Regulation of Aromatic Amino Acid Transport Systems in

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1 JOURNAL OF BACTRIOLOGY, Nov. 1977, p Copyright 1977 American Society for Microbiology Vol. 13, No. Printed in U.S.A. Regulation of Aromatic Amino Acid Transport Systems in scherichia coli K-1 MARGART J. WHIPP* AND A. J. PITTARD Microbiology Department, Uniuersity of Melbourne, Parkuille, 35, 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. In scherichia coli there are four systems concerned with the transport of the aromatic amino acids: a common (general) aromatic transport system, which transports each of the three aromatic amino acids, and three specific systems, each of which transports a single aromatic amino acid, either phenylalanine, tyrosine, or tryptophan (). Mutants that are affected in each of these systems have been isolated (, 1, 17; M. J. Whipp, D. M. Hallsall, and A. J. Pittard, Proc. Aust. Biochem. Soc. 9:, 197). In addition to the above four systems, there is also an inducible system for transporting tryptophan (5, 9). This system is subject to catabolite repression and is not induced in the presence of glucose. It is not expressed under the conditions used in these studies. It has previously been shown that cells grown in the presence of tyrosine or phenylalanine have decreased levels of the common transport system (11, 1) and, in addition, that growth in the presence of tyrosine results in a decreased level of the tyrosine-specific transport system (1). In this study, we confirm these findings and look at further regulatory effects of the aromatic amino acids on the transport systems. The product of the regulator gene, tyrr, has been shown to be essential for the control of the synthesis of a number of the enzymes involved in aromatic biosynthesis (8, 1, 13, ; B. K. ly and J. Pittard, Proc. Aust. Biochem. Soc. 8:5, 1975). We show here that this regulator gene also plays an essential role in the regulation of three of the aromatic transport systems. A preliminary report of part of the work has been given (Whipp et al., Proc. Aust. Biochem. Soc. 9:, 197). MATRIALS AND MTHODS Organisms. Strains used in this work are all derivatives of. coli K-1 and are described in Table 1. Materials. The chemicals used were obtained commercially and were not further purified. L-[U- 1C]tyrosine ( mci/mmol) and L-[U-'C]phenylalanine (37 mci/mmol) were purchased from Commissariat a l'nergie Atomique, France. L- [methylene-'c]tryptophan (51.8 mci/mmol) was purchased from the Radiochemical Centre, Amersham, ngland. The isotopes were diluted appropriately with nonradioactive amino acids for use. Growth of cells. The minimal medium used was half-strength medium 5, described by Monod et al. (15), supplemented with.% glucose or other carbon sources as indicated, thiamine, and appropriate growth factors. When minimal medium was supplemented with the aromatic amino acids and vitamins, these supplements were added in the following concentrations, except where otherwise stated: L-phenylalanine, 1-3 M; L-tryptophan, 5 x 1 M; L-tyrosine, 1-3 M; p-aminobenzoic acid, 1- M; p- hydroxybenzoic acid, x 1- M; and,3-dihydroxybenzoic acid, 5 x 1-5 M. Casamino Acids (casein hydrolysate, Difco) was used at a final concentration of.5%. Cultures were grown in a rotary shaker at 37 C, using as an inoculum an unwashed 1-h culture in the same medium. The cells were grown for at least two mass doublings. Turbidity was monitored by using a Klett-Summerson photoelectric colorimeter with a no. 5 filter (.1 mg [dry weight] of cells/ml = 1 Klett units). Cells were harvested in the midexponential phase of growth by centrifugation, washed twice in an equivalent volume of half- 53 Downloaded from on September, 18 by guest

