JOURNAL OF BACTERIOLOGY, Apr. 1970, p. 6-13Copyright © 1970 American Society for Microbiology

Vol. 102, No. 1Printed In US.A.

Amino Acid Transport in Mycobacterium smegmatisKUNIHIKO YABU

Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Kiyose-Machi,Kitatama-Gun, Tokyo, Japan

Received for publication 30 December 1969

The transport of D-alanine, D-glutamic acid, and D-valine in Mycobacteriumsmegmatis was compared quantitatively with that of their L-isomers. It appearedthat the uptake of D-alanine was mediated by an active process displaying saturationkinetics characteristic of enzyme function, whereas the uptake of D-glutamic acid wasaccomplished by a passive process showing diffusion kinetics. Both processes wereinvolved in the uptake of L-alanine, L-glutamic acid, D-valine, and L-valine. D-Valine competed with L-valine for entry into the cell through a single active process.D-Alanine and L-alanine also utilized the same active process, but the D-isomercould not enter the cell through the passive process. The passive process exhibitedcharacteristics of diffusion, but was sensitive to sulfhydryl-blocking reagents andshowed competition among structurally related amino acids. These last findings sug-gested that the passive process is a facilitated diffusion.

In the previous paper (27), evidence waspresented indicating that the uptake of D-glu-tamic acid and that of L-glutamic acid by Myco-bacterium avium differ with regard to the systemby which external glutamic acid is taken up intothe cell. The uptake of L-glutamic acid conformsto Michaelis-Menten kinetics. On the otherhand, the uptake of D-glutamic acid resembleslysine accumulation by diffusion (7,8,9) in itsconcentration dependence, but it differs in thecompetitive effect by structurally related aminoacids. The present communication extends thecomparison of the rates of uptake of D- and L-isomers, by M. smegmatis, to alanine, glutamicacid, and valine, and attempts a further charac-terization of the process of amino acid transportby facilitated diffusion.

MATERIALS AND METHODSOrganism. M. smegmatis (ATCC 607) was used

throughout this work. The cultures were maintainedby biweekly transfers in a Sauton synthetic mediumwhich contained per liter: 4.0 g of asparagine, 2.0 g ofcitric acid, 0.5 g of K2HPO4, 0.5 g of MgSO4-7H20,0.05 g of ferric ammonium citrate, and 60.0 g ofglycerol (final pH 7.0).

Preparation of cell suspension. The cells requiredfor experimental procedures were grown from a 20-hrinoculum at 37 C in Sauton synthetic medium. Theywere harvested in the late exponential phase onfilter paper in a funnel, washed thoroughly with dis-tilled water, and then suspended in ice-cold distilledwater by grinding in a mortar. The cell suspensionwas added to the uptake mixture to bring the con-centration to 1.0 to 2.0 mg (dry weight) per ml.Freshly harvested cells were used for each experiment.

6

Uptake and extraction procedure. Uptake wasmeasured at 37 C on a reciprocal shaking-water bathin 50-ml Erlenmeyer flasks, each containing a totalvolume of 10 ml. The uptake mixture contained 0.04M phosphate buffer (pH 7.0), 0.025 M glucose, and14C-labeled amino acid in varying amounts. Atintervals, the flasks were placed in iced water. Thecells were centrifuged for 7 min in the cold at 15,000 Xg, and were washed twice with ice-cold phosphatebuffer containing glucose alone by a similar cen-trifugation. The centrifuged tubes were swabbed dry,and the cell pellet was extracted with 6 ml of boilingwater for 15 min, the cell debris was then removed bycentrifugation. The resulting cell extract was evapo-rated to dryness and then redissolved in 1 ml ofdistilled water. An appropriate portion of the extractsample was plated onto a stainless-steel planchet, andthe radioactivity was determined to give the value for"pool" content. All radioactive counts were carriedout with an automatic gas-flow counter (JapanRadiation and Medical Electronics Inc., Tokyo), andto reduce statistical deviation, at least 2,000 countswere recorded.

This procedure gives the reproducible data ratherthan that measuring transport into the whole cell,although it uses rather a great amount of the cellbecause of a hydrophobic cell surface of mycobac-terial cell. None of the amino acids tested is signifi-cantly incorporated into the cell debris under theexperimental conditions used.

