Vol. 132, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Nov. 1977, p. 453-461 Copyright © 1977 American Society for Microbiology
Regulation of Aromatic Amino Acid Transport Systems in Escherichia coli K-12 MARGARET J. WHIPP* AND A. J. PITTARD Microbiology Department, Uniuersity of Melbourne, Parkuille, 3052, Victoria, Australia Received for publication 19 May 1977
The regulation of the aromatic amino acid transport systems was investigated. The common (general) aromatic transport system and the tyrosinespecific transport system were found to be subject to repression control, thus confirming earlier reports. In addition, tyrosine- and tryptophan-specific transport were found to be enhanced by growth of cells with phenylalanine. The repression and enhancement of the transport systems was abolished in a strain carrying an amber mutation in the regulator gene, tyrR. This indicates that the tyrR gene product, which was previously shown to be involved in regulation of aromatic biosynthetic enzymes, is also involved in the regulation of the aromatic amino acid transport systems.
A preliminary report of part of the work has In Escherichia coli there are four systems concerned with the transport of the aromatic been given (Whipp et al., Proc. Aust. Biochem. amino acids: a common (general) aromatic Soc. 9:62, 1976). transport system, which transports each of the three aromatic amino acids, and three specific MATERIALS AND METHODS systems, each of which transports a single Organisms. Strains used in this work are all aromatic amino acid, either phenylalanine, ty- derivatives of E. coli K-12 and are described in rosine, or tryptophan (6). Mutants that are Table 1. affected in each of these systems have been Materials. The chemicals used were obtained isolated (6, 12, 17; M. J. Whipp, D. M. Hallsall, commercially and were not further purified. L-[Uand A. J. Pittard, Proc. Aust. Biochem. Soc. 14C]tyrosine (400 mCi/mmol) and L-[U-'4C]phenylalanine (374 mCi/mmol) were purchased from 9:62, 1976). In addition to the above four systems, there Commissariat a l'Energie Atomique, France. L[methylene-'4C]tryptophan (51.8 mCi/mmol) was is also an inducible system for transporting purchased from the Radiochemical Centre, Amertryptophan (5, 9). This system is subject to sham, England. The isotopes were diluted appropricatabolite repression and is not induced in the ately with nonradioactive amino acids for use. presence of glucose. It is not expressed under Growth of cells. The minimal medium used was the conditions used in these studies. half-strength medium 56, described by Monod et al. It has previously been shown that cells grown (15), supplemented with 0.2% glucose or other carin the presence of tyrosine or phenylalanine bon sources as indicated, thiamine, and appropriate have decreased levels of the common transport growth factors. When minimal medium was supplewith the aromatic amino acids and vitasystem (11, 14) and, in addition, that growth mented these supplements were added in the followmins, in the presence of tyrosine results in a de- ing concentrations, except where otherwise stated: creased level of the tyrosine-specific transport L-phenylalanine, 10-3 M; L-tryptophan, 5 x 104 M; system (14). In this study, we confirm these L-tyrosine, 10-3 M; p-aminobenzoic acid, 10-6 M; pfindings and look at further regulatory effects hydroxybenzoic acid, 4 x 10-6 M; and 2,3-dihydroxof the aromatic amino acids on the transport ybenzoic acid, 5 x 10-5 M. Casamino Acids (casein hydrolysate, Difco) was used at a final concentration systems. The product of the regulator gene, tyrR, has of 0.5%. Cultures were grown in a rotary shaker at 37°C, been shown to be essential for the control of the synthesis of a number of the enzymes using as an inoculum an unwashed 16-h culture in the same medium. The cells were grown for at least involved in aromatic biosynthesis (8, 10, 13, 24; two doublings. Turbidity was monitored by B. K. Ely and J. Pittard, Proc. Aust. Biochem. usingmass a Klett-Summerson photoelectric colorimeter Soc. 8:56, 1975). We show here that this regu- with a no. 54 filter (0.21 mg [dry weight] of cells/ml lator gene also plays an essential role in the = 100 Klett units). Cells were harvested in the midregulation of three of the aromatic transport exponential phase of growth by centrifugation, washed twice in an equivalent volume of halfsystems. 453