2 5 WHIPP AND PITTARD J. BACTRIOL. TABL 1. Description of. coli K-1 strains Strain designa- Relevant genetic locia Source or reference tion JP31 his-9 tyrr37 From JP1 (described in ref. 1) by Plkc transduction JP311 his-9 tyrr- From JP1 (1) by Plkc transduction JP5 arob351 his-9 tyrr37 From a malt derivative of JP31 by Plkc transduction JP arob351 his-9 tyrr+ From a malt derivative of JP311 by Plkc transduction JP5 his- tyrp7 From JP311 by F-prime mobilization JP his+ tyrp+ From JP311 by F-prime mobilization JC11 argg Obtained from A. J. Clark (described in ref. 1) JP11 argg mtr- From JC11 by Plkc transduction (mtr- described in ref. 1). AB353 arog35 aroh37 () AB371 arog35 aroh37 () tyrr35 a Symbols: argg is the structural gene for argininosuccinate synthetase (C.3..5); arob is the structural gene for 3-dehydroquinate synthetase (as yet not given an C number); arog is the structural gene for DAHP synthetase (C.1..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 5 buffer, and resuspended in this buffer supplemented with glucose (.%), chloramphenicol (8,ug/ml), and specific growth requirements. The cells were incubated at 37TC for 1 min and then stored at cc until used. Transport assay. Cells were brought to 3'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 (.1 ml) were withdrawn from the assay mixture at various times after initiation of the assay, passed through filter membranes of pore size.5 Am, and immediately washed with two - ml volumes of half-strength 5 buffer at 3 C. The filters were dried at 'C, 5 ml of a solution of toluene with.5%,5-diphenyloxazole and.% 1,-bis-L-(-methyl-5-phenyloxazolyl]benzene was added, and radioactivity was counted in a Packard liquid scintillation spectrometer, model 33. Radioactively labeled aromatic amino acids were used at 1 jam final concentration. This concentration is saturating for both the common and specific aromatic transport systems (7). Cold competing amino acid was used in -fold excess (,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. RSULTS 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 (). 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 - a) w ldj y FIG. 1. Uptake of tyrosine by the common aromatic transport system of JP311 after growth in minimal medium without aromatic amino acid supplement (MIN) and in minimal medium supplemented with L-phenylalanine (PH), L-tryptophan (TRP), and L-tyrosine (TYR). Downloaded from on September, 18 by guest

3 VOL. 13, 1977 in the presence of each aromatic amino acid on the uptake due to the common transport system. In this case, transport of L-[PC]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 1C-labeled amino acid in the presence of a -fold excess of one other amino acid capable of completely saturating the common transport system. Hence [1C]phenylalanine and [1C]tyrosine uptake was measured in the presence of a - fold excess of unlabeled tryptophan, and uptake of [L1C]tryptophan was measured in the presence of a -fold excess of unlabeled phenylalanine. The results in Fig. 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. ). 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 AMINO ACID TRANSPORT RGULATION 55 C H- ~ Lli "V 5 3 PH -TRP 3MIN 1 FIG.. Uptake of tyrosine by the tyrosine-specific transport system of JP311 after growth in minimal medium without aromatic amino acid supplement (MIN) and in minimal medium supplemented with L-phenylalanine (PH), L-tryptophan (TRP), and L- tyrosine (TYR). 3 5 PH >1 ~~~~~~TRP U,> 3 c a- :D PH ^(o o TYR 1 TIM (min) FIG. 3. Uptake of tyrosine by the tyrosine-specific transport system of JP311 after growth in minimal medium supplemented with L-phenylalanine and L-tryptophan (PH TRP) or L-phenylalanine and L-tyrosine (PH TYR). in phenylalanine-grown cells did in fact involve the previously characterized tyrosine-specific system, these experiments were repeated with a strain of. coli carrying a mutation in the gene tyrp and shown to be unable to take up Downloaded from on September, 18 by guest