Chemicals. D-(1-14C)glutamic acid was isolatedfrom the labeled racemate, as previously described(27). Uniformly 14C-labeled L-alanine (39.6 mc/mmole), L-glutamic acid (179 mc/mmole), andDL-(1-14C)glutamic acid (16 mc/mmole) were ob-tained from Daiichi Pure Chemical Co., Tokyo.D-(1-'4C)alanine (13.5 mc/mmole), D-(1-'4C)valine(18.1 mc/mmole), and L-(1-14C)valine (34.2 mc/

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AMINO ACID TRANSPORT IN M. SMEGMATIS

mmole) were obtained from New England NuclearCorp., Boston, Mass. The purity of these compoundswas checked by paper chromatography in each oftwo different solvents: (i) n-butyl alcohol-aceticacid-water (4:1:2, v/v) and (ii) phenol-water (4:1,v/v). Only a single radioactive and ninhydrin-positivespot was found in all cases. Unlabeled amino acidsused as carriers for "4C-amino acid were productsof Nutritional Biochemicals Corp., Cleveland, Ohio.All other chemicals used were of reagent grade.

RESULTSTime course of D- and L-amino acid uptake.

The time course of the uptake of D- and L-isomers of alanine, glutamic acid, and valine isshown in Fig. 1. The uptake of most of theseamino acids was linear with time for about 15min, and then declined, reaching a plateau afterabout 20 min. L-Glutamic acid uptake continuedat a linear rate for at least 20 min, whereasD-glutamic acid entered the cell very slowly. Forthese amino acids the uptake of the L-isomerexceeded that of the D-isomer.

Considering that this organism is a slowgrower as compared with Escherichia coli andStaphylococcus aureus, and that it possesses awaxy cell surface which could confer on ithydrophobic characteristics, the initial rate ofentry was estimated from a 10-min incubation.

Kinetics of glutamic acid uptake. The rates ofD- and L-glutamic acid uptake as a function ofthe external concentration are shown in Fig. 2.The rate of D-glutamic acid uptake increasedslowly and linearly with the external concentra-tion.This observation indicates that the entry of

D-glutamic acid into the cell is accomplished bya process similar to simple diffusion. In contrast,the rate of L-glutamic acid uptake, at externalL-glutamic acid concentrations from 0.08 mM toapproximately 0.4 mm, increased rapidly withincreasing external concentrations. Above 0.4

EXTERNAL CONCENTRATION (mM)FIG. 2. Rates ofthe uptake ofD- and L-glutamic acid

as a function of the external glutamic acid concentra-tion. The cells were incubated at 37 C for 10 min andwere then tested for glutamic acid uptake as describedin Materials and Methods.

X / LDValine

10 ~

%w

D 1 /z

01

0 5 10 15 25

FIG. 1. Time course of the uptake Of D- and L-isomers of alanine, glutamic acid, and valine. The cellswere incubated at 37 C for the time shown and were then tested for amino acid uptake as described in Ma-terials and Methods. The final concentrations were as follows: D- and L-alanine, 0.4 mM; D- and L-glutamicacid, 0.25 mM; D- and L-valine, 0.1 mM.

I I _-Alni no

-o

..L.

iNbb~ I/Z,0

*1

90.0.Z

W10. D-Alanino

0 5 10 15 20

INCUBATION T I ME (min)

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mM, further increases in external L-glutamic acidconcentrations produced a rather small but linearincrease in the rate.The shape of this biphasic curve is considered

to be the result of two component curves, oneindicating saturation kinetics characteristic ofan enzyme, and the other showing no saturationand assumed to reflect the process of diffusion.The first of these components is believed torepresent saturation of the permease system forL-glutamic acid (13, 15), and appearance of thesecond component shows that, after saturationof the permease system, L-glutamic acid is takenup by a nonsaturable, diffusion-like process, andthat the accumulation of L-glutamic acid to levelsexceeding the external concentration does notoccur through this process. The presence of thesetwo components is even more strikingly indicatedby the discontinuity in the Lineweaver-Burk plotof Fig. 3. The Km value calculated from curve Ais 2.4 X 10-4 M, and curve B would represent ahigh Km value.

