Marquette University e-Publications@Marquette Biomedical Sciences Faculty Research and Biomedical Sciences, Department of Publications 10-1-1978 Effect of Tryptophan Analogs on Derepression of the Escherichia coli Tryptophan Operon by Indole-3-Propionic Acid Robert J. Pauley Marquette University Walter W. Fredricks Marquette University Oliver H. Smith Marquette University Published version. Journal of Bacteriology, Vol. 136, No. 1 (October 1978): 219-226. Permalink. © 1978 American Society for Microbiology. Used with permission. JouRNAL oF BACTFROLOGY, Oct. 1978, p. 219-226 Vol. 136, No. 1 0021-9193/78/0136-0219$02.00/0 Copyright i 1978 American Society for Microbiology Printed in U.S.A. Effect of Tryptophan Analogs on Derepression of the Escherichia coli Tryptophan Operon by Indole-3-Propionic Acid ROBERT J. PAULEY,t WALTER W. FREDRICKS, AND OLIVER H. SMITH* Department ofBiology, Marquette University, Milwaukee, Wisconsin 53233 Received for publication 17 April 1978 The abilities of 14 tryptophan analogs to repress the tryptophan (tip) operon have been studied in Escherichia coli cells derepressed by incubation with 0.25 mM indole-3-propionic acid (IPA). tip operon expression was monitored by measuring the specific activities of anthranilate synthase (EC 4.1.3.27) and the tryptophan synthase (EC 4.2.1.20) , subunit. Analogs characterized by modifi- cation or removal of the a-amino group or the a-carboxyl group did not repress the tip operon. The only analogs among this group that appeared to interact with the tip aporepressor were IPA, which derepressed the tip operon, and D-trypto- phan. Analogs with modifications of the indole ring repressed the tip operon to various degrees. 7-Methyl-tryptophan inhibited anthranilate synthase activity and consequently derepressed the tip operon. Additionally, 7-methyltryptophan prevented IPA-mediated derepression but, unlike tryptophan, did so in a non- coordinate manner, with the later enzymes of the operon being relatively more repressed than the early enzymes. The effect of 7-methyltryptophan on IPA- mediated derepression was likely not due to the interaction of IPA with the allosteric site of anthranilate synthase, even though feedback-resistant mutants of anthranilate synthase were partially resistant to derepression by IPA. The effect of 7-methyltryptophan on derepression by IPA was probably due to the effect of the analog-aporepressor complex on trp operon expression. The efficiency ofregulation oftryptophan bio- 10, 14, 15, 18). These studies precluded the in- synthesis in Escherichia coli resides in the abil- volvement of tryptophanyl-tRNA in repression ity of tryptophan to interact at three different control, although similar studies have indicated controlling sites. Negative control oftryptophan that tryptophanyl-tRNA may be involved in the (trp) operon mRNA synthesis is mediated by control of tip mRNA elongation through the the interaction of tryptophan and the tipR gene attenuator site (12). Experiments with 7-methyl- product, the aporepressor (10, 15, 20). Trypto- tryptophan (7-MT) have demonstrated that this phan or a metabolic derivative of tryptophan analog affects tip operon expression as a result modulates the fiequency ofelongation ofmRNA of its interaction with the first enzyme in the throigh the attenuator region (3, 12). In addi- tryptophan biosynthetic pathway (8). tion, tryptophan functions as an allosteric inhib- The aim ofthe studies described in this report itor of anthranilate synthase, the first enzyme in was to evaluate tryptophan analogs for their the tryptophan biosynthetic pathway (2). ability to repress the in vivo synthesis of the Structural analogs of L-tryptophan have had tipE and tipB gene products, anthranilate syn- an important role in the development of our thase and the ,B subunit of tryptophan synthase, current knowledge of the mechanisms by which respectively. Therefore these experiments re- tryptophan regulates its own biosynthesis. Stud- quired a system in which repression of tip op- ies with tryptophan analogs have demonstrated eron expression by tryptophan analogs could be that repression control of tip operon mRNA reliably quantitated. synthesis, both in vivo and in vitro, requires the Repression of tip operon expression in E. coli formation of a functional repressor by the inter- cells growing in minimal medium is slight, be- action of the aporepressor and L-tryptophan or cause the tip operon is partially repressed under certain analogs, such as 6-methyltryptophan (5, these conditions. However, treatment of cells with indole-3-propionic acid (IPA) causes rapid t Present Address: Department of Cell Biology, Baylor and coordinate derepression of the trp operon College of Medicine, Houston, TX 77030. that is prevented by L-tryptophan (1, 11, 13). In 219 220 PAULEY, FREDRICKS, AND SMITH J. BACTERIOL. vitro studies have shown that derepression of rupted by intermittent sonic oscillation for 2 min. Cell trp operon expression by IPA was due to a direct