JOURNAL OF BACTERIOLOGY, June 1975, p. 1045-1052 Copyright 0 1975 American Society for Microbiology
Vol. 122, No. 3 Printed in U.S.A.
Aromatic Amino Acid Transport in Yersinia pestis P. BLAISE SMITH1 AND THOMAS C. MONTIE* Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37916
Received for publication 21 February 1975
The uptake and concentration of aromatic amino acids by Yersinia pestis TJW was investigated using endogenously metabolizing cells. Transport activity did not depend on either protein synthesis or exogenously added energy sources such as glucose. Aromatic amino acids remained as the free, unaltered amino acid in the pool fraction. Phenylalanine and tryptophan transport obeyed Michaelis-. Menten-like kinetics with apparent Km values of 6 x 10-7 to 7.5 x 10-7 and 2 x 10-1 M, respectively. Tyrosine transport showed biphasic concentration-dependent kinetics that indicated a diffusion-like process above external tyrosine concentrations of 2 x 10-8 M. Transport of each aromatic amino acid showed different pH and temperature optima. The pH (7.5 to 8) and temperature (27 C) optima for phenylalanine transport were similar to those for growth. Transport of each aromatic amino acid was characterized by Q10 values of approximately 2. Cross inhibition and exchange experiments between the aromatic amino acids and selected aromatic amino acid analogues revealed the existence of three transport systems: (i) tryptophan specific, (ii) phenylalanine specific with limited transport activity for tyrosine and tryptophan, and (iii) general aromatic system with some specificity for tyrosine. Analogue studies also showed that the minimal stereo and structural features for phenylalanine recognition were: (i) the L isomer, (ii) intact a amino and carboxy group, and (iii) unsubstituted aromatic ring. Aromatic amino acid transport was differentially inhibited by various sulfhydryl blocking reagents and energy inhibitors. Phenylalanine and ty-rosine transport was inhibited by 2,4-dinitrophenol, potassium cyanide, and sodium azide. Phenylalanine transport showed greater sensitivity to inhibition by sulfhydryl blocking reagents, particularly N-ethylmaleimide, than did tyrosine transport. Tryptophan transport was not inhibited by either sulfhyd,ryl reagents or sodium azide. The results on the selective inhibition of arom#tic amino acid transport provide additional evidence for multiple transport systems. These results further suggest both specific mechanisms for carrier-mediated active transport and coupling to metabolic energy. The properties of aromatic amino acid transport in some gram-negative species of bacteria have been characterized. Studies on Salmonella typhimurium (1), Pseudomonas aeruginosa (7), and Escherichia coli K-12 (4, 5) have revealed enzyme-like, active transport systems for the uptake of aromatic amino acids. Kinetic studies and cross competition and exchange experiments have further shown the presence of multiple transport systems in each species. S. typhimurium (1) and E. coli K-12 (4) possess general systems, which show equal transport activity for any one of the aromatic amino acids, and specific systems which have greatest specificity and transport activity for individual aromatic amino acids. P. aeruginosa has two transport systems, designated transport system I and II,
which are functional with phenylalanine, tyrosine, and tryptophan (I) and tryptophan, phenylalanine, and tyrosine (II) (7). Division of aromatic amino acid transport systems has been further clarified by the isolation of mutants defective in transport over a designated system. The data presented here is a characterization of aromatic amino acid uptake in Yersinia pestis TJW. It was hypothesized that since Y. pestis TJW requires phenylalanine for growth, the organism would possess a selective and high affinity transport system for phenylalanine. Evidence is presented that is consistent with this hypothesis. As a portion of this study, the relationship between phenylalanine transport and other aromatic amino acid transport sys' Present address: Division of Neurology, Duke University tems was investigated. Evidence is presented Medical Center, Durham, N.C. 27710. indicating the existence of multiple transport 1045
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SMITH AND MONTIE
J. BACMEIOL.
Studies on the inhibition of aromatic amino acid systems which differ with respect to: (i) concentration-dependent kinetics, (ii) pH and temper- uptake by the natural amino acid and amino acid ature conditions optimal for transport activity, analogues were done by incubating a cell suspension (iii) stereo and structural specificity, and (iv) simultaneously with labeled amino acid (1 x 10-' M) and either natural amino acid or analogue (1 x 10-4 response to sulfhydryl blocking reagents and M) for a 2-min time period followed by rapid filtration metabolic inhibitors. of 0.5 ml of the cell suspension as described above. Identification of radioactivity in the pool. A cell MATERIALS AND METHODS suspension was incubated 5 min with a radioactively Organism. Yersinia pestis TJW (previously named Pasteurella pestis) is the wild-type, phenylalaninerequiring, avirulent strain used in these studies. Stock cultures of this organism were maintained on casein hydrolysate enzymatic (General Biochemicals, Chagrin Falls, Ohio) agar slants at 4 C. Growth of Y. petis TJW. Cells employed for transport studies were grown to late logarithmic phase at 27 C with shaking (Gyrotary water bath shaker, New Brunswick Scientific, New Brunswick, N.J.) on Casamino Acids medium (Difco Laboratories, Detroit, Michigan). This medium contained (per liter): 30 g of Casamino Acids, 2 g of glucose, 1 g of ammonium chloride, 0.5 g of magnesium sulfate, 10 mg of calcium chloride, 2.5 mg of ferric chloride, 5.8 g of monobasic potassium phosphate, and 11.6 g of dibasic potassium phosphate. Preparation of cells for transport studies. Cells were collected by centrifugation (12,000 x g for 10 min) at 4 C and washed twice at 4 C with potassium phosphate (0.05 M)-magnesium chloride (0.005 M) suspension buffer, pH 7. The cells finally were suspended to an optical density of 140 Klett units (no. 62 filter, 250 ug of cell protein/ml). The cell suspension was pipetted into a 50-ml Erlenmeyer flask, and 200 pg of chloramphenicol (Sigma Chemical Company, St. Louis, Mo.) per ml was added unless otherwise noted. This mixture was incubated with shaking at 27 C for 15 min and then used immediately for individual experiments. Each experiment was performed with a freshly prepared cell suspension. Measurement of aromatic amino acid uptake. Measurement of the aromatic amino acid pool was made by the method of Britten and McClure (3). Cell suspensions (10 ml) were incubated with shaking at 27 C on a Dubnoff metabolic shaker (Precision Scientific, Chicago, Ill.). Tritium-labeled aromatic amino acids were added to the cell suspension to a final concentration of 1 x 10-' M. At various times two 0.5-ml portions of the cell suspension were withdrawn and filtered immediately through a membrane filter (Schleicher and Schuell, 0.45-pm pore size). For measurement of pool formation, 0.5 ml of cells was added to 0.5 ml of cold 10% trichloroacetic acid in an ice bath. The trichloroacetic acid samples were incubated 15 min and then filtered. Filters were washed once with 20 volumes of 27 C suspension buffer and then dried in a vacuum oven at 80 C for 10 min. The dried filters were placed in scintillation vials to which 5 ml of scintillation fluid [2,5-bis-2-(tert-butyl-benzoxazoly)-thiophene-toluene, 4 g/literl was added. Samples were counted in a Beckman LS-100 liquid scintillation spectrometer with an efficiency for tritium of 13%. Background counts on the filters were less than 10% of the total and were subtracted from the total of each sample.
labeled aromatic amino acid (1 x 10-6 M). The suspension was centrifuged (12,000 x g for 10 min) and washed once with suspension buffer at a temperature of 27 C. Then the pellet was suspended in 5% cold trichloroacetic acid and incubated in an ice bath for 15 min. The resulting precipitate was removed by filtration, and the trichloroacetic acid was extracted with cold ethyl ether five times. The remaining solution was lyophilized to dryness, and the resulting residue was resuspended in 1 ml of suspension buffer. Cell extract and appropriate standards were applied in 40-ul amounts to preformed cellulose thin layer sheets (Eastman Kodak no. 6065) and chromatographed in n-butyl alcohol-acetic acid-water (120:30:50, vol/vol/vol). Chromatograms were developed with ninhydrin (0.2%, wt/vol, in acetone). Onecentimeter sections of the chromatogram were scraped off and placed in scintillation vials with 10 ml of scintillation fluid for counting. Chemicals. L- [(HIphenylalanine (specific activity, 7.0 Ci/mmol), L-['HJtyrosine (specific activity, 7.0 Ci/mmol), and L-['H]tryptophan (specific activity, 5.0 Ci/mmol) were purchased from Schwarz/Mann, Orangeburg, N.Y. Chloramphenicol, 2,4-dinitrophenol, L-phenylalanine methyl ester, ,-2-thienylalanine, phenylpyruvic acid, p-hydroxy phenylpyruvic acid, DL-p-fluorophenylalanine, DL-m-fluorophenylalanine, DL-o-fluorophenylalanine, DL-m-tyrosine, and DL-o-tyrosine were purchased from Sigma Chemical Co., St. Louis, Mo. D.Phenylalanine, Dtyrosine. D-tryptophan, L-monoidotyrosine, and L, 3,5-diiodotyrosine were purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. 4-Methyltryptophan, 5-methyltryptophan, 6-methyltryptophan, 5-fluorotryptophan, N-ethylmaleimide, and pchloromercuribenzoate were obtained from Mann Research Laboratories, New York, N.Y. Potassium cyanide and sodium azide were purchased from Matheson Coleman and Bell, Norwood, Ohio. Casamino Acids medium (technical) was purchased from Difco Laboratories, Detroit, Mich.