Kinetics of alanine uptake. The rates of D- andL-alanine uptake as a function of the externalconcentration are shown in Fig. 4. At low ex-ternal concentrations of D-alanine, the rate ofD-alanine uptake increased proportionally withincreasing external concentrations, but, at higherconcentrations of D-alanine above approximately0.4 mm, the uptake system became saturated andthe rate of uptake was independent of the externalconcentration. The data exhibit solely saturationkinetics, indicating the existence of permease

0.081

A

10

C

0 L-Alanili

75-

_. 5

B 25z

O-AIanin.0 0 * ' _0

O 2 6EXTERNAL CONCENTRATION (mM)

Fio. 4. Rates of the uptake ofD- and L-alanine as afunction ofthe external alanine concentration. The cellswere incubated at 37 Cfor 10 min and were then testedfor alanine uptake as described in Materials andMethods.

system responsible for D-alanine uptake. On theother hand, the rate of L-alanine uptake at lowexternal concentrations of L-alanine increasedrapidly with increasing external concentrations.At external L-alanine concentrations aboveapproximately 0.4 mm, the increase in the rateof L-alanine uptake with increasing externalconcentrations decreased and exhibited a linearcurve. The Lineweaver-Burk plot of the datayields two components similar to those obtainedin the experiment measuring the rate of L-glutamic acid uptake.The results indicate that two processes, a

permease system (21, 22) and a diffusion-likeprocess, are involved in the uptake of L-alanine,as in that of L-glutamic acid. The Vmaz of thepermease system for L-alanine is higher than thatfor D-alanine. The Km values for D- and L-alanine are 1.9 x 10- and 7.0 x 10- M, re-spectively.

In Lactobacillus casei (21) and Streptococcusfaecalis (22), it has been reported that D- andL-alanine are taken up by the same transportsystem. It is therefore of interest to know whetherD- and L-alanine utilize the same permeasesystem for entering M. smegmatis cells, sinceonly a single transport process operates for theentry of D-alanine, but two processes operate forthe entry of L-alanine. Competition experimentswere undertaken to elucidate the relationship

10-34L-GLUTAMIC ACID]FIG. 3. Lineweaver-Burk plot of the data on L-glu-

tamic acid uptake of Fig. 1.

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AMINO ACID TRANSPORT IN M. SMEGMATIS

between D- and L-alanine uptake. D-Alaninecompetitively inhibited L-alanine uptake, andconversely, L-alanine competitively inhibitedD-alanine uptake (Fig. 5).

These findings emphasize that D-alaninecompetes with L-alanine for entry into the cellthrough a single permease system, but this D-isomer cannot enter the cell through the diffusion-like process. A stereospecific difference existsbetween D- and L-alanine for the operation ofdiffusion-like process.

Kinetics of valine uptake. The rates of D- andL-valine uptake as a function of the externalconcentration are shown in Fig. 6. The rate ofD-valine uptake at external D-valine concentra-tions from 0.0125 mm to approximately 0.1 mMincreased rapidly with increasing external con-centrations. At D-valine concentrations above0.1 mM, the increase in the rate of D-valineuptake with increasing external concentrationsdecreased and showed a linear curve. The rate ofL-valine uptake, on the other hand, at lowexternal concentrations of L-valine, increasedalmost linearly with increasing external concen-trations, but at higher external concentrations ofL-valine above approximately 0.1 mM, theincrease in the rate of L-valine uptake decreasedand approached that of D-valine uptake.The Lineweaver-Burk plot of the data presents

two component curves, indicating that twoprocesses, a permease system (6, 20) and adiffusion-like process, are involved in the entryof both D- and L-valine into the cell. The Vmaxof permease system for L-valine is approxi-mately twice that for D-valine. The Km valuesfor D- and L-valine were 6.0 X 10-5 and 2.0 X10- M, respectively. Although the Km and V.ax

0 5 10 a 20 0 S 10 20

10tW/(-ALMAINE 10'2/(L-ALANINE]

FiG. 5. (A) Competitive inhibition of D-alanine up-take by L-alanine. D-Alanine uptake at external D-ala-nine concentrations from 0.05 to 2.0 mM was examinedin the presence and absence of L-alanine. Other condi-tions were as in Fig. 4. (B) Competitive inhibition ofL-alanine uptake by D-alanine. L-Alanine uptake atexternal L-alanine concentrations from 0.05 to 0.8 mmwas examined in the presenice and absence of D-alanine.Other conditions were as in Fig. 4.

