A1.MIcRoBiAL AGENTs AND CHEZMOCrRAPY, Sept. 1977, p. 322-327 Copyright C 1977 American Society for Microbiology

Vol. 12, No. 3 U.S.A.

Printed in

Detection of Antimetabolite Activity: Effects and Transport of Tryptophan Analogs in Escherichia coli JONATHAN KUHNt

Department of Biology, Ben Gurion University of the Negev, Beersheba, Israel

Received for publication 23 June 1977

Simple and rapid techniques were developed that allow detection of the effects of a wide range of tryptophan analogs in Escherichia coli. (i) By using certain supersensitive mutant strains, analogs that were without effect on a wild-type strain were shown to be inhibitory. (ii) Many other analogs could inhibit the utilization of o-tryptophan when present as L-tryptophan replacements in a trp dadR strain. (iii) Another approach was to test the ability of a given analog to reverse the inhibition caused by an inhibitory analog. These combined approaches revealed activities in 26 analogs out of a total of 40 that were inactive by testing solely on a wild-type strain. The route of entry of inhibitory analogs was determined unambiguously by comparing their effect on aroP+ (aromatic permease) and aroP strains. Uptake studies were also performed to determine whether various analogs compete for entry via the aromatic permease system. Many tryptophan analogs enter the cell via this system. The methods developed here should have general applicability to the testing of analogs of a variety of other metabolites. Metabolic analogs have been widely used in biology for biochemical and genetic purposes (14). In the tryptophan system of Escherichia coli, for example, a number of mutant types with altered sensitivities to analogs have been isolated. Among them are those with mutations leading to the loss of functional repressor (4, 11), to operators no longer recognized by the repressor (7), to feedback-insensitive anthranilate synthetase (13), and to an inability of the subunits of the anthranilate synthetase-phosphoribosyl transferase complex to aggregate (9). However, the choice of analogs used in this system and many others has been haphazard in the sense that their sites of action were not known. The present investigation was undertaken to develop general methods for detecting which analogs affect a specific pathway and on which step(s) they exert action. The initial studies were directed towards improving the detection of analog activity. To this end, mutant strains selected on the basis of increased sensitivity to analogs were used as tester strains. A second approach involved testing various analogs for their ability to reverse the inhibition caused by an inhibitory analog. In addition, analogs were examined for their ability to inhibit a secondary pathway, namely, the -tryptophan utiliza-

tion pathway present in dadR strains, in which D-tryptophan is converted to L-tryptophan. These three techniques allowed the activity of many seemingly inactive analogs to be demon-

strated.

Many aromatic amino acid analogs are thought to enter the cell via the aromatic permease system (1). Comparisons between aroP+ and aroP (lacking aromatic permease activity) strains allowed us to decide which analogs are transported by this system. Classical uptake studies have also been performed on many of these analogs.

MATERIALS AND METHODS Bacterial strains. The relevant genotypes and origins of the strains are presented in Table 1. MTS6B has been shown to contain a mutation in the trpE gene, the product of which, anthranilate synthetase, has become much more sensitive to feedback inhibition by L-tryptophan. MTS21 contains a trpD mutation, which blocks aggregation of the two different subunits of anthranilate synthetase. The net result of these mutations is to lower the cell's ability to synthesize tryptophan, thereby rendering them more sensitive to inhibition. Strain JK222 contains mutations that lead to the loss of functional tryptophanase (tna) and that block the synthesis of L-tryptophan from chorismic acid at two steps (trpE and trpA). A fourth mutation (dadR [8) allows Dtryptophan to be converted to L-tryptophan via deamination and transamination. Hence, JK222 can t Present address: Department of Biology, Technion-Is- grow with D-tryptophan as a source of L-tryptophan. rael Institute of Technology, Haifa, Israel. Strain JK271 was constructed by transducing strain 322

TRYPTOPHAN ANALOGS

VOL. 12, 1977 TABLE 1. Bacterial strainsa Origin Genotype Strain C. Yanofsky (2) Wild type W3110 R. Somerville Wild type, F+ W1485 K. Brown (3) KB3100 aroP (9) MTS6B trpE (MTS) (9) MTS21 trpD (MTS) This paper aroP trpE (MTS) JK271 (8) trpE trpA dadR tna JK222 a List of those strains used in this work. Abbreviations: aroP, aromatic permease minus; trp, L-tryptophan requirement; MTS, phenotypic supersensitivity to 5-methyl-DL-tryptophan inhibition; dadR, ability to use D-tryptophan; tna, tryptophanase negative.

