampleswere removed and centrifuged at 20,000 x g for 5 min,and the supernatant was stored at -20°C before assay.For molar growth yield determination, three 20-mlsamples were taken after the maximum cell densityhad been reached. The bacterial density in thesesamples was determined directly by using membranefilters (29).

Substrate and product analysis. Glucose was deter-mined with Glucostat reagents (Worthington Diagnos-tics). For lactose measurement, the Glucostat reagentswere modified by the addition of MgCl2 (5 mM finalconcentration) and 0.8 U of ,B-galactosidase per ml ofreconstituted Glucostat reagent. Galactose was as-sayed enzymatically by the method of Kurz and Wal-lenfels (11). Ammonia was measured via glutamatedehydrogenase in the following reaction mixture (1 ml)incubated at 25°C for 45 min: 0.25 mM NADH, 25 mMa-ketogluturate, 200 mM triethanolamine-hydrochlo-

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1026 CROW AND THOMAS

ride buffer (pH 7.8), 9.6 U of glutamate dehydroge-nase, and the sample. Arginine and ornithine were

measured with a Locarte amino acid analyzer, al-though a more convenient arginine assay was devel-oped by coupling the reaction of arginine kinase topyruvate kinase and lactate dehydrogenase. The reac-

tion mixture (1 ml) contained 2 mM phosphoenolpyr-uvate, 5 mM ATP, 10mM MgCl2, 50mM KCl, 0.4mMNADH, 200mM triethanolamine-hydrochloride buffer(pH 7.8), 7 U of pyruvate kinase, 10 U of lactatedehydrogenase, 2.8 U of arginine kinase, and 0.1 to 0.2mM arginine. After incubation at 25C for 30 min, theabsorbance was measured at 340 nm.

Materials. All biochemicals were obtained from Sig-ma Chemical Co., St. Louis, Mo., as the grades withhighest analytical purity.

RESULTSProperties of enzymes involved in agnine me-

tabolism. The kinetic properties of arginine dei-minase, ornithine transcarbamylase, and carba-mate kinase were examined to ensure thatsaturating, noninhibiting substrate concentra-tions were chosen for routine assays. With cell-free extracts from S. lactis strains C10 and ML3,a km value of 2.5 to 5.0 mM arginine wasobtaiped from Lineweaver-Burk plots for argi-nine deiminase; 50 mM arginine did not inhibitactivity. With carbamate kinase the Km valueswere 1.2 to 1.5 mM for carbamyl phosphate and0.4 to 0.5 mM for ADP; the near-saturatingconcentrations of substrates used in the stan-dard assay caused no apparent inhibition. Withornithine transcarbamylase the Km values were2.1 to 2.5 mM for carbamyl phosphate and 3 to 5

mM for ornithine. However, saturating ornithinewas not used in routine assays, as -12 mMornithine gave 50%o inhibition of activity. Orni-thine also inhibited the ornithine transcarbamy-lase from S. faecalis (19), but this substrate didnot inhibit the enzyme from S. lactis strain 8039

(24), in contrast to the present results for strainsCI0 and ML3.The arginine deiminase activity from S. lactis

was the least stable of the three enzymes stud-ied. The activity of this enzyme in cell-freeextracts maintained at 4°C had decreased by40%o after 8 h. In contrast, ornithine transcarba-mylase activity fell by only 10%o, and the carba-mate kinase showed no loss of activity afterstorage for 8 h at 4°C. Arginine deiminase andornithine transcarbamylase were assayed within2 h of preparing the cell-free extracts. Carba-mate kinase was assayed within 4 h of extractpreparation.Enzyme activities in S. lacis stranl. (i)