454
J. BACTERIOL.
WHIPP AND PITTARD TABLE 1. Description of E. coli K-12 strains
Strain designa-
Source or reference
Relevant genetic locia
tion
JP2310 JP2311 JP2625 JP2626 JP2665 JP2666 JC411 JP2112
his-29 tyrR370 his-29 tyrRaroB351 his-29 tyrR370 aroB351 his-29 tyrR+ his- tyrP474 his+ tyrP+
AB3253 AB3271
aroG365 aroH367 aroG365 aroH367 tyrR352
argG6 argG mtr-24
From JP2140 (described in ref. 10) by Plkc transduction From JP2140 (10) by Plkc transduction From a malT derivative of JP2310 by Plkc transduction From a malT derivative of JP2311 by Plkc transduction From JP2311 by F-prime mobilization From JP2311 by F-prime mobilization Obtained from A. J. Clark (described in ref. 1) From JC411 by Plkc transduction (mtr-24 described in ref. 12). (24) (24)
a Symbols: argG is the structural gene for argininosuccinate synthetase (EC 6.3.4.5); aroB is the structural gene for 3-dehydroquinate synthetase (as yet not given an EC number); aroG is the structural gene for DAHP synthetase (EC 4.1.2.15) (phe); aroH is the structural gene for DAHP synthetase (trp); his is any one of the structural genes for histidine biosynthesis; mtr is a gene involved in the tryptophanspecific transport system; tyrP is a gene involved in the tyrosine-specific transport system; tyrR is a regulator gene controlling the expression of a number of the aromatic biosynthetic enzymes.
strength 56 buffer, and resuspended in this buffer supplemented with glucose (0.2%), chloramphenicol (80 ,ug/ml), and specific growth requirements. The cells were incubated at 37TC for 10 min and then stored at 4cC until used. Transport assay. Cells were brought to 30'C, and the assay was initiated by adding a sample of the cells to a flask containing radioactively labeled amino acids plus cold competing amino acid if appropriate. Samples (0.1 ml) were withdrawn from the assay mixture at various times after initiation of the assay, passed through filter membranes of pore size 0.45 Am, and immediately washed with two 2ml volumes of half-strength 56 buffer at 30°C. The filters were dried at 60'C, 5 ml of a solution of toluene with 0.5% 2,5-diphenyloxazole and 0.02% 1,4-bis-L2-(4-methyl-5-phenyloxazolyl]benzene was added, and radioactivity was counted in a Packard liquid scintillation spectrometer, model 3320. Radioactively labeled aromatic amino acids were used at 10 jAM final concentration. This concentration is saturating for both the common and specific aromatic transport systems (7). Cold competing amino acid was used in 20-fold excess (200 ,uM). Standard counts were carried out with each batch of stock solution. Control filtrations (without cells) were performed with each set of experiments to correct for background radiation and nonspecific adsorption of radioactive material to the filters. Corrected values of counts of cellular radioactivity are expressed as nanomoles of amino acid taken up per milligram (dry weight) of bacteria.
RESULTS Regulation of the common transport system. The uptake of each aromatic amino acid by the common transport system can be completely inhibited by adding either of the other two aromatic amino acids in excess (6). The uptake that occurs under these conditions is
due to the relevant specific transport system. The uptake due to the common transport system can be calculated as the difference between the total uptake for a particular amino acid and the uptake due to its specific system. In the experiments that follow, we are examining the effect that the presence or absence of each of the amino acids in the growth medium has on the expression of each of these various transport systems. The results in Fig. 1 show the effect of growth
5 -
a)
3 0
E w
2
lDJ
y
1
2
TIME (min) FIG. 1. Uptake of tyrosine by the common aromatic transport system of JP2311 after growth in minimal medium without aromatic amino acid supplement (MIN) and in minimal medium supplemented with L-phenylalanine (PHE), L-tryptophan (TRP), and L-tyrosine (TYR).