KB3100 with bacteriophage Plkc propagated on a strain deleted for the tonB-trp region (the entire trp operon is deleted). After allowing time for phenotypic expression, resistance to phage Ti was selected. Such a resistant strain was, in turn, transduced, using MTS6B as a donor, with selection for Trp+. The resulting strain, JK271, contains the aroP mutation of strain KB3100 and the trp operon of strain MTS6B. Medium and chemicals. The minimal medium E of Vogel and Bonner (15) supplemented with 0.2% glucose and thiamine (1 ,ug/ml) was used in all experiments. Composition of top layer agar and preparation of plates have been described previously (9). L-Tryptophan, D-tryptophan, or glycyl-L-tryptophan at 20 ,g/ml was added as indicated. All were purchased from Sigma Chemical Co. (For sources of other compounds, see Table 2.) Disk tests. Before plating, the strains were grown to stationary phase with shaking at 37°C in minimal medium, except for JK222, which was supplemented with r-tryptophan (20 ug/ml). Disk tests were performed as previously described (9), except the cells were diluted only 10-fold rather than 100-fold. Less dilution renders heavier and more uniform bacterial lawns. A 0.025-ml portion of an analog was applied to the disk. The plates were scored after 1 day of incubation at 37°C. Transduction with bacteriophage PLkc. The techniques used for transduction with phage Plkc were those of Yanofsky and Lennox (17).

RESULTS Analogs inhibitory to wild-type and supersensitive mutants. The initial phase of any study of a group of related analogs is to determine which analogs inhibit growth. A rapid and easy method for such determinations is the disk test. A total of 44 aromatic acid analogs were screened by the disk test for their ability to inhibit the wild-type strain, W3110, and its 5methyltryptophan-supersensitive (MTS) derivatives, strains MTS6B and MTS21. The results of these tests are presented in

323

Table 2 (columns A, C, and E). Those analogs that inhibited the wild-type strain (column A, types I and II) had been shown to do so previously. The value of using supersensitive derivatives as testers was immediately apparent (columns C and E); the inhibitory effects of a number of compounds (types III through V) that did not affect the wild type were revealed. Thus, the detection of inhibitory analogs can be improved through the use of special tester strains. That 6- and 7-methyltryptophan could also cause inhibition indicates that, whereas methyltryptophans are inhibitory, there is a definite effect of the methyl group's position. Indoleacrylic acid has been shown to cause derepression of the trp operon (10), whereas Dtiyptophan has been reported to reduce the growth rate of wild-type E. coli (6). The effect of indolepropionic acid was somewhat unexpected because this compound was found (16) to be a degradation product of L-tryptophan when E. coli are grown anaerobically. The response of strain MTS21 to type V compounds was unexpected because these three compounds are not tryptophan analogs. The inhibition produced by these compounds was weak. L-Phenylalanine, for example, caused a large zone of inhibition, but the zone was completely filled with more slowly growing cells. The underlying mechanism of inhibition is unclear for these compounds. They may inhibit tryptophan synthesis indirectly by affecting the aromatic pathway and lowering the amount of chorismic acid available for this synthesis. For the sake of brevity, only the size of the zone (in centimeters) in which noticeable inhibition of growth occurred is given in Table 2. A description of the type of zone has been omitted. The zones ranged from ones in which there was no visible growth (e.g., 5-fluorotryptophan) to those with gradients of more slowly growing cells. In all cases the point of measurement was that where inhibition became apparent, regardless of the type of inhibition. These points were unambiguous and reproducible. Unfortunately, the size of a zone only indicates the relative strength of inhibition (assuming that all analogs diffuse equally rapidly) rather than the lowest concentration producing it. One way to determine whether these inhibitory analogs act exclusively on a specific pathway is to examine whether the addition of the pathway's end product can overcome the inhibition. The ability of L-tryptophan and glycyl-Ltryptophan (at 20 p,g/ml in the plates) to reverse the inhibition caused by analog types I through V was checked by using the concentrations of analogs listed in Table 2. Tests with glycyl-L-tryptophan were also included because

324

ANTIMICROB. AGENTS CHZMOTHER.