Batch culture experiments. With strain ML3, thespecific activities of arginine deiminase, orni-thine transcarbamylase, and carbamate kinasewere measured in extracts from cells grown inmedia of different composition (Table 1). Thespecific activity of carbamate kinase was inde-pendent of medium composition, whereas thespecific activities of arginine deiminase and orni-thine transcarbamylase varied considerably.Specific activities of both the latter enzymeswere higher (i) when galactose, rather than glu-cose, was the growth sugar, and (ii) when argi-nine was added to the medium. Much lowerspecific activities of arginine deiminase and orni-thine transcarbamylase were usually found incells grown in defined medium compared withthose in cells grown in the complex broth. Thisbroth medium was therefore chosen for moststudies. Use of broth also allowed examinationof S. cremoris strains which do not grow in thedefined medium.Three other S. lactis strains (C1o, H1, ML8)

grown in complex broth showed enzyme pat-terns (data not shown) similar to those of strainML3 (Table 1). In addition, the pattern of en-

TABLE 1. Effect of growth medium composition on activities of enzymes involved in arginine metabolism inS. lactis ML3a

Growth medium Enzyme sp act

Armginne Arginine Ornithine CarbamateType Sugar (Mid) deiminaseb trn bkiae

Defined Galactose 0.4 0.06 3.3 7.1Galactose 47.0 0.09 5.3 7.1Glucose 0.4 0.001 2.9 7.2Glucose 47.0 0.05 5.0 7.2

Complex broth Galactose 0.6 0.05 7.9 5.6Galactose 47.0 0.28 59.3 6.6Glucose 0.6 0.01 0.9 6.0Glucose 47.0 0.14 25.5 5.0

a Culture conditions and enzyme assays were as described in the text. Mean values of enzyme specific activityfrom at least two separate experiments are given.

b Micromoles of citrulline formed per milligam of protein per minute.c Micromoles of ATP formed per milligram of protein per minute.

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ARGININE METABOLISM IN LACTIC STREPTOCOCCI 1027

E.C_ D50<

ZZ 82

w .SZ

a: to0E

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0

0-r

z z4 Z O0 Fa: << cc

3 co ZO L)L) ZD) O-J (

2 (D-j

a1ICl)Xc

DILUTION RATE (h-W)

FIG. 1. Effect of residual glucose concentration (A) on the level of arginine deiminase (0) and on themetabolism of arginine (0) to ornithine (0) in steady-state continuous cultures of S. lactis ML3 growing indefined medium containing 5.5 mM arginine.

zyme specific activities with all four S. lactisstrains was the same on substituting lactose forglucose.

Arginine-negative mutants of strains H1 andML8 grown in complex broth containing galac-tose, lactose or glucose, and 47 mM argininecontained no detectable arginine deiminase orornithine transcarbamylase activity (data notshown). The activity of carbamate kinase inthese mutants was similar to that found in strainML3.

Preliminary experiments (data not shown) inbatch cultures indicated that the specific activi-ties of arginine deiminase and ornithine transcar-bamylase, but not carbamate kinase, were influ-enced by the concentration of either glucose orgalactose in the medium at the point of cellharvest. Enzyme specific activities were higherin cells harvested when the residual sugar con-centration was low. This trend was more markedfor the glucose-grown cells. More precise ex-periments were therefore undertaken with con-tinuous cultures where cells can be grown atdefined sugar concentrations.

(U) Continuous culture experiments. At highdilution rates (0.74 and 0.67 h-1) where glucosewas in excess (7 and 4 mM, respectively), thelevel of arginine deiminase was 0.06 Fmol ofcitrulline formed per mg of protein per min (Fig.1). At lower dilution rates, where glucose waslimiting, the level of arginine deiminase in-creased markedly to reach a maximum of 0.6

gLmol of citrulline formed per mg of protein permin.Enzyme specific activities in S. cremons strains.