VOL. 132, 1977
AMINO ACID TRANSPORT REGULATION
in the presence of each aromatic amino acid on the uptake due to the common transport system. In this case, transport of L-[P4C]tyrosine is being used to estimate common transport activity. When either phenylalanine or tyrosine was present in the growth medium, the cell's ability to transport the aromatic amino acids via the common transport system was considerably depressed. Initial rates of uptake were six- to eightfold lower than those observed in cells grown in minimal medium. Growing the cells in the presence of tryptophan also caused a decrease in the level of the common transport system. In this case, however, the extent of the change was smaller than that produced by either tyrosine or phenylalanine. Regulation of the specific transport systems. An estimate of the levels of the specific transport system for phenylalanine, tyrosine, or tryptophan was made by measuring the cells' ability to transport the relevant 14C-labeled amino acid in the presence of a 20-fold excess of one other amino acid capable of completely saturating the common transport system. Hence [14C]phenylalanine and [14C]tyrosine uptake was measured in the presence of a 20fold excess of unlabeled tryptophan, and uptake of [L14C]tryptophan was measured in the presence of a 20-fold excess of unlabeled phenylalanine. The results in Fig. 2 and 5 show the effects of different growth conditions on the tyrosine- and tryptophan-specific transport systems. No results are shown for the phenylalanine-specific system, which was found not to vary regardless of the medium in which the cells were grown. However, the level of the tyrosine-specific system changed dramatically in response to the presence of specific amino acids in the medium (Fig. 2). Whereas tryptophan had no effect, tyrosine repressed levels to approximately zero, and the presence of phenylalanine in the growth medium resulted in a six- to eightfold increase in the level of the tyrosine-specific system. Cells grown in the presence of both phenylalanine and tyrosine exhibited the same low level of activity as cells grown in the presence of tyrosine alone (Fig. 3), indicating that the tyrosine-mediated repression was dominant over the enhancing effect of phenylalanine. Cells grown in the presence of phenylalanine and tryptophan, however, showed the same high level of activity as in the phenylalaninegrown cells (Fig. 3). A functional common transport system is not required for this phenylalanine-mediated effect, since aroP cells gave the same result as aroP+ cells. To determine whether the increased uptake
455 PHE
6 5 E C
4
0 0
3 E H0~ Lli
TRP 3MIN
2
-
"V
1 2 TIME (min) FIG. 2. Uptake of tyrosine by the tyrosine-specific transport system of JP2311 after growth in minimal medium without aromatic amino acid supplement (MIN) and in minimal medium supplemented with L-phenylalanine (PHE), L-tryptophan (TRP), and L-
tyrosine (TYR). 3
5
PHE
~~~~~~TRP
>1
E
4
U,>
3
0
E c
2
a-
:D
PHE
o ^ ( 1
o TYR
2
TIME (min) FIG. 3. Uptake of tyrosine by the tyrosine-specific transport system of JP2311 after growth in minimal medium supplemented with L-phenylalanine and L-tryptophan (PHE TRP) or L-phenylalanine and L-tyrosine (PHE TYR). in phenylalanine-grown cells did in fact involve
the previously characterized tyrosine-specific system, these experiments were repeated with a strain of E. coli carrying a mutation in the gene tyrP and shown to be unable to take up
456
WHIPP AND PITTARD
J. BACTERIOL.
tyrosine by the tyrosine-specific system (Whipp et al., Proc. Aust. Biochem. Soc. 9:62, 1976). In conjugation experiments, the gene tyrP exhibits close linkage to his. Figure 4 shows the results with a tyrP and an isogenic tyrP+ strain. The increased transport in phenylalanine-grown cells did depend on a functional tyrosine-specific system, since the introduction of the tyrP mutation severely reduced tyrosine uptake in this system. Phenylalanine also caused an increase in tryptophan uptake by the tryptophan-specific transport system above that seen in minimally grown cells (Fig. 5). The apparent variation in uptake of cells grown in minimal media without aromatic supplements, with tyrosine and with tryptophan, was not marked and was considered to be due to the variability inherent in these assays. Mutations in the mtr gene have been isolated by Hiraga et al. (12). These mutations have been found by Yanofsky, as cited by Oxender (17), to result in loss of the t;ryptophan-specific transport system. Strain J]P2112, which possesses such an mtr- mutati on, failed to show enhanced transport of tryptc)phan in phenylalanine-grown cells, whereas JC411, the strain from which JP2112 was deriived, did show enhanced transport of trypt tophan (data not shown). This confirms that t]he increased activity in tryptophan transport iinvolves the previously characterized tryptoph an-specific system.
_oPHIE ::E C-
L.
/O
ci
3 0 0
2
- _.MIN
0
L,-J
y
/
-*-
*.o
-----.x PH E 2
TIME (m in) FIG. 4. Uptake of tyrosine by the tyrosine-specific transport system of JP2665 (t)'rP474} (solid lines) and JP2666 (tyrP+) (broken li,nes) after growth in minimal medium without aromi.atic amino acid supplement (MIN) and in minimtal medium supplemented with L-phenylalanine (FWE).