RESULTS Kinetics of aromatic amino acid pool formation. The time course of phenylalanine uptake and pool formation in the presence or absence of combinations of chloramphenicol (200 ,ug/ml) and glucose (0.2%) is shown in Fig. 1. Phenylalanine was taken up and incorporated into cell protein in cell suspensions that were incubated with glucose 15 min prior to the addition of radioactively labeled phenylalanine (Fig. 1A). The phenylalanine pool formed under these conditions reached maximum concentra-
1.4 A*
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1047
AMINO ACID TRANSPORT IN Y. PESTIS
VOL. 122, 1975
(MN
Kinet cofaphenylain uptake. Cel sus-
15 0os5lu 10oamhnio 200
5g)10)15l20
thelsindticatedrmannert1im pridinorltobte faddtion; of ( eido linabeled -i phenylalanine 06M.Penuae with:e(A) 0.2% glucse,mnus luoreaphni ch col;r(B) (C) mienuslglumienusglcose(ig ichoraphenticon; ove an9 inta and-mpro FgD.Atrti tieutk ese n rg/mI); oso tepeyaa (200 cose plunhoprampeinio (nd) plusate niepo a hor bevd Guoeddntsiu gluosertand heampeio. Symbolas:0,un wholhe laeutk vrenoeosrts chelyls;a,atrichporoatcai-noul fraction ; U,tepesneo trchloroacpetico acid-oul fabection.lcoe(Fg tinatrC)-mpeido phenylalanineupaeocrdataon utake.iniilpro the absene ofgucoske adchiream-d Chloramphenicol-andascelo gucons (ig. iB) incoprtion phenirolx(ig.el fh oftePhenylalalnineinto proteincwasreduted andoael greatern prpotono the amino acid wxstdalmstfondie inth Ifraction. phenllne prchoroltcai-oul h rsneo
The uptake of tryptophan and tyrosine in chloramphenicol-treated cell suspensions is shown in Fig. 2. Each amino acid was taken up into a pool fraction with little incorporation into acid-precipitable material. The rate of uptake of tryptophan and tyrosine was approximately 50% less than that of phenylalanine uptake. In the presence of glucose and chloramphenicol no uptake of tryptophan or tyrosine was observed (data not shown). Each amino acid was concentrated in the cell based on results from uptake studies over a 15-min period with chloramphenicol-treated cell suspensions. Assuming a dry weight and cell volume of 2 x 10- 7gg and 1 x 10-12 ml (6), the ratio of the internal concentration to the external concentration was approximately 100:1, 50:1, and 50:1 for phenylalanine, tyrosine, and tryptophan. Extraction and chromatography of the amino acid pool showed that the amino acid was not altered or degraded in the pool fraction. Concentration-dependent kinetics of aromatic amino acid uptake. The uptake of aromatic amino acids as a function of the extemal amino acid concentration is shown in Fig. 3. Phenylalanine uptake was saturable with a Km of 6 x 10-7 to 7.5 x 10-7 M. Tryptophan uptake was not strictly saturable, with a gradual increase in the rate of uptake occurring over the concentration range of 1.5 x 10-6 to 5.0 x 10-6 M. The apparent Km for tryptophan uptake was 2 x 10-6 M. Tyrosine uptake (Fig. 3, inset) showed a biphasic saturation curve. The initial rate of tyrosine uptake was directly proportional to the external concentration of tyrosine above concentrations of 2 x 10-6 M. Effect of pH on aromatic amino acid uptake. The initial rate of aromatic amino acid uptake was dependent on the pH of the incubation medium. Phenylalanine uptake had an F
0.8
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B
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oC) pheyanie uptake occyacuurrtedpeyatlanconstntrtefr an initial 2--tm3-m period(Fg1D. Afterti tomapprxmtael 30%d n ls ofitalre.phenylalthe alninepolwasnonorporatedintcosel prdotesin,ofathe uptake assay acgenumulatedshnyaann
a
10
15
20 TIME
0
5
10
15
20
(MIN)
FIG. 2. Kinetics of tryptophan and tyrosine uptake. Cell suspensions were preincubated with chloramphenicol for 15 min prior to the addition of either labeled tryptophan (A; 1 x 10-6 A) or tyrosine (B; 1 x 10-6 A). Symbols: 0, whole cells; A, trichloroacetic acid-insoluble fraction; U, trichloroacetic acidsoluble fraction.
SMITH AND MONTIE
1048
J. BACZRIOL.
04
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0.2
0.20
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0.1
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FiG. 3. Concentration-dependent kinetics of aromatic amino acid uptake. Cell suspensions were incubated for 2 min with the indicated concentrations of labeled amino acids. Symbols: 0, phenylalanine; tyrosine. A, tryptophan; U,
optimum over the pH range of 7.5 to 8.0 (Fig. 4). Tyrosine and tryptophan uptake showed optima in the more basic pH range of 8 to 8.5 and 8 to 9, respectively. Effect of temperature on aromatic amino acid uptake. The uptake of each amino acid differs with respect to the temperature-dependent kinetics of uptake (Fig. 5). The optimum temperature for phenylalanine uptake was 27 C. At 37 C uptake was linear for 3 min. Tryptophan uptake had a broad temperature optimum over the range of 22 to 37 C. Tyrosine uptake was optimal at a temperature of 45 C, a z
z
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FIG. 4. Effect of pH on the rate of aromatic amino acid uptake. Cell suspensions were in buffer adjusted to the indicated pH values with either hydrochloric acid or potassium hydroxide solutions and incubated 10 min at 27 C. Labeled amino acid (1 x 10-6 M) was added to each cell suspension, and the incubation was continued for 3 min. One-half milliliter of the cell suspension was filtered and washed with 20 volumes of potassium phosphate (0.05 M) buffer adjusted to the pH of each reaction mixture. Symbols: 0, phenylalanine; *, tyrosine; A, tryptophan.
60 50 40 30 TEMP OC FIG. 5. Effect of temperature on the rate of aromatic amino acid uptake. Cell suspensions were incubated at the indicated temperatures for 10 min. Labeled amino acid (1 x 10-6 M) was added to each cell suspension, and uptake was terminated and measured at 3 min. Symbols: *, phenylalanine; *, 10
20
tyrosine; A, tryptophan.