Sc ~~~~~~~~~~~L-Va&lino z

o2

0~~~~~~~~~~~~~

k /.. 31 X

12w

0.

z

0 0.5 1.0 1.5 20EXTERNAL CONCENTRATION (mM)

FIG. 6. Rates of the uptake of D- and L-valine as afunction of the external valine concentration. The cellswere incubated at 37 Cfor 10 min and were then testedfor valine uptake as described in Materials and Methods.

values differ for each of these isomers, a singlepermease system appears to be involved in theiruptake, as in the uptake of D- and L-alanine.This conclusion is based on the competitionexperiments, which show that D-valine competi-tively inhibits L-valine uptake, and conversely,L-valine competitively inhibits D-valine uptake(Fig. 7).

Effect of temperature. It is of great interest tocharacterize the diffusion-like process observedin the uptake of L-alanine, L-glutamic acid, andD-valine. Both permease system and diffusion-like process, however, are involved in the uptakeof these amino acids. If a permeaseless mutant,showing only a linear rate response with theincreasing external amino acid concentration,could be isolated, the operation of the permeasesystem can be excluded. Attempts to isolate sucha permeaseless mutant have been numerous butunsuccessful. Therefore, in order to compare thediffusion-like process with the permease system,the rates of the uptake of L-alanine, L-glutamicacid, and D-valine, at two external concentrationsat which the permease system dominates and thediffusion-like process dominates, were examinedfor temperature dependence, inhibition bymetabolic inhibitors, and competitive effect byamino acids.

Figure 8 shows the temperature dependenceof the uptake of L-alanine, L-glutamic acid, andD-valine at low and high external concentrations.

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(a)

10

Z0NOD-21m

0 D so 71

V CL-VALI

FIG. 7. (A) Competitive inhibition of D-valine up-take by L-valine. D-Valine uptake at external D-valineconcentrations from 0.025 to 0.2 mM was examined inthe presence and absence of L-valine. Other conditionswere as in Fig. 6. (B) Competitive inhibition of L-Valineuptake by D-valine. L-Valine uptake at external -valineconcentrations from 0.01 to 0.1 mM was examined inthe presence and absence of D-valine. Other conditionswere as in Fig. 6.

(A) L-AlWAs

10

LGamoic10 0-V4alise

' Rl1 -./;-. I

I Ia

eL

II

I L-SeIA

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INClUATION TEMPETUE V'C)

FIG. 8. (A) Temperature dependence of the uptakeof L-alanine, L-glutamic acid, and D-valine at low ex-

ternal concentrations. The external concentrationswere as follows: L-alanine, 0.4 mM; L-glutamic acid,0.25 mM; D-valine, 0.1 mm. The cells were incubatedfor 10 min at the indicated temperature and were thentested for amino acid uptake as described in Materialsand Methods. Amino acid uptake (ordinate) is expressedin micromoles per gram (dry weight) per 10 min forL-alanine and L-glutamic acid and in micromoles per10 g (dry weight) per 10 min for D-valine. (B) Tem-perature dependence of the uptake of L-adanine, L-glu-tamic acid, and D-valine at high external concentra-tions. The external concentrations were as follows:L-alanine, 8.0 mM; L-glutamic acid, 8.0 mi; D-valine,2.0 mM. Amino acid uptake was measured as in (A).

The external concentrations of each amino acidare listed in the legend of the figure. The rates ofamino acid uptake at low external concentrationswere negligible below 10 C, but they increasedrapidly with the increasing temperature to themaximum rate exhibited at 38 C. At tempera-tures exceeding 38 C, the rates in all cases de-clined sharply, showing a characteristic commonto many enzyme systems. On the other hand, at

high external concentrations, the increase in therates of uptake with the increasing temperaturewas approximately linear to 38 C.At the high external concentrations used, the

permease systems are responsible for only about20 to 25% of the uptake of the amino acids, as

can be seen in Fig. 2, 4, and 6. The remaining 75to 80% of the uptake is due to the diffusion-likeprocess. Therefore, the results obtained are

considered to exhibit solely a feature of thediffusion-like process.