No arginine deiminase activity was detected incell-free extracts from nine S. cremoris strainsgrown in complex broth, even when cells weregrown on galactose in the presence of a higharginine concentration (Table 2). The possibilityof there being inhibitory material in the S. cre-moris extracts was checked. The addition ofcell-free extract (0.5 to 2 mg of protein) from S.cremoris strains ML1, E8, HP, or 166 to assaysystems containing extract from S. lactis C10,H1, or ML3 did not alter the arginine deiminaseactivity. To check for possible enzyme instabil-ity, four other buffers having different pH valuesand containing 2 mM dithioerythritol were usedto prepare cell-free extracts from S. cremorisstrains ML1, E8, and 166. However, no argininedeiminase activity was detected in any of thefreshly prepared extracts.The nine S. cremoris strains were divided into

two groups according to the specific activity ofornithine transcarbamylase found (Table 2). Noactivity of this enzyme was detected in the groupII strains. The extracts from these strains did notalter the ornithine transcarbamylase activity ofextracts from S. lactis strains C1o, H1, and ML3,and different procedures for the preparation ofcell-free extracts failed to elicit any activity. Thefive S. cremoris strains in group I had lowspecific activities of ornithine transcarbamylase

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1028 CROW AND THOMAS

TABLE 2. Activities of enzymes involved in arginine metabolism in S. cremoris strains grown in complexbrotha

Enzyme sp act

Strain Sugar in OrnithineStFSM1 ~~~mediuMb Arginine t~-Carbamatedeiminasec rbamylasec kinased

Group 1c Galactose NDf 0.1-0.3 1.0-3.0Glucose' ND 0.02-0.08 0.8-1.5

Group II' Galactose ND ND 1.0-3.0Glucoseg ND ND 1.0-3.0

a Culture conditions and enzyme assays were as described in the text. Mean values of enzyme specific activityfrom at least two separate experiments are given.

b Similar data were obtained with 0.6 and 47 mM arginine in the growth media.Micromoles of citrulline formed per millim of protein per minute.

d Micromoles of ATP formed per miligam of protein per minute.Group I strains are 104, 266, AM2, E8, and ML1. Group II strains are 114, 166, C13, and HP.

f ND, Not detectable Oess than 0.001).TThe same results were obtained when glucose was replaced by lactose.

activity when grown on glucose or lactose. Thespecific activity increased when cells weregrown on galactose but, in contrast to the S.lactis strains, activity was still low and was notincreased by the presence of arginine.The S. cremoris strains had specific activities

of carbamate kinase (Table 2) similar to thosefound in S. lactis strains. Citrulline at concentra-tions up to 50mM in the growth medium was notan inducer of the enzymes for arginine metabo-lism in either S. cremoris or S. lactis strains. Thearginine-positive mutant of S. cremoris 166showed high levels of arginine deiminase andornithine transcarbamylase (specific activities of0.3 and 14.7, respectively) when grown in com-plex broth containing galactose and 47 mMarginine. As in strain ML3 (Table 1), the specificactivities of these two enzymes decreased whenthe mutant was grown on glucose or with a lowarginine concentration (0.6 mM), whereas the

TABLE 3. Rates of arginine metabolism bynngrowing S. lactis ML3 cellsa

Growth medium composition Arginine utilized(Lmol/mg [dry

Sugar AArginine wtJ of bacteria/

Galactose 0.6 0.011Galactose 47.0 0.092Glucose 0.6 0.003Glucose 47.0 0.006Lactose 0.6 0.003Lactose 47.0 0.006

a Cells were grown in complex broth containing 28mM galactose or glucose or 14 mM lactose witharginine at the concentrations indicated. Harvestedcells were held in buffer, and the pH was maintained at6.5 using the pH-stat apparatus described in the text.

specific activity of carbamate kinase was notaltered by the different growth conditions.Nongrowing cell experiments. The rate of argi-

nine metabolism by nongrowing S. lactis ML3cells (Table 3) was consistent with the specificactivities of arginine deiminase and ornithinetranscarbamylase present in cell-free extracts(Table 1). Similar findings were made with theother three S. lactis strains (data not shown).With S. cremoris strains E8 and ML1 grown

under a variety of conditions, the nongrowingcells were unable to produce NH4' from argi-nine. This is consistent with the absence ofdetectable arginine deiminase activity in cell-free extracts.