nonpermissive growth temperature for Y. pestis TJW. The uptake of each aromatic amino acid showed a marked decrease in activity at 55 C. Q1O values of approximately 2 were determined for the uptake of each amino acid over a temperature range of 10 to 20 C. Cross inhibition between aromatic amino acids. The uptake of aromatic amino acids was specific for this group of amino acids since the other 17 naturally occurring amino acids did not inhibit the uptake of phenylalanine, tyrosine, or tryptophan. The results of cross inhibition experiments between the aromatic amino acids are shown in Table 1. When unlabeled amino acids were present at a concentration 100-fold greater than the labeled amino acid, (i) tyrosine uptake was inhibited to a greater degree by phenylalanine (72%) than by tryptophan (60%), and (ii) tryptophan uptake was not inhibited by phenylalanine or tyrosine. Histidine and proline had no significant inhibitory effect on aromatic amino acid uptake. The inhibition of phenylalanine uptake by tyrosine and trytophan was strictly competitive (Fig. 6). When unlabeled amino acid was present at a 10-fold excess (1 x 10-5 M) above the concentration of the labeled aromatic amino acid, tyrosine and tryptophan did not inhibit phenylalanine uptake (Table 1). Tyrosine uptake was inhibited 65% by phenylalanine and 45% by tryptophan.These data indicate that phenylalanine and tryptophan uptake show greater substrate specificity than tyrosine
1049
AMINO ACID TRANSPORT IN Y. PESTIS
VOL. 122, 1975
uptake, since at low amino acid concentrations (10-fold excess) phenylalanine and tyrosine uptake were not inhibited by aromatic amino acids, but tyrosine uptake was inhibited by phenylalanine and tryptophan. Amino acid pool exchange experiments (Table 2) also separated the presence of a phenylalanine tyrosine system from a tryptophan system. Tryptophan did not exchange with the tyrosine and phenylalanine pool. Exchange of phenylalanine and tyrosine with the tryptophan pool probably reflects exchange over a general aromatic transport system. Effect of analogues on aromatic amino acid uptake. Table 3 summarizes the results of experiments on the effect of stereoisomers and structural analogues of the aromatic amino acids on the uptake of phenylalanine, tyrosine, and tryptophan. Structural analogues had no inhibitory effect on the uptake of tryptophan except for the iodinated derivatives of tyrosine. The D isomers had little or no effect on the uptake of phenylalanine or tryptophan, indicating that the uptake of these two amino acids is specific for the natural L isomer. Surprisingly, and in contrast to the latter results, each of the D isomers of the aromatic amino acids inhibited the uptake of tyrosine equally as well (37%). The data indicate that the uptake of tyrosine is less stereo specific than phenylalanine or tryp-
50 45
z 35 04
z w 0
25 ,, E
(I 0
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5
2
4
6
[PHE)
0
8
10
TAaz 1. Cross inhibition of aromatic amino acid uptake % Inhibition in rate of uptake of labeled amino acida Amino acid
Phenylalanine
Phenylalanine 10-' M 10-'M
Tyrosine
tophan tpa
72 65
0 0
FiG. 6. Kinetics of competitive inhibition of phenylalanine uptake by tyrosine and tryptophan. Cell suspensions were incubated for 2 min with the indicated concentrations of labeled phenylalanine plus either 1 x 10-4 M tyrosine (-) or 1 x 10-4 M tryptophan (A).
tophan uptake. The placement of a fluorine atom on the ortho, meta, or para position of the Tyrosine phenylalanine ring did not abolish recognition 10-4M 65 0 of phenylalanine since these compounds 10-'M 7 0 strongly inhibited the uptake of phenylalanine, whereas p-chlorophenylalanine was apparently Tryptophan less acceptable for recognition due to the pres10-'M 46 60 ence of the larger chlorine atom on the para 10-, M 5 45 position of the phenylalanine ring. Ortho and Histidine meta tyrosine inhibited the uptake of phenylal0 10-4 M 14 14 anine to the same degree (67 and 64%) as natural tyrosine, but only inhibited tyrosine Proline uptake 50 and 36%, respectively. This result 10-4M 5 14 8 indicates that tyrosine uptake shows specificity aLabeled amino acid (1 x 10-' M) and unlabeled for the para hydroxyl group on the tyrosine ring. The fluoro and methyl derivatives of tryptoamino acid were added together to cell suspensions at zero time. The incubation period for the inhibition phan inhibited the uptake of tryptophan more assay was 2 min. effectively than the uptake of phenylalanine or
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J. BACMEIOL.
SMITH AND MONTIE TABLE 2. Exchange of aromatic amino acid pools Pool exchanges in 10 min with unlabeled: Before
Pool labeled
amino acida
Phenylalanine ....... Tyrosine ............ Tryptophan .........
exchange (nmol)
Phenylalanine (no)
(nmol)
TYrnoline
0.72 0.56 0.43
0.201 0.310 0.228
72 45 48
0.388 0.322 0.308
-
Tyrosine
_____
_____
Tryptophan (nmol)
46 44 31
10
0.652 0.580 0.186
0
56
a Amino acid pools were formed by incubating chloramphenicol-treated cell suspensions with labeled amino acid for 10 min. The cell suspension was centrifuged, and the excess labeled amino acid was decanted. The cell pellet was resuspended in potassium phosphate (0.05 M)-magnesium chloride (0.005 M) buffer, pH 7.0, 27 C. Unlabeled amino acids (1 x 10- 4 M) were added to the cell suspension and incubated 10 min at 27 C. The reaction was terminated by rapid filtration as previously described.
TABLE 3. Inhibition of aromatic amino acid uptake by structural analoguesa
TABLE 4. Inhibition of aromatic amino acid uptake by structural analogues of tryptophan
% Inhibition of rate of uptake
% Inhibition of rate of uptake
Analoguea
Analogue
Phenyl- Tyroalanine sine
o-Fluorophenylalanine p-Fluorophenylalanine .... m-Fluorophenylalanine . p-Chlorophenylalanine ... ,-Thienylalanine ......... m-Tyrosine .............. o-Tyrosine ................ Monoiodotyrosine ........ 3,5-Diiodotyrosine ........ ....
74 67 85 53 79 64 67 72 28 28 0
D-Phenylalanine ......... D-Tyrosine ............... D-Tryptophan ...........0.
51 41 81 77 40 36 50 80 50 37 37 37
Phenyl- Tyroalanine sine
Tryptophan
0 0 0 0 0 0 0 54 0 0 0 0
a Analogues (1 x 10-4 M) and labeled amino acids (1 x 10-6 M) were added together at zero time. The incubation period for the inhibition assay was 2 min.
tyrosine (Table 4). The effectiveness of inhibition in descending order was 5-fluorotryptophan, 5-methyltryptophan, 6-methyltryptophan, and 4-methyltryptophan. These results suggest that the 4 position of the indole ring of tryptophan is important for the recognition of this amino acid by the transport system since the placement of a large methyl group in the 4 position alters the molecule so that it can no longer inhibit the uptake of natural tryptophan. The a amino and carboxyl groups are important for recognition of phenylalanine by the transport system since 100-fold excess of phenylpyruvic acid and phenylalanine methylester inhibited phenylalanine uptake only 45 and 47%, respectively (Table 5). Alanine did not inhibit phenylalanine uptake, indicating that the aromatic ring is an essential structure of the phenylalanine molecule needed for substrate recognition. The dipeptide, L-seryl-L-phenylala-
4-Methyltryptophan ...... 5-Methyltryptophan ...... 6-Methyltryptophan ...... 5-Fluorotryptophan .......
9 23 18 43
Tryptophan
20 59 52 75
14 39 37 44
a Analogues (1 x 10-I M) and labeled amino acids (1 x 10-6 M) were added together to cell suspensions at zero time. The incubation period for the inhibition assay was 2 min.
TABLE 5. Inhibition of phenylalanine uptake of structurally related compounds % Inhibition of rate of phenylalanine uptake
Compounda
L-Seryl-L-phenylalanine
.......
85
47 Phenylpyruvic acid ............. 5 Hydroxyphenylpyruvic acid .... 45 Phenylalanine methyl ester ..... 5 Alanine ...................... a Compounds (1 x 10-4 M) and labeled phenylalanine (1 x 10-6 M) were added together to cell suspensions at zero time. The incubation period for the inhibition assay was 2 min.
nine, inhibited phenylalanine uptake 85% suggesting that the phenylalanine transport system can recognize phenylalanine with the amino group blocked by the serine residue. These results together with the finding that the D isomer of phenylalanine did not significantly inhibit the uptake of the natural L isomer show that the L form of the amino acid, an intact a amino and carboxy group, and the aromatic ring are required for maximum recognition of
AMINO ACID TRANSPORT IN Y. PESTIS
VOL. 122, 1975
this amino acid by the transport system. Effect of sulfhydryl blocking reagents and metabolic inhibitors on aromatic amino acid uptake. The uptake of aromatic amino acids showed some selectivity to inhibition by sulfhydryl blocking reagents and metabolic inhibitors (Table 6). Cell suspensions preincubated either 1 or 15 min with the indicated compound were assayed for amino acid uptake over a 2-min period. Sulfhydryl reagents, p-chloromercuribenzoate, and N-ethylmaleimide strongly inhibited phenylalanine and tyrosine uptake after a brief (1 min) or extended pretreatment (15 min). Notably, tryptophan uptake was only slightly inhibited by p-chloromercuribenzoate (18%, 15 min) and was not inhibited at all by N-ethylmaleimide. A comparison of the inhibition by sulfhydryl reagents over the 1-min preincubation period shows phenylalanine uptake to be somewhat more sensitive to these compounds than tyrosine uptake. N-ethylmaleimide inhibited phenylalanine uptake 78%, whereas tyrosine uptake was inhibited only 41% under these conditions. TABLE 6. Inhibition of aromatic amino acid uptake by sulfhydryl blocking reagents and metabolic inhibitorsa % Inhibition of rate of uptake
Inhibitor
Phenyl.