Effect of some inhibitors. The effect of sulfhy-dryl-blocking reagents on the uptake of L-alanine,L-glutamic acid, and D-valine at low and highexternal. concentrations was investigated. Asshown in Fig. 9, the uptake of the amino acids athigh external concentrations was consistentlysensitive to p-chloromercuribenzoate, althoughnot so great as the uptake at low external con-centrations. The inhibition of L-glutamic aciduptake at the high external. concentration,however, was no less than that of D-glutamic aciduptake in which no permease system was involved.The results obtained with iodoacetate wereessentially similar to those recorded with p-chloromercuribenzoate. These findings demon-strate the involvement of the sulfhydryl group inthe diffusion-like process.

Competitive effect of amino acids. The rates oflabeled L-alanine uptake at low and high externalL-alanine concentrations were measured in thepresence of other unlabeled amino acids at an

external concentration of 32 mm. The results aresummarized in Table 1.

Since, at an external L-alanine concentrationof 8.0 mm, approximately 25% of the rate ofL-alanine uptake was due to the permease

2Z

i

p.HLORD E1 IDW20^ATE ({1")

FiG. 9. Effect of p-chloromercuribenzoate on theuptake of D-valine, L-alanine, and L-glutamic acid atlow and high external concentrations. External aminoacid concentrations were as described in Fig. 8. Thedotted line indicates the uptake of D-glutamkc acid(0.25 mm) under the same experimental conditions. Thecells were preincubated with p-chloromericuribenzoatefor 20 min prior to the addition of amino acid. The up-take wasfollowedfor an additional 10 min.

(A)

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10"

20

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He L-VeAiew

0 10 2 0 4

I'i,

0

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I

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!40

20-

1. 2 1I iS * tO I a 11 ta laI * 10

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AMINO ACID TRANSPORT IN M. SMEGMATIS

system, the inhibition of less than 25% would notbe considered as real inhibition of the diffusion-like process, as described earlier.Although D-alanine inhibited L-alanine uptake

by the permease system, it did not inhibit theentry of L-alanine through the diffusion-likeprocess. L-Glutamic acid, L-leucine, and L-valinealso had no significant effect on the entry ofL-alanine through the diffusion-like process.D-Glutamic acid and D-valine showed no inhibi-tion on both processes. Only glycine producedsignificant inhibition.The effect of several amino acids (32 mm) on

the rates of L-glutamic acid uptake at low andhigh external concentrations is shown in Table 2.The entry of L-glutamic acid through the diffu-sion-like process was markedly inhibited byD- and L-alanine. L-Aspartic acid showed a small

TABLE 1. Effect of other amino acids on L-alanineuptake at external L-alanine concentrations of

0.4 and 8.0 mM

Per cent inhibition of L-alanineuptake at external L-alanine

Unlabeled amino acid added concn of(32 mm)

0.4 mm 8.0 ml&

D-Alanine ............. 83 22D-Glutamic acid ....... 0 0L-Glutamic acid. 10 7Glycine................ 83 49L-Leucine.............. 32 8D-Valine............... 1 0L-Valine............... 24 19

Owing to the involvement of approximately25% of the permease system, a value of less than25% is not considered to be inhibitory.

TABLE 2. Effect of other amino acids on L-glutamicacid uptake at external L-glutamic acidconcentrations of 0.25 and 8.0 mm

Per cent inhibition of L-glutamicacid uptake at external L-glu-

Unlabeled amino acid added tamic acid concn of(32 mm)

0.25 mm 8.0 mxa

D-Alanine ............. 45 55L-Alanine.............. 54 57D-Aspartic acid........ 30 0L-Aspartic acid ........ 83 37D-Glutamic acid ....... 14 3D-Valine............... 41 29L-Valine............... 45 35

Owing to the involvement of approximately20% of the permease system, a value of less than20%o is not considered to be inhibitory.

inhibitory effect, whereas D-aspartic acid andD-glutamic acid were inactive in inhibiting.