Various combinations of carbohydrates andarginine were incubated with S. lactis C1o cellswhich had been grown in a medium containinggalactose plus arginine (Table 4). The presenceof carbohydrate reduced the rate of argininemetabolism and vice versa.Molar growth yield for arginin. To investigate

the coupling of energy from arginine metabolismto growth, molar growth yields for arginine(Y.,ww,) were measured with different ferment-able sugars. The results for ML3 on definedmedium are shown in Fig. 2. With galactose asthe fermentable carbohydrate the Y||il,e de-rived from the data shown in Fig. 2 was 16 g (dryweight) of bacteria per mol of arginine utilized.In contrast there was only a slight increase in theyield of bacteria due to arginine metabolismwhen glucose was the fermentable carbohydrateand the apparent Yain was <2.0 g/mol. Lac-tose gave similar results to glucose with strainML3 (data not shown).With complex medium containing either ga-

lactose or glucose, all four S. lactis strains gaveresults similar to those shown in Fig. 2. Howev-

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ARGININE METABOLISM IN LACTIC STREPTOCOCCI 1029

TABLE 4. Initial rates of arginine and carbohydratemetabolism by nongrowing S. lactis Clo cells'

Rate of metabolismAddition to buffer

(mM) Arginineb hydrte

Arginine (10) 0.088Galactose (20) plus 0.050 0.047

arguinine (10)Galactose (20) 0.081Glucose (20) plus 0.050 0.055

argnine (10)Glucose (20) 0.089Lactose (20) plus 0.050 0.030

arginine (10)Lactose (20) 0.043

a Cells were grown in complex broth containing 28mM galactose and 47 mM arginine. Harvested cellswere held in buffer, and the pH was maintained at 6.50using the pH-stat apparatus described in the text.

b Micromoles of arginine utilized per milligram (dryweight) of bacteria per minute.

c Micromoles of carbohydrate utilized per milligram(dry weight) of bacteria per minute.

er, on lactose Y,i,, values varied from 2.5 to6.0 g/mol. All molar growth yield values wereobtained in the absence of autolysis.

Concurrent or sequential metabolism of argi-nine. (i) Batch culture experiments. The previousdata indicating that the apparent Yargnie varieswith the growth sugar raises the question as towhether metabolism of sugar and arginine wasconcurrent or sequential in batch cultures. WithS. lactis H1 growing on glucose, the concentra-tion of sugar dropped from an initial value of 28

0.7

E

2 0.6

E

0.5

z

mM to between 0 and 2 mM before argininemetabolism was initiated (Fig. 3A).

In contrast to the sequential metabolism ofglucose and arginine, both galactose and argi-nine were metabolized concurrently (Fig. 3B).

(i) Continuous culture experiments. In contin-uous cultures containing excess glucose, argi-nine was not appreciably metabolized to orni-thine (Fig. 1). The decrease in arginineconcentration in the glucose-excess cultures(i.e., from 5.5 mM in the medium to -4 mM inthe culture) presumably reflects the growth re-quirement for this essential amino acid. Howev-er, as the dilution rate was dropped and glucoselimitation occurred, then arginine was metabo-lized until at a dilution rate of -0.35 h-1 argininewas not detectable in the culture. Results similarto those shown in Fig. 1 were obtained with S.lactis strains H1 and ML8.Determination of arginine, ornithine and

NH4' concentrations in samples from experi-ments described in Fig. 1, 2, and 3 and in Tables3 and 4 showed the expected stoichiometry forarginine metabolism, i.e., 1 mol of ornithine plus2 mol of NH4' produced per mol of arginineutilized.

DISCUSSIONIn S. lactis cells growing in batch cultures, the

specific activities of the first two enzymes of thearginine deiminase pathway were dependentupon both the nature of the sugar present and onthe arginine concentration. In contrast to theinducible nature of arginine deiminase and orni-thine transcarbamylase (and the carbamate ki-

10 20 30 40INITIAL ARGININE CONCENTRATION IN MEDIUM (mM)

50

FIG. 2. Effect of the initial arginine concentration on the yield of S. lactis ML3 cells growing in definedmedium containing an initial sugar concentration of 11 mM galactose (0) or 11 mM glucose (A). When celldensities were determined, sugar was not detectable in the medium, whereas residual arginine concentrationswere <0.5 mM.