~--
0>
0 6 E LL
0
,,
0 4
JOURNAL OF BACTERIOLOGY, Nov. 1977, p. 453-461 Copyright © 1977 American Society for Microbiology
Regulation of Aromatic Amino Acid Transport Systems in Escherichia coli K-12 MARGARET J. WHIPP* AND A. J. PITTARD Microbiology Department, Uniuersity of Melbourne, Parkuille, 3052, Victoria, Australia Received for publication 19 May 1977
The regulation of the aromatic amino acid transport systems was investigated. The common (general) aromatic transport system and the tyrosinespecific transport system were found to be subject to repression control, thus confirming earlier reports. In addition, tyrosine- and tryptophan-specific transport were found to be enhanced by growth of cells with phenylalanine. The repression and enhancement of the transport systems was abolished in a strain carrying an amber mutation in the regulator gene, tyrR. This indicates that the tyrR gene product, which was previously shown to be involved in regulation of aromatic biosynthetic enzymes, is also involved in the regulation of the aromatic amino acid transport systems.
A preliminary report of part of the work has In Escherichia coli there are four systems concerned with the transport of the aromatic been given (Whipp et al., Proc. Aust. Biochem. amino acids: a common (general) aromatic Soc. 9:62, 1976). transport system, which transports each of the three aromatic amino acids, and three specific MATERIALS AND METHODS systems, each of which transports a single Organisms. Strains used in this work are all aromatic amino acid, either phenylalanine, ty- derivatives of E. coli K-12 and are described in rosine, or tryptophan (6). Mutants that are Table 1. affected in each of these systems have been Materials. The chemicals used were obtained isolated (6, 12, 17; M. J. Whipp, D. M. Hallsall, commercially and were not further purified. L-[Uand A. J. Pittard, Proc. Aust. Biochem. Soc. 14C]tyrosine (400 mCi/mmol) and L-[U-'4C]phenylalanine (374 mCi/mmol) were purchased from 9:62, 1976). In addition to the above four systems, there Commissariat a l'Energie Atomique, France. L[methylene-'4C]tryptophan (51.8 mCi/mmol) was is also an inducible system for transporting purchased from the Radiochemical Centre, Amertryptophan (5, 9). This system is subject to sham, England. The isotopes were diluted appropricatabolite repression and is not induced in the ately with nonradioactive amino acids for use. presence of glucose. It is not expressed under Growth of cells. The minimal medium used was the conditions used in these studies. half-strength medium 56, described by Monod et al. It has previously been shown that cells grown (15), supplemented with 0.2% glucose or other carin the presence of tyrosine or phenylalanine bon sources as indicated, thiamine, and appropriate have decreased levels of the common transport growth factors. When minimal medium was supplewith the aromatic amino acids and vitasystem (11, 14) and, in addition, that growth mented these supplements were added in the followmins, in the presence of tyrosine results in a de- ing concentrations, except where otherwise stated: creased level of the tyrosine-specific transport L-phenylalanine, 10-3 M; L-tryptophan, 5 x 104 M; system (14). In this study, we confirm these L-tyrosine, 10-3 M; p-aminobenzoic acid, 10-6 M; pfindings and look at further regulatory effects hydroxybenzoic acid, 4 x 10-6 M; and 2,3-dihydroxof the aromatic amino acids on the transport ybenzoic acid, 5 x 10-5 M. Casamino Acids (casein hydrolysate, Difco) was used at a final concentration systems. The product of the regulator gene, tyrR, has of 0.5%. Cultures were grown in a rotary shaker at 37°C, been shown to be essential for the control of the synthesis of a number of the enzymes using as an inoculum an unwashed 16-h culture in the same medium. The cells were grown for at least involved in aromatic biosynthesis (8, 10, 13, 24; two doublings. Turbidity was monitored by B. K. Ely and J. Pittard, Proc. Aust. Biochem. usingmass a Klett-Summerson photoelectric colorimeter Soc. 8:56, 1975). We show here that this regu- with a no. 54 filter (0.21 mg [dry weight] of cells/ml lator gene also plays an essential role in the = 100 Klett units). Cells were harvested in the midregulation of three of the aromatic transport exponential phase of growth by centrifugation, washed twice in an equivalent volume of halfsystems. 453