Tryp-
alanine
Tsme Tyrosine
1 min
76
61
0
15 min
95
91
18
1 min
78
41
7
15 min
100
86
0
1 min
48
67
60
15 min
83
95
95
pCMB (5 x 10-4M)
tophan
NEM (2 x 10-3 M)
DNP (1 x 1o- M)
KCN (1 x 10-2M) 1 min
15 min
NaN, (1 x 10-2M) 1 min 15 min
30
14
15
76
47
49
36 80
13 59
0 0
aCell suspensions were incubated with the inhibitors at the indicated concentrations for either 1 or 15 min. Labeled amino acids (1 x 10-6 M) were then added and the incubation was continued for a 2-min period. pCMB, p-Chloromercuribenzoate; NEM, Nethylmaleimide; DNP, 2,4-dinitrophenol; KCN, potaaaium cyanide; NaN,, sodium azide.
1051
Metabolic inhibitors, potassium cyanide, and sodiom azide inhibited phenylalanine and tyrosine uptake. Dinitrophenol was the most effective inhibitor of these compounds. Tryptophan uptake was not inhibited by sodium azide but was inhibited by 2,4-dinitrophenol and potassium cyanide. Inhibition of transport by metabolic inhibitors plus the finding that aromatic amino acid transport in Y. pestis is concentrative indicate that this group of amino acids is actively transported by this organism. DISCUSSION The results show that aromatic amino acid transport in Y. pestis exhibits concentrative uptake, depends on the extemal concentration, and requires metabolic energy. The observations that glucose depressed aromatic amino acid uptake in the presence of chloramphenicol were unexpected. The glucose effect occurs very likely because the cell is vigorously synthesizing tryptophan and tyrosine as end products in the absence of protein synthesis. Consequently, these compounds in effect dilute the labeled aromatic pool directly and by counterfluxing dilute extemal aromatic label. In minus chloramphenicol experiments protein synthesis effectively reduced the pool, thereby allowing rapid uptake of phenylalanine. Experiments were performed to delineate separate transport systems by examining their substrate specificity. Cross inhibition experiments between the aromatic amino acids and selected analogues showed that the tryptophan transport system was highly specific. These data also indicate that phenylalanine and tryptophan transport show greater substrate specificity than tyrosine transport. The recognition of phenylalanine or tyrosine showed a high degree of substrate specificity overlap in experiments where analogues or natural aromatic amino acids were present at a 100-fold excess above the concentration of the labeled amino acid. These studies showed that the phenylalanine transport system was capable of recognizing tyrosine and tryptophan only when these amino acids were present at a high concentration relative to the phenylalanine concentration. Phenylalanine and tryptophan transport was not significantly inhibited by their respective D isomers, indicating that the transport of phenylalanine and tryptophan is stereo specific for the natural L isomer. Tyrosine transport did not show the same degree of stereo specificity as phenylalanine and tryptophan transport since tyrosine transport was inhibited significantly by each of the D aromatic amino acids. These
1052
SMITH AND MONTIE
findings are similar to those made for tyrosine transport in P. aeruginosa (7), where Dphenylalanine and D-tyrosine inhibited the transport of L-tyrosine. The fluoro analogues of phenylalanine were effective inhibitors of phenylalanine and tyrosine transport, indicating that the fluorine atom does not appreciably alter the phenylalanine molecule with respect to recognition by the transport system. This finding is in agreement with studies on the inhibition of phenylalanine transport by fluoro analogues in Salmonella typhimrium (1) and inhibition of tyrosine transport in P. aeruginosa by the fluoro derivatives of tyrosine and phenylalanine (7). Data showed decreased inhibition of tyrosine by ortho and meta tyrosine. These results indicate that tyrosine recognition is dependent on the position of the hydroxyl group on the tyrosine ring. The methyl and fluoro analogues of tryptophan were effective in inhibiting the transport of tryptophan, phenylalanine, and tyrosine. These findings are in contrast to that observed in P. aeruginosa in which 5-methyltryptophan did not inhibit tryptophan transport and 5-fluorotryptophan did not inhibit tyrosine transport (7). The results of cross inhibition experiments between the aromatic amino acids and their analogues suggest that three transport systems exist in Y. pestis TJW for the transport of aromatic amino acids: (i) tryptophan specific, (ii) phenylalanine specific with transport activity for tyrosine and tryptophan when present at high external concentrations, and (iii) a general system which recognizes each amino acid at a high (1 x 10-4 M) or low (1 x 10- M) external concentration but shows a degree of substrate specificity for tyrosine transport. The high-affinity phenylalanine transport system provides a mechanism whereby wildtype Y. pestis TJW can take up and concentrate a required nutrient for biosynthesis and growth. Also the maximum efficiency of the transport system corresponds with the optimum pH and temperature requirements for growth. Similar studies on tyrosine and tryptophan transport did not show this correlation between pH and temperature optimum for growth. The above discussed characteristics of phenylalanine transport suggests that this system serves to supply an amino acid which is required for the growth and maintenance of this organism. Division of aromatic amino acid transport into multiple systems in Y. pestis TJW was also substantiated by the finding that the uptake of amino acid was differentially inhibited by sulfhydryl blocking reagents and energy inhibitors. Initial phenylalanine transport (1 min) was
J. BACTERIOL.
inhibited somewhat more by sulfhydryl reagents than tyrosine transport. Tryptophan transport was not significantly inhibited by these compounds during a 1- or 15-min preincubation period. It is very likely that the tryptophan transport system does not depend on active sulfhydryl groups for transport activity. It is interesting to note that binding proteins isolated by osmotic shock procedures are not inactivated by sulfhydryl reagents (2). We have found that extraction of Y. pestis membranes with the detergent, Brij 36T, yields tryptophan but not phenylalanine or tyrosine binding activity, but we could not identify a "shockable" activity for tryptophan binding. The lack of inhibition by azide would also suggest an adenosine 5'-triphosphate-dependent periplasmic system (2). It is possible that activity is lost or diluted in the shock procedure but retained in the Brij procedure, since a greater quantity of loosely bound protein might be obtained. Alternatively, the tryptophan transport system may be "unavailable" in the cell membrane so that sulfhydryl reagents cannot penetrate to an essential site (9). The lack of sensitivity of tryptophan transport to NaN, may indicate either positional differences in the anatomical location of the transport system in the membrane or a difference in sensitivity of a given transport system to respiratory inhibition. ACKNOWLEDGMENTS P. B. S., a predoctoral trainee, was supported by Public Health Service grant no. TO1-AI00435 from the National Institute of Allergy and Infectious Diseases. We would like to thank Diane Montie for her critical evaluation of the manuscript.
LITERATURE CITED 1. Ames, G. F. 1964. Uptake of amino acids by Salmonella typhimurium. Arch. Biochem. Biophys. 104:1-18. 2. Berger, E. A. 1973. Different mechanisms of energy coupling for the active transport of proline and glutamine in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 70:1514-1518. 3. Britten, R. J., and F. T. McClure. 1962. The amino acid pool in Escherichia coli. Bacteriol. Rev. 26:292-335. 4. Brown, K. D. 1970. Formation of aromatic amino acid pools in Escherichia coli K-12. J. Bacteriol. 104:177-188. 5. Brown, K. D. 1971. Maintenance and exchange of the aromatic amino acid pool in Escherichia coli. J. Bacteriol. 106:70-81. 6. Burrous, S. E., and R. D. Demoss. 1963. Studies on tryptophan permease in Escherichia coli. Biochim. Biophys. Acta 73:623-637. 7. Kay, W. W., and A. Gronlund. 1971. Transport of aromatic amino acids by Pseudomonas aeruginosa. J. Bacteriol. 105:1039-1046. 8. Roseman, S. 1969. The transport of carbohydrates by a bacterial phosphotransferase system. J. Gen. Physiol.
54(Suppl.):138-184.
9. Vansfeveninck, J., R. I. Weed, and A. Rothstein. 1965. Localization of erythrocyte membrane sulfhydryl groups essential for glucose transport. J. Gen. Physiol. 48:617-632.
Vol. 122, No. 3 Printed in U.S.A.