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cc ~~~arginine AH4w zz zz a(3rgluoea alactose

U,. 0-

LL200 z LL z

0 z~~~~~~~~~<Z5 ~~~~~~Z5-cc~~~~~~~~~~~~~~~~~~~'z Iw ~~~~~~~~~zo w

z 0o zo 0

2 4 6 8 2 4 6 8INCUBATION TIME (h) INCUBATION TIME (h)

FIG. 3. Metabolism of glucose (A) or galactose (B) and arginine, together with NH4+ production, when S.lactis H1 was grown in batch culture in complex broth medium. Symbols: 0, glucose; 0, galactose; A, arginine;0, NH(+.

nase from S. faecalis [8]), the carbamate kinasewas constitutive in all four S. lactis strains.

Continuous culture experiments indicated thatthe glucose concentration in the growth mediumhas a marked effect on arginine deiminase withglucose-limited cells containing up to 10 timesthe specific activity of cells growing with excessglucose. It is interesting that growth of S. lactisunder glucose-limiting (energy-limiting) condi-tions causes two phenotypic changes which bothresult in an increase in energetic efficiency,namely, (i) the induction of arginine metabolism,as shown in the present study, and (ii) a changefrom homo- to heterolactic fermentation withmost strains (29). The results for continuouscultures are consistent with the finding that inbatch cultures of S. lactis the metabolism ofglucose and arginine was sequential (Fig. 3A). Inthese cultures, the energy derived from argininemetabolism was not eficiently coupled togrowth, as shown by the low Y Xwe values.This is presumably because the carbon source(glucose) was virtually exhausted before argi-nine metabolism commenced. Arginine cannotserve as the sole source of carbon; for itsmetabolism to proceed in the nongrowing cells,energy must be dissipated to regenerate ADP.Myokinase activity (1 to 4 ,umol of ATP formedper mg of protein per min) was found in the

strains of S. lactis studied and could play a rolein the regeneration of ADP. However, whenarginine metabolism was concurrent with sugarmetabolism (i.e., in cells growing on galactose)energy was efficiently coupled to growth asshown by the Ya,e (YATP) of 16 g/mol. This issimilar to the Yrgini value (17.8) reported forS. faecalis (18).The inability of S. cremoris strains to metabo-

lize arginine appears to be due to the absence ofarguiine deiminase. Some of the S. cremorisstrains examined also lacked ornithine transcar-bamylase activity (group II, Table 2). It is con-sidered unlikely that the absence of detectableactivity was due to enzyme instability as differ-ent extraction procedures were used and en-zymes were assayed immediately after prepara-tion of cell-free extracts.The specific activity of carbamate kinase was

similar in S. cremoris and S. lactis. It is not clearwhether this activity was due to the enzymecarbamate kinase or to a nonspecific kinase witha broad substrate specificity. For example,Thorne and Jones (33) separated two proteinsfrom S. faecalis which both had activity withacetyl phosphate. One of these proteins, whichhad an unusually high Km for acetate, also hadcarbamate kinase activity, whereas in E. coli asingle protein appeared to have both kinase

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ARGININE METABOLISM IN LACTIC STREPTOCOCCI 1031

activities (33). If carbamyl phosphate was asubstrate for the acetate kinase in lactic strepto-cocci and the specific activity varied withgrowth conditions, then the observed lack ofregulation of carbamate kinase could be an arti-fact. However, the specific activity of acetatekinase did not vary with the sugar in the growthmedium (31) or with various arginine concentra-tions (V. L. Crow, unpublished data), and it isreasonable to assume that the acetate kinasefrom lactic streptococci is similar to that from S.faecalis in not utilizing carbamyl phosphate as asubstrate.The activity of ornithine transcaroamylase in