4 5 WHIPP AND PITTARD J. BACTRIOL. tyrosine by the tyrosine-specific system (Whipp et al., Proc. Aust. Biochem. Soc. 9:, 197). In ~-- conjugation experiments, the gene tyrp > exhibits close linkage to his. Figure shows the results with a tyrp and an isogenic tyrp+ LL 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 c- uptake in this system. Phenylalanine also caused an increase in tryptophan uptake by the tryptophan-specific transport system above that seen in minimally 1 grown cells (Fig. 5). The apparent variation in uptake of cells grown in minimal media without aromatic supplements, with tyrosine and specific transport system of JP311 after growth in FIG. 5. Uptake of tryptophan by the tryptophan- with tryptophan, was not marked and was minimal medium without aromatic amino acid supplement (MIN) and in minimal medium supple- considered to be due to the variability inherent in these assays. mented with L-phenylalanine (PH), L-tryptophan Mutations in the mtr gene have been isolated (TRP), and L-tyrosine (TYR). by Hiraga et al. (1). These mutations have been found by Yanofsky, as cited by Oxender This effect of phenylalanine was not altered (17), to result in loss of the t;ryptophan-specific by the presence of tyrosine in the medium, but transport system. Strain J] P11, which pos- was completely negated if tryptophan was also sesses such an mtr- mutation, failed to show added to the growth medium (data not shown). enhanced transport of tryptc)phan in phenylal- ffect of a tyrr mutation on transport anine-grown cells, whereas JC11, the strain regulation. The tyrr gene has been shown to from which JP11 was deriived, did show en- code for an aporepressor that is involved in the hanced transport of trypttophan (data not control of the expression of at least four differhe increased activ- ent genetic loci concerned with aromatic bio- shown). This confirms that t] ity in tryptophan transport iinvolves the previ- synthesis (8, 1, 13, ; ly and Pittard, Proc. ously characterized tryptophan-specific system. Aust. Biochem. Soc. S:5, 1975). To see whether the tyrr gene was also involved in regulating these transport systems, a strain isogenic with JP311 except for an amber mutation in the _ophi tyrr gene was examined for its ability to transport the aromatic amino acids. That the tyrr37 allele results in a nonfunctional gene :: L. product is indicated by the data of Camakaris C- /O and Pittard (1). For each of the transport systems, the regulation resulting from growth ci 3 in the presence of the aromatic amino acids was abolished in the tyrr strain (Fig. ). The levels of the transport systems in the tyrr strain (JP31) were the same as those observed when the tyrr+ strain (JP311) was - _.MIN grown in minimal medium. It is interesting to L,-J note that not only repression effects, but also y / -*- the phenylalanine-mediated increase in levels *.o x PH of activity, were missing in the tyrr strain. ffect of endogenous synthesis on uptake. Strains unable to produce functional tyrr prodin) uct have been shown to overproduce tyrosine TIM (m FIG.. Uptake of tyrosine by the tyrosine-specific (R. R. B. Russell, Ph.D. thesis, University of transport system of JP5 (t)'rp7} (solid lines) Melbourne, Melbourne, Australia, 197). To and JP (tyrp+) (broken li, nes) after growth in exclude the possibility that the failure to obminimal medium without aromi.atic amino acid sup- serve specific changes in the levels of the trans- medium supple- port systems in the tyrr strain was an indirect plement (MIN) and in minimtal mented with L-phenylalanine (FW). result of change in the intracellular concentra- Downloaded from on September, 18 by guest

5 VOL. 13, 1977 a- :D 1 FIG.. Uptake of tyrosine by the common and tyrosine-specific transport systems and uptake of tryptophan by the tryptophan-specific transport system in the tyrr strain, JP31. Abbreviations: Growth in minimal medium without aromatic amino acid supplement (MIN); in minimal medium supplemented with L-phenylalanine (PH), L-tryptophan (TRP), and L-tyrosine (TYR); and L-phenylalanine, L-tryptophan, L-tyrosine, p-aminobenzoic acid, p-hydroxybenzoic acid, and,3-dihydroxybenzoic acid (P). tion of tyrosine or another aromatic amino acid, resulting from a greatly increased rate of endogenous synthesis, the following experiment was carried out. The mutant allele arob351 specifies a nonfunctional protein for the second step in aromatic biosynthesis. Strains possessing this mutation are unable to synthesize the aromatic amino acids and vitamins and require tyrosine, phenylalanine, tryptophan, and the three aromatic vitamins for growth. The mutant allele arob351 was therefore introduced into strains JP311 and JP31. The resulting strains, JP and JP5, were grown in minimal