Aromatic Amino Acid Transport in Yersinia pestis P. BLAISE SMITH1 AND THOMAS C. MONTIE* Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37916
Received for publication 21 February 1975
The uptake and concentration of aromatic amino acids by Yersinia pestis TJW was investigated using endogenously metabolizing cells. Transport activity did not depend on either protein synthesis or exogenously added energy sources such as glucose. Aromatic amino acids remained as the free, unaltered amino acid in the pool fraction. Phenylalanine and tryptophan transport obeyed Michaelis-. Menten-like kinetics with apparent Km values of 6 x 10-7 to 7.5 x 10-7 and 2 x 10-1 M, respectively. Tyrosine transport showed biphasic concentration-dependent kinetics that indicated a diffusion-like process above external tyrosine concentrations of 2 x 10-8 M. Transport of each aromatic amino acid showed different pH and temperature optima. The pH (7.5 to 8) and temperature (27 C) optima for phenylalanine transport were similar to those for growth. Transport of each aromatic amino acid was characterized by Q10 values of approximately 2. Cross inhibition and exchange experiments between the aromatic amino acids and selected aromatic amino acid analogues revealed the existence of three transport systems: (i) tryptophan specific, (ii) phenylalanine specific with limited transport activity for tyrosine and tryptophan, and (iii) general aromatic system with some specificity for tyrosine. Analogue studies also showed that the minimal stereo and structural features for phenylalanine recognition were: (i) the L isomer, (ii) intact a amino and carboxy group, and (iii) unsubstituted aromatic ring. Aromatic amino acid transport was differentially inhibited by various sulfhydryl blocking reagents and energy inhibitors. Phenylalanine and ty-rosine transport was inhibited by 2,4-dinitrophenol, potassium cyanide, and sodium azide. Phenylalanine transport showed greater sensitivity to inhibition by sulfhydryl blocking reagents, particularly N-ethylmaleimide, than did tyrosine transport. Tryptophan transport was not inhibited by either sulfhyd,ryl reagents or sodium azide. The results on the selective inhibition of arom#tic amino acid transport provide additional evidence for multiple transport systems. These results further suggest both specific mechanisms for carrier-mediated active transport and coupling to metabolic energy. The properties of aromatic amino acid transport in some gram-negative species of bacteria have been characterized. Studies on Salmonella typhimurium (1), Pseudomonas aeruginosa (7), and Escherichia coli K-12 (4, 5) have revealed enzyme-like, active transport systems for the uptake of aromatic amino acids. Kinetic studies and cross competition and exchange experiments have further shown the presence of multiple transport systems in each species. S. typhimurium (1) and E. coli K-12 (4) possess general systems, which show equal transport activity for any one of the aromatic amino acids, and specific systems which have greatest specificity and transport activity for individual aromatic amino acids. P. aeruginosa has two transport systems, designated transport system I and II,
which are functional with phenylalanine, tyrosine, and tryptophan (I) and tryptophan, phenylalanine, and tyrosine (II) (7). Division of aromatic amino acid transport systems has been further clarified by the isolation of mutants defective in transport over a designated system. The data presented here is a characterization of aromatic amino acid uptake in Yersinia pestis TJW. It was hypothesized that since Y. pestis TJW requires phenylalanine for growth, the organism would possess a selective and high affinity transport system for phenylalanine. Evidence is presented that is consistent with this hypothesis. As a portion of this study, the relationship between phenylalanine transport and other aromatic amino acid transport sys' Present address: Division of Neurology, Duke University tems was investigated. Evidence is presented Medical Center, Durham, N.C. 27710. indicating the existence of multiple transport 1045
1046
SMITH AND MONTIE
J. BACMEIOL.
Studies on the inhibition of aromatic amino acid systems which differ with respect to: (i) concentration-dependent kinetics, (ii) pH and temper- uptake by the natural amino acid and amino acid ature conditions optimal for transport activity, analogues were done by incubating a cell suspension (iii) stereo and structural specificity, and (iv) simultaneously with labeled amino acid (1 x 10-' M) and either natural amino acid or analogue (1 x 10-4 response to sulfhydryl blocking reagents and M) for a 2-min time period followed by rapid filtration metabolic inhibitors. of 0.5 ml of the cell suspension as described above. Identification of radioactivity in the pool. A cell MATERIALS AND METHODS suspension was incubated 5 min with a radioactively Organism. Yersinia pestis TJW (previously named Pasteurella pestis) is the wild-type, phenylalaninerequiring, avirulent strain used in these studies. Stock cultures of this organism were maintained on casein hydrolysate enzymatic (General Biochemicals, Chagrin Falls, Ohio) agar slants at 4 C. Growth of Y. petis TJW. Cells employed for transport studies were grown to late logarithmic phase at 27 C with shaking (Gyrotary water bath shaker, New Brunswick Scientific, New Brunswick, N.J.) on Casamino Acids medium (Difco Laboratories, Detroit, Michigan). This medium contained (per liter): 30 g of Casamino Acids, 2 g of glucose, 1 g of ammonium chloride, 0.5 g of magnesium sulfate, 10 mg of calcium chloride, 2.5 mg of ferric chloride, 5.8 g of monobasic potassium phosphate, and 11.6 g of dibasic potassium phosphate. Preparation of cells for transport studies. Cells were collected by centrifugation (12,000 x g for 10 min) at 4 C and washed twice at 4 C with potassium phosphate (0.05 M)-magnesium chloride (0.005 M) suspension buffer, pH 7. The cells finally were suspended to an optical density of 140 Klett units (no. 62 filter, 250 ug of cell protein/ml). The cell suspension was pipetted into a 50-ml Erlenmeyer flask, and 200 pg of chloramphenicol (Sigma Chemical Company, St. Louis, Mo.) per ml was added unless otherwise noted. This mixture was incubated with shaking at 27 C for 15 min and then used immediately for individual experiments. Each experiment was performed with a freshly prepared cell suspension. Measurement of aromatic amino acid uptake. Measurement of the aromatic amino acid pool was made by the method of Britten and McClure (3). Cell suspensions (10 ml) were incubated with shaking at 27 C on a Dubnoff metabolic shaker (Precision Scientific, Chicago, Ill.). Tritium-labeled aromatic amino acids were added to the cell suspension to a final concentration of 1 x 10-' M. At various times two 0.5-ml portions of the cell suspension were withdrawn and filtered immediately through a membrane filter (Schleicher and Schuell, 0.45-pm pore size). For measurement of pool formation, 0.5 ml of cells was added to 0.5 ml of cold 10% trichloroacetic acid in an ice bath. The trichloroacetic acid samples were incubated 15 min and then filtered. Filters were washed once with 20 volumes of 27 C suspension buffer and then dried in a vacuum oven at 80 C for 10 min. The dried filters were placed in scintillation vials to which 5 ml of scintillation fluid [2,5-bis-2-(tert-butyl-benzoxazoly)-thiophene-toluene, 4 g/literl was added. Samples were counted in a Beckman LS-100 liquid scintillation spectrometer with an efficiency for tritium of 13%. Background counts on the filters were less than 10% of the total and were subtracted from the total of each sample.
labeled aromatic amino acid (1 x 10-6 M). The suspension was centrifuged (12,000 x g for 10 min) and washed once with suspension buffer at a temperature of 27 C. Then the pellet was suspended in 5% cold trichloroacetic acid and incubated in an ice bath for 15 min. The resulting precipitate was removed by filtration, and the trichloroacetic acid was extracted with cold ethyl ether five times. The remaining solution was lyophilized to dryness, and the resulting residue was resuspended in 1 ml of suspension buffer. Cell extract and appropriate standards were applied in 40-ul amounts to preformed cellulose thin layer sheets (Eastman Kodak no. 6065) and chromatographed in n-butyl alcohol-acetic acid-water (120:30:50, vol/vol/vol). Chromatograms were developed with ninhydrin (0.2%, wt/vol, in acetone). Onecentimeter sections of the chromatogram were scraped off and placed in scintillation vials with 10 ml of scintillation fluid for counting. Chemicals. L- [(HIphenylalanine (specific activity, 7.0 Ci/mmol), L-['HJtyrosine (specific activity, 7.0 Ci/mmol), and L-['H]tryptophan (specific activity, 5.0 Ci/mmol) were purchased from Schwarz/Mann, Orangeburg, N.Y. Chloramphenicol, 2,4-dinitrophenol, L-phenylalanine methyl ester, ,-2-thienylalanine, phenylpyruvic acid, p-hydroxy phenylpyruvic acid, DL-p-fluorophenylalanine, DL-m-fluorophenylalanine, DL-o-fluorophenylalanine, DL-m-tyrosine, and DL-o-tyrosine were purchased from Sigma Chemical Co., St. Louis, Mo. D.Phenylalanine, Dtyrosine. D-tryptophan, L-monoidotyrosine, and L, 3,5-diiodotyrosine were purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. 4-Methyltryptophan, 5-methyltryptophan, 6-methyltryptophan, 5-fluorotryptophan, N-ethylmaleimide, and pchloromercuribenzoate were obtained from Mann Research Laboratories, New York, N.Y. Potassium cyanide and sodium azide were purchased from Matheson Coleman and Bell, Norwood, Ohio. Casamino Acids medium (technical) was purchased from Difco Laboratories, Detroit, Mich.