KUHN

TABLz 2. Disk tests on aromatic amino acids and their analogsa Compound

DL-5-Fluorotryptophan

C S S

(C) (D) (E) (A) (B) JK271 MTS21 W31103 KB3100 MTS6B aroP aroP+ araP+ aroP arPMS MTS MTS 5.6 5.8 3.5 5.0 125 4.6 4.2 4.2 3.0 5.1 125 4.2 2.5 3.5 5.3 125

DL-6Fluorotryptophan

C S C N 5 C

125 50 50 50 125 125

4.4 3.9 3.6 3.7 4.9 2.0

S S S S S

50 25 50 125 50

NE NE NE NE NE

S S S S

250 125 125 125

NE NE NE NE

-

D-sTryptophan methyl ester Indole-3-acetic acid Indole-3-aldehyde L-Phenylalanine Glycyl-L-phenylalanine 3-Nitrotyrosine

S S S

50 125 50

D-Phenylalanine DL4-Ammnophenylalamne DL-4-Bromophenylalanine L-Tyrosine

S S 5 S S S S 5 S S

C B S S S S s S S S A S S S S S S S S S

Type Name

I

DL-3Fluorophenylalanine DL-2-Fluorophenylalanine R

DL4-Methyltryptophan DL-5-Methyltryptophan DL-7-Azatryptophan

2-(Thienyl)-p-alanine

DL4.Fluorophenylalanine EII

DLA-Methyltryptophan DL-7-Methyltryptophan

L-5-Hydroxytryptophan

3p-Indoleacrylic acid

Indole-3-propionic acid

IV

V

VI

s-Tryptophan

DL-m-Tyrosine

DL-0-Tyrosine L4-Aminotyrosine Glycyl-L-tyrosine p-Aminobenzoic acid Shikimic acid Inactive with all strains

Zone of inhibition (cm) of strain:

Indole Anthranilic acid

Indole-3-butyric acid L-Tryptophan methyl ester Tryptophol D-Histidine

3-Methylindole DL-Indole-3-lactic acid

Tryptamine

Glycyl-L-tryptophan Glycyl-D-tryptophan Glycyl-D-phenylalanine D-Tyrosine

Amtc Source,,At (aUg)

(F)

JK222Ote trp dadR 3.6 4.6 4.8

Inh Inh

NE NE NE NE TR NE

4.9 5.4 6.7 5.0 4.4 NE

4.5 5.0 6.0 3.3 NE NE

5.2 6.2 6.7 5.7 5.2 4.2

3.9 3.6 3.6 3.0 4.3 5.1

-

5.5 5.8 4.7 3.2 2.7

4.6 4.1 3.2 2.7 1.9

5.9 5.2 5.2 1.9 2.5

2.8 3.6 2.7 NE NE

NE NE NE NE

4.9 4.1 2.3 NE

NE 2.0 NE

-

-

3.5 2.5 1.8 1.6

NE NE NE

_ _

NE NE NE

-

4.7 5.1 1.6

4.2 4.9

Rev Rev

1.7

-

125 125 50 50 50 50 50 125 50 50

NE NE NE NE NE NE NE NE NE NE

_ _ _ _ _ -

NE NE NE NE NE NE NE NE NE NE

_ _ _ -

NE NE NE NE NE NE NE NE NE NE

3.2 4.9 4.4 4.2 3.3 4.0 3.6 4.7 2.6 2.9

50 50 125 125 125 250 50 50 125 125 125 125 50 250 50 125 125 125 125 125

-

-

-

-

Inh Inh -

-

-

Rev Rev Rev

Rev Rev Rev Rev Rev Rev Rev Rev Rev Rev

-

-

L-Histidine p-Hydroxybenzoic acid 3-Indole acetone 3-Indole acetamide 3-Indole glyoxylic acid N-Acetyl-L-tryptophan N-Acetyl-D-tryptophan a NE, No effect; TR, trace, very weak effect; Inh, Inhibitory in the presence of L-tryptophan, glycyl-iAtryptophan, and D-tryptophan; Rev, reversal Dtryptophan inhibition of strain MTS6B; -, Not tested. (Numbers represent the zone diameter of growth inhibition in centimeters.) b A, Cyclo Chemical Co.; B, BDH Pharmaceuticals Ltd.; C, Calbiochem; N, Nutritional Biochemicals Corp.; S, Sigma Co. Chemical c Amount placed on the disk.