medium supplemented with the aromatic amino acids and vitamins and harvested, and the cells were tested for their ability to transport the aromatic amino acids (Fig. 7). The results obtained were similar to those already found in the arob+ strains JP311 and JP31. In the tyrr+ strain, AMINO ACID TRANSPORT RGULATION 57 both the common transport system and the tyrosine-specific transport system were repressed to very low levels in the presence of the aromatic end products. This repression was completely missing in the corresponding tyrr strain, showing that the failure to observe repression in the tyrr strains is not due to any effects of endogenous synthesis. ffect of a tyrr35 mutation on regulation. The mutant allele tyrr35 is unusual in that strains possessing this mutation, although unable to repress the synthesis of enzymes in which both the tyrr product and tyrosine or tryptophan are involved, retain the ability to repress the synthesis of an enzyme (3-deoxy-Darabinoheptulosonic acid-7-phosphate [DAHP]- synthetase [phe]) in which the tyrr product appears to act in conjunction with phenylalanine as the effector molecule (13; B. K. ly, personal communication). It has been postulated that in such strains either the ability of the tyrr product to bind tyrosine or the conformational shifts resulting from tyrosine binding have been altered without any similar change occurring in the interaction between the tyrr product and phenylalanine. It is not true to say that there is no change at all, since in fact strains carrying the tyrr35 allele exert a cr - "I H- C a- D ocommon ospcific 1// COMMON -A_ -e *SPCIFIC l FIG. 7. Uptake of tyrosine by the common aromatic transport system (COMMON) and tyrosinespecific transport system (SPCIFIC) of JP5 (arob tyrr) (solid lines) and JP (arob tyrr) (broken lines) after growth in minimal medium supplemented with L-phenylalanine, L-tryptophan, L-tyrosine, p-aminobenzoic acid, p-hydroxybenzoic acid, and,3-dihydroxybenzoic acid. Downloaded from on September, 18 by guest

6 58 WHIPP AND PITTARD more pronounced phenylalanine-mediated repression on the gene in question (13). One might predict, therefore, that in a strain carrying the tyrr35 allele one should no longer see tyrosine-mediated repression of either the tyrosine-specific or the common transport system. One would expect, however, that the phenylalanine-mediated repression of the common transport system would still occur and that in the presence of both phenylalanine and tyrosine, levels of the tyrosine-specific system would be enhanced and not repressed. These predictions are, in fact, met (Fig. 8 and 9). This evidence further supports the involvement of the tyrr gene product in the control of the common and tyrosine-specific transport systems. ffect of growth conditions on total tryptophan uptake. Thorne and Corwin (3) reported that growth of. coli in the presence of tryptophan ( jig/ml) resulted in a large increase in the total uptake of tryptophan. This contrasts with the overall decrease in total C ch- ~ 8 8 t yr PR,SPCIFIC,COMMO)N - { S c a- 8 L tyLrR tyrr 35 J. BACTRIOL. COMMON -xcspcific x SPCIFIC COMMON 1 FIG. 9. Uptake of tyrosine by the common and tyrosine-specific transport systems in the tyrr' strain, AB353, and the tyrr35 strain, AB371. For both experiments, the cells were grown in minimal medium supplemented with the aromatic amino acids and vitamins (L-phenylalanine, L-tryptophan, L-tyrosine, p-aminobenzoic acid, p-hydroxybenzoic acid, and,3-dihydroxybenzoic acid). 'SPCIFIC tryptophan uptake we observed. In the study of Thorne and Corwin, 1- to 17-h cultures rather than exponentially growing cultures were used for uptake assays. Under these conditions, it seemed possible that exhaustion of glucose in the medium while tryptophan was LL still present would give rise to induction of the inducible tryptophan transport system (9). Cultures of 1 to 17 h grown in the presence of tryptophan with (i).% glucose or (ii).1% glucose (at this concentration, growth ceases in late exponential phase due to exhaustion of DCOMMON glucose) were assayed for tryptophan uptake and compared with cells grown to mid-exponential phase in glucose minimal media with and without tryptophan (Table ). These results FIG. 8. Uptake of tyrosine by the corn.mon and indicate that induction of the inducible tryptothe tyrr phan transport system was occurring in the tyrosine-specific transport systems in strain, AB353, and the tyrr35 strain AB371. cells grown to stationary phase. The level of For both experiments, the cells were grouw'n in mini- tryptophan uptake in the stationary-phase cells mal medium supplemented with L-phenylo'alanine. was not as high as in the Casamino Acids- Downloaded from on September, 18 by guest