454
J. BACTERIOL.
WHIPP AND PITTARD TABLE 1. Description of E. coli K-12 strains
Strain designa-
Source or reference
Relevant genetic locia
tion
JP2310 JP2311 JP2625 JP2626 JP2665 JP2666 JC411 JP2112
his-29 tyrR370 his-29 tyrRaroB351 his-29 tyrR370 aroB351 his-29 tyrR+ his- tyrP474 his+ tyrP+
AB3253 AB3271
aroG365 aroH367 aroG365 aroH367 tyrR352
argG6 argG mtr-24
From JP2140 (described in ref. 10) by Plkc transduction From JP2140 (10) by Plkc transduction From a malT derivative of JP2310 by Plkc transduction From a malT derivative of JP2311 by Plkc transduction From JP2311 by F-prime mobilization From JP2311 by F-prime mobilization Obtained from A. J. Clark (described in ref. 1) From JC411 by Plkc transduction (mtr-24 described in ref. 12). (24) (24)
a Symbols: argG is the structural gene for argininosuccinate synthetase (EC 6.3.4.5); aroB is the structural gene for 3-dehydroquinate synthetase (as yet not given an EC number); aroG is the structural gene for DAHP synthetase (EC 4.1.2.15) (phe); aroH is the structural gene for DAHP synthetase (trp); his is any one of the structural genes for histidine biosynthesis; mtr is a gene involved in the tryptophanspecific transport system; tyrP is a gene involved in the tyrosine-specific transport system; tyrR is a regulator gene controlling the expression of a number of the aromatic biosynthetic enzymes.
strength 56 buffer, and resuspended in this buffer supplemented with glucose (0.2%), chloramphenicol (80 ,ug/ml), and specific growth requirements. The cells were incubated at 37TC for 10 min and then stored at 4cC until used. Transport assay. Cells were brought to 30'C, and the assay was initiated by adding a sample of the cells to a flask containing radioactively labeled amino acids plus cold competing amino acid if appropriate. Samples (0.1 ml) were withdrawn from the assay mixture at various times after initiation of the assay, passed through filter membranes of pore size 0.45 Am, and immediately washed with two 2ml volumes of half-strength 56 buffer at 30°C. The filters were dried at 60'C, 5 ml of a solution of toluene with 0.5% 2,5-diphenyloxazole and 0.02% 1,4-bis-L2-(4-methyl-5-phenyloxazolyl]benzene was added, and radioactivity was counted in a Packard liquid scintillation spectrometer, model 3320. Radioactively labeled aromatic amino acids were used at 10 jAM final concentration. This concentration is saturating for both the common and specific aromatic transport systems (7). Cold competing amino acid was used in 20-fold excess (200 ,uM). Standard counts were carried out with each batch of stock solution. Control filtrations (without cells) were performed with each set of experiments to correct for background radiation and nonspecific adsorption of radioactive material to the filters. Corrected values of counts of cellular radioactivity are expressed as nanomoles of amino acid taken up per milligram (dry weight) of bacteria.
RESULTS Regulation of the common transport system. The uptake of each aromatic amino acid by the common transport system can be completely inhibited by adding either of the other two aromatic amino acids in excess (6). The uptake that occurs under these conditions is
due to the relevant specific transport system. The uptake due to the common transport system can be calculated as the difference between the total uptake for a particular amino acid and the uptake due to its specific system. In the experiments that follow, we are examining the effect that the presence or absence of each of the amino acids in the growth medium has on the expression of each of these various transport systems. The results in Fig. 1 show the effect of growth
5 -
a)
3 0
E w
2
lDJ
y
1
2
TIME (min) FIG. 1. Uptake of tyrosine by the common aromatic transport system of JP2311 after growth in minimal medium without aromatic amino acid supplement (MIN) and in minimal medium supplemented with L-phenylalanine (PHE), L-tryptophan (TRP), and L-tyrosine (TYR).