RESULTS Kinetics of aromatic amino acid pool formation. The time course of phenylalanine uptake and pool formation in the presence or absence of combinations of chloramphenicol (200 ,ug/ml) and glucose (0.2%) is shown in Fig. 1. Phenylalanine was taken up and incorporated into cell protein in cell suspensions that were incubated with glucose 15 min prior to the addition of radioactively labeled phenylalanine (Fig. 1A). The phenylalanine pool formed under these conditions reached maximum concentra-
1.4 A*
B
1.2
09 z w
003 w
w121
E >'06 wt 0.3
_
w0
E~~~~~TM
Fg.1.e
1047
AMINO ACID TRANSPORT IN Y. PESTIS
VOL. 122, 1975
(MN
Kinet cofaphenylain uptake. Cel sus-
15 0os5lu 10oamhnio 200
5g)10)15l20
thelsindticatedrmannert1im pridinorltobte faddtion; of ( eido linabeled -i phenylalanine 06M.Penuae with:e(A) 0.2% glucse,mnus luoreaphni ch col;r(B) (C) mienuslglumienusglcose(ig ichoraphenticon; ove an9 inta and-mpro FgD.Atrti tieutk ese n rg/mI); oso tepeyaa (200 cose plunhoprampeinio (nd) plusate niepo a hor bevd Guoeddntsiu gluosertand heampeio. Symbolas:0,un wholhe laeutk vrenoeosrts chelyls;a,atrichporoatcai-noul fraction ; U,tepesneo trchloroacpetico acid-oul fabection.lcoe(Fg tinatrC)-mpeido phenylalanineupaeocrdataon utake.iniilpro the absene ofgucoske adchiream-d Chloramphenicol-andascelo gucons (ig. iB) incoprtion phenirolx(ig.el fh oftePhenylalalnineinto proteincwasreduted andoael greatern prpotono the amino acid wxstdalmstfondie inth Ifraction. phenllne prchoroltcai-oul h rsneo
The uptake of tryptophan and tyrosine in chloramphenicol-treated cell suspensions is shown in Fig. 2. Each amino acid was taken up into a pool fraction with little incorporation into acid-precipitable material. The rate of uptake of tryptophan and tyrosine was approximately 50% less than that of phenylalanine uptake. In the presence of glucose and chloramphenicol no uptake of tryptophan or tyrosine was observed (data not shown). Each amino acid was concentrated in the cell based on results from uptake studies over a 15-min period with chloramphenicol-treated cell suspensions. Assuming a dry weight and cell volume of 2 x 10- 7gg and 1 x 10-12 ml (6), the ratio of the internal concentration to the external concentration was approximately 100:1, 50:1, and 50:1 for phenylalanine, tyrosine, and tryptophan. Extraction and chromatography of the amino acid pool showed that the amino acid was not altered or degraded in the pool fraction. Concentration-dependent kinetics of aromatic amino acid uptake. The uptake of aromatic amino acids as a function of the extemal amino acid concentration is shown in Fig. 3. Phenylalanine uptake was saturable with a Km of 6 x 10-7 to 7.5 x 10-7 M. Tryptophan uptake was not strictly saturable, with a gradual increase in the rate of uptake occurring over the concentration range of 1.5 x 10-6 to 5.0 x 10-6 M. The apparent Km for tryptophan uptake was 2 x 10-6 M. Tyrosine uptake (Fig. 3, inset) showed a biphasic saturation curve. The initial rate of tyrosine uptake was directly proportional to the external concentration of tyrosine above concentrations of 2 x 10-6 M. Effect of pH on aromatic amino acid uptake. The initial rate of aromatic amino acid uptake was dependent on the pH of the incubation medium. Phenylalanine uptake had an F
0.8
A
B
A
l'
XJ 0.6
-
0
oC) pheyanie uptake occyacuurrtedpeyatlanconstntrtefr an initial 2--tm3-m period(Fg1D. Afterti tomapprxmtael 30%d n ls ofitalre.phenylalthe alninepolwasnonorporatedintcosel prdotesin,ofathe uptake assay acgenumulatedshnyaann
a
10
15
20 TIME
0
5
10
15
20
(MIN)
FIG. 2. Kinetics of tryptophan and tyrosine uptake. Cell suspensions were preincubated with chloramphenicol for 15 min prior to the addition of either labeled tryptophan (A; 1 x 10-6 A) or tyrosine (B; 1 x 10-6 A). Symbols: 0, whole cells; A, trichloroacetic acid-insoluble fraction; U, trichloroacetic acidsoluble fraction.
SMITH AND MONTIE
1048
J. BACZRIOL.
04
zi,
z
z
C\3
w i.0
0.3
a:a.
w
-j -J w LAJ
0.2
0.20
E w 2010
10
50
040
0
W oII
a-
D U)
0.1
w [AMINO ACID] 10-7M
FiG. 3. Concentration-dependent kinetics of aromatic amino acid uptake. Cell suspensions were incubated for 2 min with the indicated concentrations of labeled amino acids. Symbols: 0, phenylalanine; tyrosine. A, tryptophan; U,
optimum over the pH range of 7.5 to 8.0 (Fig. 4). Tyrosine and tryptophan uptake showed optima in the more basic pH range of 8 to 8.5 and 8 to 9, respectively. Effect of temperature on aromatic amino acid uptake. The uptake of each amino acid differs with respect to the temperature-dependent kinetics of uptake (Fig. 5). The optimum temperature for phenylalanine uptake was 27 C. At 37 C uptake was linear for 3 min. Tryptophan uptake had a broad temperature optimum over the range of 22 to 37 C. Tyrosine uptake was optimal at a temperature of 45 C, a z
z
d0.3
C 5.
_J
02
402 _J~
6
9
1
9
10
~~~P
0.1 w
-J
C
5
6
7
pH
FIG. 4. Effect of pH on the rate of aromatic amino acid uptake. Cell suspensions were in buffer adjusted to the indicated pH values with either hydrochloric acid or potassium hydroxide solutions and incubated 10 min at 27 C. Labeled amino acid (1 x 10-6 M) was added to each cell suspension, and the incubation was continued for 3 min. One-half milliliter of the cell suspension was filtered and washed with 20 volumes of potassium phosphate (0.05 M) buffer adjusted to the pH of each reaction mixture. Symbols: 0, phenylalanine; *, tyrosine; A, tryptophan.
60 50 40 30 TEMP OC FIG. 5. Effect of temperature on the rate of aromatic amino acid uptake. Cell suspensions were incubated at the indicated temperatures for 10 min. Labeled amino acid (1 x 10-6 M) was added to each cell suspension, and uptake was terminated and measured at 3 min. Symbols: *, phenylalanine; *, 10
20
tyrosine; A, tryptophan.