In the presence of several amino acids at twoexternal concentrations of 1.0 and 8.0 mm, therates of D-valine uptake at low and high externalD-valine concentrations were measured (Table 3).Both isomers of glutamic acid did not affect theentry of D-valine, but all other amino acids triedwere effective competitors.

DISCUSSIONThe experiments described in this paper

demonstrate that both D- and L-isomers ofalanine, glutamic acid, and valine are taken upinto the pool of M. smegmatis cells, but theuptake of L-amino acids is greater than that ofcorresponding D-amino acids. The kineticstudies indicate the existence of two modes ofamino acid transport: one, a process concentra-ting amino acid in the cell in excess of the externalconcentration, and the other, a process in whichthe uptake is directly proportional to the externalconcentration. The first process displays satura-tion kinetics characteristic of enzyme functionthat is assumed to reflect the operation of per-mease which catalyzes the entry of specificsubstrate across a cellular osmotic barrier intothe cell (5). D-Alanine uptake is mediated solelyby this permease system. The second processexhibits linear, nonsaturation kinetics. Theentry of D-glutamic acid into the cell is ac-complished by the diffusion-like process. M.smegmatis cell is defective in a permease system

TABLE 3. Effect of other amino acids on D-valineuptake at external D-valine concentrations of

0.1 and 2.0 mm

Per cent inhibition ofD-valine uptake at external

Unlabeled amino acid added D-valine concn of

0.1 mm 2.0 mMG

D-Alanine, 1 mm........... 89 61D-Alanine, 8 mm........... 91 71L-Alanine, 1 mm........... 89 63L-Alanine, 8 mM ........... 92 71D-Glutamic acid, 1 mm..... 21 14D-Glutamic acid, 8 mm..... 21 17L-Glutamic acid, 1 mm ..... 42 21L-Glutamic acid, 8 mm ..... 61 37Glycine, 1 mM ............. 87 51Glycine, 8 mM ............. 91 63L-Leucine, 1 mm........... 86 60L-Valine, 8 mM ............ 91 65

v Owing to the involvement of approximately20% of the permease system, a value of less than20% is not considered to be inhibitory.

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for D-glutamic acid uptake. The biphasic curvesfound in the uptake of L-alanine, L-glutamicacid, and D- and L-valine indicate the involve-ment of these two processes. When the externalamino acid is increased at levels beyond thosenecessary to saturate a specific permease system,the diffusion-like process becomes evident. Incontrast to L-alanine uptake, D-alanine does notenter the cell through the diffusion-like process atany external D-alanine concentration. Theabsence of a diffusion-like process for D-alanineimplies the probability that there exists a stereo-specific carrier (26) which forms a complex withthe amino acid.The diffusion-like process that is involved in

the uptake of L-alanine, L-glutamic acid, andD-valine is probably carrier-mediated, facilitateddiffusion (23). This assumption is supported bythe following observations. (i) The uptake isdirectly proportional to the external concentra-tion, and exhibits non-enzymatic temperaturedependence. (ii) This process is relatively sensi-tive to sulfhydryl-blocking reagents, and presentsa rather strict substrate specificity; only glycinecompetes with L-alanine, and D- and L-alanineand L-aspartic acid compete with L-glutamicacid. Glycine, L-leucine, L-valine, and D- andL-alanine are all inhibitory to the entry of D-valine through the diffusion-like process. Thecompetition among these amino acids occurseven at 1 mm, that is, only one-half the externalD-valine concentration (Table 3). Furthermore,amino acids which affect the diffusion-likeprocess are always inhibitory to the permeasesystem, suggesting that the diffusion-like processmay be similar to the permease system in havinga stereospecific element.

Recently several studies have reported that,in the cells and the membrane preparation ofE. coli, an entry process with a rate which islinear with the external concentration is involvedin the uptake of amino acid or amino acidanalogue (16, 17, 19, 24). Their findings are

consistent with the concentration dependence ofdiffusion-like process in the present work.