VOL. 132, 1977
AMINO ACID TRANSPORT REGULATION
in the presence of each aromatic amino acid on the uptake due to the common transport system. In this case, transport of L-[P4C]tyrosine is being used to estimate common transport activity. When either phenylalanine or tyrosine was present in the growth medium, the cell's ability to transport the aromatic amino acids via the common transport system was considerably depressed. Initial rates of uptake were six- to eightfold lower than those observed in cells grown in minimal medium. Growing the cells in the presence of tryptophan also caused a decrease in the level of the common transport system. In this case, however, the extent of the change was smaller than that produced by either tyrosine or phenylalanine. Regulation of the specific transport systems. An estimate of the levels of the specific transport system for phenylalanine, tyrosine, or tryptophan was made by measuring the cells' ability to transport the relevant 14C-labeled amino acid in the presence of a 20-fold excess of one other amino acid capable of completely saturating the common transport system. Hence [14C]phenylalanine and [14C]tyrosine uptake was measured in the presence of a 20fold excess of unlabeled tryptophan, and uptake of [L14C]tryptophan was measured in the presence of a 20-fold excess of unlabeled phenylalanine. The results in Fig. 2 and 5 show the effects of different growth conditions on the tyrosine- and tryptophan-specific transport systems. No results are shown for the phenylalanine-specific system, which was found not to vary regardless of the medium in which the cells were grown. However, the level of the tyrosine-specific system changed dramatically in response to the presence of specific amino acids in the medium (Fig. 2). Whereas tryptophan had no effect, tyrosine repressed levels to approximately zero, and the presence of phenylalanine in the growth medium resulted in a six- to eightfold increase in the level of the tyrosine-specific system. Cells grown in the presence of both phenylalanine and tyrosine exhibited the same low level of activity as cells grown in the presence of tyrosine alone (Fig. 3), indicating that the tyrosine-mediated repression was dominant over the enhancing effect of phenylalanine. Cells grown in the presence of phenylalanine and tryptophan, however, showed the same high level of activity as in the phenylalaninegrown cells (Fig. 3). A functional common transport system is not required for this phenylalanine-mediated effect, since aroP cells gave the same result as aroP+ cells. To determine whether the increased uptake
455 PHE
6 5 E C
4
0 0
3 E H0~ Lli
TRP 3MIN
2
-
"V
1 2 TIME (min) FIG. 2. Uptake of tyrosine by the tyrosine-specific transport system of JP2311 after growth in minimal medium without aromatic amino acid supplement (MIN) and in minimal medium supplemented with L-phenylalanine (PHE), L-tryptophan (TRP), and L-
tyrosine (TYR). 3
5
PHE
~~~~~~TRP
>1
E
4
U,>
3
0
E c
2
a-
:D
PHE
o ^ ( 1
o TYR
2
TIME (min) FIG. 3. Uptake of tyrosine by the tyrosine-specific transport system of JP2311 after growth in minimal medium supplemented with L-phenylalanine and L-tryptophan (PHE TRP) or L-phenylalanine and L-tyrosine (PHE TYR). in phenylalanine-grown cells did in fact involve
the previously characterized tyrosine-specific system, these experiments were repeated with a strain of E. coli carrying a mutation in the gene tyrP and shown to be unable to take up
456
WHIPP AND PITTARD
J. BACTERIOL.
tyrosine by the tyrosine-specific system (Whipp et al., Proc. Aust. Biochem. Soc. 9:62, 1976). In conjugation experiments, the gene tyrP exhibits close linkage to his. Figure 4 shows the results with a tyrP and an isogenic tyrP+ strain. The increased transport in phenylalanine-grown cells did depend on a functional tyrosine-specific system, since the introduction of the tyrP mutation severely reduced tyrosine uptake in this system. Phenylalanine also caused an increase in tryptophan uptake by the tryptophan-specific transport system above that seen in minimally grown cells (Fig. 5). The apparent variation in uptake of cells grown in minimal media without aromatic supplements, with tyrosine and with tryptophan, was not marked and was considered to be due to the variability inherent in these assays. Mutations in the mtr gene have been isolated by Hiraga et al. (12). These mutations have been found by Yanofsky, as cited by Oxender (17), to result in loss of the t;ryptophan-specific transport system. Strain J]P2112, which possesses such an mtr- mutati on, failed to show enhanced transport of tryptc)phan in phenylalanine-grown cells, whereas JC411, the strain from which JP2112 was deriived, did show enhanced transport of trypt tophan (data not shown). This confirms that t]he increased activity in tryptophan transport iinvolves the previously characterized tryptoph an-specific system.
_oPHIE ::E C-
L.
/O
ci
3 0 0
2
- _.MIN
0
L,-J
y
/
-*-
*.o
-----.x PH E 2
TIME (m in) FIG. 4. Uptake of tyrosine by the tyrosine-specific transport system of JP2665 (t)'rP474} (solid lines) and JP2666 (tyrP+) (broken li,nes) after growth in minimal medium without aromi.atic amino acid supplement (MIN) and in minimtal medium supplemented with L-phenylalanine (FWE).
~--
0>
0 6 E LL
0
,,
0 4