nonpermissive growth temperature for Y. pestis TJW. The uptake of each aromatic amino acid showed a marked decrease in activity at 55 C. Q1O values of approximately 2 were determined for the uptake of each amino acid over a temperature range of 10 to 20 C. Cross inhibition between aromatic amino acids. The uptake of aromatic amino acids was specific for this group of amino acids since the other 17 naturally occurring amino acids did not inhibit the uptake of phenylalanine, tyrosine, or tryptophan. The results of cross inhibition experiments between the aromatic amino acids are shown in Table 1. When unlabeled amino acids were present at a concentration 100-fold greater than the labeled amino acid, (i) tyrosine uptake was inhibited to a greater degree by phenylalanine (72%) than by tryptophan (60%), and (ii) tryptophan uptake was not inhibited by phenylalanine or tyrosine. Histidine and proline had no significant inhibitory effect on aromatic amino acid uptake. The inhibition of phenylalanine uptake by tyrosine and trytophan was strictly competitive (Fig. 6). When unlabeled amino acid was present at a 10-fold excess (1 x 10-5 M) above the concentration of the labeled aromatic amino acid, tyrosine and tryptophan did not inhibit phenylalanine uptake (Table 1). Tyrosine uptake was inhibited 65% by phenylalanine and 45% by tryptophan.These data indicate that phenylalanine and tryptophan uptake show greater substrate specificity than tyrosine
1049
AMINO ACID TRANSPORT IN Y. PESTIS
VOL. 122, 1975
uptake, since at low amino acid concentrations (10-fold excess) phenylalanine and tyrosine uptake were not inhibited by aromatic amino acids, but tyrosine uptake was inhibited by phenylalanine and tryptophan. Amino acid pool exchange experiments (Table 2) also separated the presence of a phenylalanine tyrosine system from a tryptophan system. Tryptophan did not exchange with the tyrosine and phenylalanine pool. Exchange of phenylalanine and tyrosine with the tryptophan pool probably reflects exchange over a general aromatic transport system. Effect of analogues on aromatic amino acid uptake. Table 3 summarizes the results of experiments on the effect of stereoisomers and structural analogues of the aromatic amino acids on the uptake of phenylalanine, tyrosine, and tryptophan. Structural analogues had no inhibitory effect on the uptake of tryptophan except for the iodinated derivatives of tyrosine. The D isomers had little or no effect on the uptake of phenylalanine or tryptophan, indicating that the uptake of these two amino acids is specific for the natural L isomer. Surprisingly, and in contrast to the latter results, each of the D isomers of the aromatic amino acids inhibited the uptake of tyrosine equally as well (37%). The data indicate that the uptake of tyrosine is less stereo specific than phenylalanine or tryp-
50 45
z 35 04
z w 0
25 ,, E
(I 0
15 c
5
2
4
6
[PHE)
0
8
10
TAaz 1. Cross inhibition of aromatic amino acid uptake % Inhibition in rate of uptake of labeled amino acida Amino acid
Phenylalanine
Phenylalanine 10-' M 10-'M
Tyrosine
tophan tpa
72 65
0 0
FiG. 6. Kinetics of competitive inhibition of phenylalanine uptake by tyrosine and tryptophan. Cell suspensions were incubated for 2 min with the indicated concentrations of labeled phenylalanine plus either 1 x 10-4 M tyrosine (-) or 1 x 10-4 M tryptophan (A).
tophan uptake. The placement of a fluorine atom on the ortho, meta, or para position of the Tyrosine phenylalanine ring did not abolish recognition 10-4M 65 0 of phenylalanine since these compounds 10-'M 7 0 strongly inhibited the uptake of phenylalanine, whereas p-chlorophenylalanine was apparently Tryptophan less acceptable for recognition due to the pres10-'M 46 60 ence of the larger chlorine atom on the para 10-, M 5 45 position of the phenylalanine ring. Ortho and Histidine meta tyrosine inhibited the uptake of phenylal0 10-4 M 14 14 anine to the same degree (67 and 64%) as natural tyrosine, but only inhibited tyrosine Proline uptake 50 and 36%, respectively. This result 10-4M 5 14 8 indicates that tyrosine uptake shows specificity aLabeled amino acid (1 x 10-' M) and unlabeled for the para hydroxyl group on the tyrosine ring. The fluoro and methyl derivatives of tryptoamino acid were added together to cell suspensions at zero time. The incubation period for the inhibition phan inhibited the uptake of tryptophan more assay was 2 min. effectively than the uptake of phenylalanine or
1050
J. BACMEIOL.
SMITH AND MONTIE TABLE 2. Exchange of aromatic amino acid pools Pool exchanges in 10 min with unlabeled: Before
Pool labeled
amino acida
Phenylalanine ....... Tyrosine ............ Tryptophan .........
exchange (nmol)
Phenylalanine (no)
(nmol)
TYrnoline
0.72 0.56 0.43
0.201 0.310 0.228
72 45 48
0.388 0.322 0.308
-
Tyrosine
_____
_____
Tryptophan (nmol)
46 44 31
10
0.652 0.580 0.186
0
56
a Amino acid pools were formed by incubating chloramphenicol-treated cell suspensions with labeled amino acid for 10 min. The cell suspension was centrifuged, and the excess labeled amino acid was decanted. The cell pellet was resuspended in potassium phosphate (0.05 M)-magnesium chloride (0.005 M) buffer, pH 7.0, 27 C. Unlabeled amino acids (1 x 10- 4 M) were added to the cell suspension and incubated 10 min at 27 C. The reaction was terminated by rapid filtration as previously described.
TABLE 3. Inhibition of aromatic amino acid uptake by structural analoguesa
TABLE 4. Inhibition of aromatic amino acid uptake by structural analogues of tryptophan
% Inhibition of rate of uptake
% Inhibition of rate of uptake
Analoguea
Analogue
Phenyl- Tyroalanine sine
o-Fluorophenylalanine p-Fluorophenylalanine .... m-Fluorophenylalanine . p-Chlorophenylalanine ... ,-Thienylalanine ......... m-Tyrosine .............. o-Tyrosine ................ Monoiodotyrosine ........ 3,5-Diiodotyrosine ........ ....
74 67 85 53 79 64 67 72 28 28 0
D-Phenylalanine ......... D-Tyrosine ............... D-Tryptophan ...........0.
51 41 81 77 40 36 50 80 50 37 37 37
Phenyl- Tyroalanine sine
Tryptophan
0 0 0 0 0 0 0 54 0 0 0 0
a Analogues (1 x 10-4 M) and labeled amino acids (1 x 10-6 M) were added together at zero time. The incubation period for the inhibition assay was 2 min.
tyrosine (Table 4). The effectiveness of inhibition in descending order was 5-fluorotryptophan, 5-methyltryptophan, 6-methyltryptophan, and 4-methyltryptophan. These results suggest that the 4 position of the indole ring of tryptophan is important for the recognition of this amino acid by the transport system since the placement of a large methyl group in the 4 position alters the molecule so that it can no longer inhibit the uptake of natural tryptophan. The a amino and carboxyl groups are important for recognition of phenylalanine by the transport system since 100-fold excess of phenylpyruvic acid and phenylalanine methylester inhibited phenylalanine uptake only 45 and 47%, respectively (Table 5). Alanine did not inhibit phenylalanine uptake, indicating that the aromatic ring is an essential structure of the phenylalanine molecule needed for substrate recognition. The dipeptide, L-seryl-L-phenylala-
4-Methyltryptophan ...... 5-Methyltryptophan ...... 6-Methyltryptophan ...... 5-Fluorotryptophan .......
9 23 18 43
Tryptophan
20 59 52 75
14 39 37 44
a Analogues (1 x 10-I M) and labeled amino acids (1 x 10-6 M) were added together to cell suspensions at zero time. The incubation period for the inhibition assay was 2 min.
TABLE 5. Inhibition of phenylalanine uptake of structurally related compounds % Inhibition of rate of phenylalanine uptake
Compounda
L-Seryl-L-phenylalanine
.......
85
47 Phenylpyruvic acid ............. 5 Hydroxyphenylpyruvic acid .... 45 Phenylalanine methyl ester ..... 5 Alanine ...................... a Compounds (1 x 10-4 M) and labeled phenylalanine (1 x 10-6 M) were added together to cell suspensions at zero time. The incubation period for the inhibition assay was 2 min.
nine, inhibited phenylalanine uptake 85% suggesting that the phenylalanine transport system can recognize phenylalanine with the amino group blocked by the serine residue. These results together with the finding that the D isomer of phenylalanine did not significantly inhibit the uptake of the natural L isomer show that the L form of the amino acid, an intact a amino and carboxy group, and the aromatic ring are required for maximum recognition of
AMINO ACID TRANSPORT IN Y. PESTIS
VOL. 122, 1975
this amino acid by the transport system. Effect of sulfhydryl blocking reagents and metabolic inhibitors on aromatic amino acid uptake. The uptake of aromatic amino acids showed some selectivity to inhibition by sulfhydryl blocking reagents and metabolic inhibitors (Table 6). Cell suspensions preincubated either 1 or 15 min with the indicated compound were assayed for amino acid uptake over a 2-min period. Sulfhydryl reagents, p-chloromercuribenzoate, and N-ethylmaleimide strongly inhibited phenylalanine and tyrosine uptake after a brief (1 min) or extended pretreatment (15 min). Notably, tryptophan uptake was only slightly inhibited by p-chloromercuribenzoate (18%, 15 min) and was not inhibited at all by N-ethylmaleimide. A comparison of the inhibition by sulfhydryl reagents over the 1-min preincubation period shows phenylalanine uptake to be somewhat more sensitive to these compounds than tyrosine uptake. N-ethylmaleimide inhibited phenylalanine uptake 78%, whereas tyrosine uptake was inhibited only 41% under these conditions. TABLE 6. Inhibition of aromatic amino acid uptake by sulfhydryl blocking reagents and metabolic inhibitorsa % Inhibition of rate of uptake
Inhibitor
Phenyl.