7 VOL. 13, 1977 tryptophan-grown cells. This may have been because the concentration of tyrptophan was suboptimal for induction at the stage when glucose was exhausted. DISCUSSION The work reported here shows that both the so-called common aromatic transport system and the tyrosine-specific transport system are subject to regulation. By contrast, the phenylalanine-specific transport system appears to be expressed constitutively, and the tryptophanspecific system, although regulated, is subject to more marginal changes. The tyrr gene and hence its gene product (apo-tyrr) have been seen to be involved in the regulation of the common, tyrosine-specific, and tryptophan-specific transport systems. Tyrosine, phenylalanine, and tryptophan all appear to have roles as effectors in one or more of these systems. Previous work has shown that apotyrr is involved in regulating the expression of (i the arof tyra operon, which codes for DAHP synthetase (tyr) and chorismate mutase T (C )-prephenate dehydrogenase (C ), respectively (), (ii) tyrb (), the structural gene for the tyrosine-repressible L- tyrosine:-oxyglutarate aminotransferase (C..1.5) activity (), (iii) arog, the structural gene for DAHP synthetase (phe) (8, 1, 13), and (iv) arol, one of the structural genes for shikimate kinase (C ) activity (ly and Pittard, Proc. Aust. Biochem. Soc. S:5, 1975). In some cases the effector molecule is tyrosine alone, but in other cases either phenylalanine or tryptophan acts as an effector. The work of Ravel et al. (1) indicates that tyrosine rather than charged tyrosine transfer ribonu- TABL. ffect of growth conditions on total tryptophan uptake of strain JP311 Total tryptophan up- Supplements to take minimal media G (nmol/ mg [dry wt] per min).% Glucose + trypto- Stationary' 9.3 phana.1% Glucose + trypto- Stationary 8.8 phan Casamino Acids + tryp- Mid-exponential 1.8 tophan.% Glucose + trypto- Mid-exponential 1. phan.% Glucose Mid-exponential 3. a Tryptophan concentration = t±g/ml. ' Stationary = 1- to 17-h culture. AMINO ACID TRANSPORT RGULATION 59 cleic acid (trnaf'r) is the co-repressor. However, in the case of these genes no such evidence exists, indicating the involvement of phenylalanine and tryptophan rather than their trna derivatives. In the case of the arof tyra operon and the arol locus, growth in minimal medium produces enough endogenously synthesized tyrosine to cause significant repression of these systems. However, in the case of the specific and the common transport systems, cells grown in minimal medium are fully derepressed, having the same levels of activity as those found in genetically derepressed strains (i.e., tyrr strains). A similar situation has been observed in the case of the tyrosine-regulated aminotransferase enzyme, whose synthesis is fully derepressed in cells grown in minimal medium but can be repressed by exogenously supplied tyrosine. A similar case has been reported by Quay and Oxender (19) for the branched-chain amino acids, where cells grown in minimal medium are fully derepressed for the branchedchain amino acid transport systems but significantly repressed for the corresponding biosynthetic enzymes. The finding that each of the aromatic amino acids appears to play a part in the repression of the common transport system and that the effective involvement of each requires functional apo-tyrr suggests that this system could fit a simple model of tyrr-mediated repression, with each of the putative complexes, apo-tyrrtyr, apo-tyrr-phe, and apo-tyrr-trp, able to function as repressor. These complexes would be formed by combination of apo-tyrr