VOL. 12, 1977

this compound is transported by different permeases than those of the inhibitory compounds (12). In contrast, L-tryptophan might reverse inhibition by competing for transport and, hence, destroy the specificity of the test. Except for the four analogs noted in Table 2, the effects of all inhibitory analogs were abolished by either of these additions to the plates. Three tester strains were used: W3110, MTS6B, and MTS21. The compounds 2-, 3-, and 4-DL-fluorophenylalanine and 2-(thienyl) (8-fluorophenylalanine, which could still inhibit in the presence of either L-tryptophan or glycyl-L-tryptophan, are phenylalanine analogs. Testing noninhibitory compounds by reversal of inhibition. Even when supersensitive strains were used as testers, the majority of the compounds in Table 2 were noninhibitory. Another approach to ascribe effects to these compounds is to examine them for their ability to reverse the inhibition caused by an inhibitory compound. The test system chosen was n-tryptophan's inhibition of strain MTS6B because Dtryptophan is known to be transported exclusively by aromatic permease. Since all the inhibitory compounds considered here almost certainly must enter the cell to exert their action, there is a distinct advantage in using a compound with a single route of entry. Reversal of inhibition can then come about through competition for this permease, and subsequent analysis will be simplified. In addition, radioactively labeled D-tryptophan for uptake studies is commercially available, whereas the other inhibitory compounds are not. The test consisted of pour-plating strain MTS6B on plates containing u-tryptophan (50 pg/ml) and applying the compound to be examined on a paper disk. This concentration of itryptophan is enough to prevent growth; reversal of inhibition is indicated by a zone of growth around the disk. As indicated in Table 2, 15 compounds were able to reverse the effect of I>tryptophan. Indole and anthranilic acid are tryptophan pathway intermediates subsequent to the mutational block in trpE and would be expected to have such an effect. The phenylalanine and tyrosine analogs are not likely to compete with D-tryptophan for the internal element(s) inhibited and probably act by reducing )-tryptophan entry. Tryptophan analogs might act at either level. Inhibitory analogs were also examined by this test. A variety of responses was observed. These included solid zones of growth, halos of growth at some distance from the disk, and even double halos. Halos possibly come about when the two inhibitors compete for a common

TRYPTOPHAN ANALOGS

325

element, such as permease, and lower the internal concentrations of each other. Route of entry of inhibitory analogs. Since inhibitory tryptophan analogs almost certainly must enter the cell to cause inhibition, the most important considerations in studying an analog's mode of action are the analog's permeation and the detennination of the internal element affected. Whereas the latter is difficult to ascertain in vivo, the route of entry may be determined by uptake experiments or by using mutants. Most of these analogs are, however, unavailable as radioactively labeled compounds. A different and more precise approach is to compare the sensitivity of a pair of strains that differ at a genetic locus specifying a permease or an element involved in permeation. The availability of a mutation, aroP, that abolishes the activity of the general aromatic amino acid permease (3) allows a comparison to be made between the sensitivities of aroP+ and aroP strains to the inhibitory analogs. The comparison between such a pair, which are in all other respects wild types, is given in Table 2, columns A and B. The lack of an active aromatic permease (KB3100, column B) abolished the effects of type II compounds and reduced those of type I compounds. These results show that all these analogs are, in part, transported via the aromatic permease. To decide whether type I compounds are exclusively transported by the aromatic permease, one must know if the mutational block caused by the aroP allele used is absolute. On the basis of uptake and growth studies, this mutation seems to totally abolish aromatic permease activity (8). Thus, it appears that hype I compounds are also transported by an additional system(s). The bulk, and perhaps all, of the transport of type II compounds must be via the aromatic permease. Since the parent strain of KB3100 is W1485, it should be mentioned that W1485 was very similar to W3110 in these tests (data not shown) and that these two strains are related. A similar analysis of type III and type IV compounds was performed by using strains MTS6B (aroP+ trpE, MTS) and JK271 (aroP trpE, MTS). The results of these tests are presented in Table 2, columns C and D. From the results of Table 2 it can be concluded that all type II through IV analogs enter through the aromatic permease, as judged by reduction in zone size, but that only type IV compounds require this permease to produce inhibition. Even the presence of an aroP mutation is not enough to make strain JK271 insensitive to some type II compounds. Therefore, either the

326

KUHN

ANTIMICROB. AGENTS CHEMOTHER.