Tryp-
alanine
Tsme Tyrosine
1 min
76
61
0
15 min
95
91
18
1 min
78
41
7
15 min
100
86
0
1 min
48
67
60
15 min
83
95
95
pCMB (5 x 10-4M)
tophan
NEM (2 x 10-3 M)
DNP (1 x 1o- M)
KCN (1 x 10-2M) 1 min
15 min
NaN, (1 x 10-2M) 1 min 15 min
30
14
15
76
47
49
36 80
13 59
0 0
aCell suspensions were incubated with the inhibitors at the indicated concentrations for either 1 or 15 min. Labeled amino acids (1 x 10-6 M) were then added and the incubation was continued for a 2-min period. pCMB, p-Chloromercuribenzoate; NEM, Nethylmaleimide; DNP, 2,4-dinitrophenol; KCN, potaaaium cyanide; NaN,, sodium azide.
1051
Metabolic inhibitors, potassium cyanide, and sodiom azide inhibited phenylalanine and tyrosine uptake. Dinitrophenol was the most effective inhibitor of these compounds. Tryptophan uptake was not inhibited by sodium azide but was inhibited by 2,4-dinitrophenol and potassium cyanide. Inhibition of transport by metabolic inhibitors plus the finding that aromatic amino acid transport in Y. pestis is concentrative indicate that this group of amino acids is actively transported by this organism. DISCUSSION The results show that aromatic amino acid transport in Y. pestis exhibits concentrative uptake, depends on the extemal concentration, and requires metabolic energy. The observations that glucose depressed aromatic amino acid uptake in the presence of chloramphenicol were unexpected. The glucose effect occurs very likely because the cell is vigorously synthesizing tryptophan and tyrosine as end products in the absence of protein synthesis. Consequently, these compounds in effect dilute the labeled aromatic pool directly and by counterfluxing dilute extemal aromatic label. In minus chloramphenicol experiments protein synthesis effectively reduced the pool, thereby allowing rapid uptake of phenylalanine. Experiments were performed to delineate separate transport systems by examining their substrate specificity. Cross inhibition experiments between the aromatic amino acids and selected analogues showed that the tryptophan transport system was highly specific. These data also indicate that phenylalanine and tryptophan transport show greater substrate specificity than tyrosine transport. The recognition of phenylalanine or tyrosine showed a high degree of substrate specificity overlap in experiments where analogues or natural aromatic amino acids were present at a 100-fold excess above the concentration of the labeled amino acid. These studies showed that the phenylalanine transport system was capable of recognizing tyrosine and tryptophan only when these amino acids were present at a high concentration relative to the phenylalanine concentration. Phenylalanine and tryptophan transport was not significantly inhibited by their respective D isomers, indicating that the transport of phenylalanine and tryptophan is stereo specific for the natural L isomer. Tyrosine transport did not show the same degree of stereo specificity as phenylalanine and tryptophan transport since tyrosine transport was inhibited significantly by each of the D aromatic amino acids. These
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SMITH AND MONTIE
findings are similar to those made for tyrosine transport in P. aeruginosa (7), where Dphenylalanine and D-tyrosine inhibited the transport of L-tyrosine. The fluoro analogues of phenylalanine were effective inhibitors of phenylalanine and tyrosine transport, indicating that the fluorine atom does not appreciably alter the phenylalanine molecule with respect to recognition by the transport system. This finding is in agreement with studies on the inhibition of phenylalanine transport by fluoro analogues in Salmonella typhimrium (1) and inhibition of tyrosine transport in P. aeruginosa by the fluoro derivatives of tyrosine and phenylalanine (7). Data showed decreased inhibition of tyrosine by ortho and meta tyrosine. These results indicate that tyrosine recognition is dependent on the position of the hydroxyl group on the tyrosine ring. The methyl and fluoro analogues of tryptophan were effective in inhibiting the transport of tryptophan, phenylalanine, and tyrosine. These findings are in contrast to that observed in P. aeruginosa in which 5-methyltryptophan did not inhibit tryptophan transport and 5-fluorotryptophan did not inhibit tyrosine transport (7). The results of cross inhibition experiments between the aromatic amino acids and their analogues suggest that three transport systems exist in Y. pestis TJW for the transport of aromatic amino acids: (i) tryptophan specific, (ii) phenylalanine specific with transport activity for tyrosine and tryptophan when present at high external concentrations, and (iii) a general system which recognizes each amino acid at a high (1 x 10-4 M) or low (1 x 10- M) external concentration but shows a degree of substrate specificity for tyrosine transport. The high-affinity phenylalanine transport system provides a mechanism whereby wildtype Y. pestis TJW can take up and concentrate a required nutrient for biosynthesis and growth. Also the maximum efficiency of the transport system corresponds with the optimum pH and temperature requirements for growth. Similar studies on tyrosine and tryptophan transport did not show this correlation between pH and temperature optimum for growth. The above discussed characteristics of phenylalanine transport suggests that this system serves to supply an amino acid which is required for the growth and maintenance of this organism. Division of aromatic amino acid transport into multiple systems in Y. pestis TJW was also substantiated by the finding that the uptake of amino acid was differentially inhibited by sulfhydryl blocking reagents and energy inhibitors. Initial phenylalanine transport (1 min) was
J. BACTERIOL.
inhibited somewhat more by sulfhydryl reagents than tyrosine transport. Tryptophan transport was not significantly inhibited by these compounds during a 1- or 15-min preincubation period. It is very likely that the tryptophan transport system does not depend on active sulfhydryl groups for transport activity. It is interesting to note that binding proteins isolated by osmotic shock procedures are not inactivated by sulfhydryl reagents (2). We have found that extraction of Y. pestis membranes with the detergent, Brij 36T, yields tryptophan but not phenylalanine or tyrosine binding activity, but we could not identify a "shockable" activity for tryptophan binding. The lack of inhibition by azide would also suggest an adenosine 5'-triphosphate-dependent periplasmic system (2). It is possible that activity is lost or diluted in the shock procedure but retained in the Brij procedure, since a greater quantity of loosely bound protein might be obtained. Alternatively, the tryptophan transport system may be "unavailable" in the cell membrane so that sulfhydryl reagents cannot penetrate to an essential site (9). The lack of sensitivity of tryptophan transport to NaN, may indicate either positional differences in the anatomical location of the transport system in the membrane or a difference in sensitivity of a given transport system to respiratory inhibition. ACKNOWLEDGMENTS P. B. S., a predoctoral trainee, was supported by Public Health Service grant no. TO1-AI00435 from the National Institute of Allergy and Infectious Diseases. We would like to thank Diane Montie for her critical evaluation of the manuscript.
LITERATURE CITED 1. Ames, G. F. 1964. Uptake of amino acids by Salmonella typhimurium. Arch. Biochem. Biophys. 104:1-18. 2. Berger, E. A. 1973. Different mechanisms of energy coupling for the active transport of proline and glutamine in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 70:1514-1518. 3. Britten, R. J., and F. T. McClure. 1962. The amino acid pool in Escherichia coli. Bacteriol. Rev. 26:292-335. 4. Brown, K. D. 1970. Formation of aromatic amino acid pools in Escherichia coli K-12. J. Bacteriol. 104:177-188. 5. Brown, K. D. 1971. Maintenance and exchange of the aromatic amino acid pool in Escherichia coli. J. Bacteriol. 106:70-81. 6. Burrous, S. E., and R. D. Demoss. 1963. Studies on tryptophan permease in Escherichia coli. Biochim. Biophys. Acta 73:623-637. 7. Kay, W. W., and A. Gronlund. 1971. Transport of aromatic amino acids by Pseudomonas aeruginosa. J. Bacteriol. 105:1039-1046. 8. Roseman, S. 1969. The transport of carbohydrates by a bacterial phosphotransferase system. J. Gen. Physiol.
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9. Vansfeveninck, J., R. I. Weed, and A. Rothstein. 1965. Localization of erythrocyte membrane sulfhydryl groups essential for glucose transport. J. Gen. Physiol. 48:617-632.