with each of the relevant aromatic amino acids. The results obtained with the tyrosine-specific system and to some extent with the tryptophan-specific system are, however, more complex. In the case of the tyrosine-specific system, tyrosine and apo-tyrr are required for repression to occur. In the absence of either functional apo-tyrr or added exogenous tyrosine in the growth medium, the system is derepressed to a level that we have termed the fully derepressed level. When, however, the cells are grown in the presence of phenylalanine but not tyrosine, the level of the tyrosine-specific system is increased six- to eightfold. This increase only occurs in the presence of functional apo-tyrr and functional tyrp product. In other words, it does not involve the induction of yet another transport system, but rather it seems to reflect an apo-tyr-phe enhancement of the fully derepressed level of the tyrosine-specific system. In the presence of apo-tyrr-tyr, this enhancement is completely negated. Functional apo-tyrr-tyr Downloaded from on September, 18 by guest

8 WHIPP AND PITTARD and not tyrosine alone is required for this effect, as can be seen by the results obtained using the specific tyrr mutant (tyrr35), in which effective interaction of apo-tyrr and tyrosine but not apo-tyrr and phenylalanine is destroyed. The simplest model to explain these results would have apo-tyrr-tyr functioning in a simple negative control system to block initiation of transcription at a putative operator locus of tyrp (assuming a single component for the tyrosine-specific system). In the absence of tyrosine or apo-tyrr, transcription of tyrp is not impeded by this interaction with operator. The presence of phenylalanine in the growth medium further augments the expression of tyrp in tyrr+ but not in tyrr cells. To explain this, we postulate (i) that apotyrr-phe acts at a promoter region to increase the frequency of transcription initiations for tyrp or (ii) that the gene tyrp possesses an attenuator locus analogous to the attenuator of the tryptophan operon () and that apo-tyrrphe specifically prevents premature termination of transcription at this attenuator locus. In either case, it would be easy to explain why the tyrosine effect was dominant over the phenylalanine effect. Further investigation of these models will involve the isolation of specific mutants in operator, promoter, or attenuator loci and the development of in vitro systems to study the expression of these genes. The regulation of the tryptophan-specific transport system cannot be entirely explained in terms of the above model. Since tryptophan does not mediate repression of this system, it is necessary in this case to postulate that tryptophan or apo-tyrr-trp acts specifically to prevent the action of apo-tyrr-phe. This, of course, may also be the way in which apo-tyrr-tyr negates the action of apo-tyrr-phe on the tyrosine-specific system. At the moment there are insufficient data on which to build useful hypotheses about the molecular interaction that would result in this postulated blocking of the apo-tyrr-phe effect. Parallel cases of one regulatory product mediating control of both the amino acid transport and biosynthetic enzyme systems have not yet been found. However, although the regulation for the transport and biosynthetic systems of the branched-chain amino acids have been found to be independent (19, ), interaction of leucine with trna1-u and leucyl-trna synthetase is required for repression of both the leucine biosynthetic enzyme systems and the branched-chain amino acid transport systems (18). It is also interesting to note that the cysb locus seems to be involved in regulation of both the sulfate transport system and the cysteine biosynthetic pathway (1). The total tyrosine uptake (common plus specific) of strain JP311 remains the same for cells grown in minimal medium or minimal medium supplemented with phenylalanine. This is because in the phenylalanine-grown cells the decreased uptake of tyrosine by the common system is compensated for by the increased uptake of tyrosine by the tyrosine-specific system. The increase in uptake by the tryptophan-specific