aroP mutational defect is not complete or there lization by a trp dadR strain. The results are exist other, less efficient transport systems for presented in Table 3 and show that 10 of 19 these analogs that only become apparent when analogs examined inhibited D-tryptophan upan MTS mutation is present. take. As expected, the two dipeptides, glycyl-LEffect of analogs on D-tryptophan uptake. phenylalanine and glycyl-L-tyrosine, did not inSince very few of the analogs are available hibit. Except for D-tryptophan methyl ester, commercially as radioactively labeled com- those compounds that did not compete for uppounds, their uptake cannot be directly stud- take either do not resemble tryptophan (e.g., ied. However, an indirect analysis of their ef- anthranilic acid) or lack a complete side chain fects on aromatic permease can be made by (e.g., indole aldehyde). examining their effect on D-tryptophan uptake Inhibition of D-tryptophan utilization. The because, as mentioned earlier, D-tryptophan is availability of strains (dadR) that have gained exclusively transported by this permease. The the ability to utilize D-tryptophan as a replaceability to inhibit D-tryptophan transport indi- ment for L-tryptophan allows the effects of cates, but does not prove, that a given analog is many of the compounds already discussed to be transported by this system. A negative result more specifically pinpointed. The steps of the signifies that either the analog is not trans- pathway of D-tryptophan utilization (5) become ported by this permeation system or it has a obligatory for a trp dadR strain growing on Dmuch greater Michaelis-Menten constant (Ki) tryptophan as an L-tryptophan source. Analogs than that for D-tryptophan. that inhibit this growth should specifically exUptake studies were performed as previously ert their effects on this pathway. described (8). Analogs were present at concenThe results of disk tests using strain JK222 trations of 10- or 100-fold that of D- (trp dadR) on t)-tryptophan (20 ug/ml)-contain[14C]tryptophan (5 ,uM). Rather than examin- ing plates are presented in Table 2, column F. ing all analogs, a selection was made based Among the many test compounds that inhibited mainly on two of the following three criteria: (i) growth were some (type VI) whose effects were the presence of an aroP mutation totally abol- undetectable by other tests. ishes inhibition; (ii) the analog's ability to reverse D-tryptophan inhibition of strain DISCUSSION MTS6B; and (iii) inhibition of n-tryptophan utiSeveral approaches for detecting the in vivo TABLE 3. Involvement of aromatic permease in the effects of analogs have been presented here. Whereas only aromatic amino acids were transport of analogsa screened here, the techniques developed should Com- aroP Repetes re- verses Blocks be applicable to many microbial systems. The D-Trp first of these, the use of strains with increased forupduces D Analog take inhi- inhibi- utilianalog sensitivity, detected the inhibitory prowith bi- tion of zation perties of 12 analogs that did not affect the wildD-Trp tionbN MTSc type strain (types III through V of Table 2). + DL-6-Fluorotryptophan + + Thus, such mutant strains can be a powerful DL-4-Methyltryptophan + + + tool in screening new analogs. The inhibition of + DL-5-Methyltryptophan + + growth produced by these weak analogs is prob+ DL-7-Azatryptophan + + 2-(Thienyl)-,-alanine + + + ably the result of the strain's limited ability to DL-4-Fluorotryptophan + + + synthesize tryptophan. A wild-type organism DL-Tryptophan methyl ester + might overcome the inhibitory effects of an anaIndole-3-acetic acid + + + Indole-3-aldehyde log by, for example, derepression of the operon L-Phenylalanine + + + and increased synthesis of the pathway enGlycyl-L-phenylalanine + + zymes. The mutant strains cannot use this op+ DL--Aminophenylalanine + + tion because they are already maximally dereL-Tyrosine + + + Glycyl-L-tyrosine pressed in their struggle to synthesize typtoShikimic acid + + phan (9). It is, therefore, likely that mutant Indole + strains selected on the basis of a leaky trp pheAnthranilic acid + notype or weak partial revertants of trp strains + L-Tryptophan methyl ester + D-Histidine + could also serve as indicator strains for weakly inhibitory analogs. A different approach was to DiTrp D-Tryptophan. b From comparison of aroP+ and aroP strains in Table 2. test which compounds could reverse the effect of See Table 2 and text. an inhibitory analog. Among the analogs found a