group I S. cremoris strains (Table 2) was in-creased when glucose was replaced by galac-tose. However, unlike the S. lactis strains, theaddition of arginine to the growth medium didnot increase the enzyme levels further. Theenzyme pattern in group I strains was similar tothat found in some S. lactis strains growing onglucose or lactose in the presence of low argi-nine concentrations. For example, no argininedeiminase activity and low ornithine transcarba-mylase activity (specific activity of 0.2) werefound in S. lactis C1O. The enzyme pattern ofarginine-negative strains of S. lactis ML8 and H1was the same as that of S. cremoris (group II)strains. S. cremoris strains may lack the struc-tural genes coding for these two enzymes. Alter-natively, the structural genes for arginine dei-minase and ornithine transcarbamylase may bepresent in some strains of S. cremoris, but arenot expressed because of some defect in astructural gene or a possible regulatory gene.This is supported by the finding of an arginine-positive mutant of S. cremoris which showedlevels of arginine deiminase and ornithine trans-carbamylase similar to those found in S. lactisstrains. As in S. lactis, the specific activities ofthese two enzymes in the mutant were highest incells grown with galactose and high arginineconcentrations in the medium.

Arginine is an essential amino acid for manylactic streptococci (3). In S. lactis, the enzymespecific activities vary with growth conditions,including arginine concentration. In cells grow-ing with low arginine concentrations the activityof the arginine catabolic pathway may be suffi-ciently low to ensure that depletion of the essen-tial amino acid does not occur.The metabolism of arginine by H1 (Fig. 3) and

C1o (data not shown) is concurrent with galac-tose fermentation, but is sequential with glucosefermentation. Only when the glucose concentra-tion is between 0 and 2 mM does argininemetabolism occur to an appreciable extent.However, these same batch-grown cells, whenharvested at 12 to 14 mM residual glucose, havehigh activities of the arginine enzymes and,

when used in nongrowing cell experiments (Ta-ble 3), do metabolize arginine at an appreciablerate. This apparent contradiction could be ex-plained if enzyme(s) of the arginine deiminasepathway were regulated by a fine control mecha-nism such as fluctuating metabolite concentra-tions. Pi is one metabolite that may be importantin regulation of arginine metabolism. Ornithinetranscarbamylase requires phosphate for thephosphorylytic cleavage of ornithine. The direc-tion is thermodynamically unfavorable, and theKm value for phosphate is high (47 mM) in S.lactis (10). Mason et al. (17) concluded that theintracellular concentration of Pi was a majorfactor in the control of glycolysis in nongrowingS. lactis cells, with phosphate being high instarved cells (47 mM) and low (4 mM) afterglucose addition. In our experiments, cellsgrowing on glucose or lactose fermented most ofthe carbohydrate before arginine was used.These cells, depleted of carbohydrate in thegrowth medium, are likely to contain high con-centrations of inorganic phosphate (17). Thepossibility that with a high arginine concentra-tion in the growth medium, cells growing ongalactose contain high phosphate concentrations(which can drive the ornithine transcarbamy-lase) will be investigated.As previously mentioned, arginine metabo-

lism was believed to be a definitive differencethat could be used to distinguish S. lactis and S.cremoris, which are otherwise closely relatedorganisms. The present work, however, makesit clear that this character is also relative in viewof the potential of S. cremoris to metabolizearginine, the regulatory nature of arginine me-tabolism in S. lactis, and the production ofargisine-negative strains of S. lactis. The resultslend further support to the view that S. cremorisis probably a variety of S. lactis better suited foruse in many milk fermentations. Regardless ofits taxonomic position, it is thus still importantto differentiate the S. cremoris group from S.lactis, and this can be achieved by joint consid-eration of a number of relative characters. Fur-ther studies involving arginine mutants of lacticstreptococci are likely to shed additional light onrelationships within this group of organisms.

ACKNOWLEDGMENTSWe are grateful to Judith Cleland for excellent technical

assistance and to Alan Matheson and Barry Richardson foroperating the amino acid analyzer, and Lindsay Pearce forhelpful discussions.Continuous culture experiments were carried out at the

PHLS Centre for Applied Microbiology and Research, PortonDown, England.

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