system in cells grown with phenylalanine is relatively small compared with the decrease in tryptophan transport by the common system. Such cells show a decrease in total tryptophan uptake when compared with cells grown in minimal medium. In other instances where an amino acid is transported by two different systems, situations have also been described in which one transport system is decreased in activity while another is increased (3, ). ACKNOWLDGMNTS This work was supported by a grant from the Australian Research Grants Committee. M.J.W. holds a Commonwealth postgraduate research award. Thanks are due to J. Davis and L. Vizard for skilled technical assistance. LITRATUR CITD 1. Bachmann, B. J Pedigrees of some mutant strains of scherichia coli K-1. Bacteriol. Rev. 3: Bachmann, B. J., K. B. Low, and A. L. Taylor Recalibrated linkage map of scherichia coli K-1. Bacteriol. Rev. : Berger,. A., and L. A. Heppel A binding protein involved in the transport of cystine and diaminopimelic acid in scherichia coli. J. Biol. Chem. 7: Bertrand, K., L. Korn, F. Lee, T. Platt, C. L. Squires, C. Squires, and C. Yanofsky New features of the regulation of the tryptophan operon. Science 189:-. 5. Boezi, J. A., and R. D. De Moss Properties of a tryptophan transport system in scherichia coli. Biochim. Biophys. Acta 9: Brown, K. D Formation of aromatic amino acid pools in scherichia coli K-1. J. Bacteriol. 1: Brown, K. D Maintenance and exchange of the aromatic amino acid pool in scherichia coli K-1. J. Bacteriol. 1: Brown, K. D., and R. L. Somerville Repression of aromatic amino acid biosynthesis in scherichia coli K-1. J. Bacteriol. 18: Burrous, S.., and R. D. De Moss Studies on tryptophan permease in scherichia coli. Biochim. Biophys. Acta 73: Camakaris, H., and J. Pittard Regulation of tyrosine and phenylalanine biosynthesis in scherichia coli K-1: properties of the tyrr gene product. J. Bacteriol. 115: Harrison, L. I., H. N. Christensen, M.. Handlogten, D. L. Oxender, and S. C. Quay Transport of L- J BACTRIOL Downloaded from on September, 18 by guest

9 VOL. 13, 1977 AMINO ACID TRANSPORT RGULATION 1 -azaleucine in scherichia coli. J. Bacteriol. 1: Hiraga, S., K. Ito, T. Matsuyama, H. Ozaki, and T. Yura Methyltryptophan-resistant mutations linked with the arginine G marker in scherichia coli. J.Bacteriol. 9: Im, S. W. K., H. Davidson, and J. Pittard Phenylalanine and tyrosine biosynthesis in scherichia coli K-1: mutants derepressed for 3-deoxy-Darabinoheptulosonic acid-7-phosphate synthetase (phe), 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A. J. Bacteriol. 18: Kuhn, J., and R. L. Somerville Uptake and utilization of aromatic D-amino acids in scherichio coli K-1. Biochim. Biophys. Acta 33: Monod, J., G. Cohen-Bazire, and M. Cohen Sur la biosynthese de la,3-galactosidase (lactase) chez scherichia coli. La specificite de l'induction. Biochim. Biophys. Acta 7: Ohta, N., P. R. Galsworthy, and A. B. Pardee Genetics of sulfate transport by Salmonella typhimurium. J. Bacteriol. 15: Oxender, D. L Genetic approaches to the study of transport systems, p In H. N. Christensen (ed.), Biological transport, nd ed. W. A. Benjamin, New York. 18. Quay, S. C.,. L. Kline, and D. L. Oxender Role of leucyl-trna synthetase in regulation of branched-chain amino acid transport. Proc. Natl. Acad. Sci. U.S.A. 7: Quay, S. C., and D. L. Oxender Regulation of branched-chain amino acid transport in scherichia coli. J. Bacteriol. 17: Quay, S. C., D. L. Oxender, S. Tsuyumu, and H.. Umbarger Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria. J. Bacteriol. 1: Ravel, J. M., and M. N. White, and W. Shive Activation of tyrosine analogues in relation to enzyme repression. Biochem. Biophys. Res. Commun. : Robbins, J. C., and D. L. Oxender Transport systems for alanine, serine, and glycine in scherichia coli K-1. J. Bacteriol. 11: Thorne, G. M., and L. M. Corwin Mutations affecting aromatic amino acid transport in scherichia coli and Salmonella typhimurium. J. Gen. Microbiol. 9:3-1.. Wallace, B. J., and J. Pittard Regulator gene controlling enzymes concerned in tyrosine biosynthesis in scherichia coli. J. Bacteriol. 97: Downloaded from on September, 18 by guest