Evidence for the existence of two differentamino acid transport systems has been shown inE. coli (14), S. aureus (10), Saccharomycescerevisiae (11, 12), and Salmonella typhimurium(1). Moreover, Benko, Wood, and Segel (2, 3)demonstrated that a specific permease and a

general permease with affinity for a broad range

of amino acids exist in Penicillium chrysogenum,and the latter system develops as a result. ofnitrogen deprivation. In the present work,however, evidence for the induction of thediffusion-like process in an induced cell and a

nitrogen-starved cell was not obtained.

Amino acids such as alanine, glutamic acid,and valine have multiple physiological roles notonly as direct precursor materials for protein andcell wall synthesis, but also as precursors forother cellular metabolites. Amino acids whichare taken up into the cell equilibrate with aminoacids synthesized de novo which reside in thepool. Glutamic acid, alanine, aspartic acid, andvaline are present in relatively high concentra-tions in the pool of M. smegmatis cell whengrown on Sauton synthetic medium. The sixisomers of amino acids taken up into the pool,for the most part, remain unchanged during theshort interval, whereas, in prolonged periods, theamino acids in the pool are converted to theother amino acids which preform the pool, as inPseudomonas aeruginosa (18), and the proportionof the conversion was nearly equal for the D- andL-isomers (unpublished data). It is not too diffi-cult to imagine the conversion of amino acids toother pool amino acids, of which the first step ismade by the individual racemase, although notyet detected in M. smegmatis.Although M. smegmatis cells have the ability

to synthesize all of the necessary amino acidsfrom inorganic salts and glucose, L-alanine andL-glutamic acid are the most utilizable aminoacids for this organism (4). D-Glutamic acid andD- and L-valine are poor nutrient amino acids.D-Alanine and D-glutamic acid are the mostabundant amino acids in the bacterial cell wall(25), although the rates of uptake are rathersmall. The difference in the availability of theseisomers seems to be associated with the rate ofuptake.

LITERATURE CITED

1. Ames, G. F. 1964. Uptake of amino acids by Salmonellatyphimuriwn. Arch. Biochem. Biophys. 104:1-18.

2. Benko, P. V., T. C. Wood, and 1. H. Segel. 1967. Specificityand regulation ofmethionine transport in filamentous fungi.Arch. Biochem. Biophys. 122:783-804.

3. Benko, P. V., T. C. Wood, and I. H. Segel. 1969. Multiplicityand regulation of amino acid transport in PenIllllumchrysogenum. Arch. Biochem. Biophys. 129:498-508.

4. Chang, Y. T., and M. I. Smith. 1953. Studies in the metabo-lisn of Mycobacterium 607. 111. The effect ofvarious sourcesof nitrogen on growth, riboflavin production, and synthesisof a pharmacologically active metabolite. Amer. Rev.Tuberc. 68:119-126.

5. Cohen, G. N., and J. Monod. 1957. Bacterial permeases.Bacteriol. Rev. 21:169-194.

6. Cohen, G. N., and H. W. Rickenberg. 1956. Concentrationspecifique reversible des aminoacides chez Eschertchla coil.Ann. Inst. Pasteur 91:693-720.

7. Gale, E. F. 1947. The assimilation of amino acids by bacteria.1. The passage of certain amino acids across the cell walland their concentration in the internal environment ofStreptococcus faecalis. J. Gen. Microbiol. 1:53-76.

8. Gale, E. F. 1951. The assimilation of amino acids by bacteria.II. The relationship between accumulation of free glutamicacid and the formation of combined glutamic acid inStaphylococcus aureus. Biochem. J. 48:290-297.

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9. Gale, E. F. 1953. Assimilation of amino acids by gram-posi-tive bacteria and some actions of antibiotics thereon. Advan.Protein Chem. 8:258-391.

10. Gale, E. F., and J. P. Folkes. 1967. The effect of lipids on theaccumulation of certain amino acids by Staphylococcusaureus. Biochim. Biophys. Acta 144:461-466.

11. Gits, J. J., and M. Grenson. 1967. Multiplicity of the aminoacid permeases in Saccharomyces cerevisiae. III. Evidencefor a specific methionine-transporting system. Biochim.Biophys. Acta 135:507-516.

12. Grenson, M. 1966. Multiplicity of the amino acid permeasesin Saccharomyces cerevisiae. II. Evidence for a specificlysine-transporting system. Biochim. Biophys. Acta 127:339-346.

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