c

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TRYPTOPHAN ANALOGS

to reverse the effect of D-tryptophan on MTS6B, all except -tryptophan methyl ester, indolebutyric acid, and tryptophol were not tryptophan analogs. These analogs almost certainly exert their effect by competing at the transport step, thereby lowering the internal D-tryptophan concentration. The tryptophan analogs might well act in the same way. Another approach was to use as a tester a mutant strain that contains dadR and can thus grow on i-tryptophan. D-Tryptophan utilization bypasses all steps involved in -tryptophan synthesis. Inhibiton of -tryptophan utilization by an analog must result from interaction with an element in the -tryptophan utilization pathway, with its reduced number of elements. This approach was successful in detecting 26 compounds capable of inhibiting the utilization of D-tryptophan by a dadR strain. Those compounds that are not tryptophan analogs act, in all probability, at the steps of transport and/or deamination, in a manner similar to L-phenylalanine and i-tyrosine (5). Some of the tryptophan analogs have been found to be substrates of -tryptophan oxidase, and this could result in growth inhibition. A likelier possibility is that the tryptophan analogs compete for transport. Thus, these combined approaches were able to uncover the activities fo 26 out of 40 analogs that are inactive on the wild type. However, the element primarily affected by a specific analog remains unknown. The route of entry of those analogs inhibiting wild-type or supersensitive strains was determined by the introduction of an aroP mutation and by uptake studies. A similar analysis of analogs inhibiting D-tryptophan utilization could not be done because of the absolute dependence of -tryptophan transport on an active aromatic permease. It is not possible from uptake studies to determine whether the analog itself is actually transported or if it only blocks the entry of the radioactively labeled metabolite. In contrast, the analysis of inhibitory analogs with permease mutants can often give unambiguous results.

ACKNOWLEDGMENT

327

This project was supported by a grant from the Israel Academy of Sciences. LITERATURE CITED 1. Ames, G. F. 1964. Uptake of amino acids by Salmonella typhimurium. Arch. Biochem. Biophys. 104:1-18. 2. Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 36:525-557. 3. Brown, K. D. 1970. Formation of aromatic amino acid pools in Escherichia coli K-12. J. Bacteriol. 104:177188. 4. Cohen, G. N., and F. Jacob. 1959. Sur la repression de la sythese des enzymes intervenant dans la formation du tryptophane chez Escherichia coli. C. R. Acad. Sci. 248:3490-3492. 5. Hadar, R., A. Slonim, and J. Kuhn. 1976. Role of Dtryptophan oxidase in D-tryptophan utilization by Escherichia coli. J. Bacteriol. 125:1096-1104. 6. Hatanaka, M. 1964. Action of D-tryptophan on E. coli. Nature (London) 204:202-203. 7. Hiraga, S. 1969. Operator mutants of the tryptophan operon inEscherichia coli. J. Mol. Biol. 39:159-179. 8. Kuhn, J., and R. L. Somerville. 1974. Uptake and utilization of aromatic D-amino acids in Escherichia coli K12. Biochim. Biophys. Acta 332:298-312. 9. Kuhn, J. C., M. J. Pabst, and R. L. Somerville. 1972. Mutant strains of Escherichia coli K-12 exhibiting enhanced sensitivity of 5-methyltryptophan. J. Bacteriol. 112:93-101. 10. Morse, D. E., R. D. Mosteller, and C. Yanofsky. 1969. Dynamics of synthesis, translation and degradation of the trp operon messenger RNA in E. coli. Cold Spring Harbor Symp. Quant. Biol. 34:725-740. 11. Morse, D. E., and C. Yanofsky. 1969. Amber mutants of the trpR regulatory gene. J. Mol. Biol. 44:185-193. 12. Payne, J. W., and C. Gilvarg. 1971. Peptide transport. Adv. Enzymol. 35:187-244. 13. Somerville, R. L., and C. Yanofsky. 1965. Studies on the regulation of tryptophan biosynthesis in Escherichia coli. J. Mol. Biol. 11:747-759. 14. Umbarger, H. E. 1971. Metabolic analogs as genetic and biochemical probes. Adv. Genet. 16:119-140. 15. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218:97-106. 16. Woods, D. D. 1935. Indole formation byBacterium coli. I. The breakdown of tryptophan by washed suspensions of Bacterium coli. Biochem. J. 29:640-648. 17. Yanofsky, C., and E. S. Lennox. 1959. Transduction and recombination study of linkage relationships among the genes controlling tryptophan synthesis in Escherichia coli. Virology 8:425-447.

Detection of antimetabolite activity: effects and transport of tryptophan analogs in Escherichia coli.

A1.MIcRoBiAL AGENTs AND CHEZMOCrRAPY, Sept. 1977, p. 322-327 Copyright C 1977 American Society for Microbiology Vol. 12, No. 3 U.S.A. Printed in De...
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