debris was removed by centrifugation at 20,000 x g for effect ofIPA on the trp aporepressor (17). There- 45 min at 4°C. The cell extracts were stored at -15°C fore, IPA apparently decreases the level of func- until assayed. Enzyme assays. Anthranilate synthase (choris- tional tip repressor in cells, causing derepression mate pyruvate-lyase [amino-accepting], EC 4.1.3.27) of the trp operon. Consequently, the abilities of was assayed by the method of Held and Smith (8). tryptophan analogs to repress the trp operon Assays for anthranilate phosphoribosytransferase [N- can be evaluated by determining, relative to L- (5'-phosphoribosyl)-anthranilate:pyrophosphate typtophan, each analog's ability to prevent phosphoribosyltransferase, EC 2.4.2.18] and indole-3- IPA-mediated derepression. The effects of var- glycerol-phosphate synthase [l-(2'-carboxyphenyl- ious modifications in tryptophan structure upon amino)-l-deoxyribulose-5-phosphate carboxy-lyase the ability of L-tryptophan to repress tip operon (cyclizing), EC 4.1.1.48] activities were performed ac- expression are described. In addition, the rela- cording to the methods of Creighton and Yanofsky (4). Tryptophan synthase, a and , subunits, (L-serine tionship of repression control of tip operon hydro-lyase (adding indole-glycerol-phosphate), EC expression and allosteric control of anthranilate 4.2.1.20) was assayed by the procedures of Smith and synthase by tryptophan analogs is discussed. Yanofsky (16). (This paper was taken in part from a thesis One enzyme unit was defined as the amount of submitted to Marquette University, Milwaukee, enzyme that catalyzed the production of one pmole of Wis., by R. J. P. in partial fulfillment of the product or the consumption of one ismole of substrate requirements for the Ph.D. degree.) in 1 min at 37°C. Specific activity is given in enzyme units per milliam of protein. Protein was determined MATERIALS AND METHODS by the method of Lowry et al. (9). Bacterial strains. The strains of E. coli K-12 used Relative repression was calculated by comparing in this study were derived from either W3110 tna or the level of a particular trp operon gene product W1485 tna, which were isolated in this laboratory and synthesized in the presence of an analog and IPA to lack detectable tryptophanase activity. Mutants de- the level of that gene product synthesized in the scribed in this study were isolated after UV mutagen- presence of L-tryptophan and IPA by the formula: esis. The anthrnilate synthase activities ofthe thpE44 [(SA [IPA] - SA [IPA + analog])/(SA [IPA] - SA tna and tipE45 tna mutants were resistant to inhibi- [IPA + L-tryptophan])] x 100 = relative repression tion by L-tryptophan. aroG13 tna was characterized (percent), by its resistance to growth inhibition by 3-methylan- thranilic acid and has 7-phospho-2-keto-3-deoxy-D-ar- where SA [IPA] was the enzyme specific activity from abinoheptonate D-erythrose-4-phosphate-lyase (py- cells treated with 0.25 mM IPA, SA [IPA + analog] ruvate-phosphorylating) (DAHP synthase) (EC was the enzyme specific activity from cells treated 4.1.2.15) activity which is resistant to allosteric inhi- with 0.25 mM IPA and the designated concentration bition by phenylalanine (8). of analog, and SA [IPA + L-tryptophan] was the Culture conditions. Cultures of E. coli strains enzyme specific activity from cells treated with 0.25 were routinely grown at 37°C with vigorous aeration mM IPA plus L-tryptophan at the same concentration on a rotary shaker in the minim medium of Vogel as indicated for the analog. and Bonner (19) supplemented with 0.2% (wt/vol) Chemicals Chorismic acid was prepared by the glucose as a carbon source. Cells from an overnight method of Gibson (6). N-Acetyl-L-, 4-methyl-DL-, 6- late log- or early stationary-phase culture were re- methyl-DL-, and 7-aza-DL-tryptophan were purchased covered by centrifugation at 5,000 x g for 15 min from Cyclo Chemical Co., Los Angeles, Calif. Trypt- (400), suspended in the same volume of cold sline amine hydrochloride, 5-fluorotryptophan, and 6-fluo- (0.9% NaCl), and centrdfuged again. The cells were rotryptophan were acquired from Aldrich Chemical resuspended in 0.1 volume of fresh medium and used Co., Milwaukee, Wis. Sigma Chemical Co., St. Louis, to inoculate fresh medium (37°C) to an initial density Mo., supplied 5-hydroxy-DL-tryptophan, 7-methyl- of 108 cells per ml. Cultures were incubated until the DL-tryptophan, and DL-tryptophan butyl ester hydro- density reached 4 x 108 to 5 x 108 cells per ml. chloride. IPA was purchased from Eastman Organic Supplements, as specified in Results, were then added Chemical Div., Eastman Kodak Co., Rochester, N. Y. to each culture, and incubation was continued for 45 L-Tryptophan, D-tryptophan, DL-tryptophan, L-tryp- min. Culture flasks were chilled in an ice bath for 5 tophan methyl ester hydrochlonde, L-tryptophan min, and then the contents were transferred to chilled ethyl ester hydrochloride, and 5-methyl-DL-trypto- centrifuge tubes.