Marquette University e-Publications@Marquette Biological Sciences Faculty Research and Biological Sciences, Department of Publications 1-1-1970 Regulation of the Escherichia coli Tryptophan Operon by Early Reactions in the Aromatic Pathway William A. Held Marquette University Oliver H. Smith Marquette University Published version. Journal of Bacteriology, Vol. 101, No. 1 (January 1970): 202-208. Permalink. © 1970 American Society for Microbiology. Used with permission. JOURNAL OF BACTERIOLOGY, Jan. 1970, p. 202-208 Vol. 101, No. I Copyright © 1970 American Society for Microbiology Printed In U.S.A. Regulation of the Escherichia coli Tryptophan Operon by Early Reactions in the Aromatic Pathway1 WILLIAM A. HELD AND OLIVER H. SMITH Department of Biology, Marquette University, Milwaukee, Wisconsin 53233 Received for publication 11 October 1969 7-Methyltryptophan (7MT) or compounds which can be metabolized to 7MT, 3-methylanthranilic acid (3MA) and 7-methylindole, cause derepression of the trp operon through feedback inhibition of anthranilate synthetase. Tyrosine re- verses 3MA or 7-methylindole derepression, apparently by increasing the amount of chorismic acid available to the tryptophan pathway. A mutant isolated on the basis of 3MA resistance (MAR 13) was found to excrete small amounts of chorismic acid and to have a feedback-resistant phenylalanine 3-deoxy-D-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase. Genetic evidence indicates that the muta- tion conferring 3MA resistance and feedback resistance is very closely linked to aroG, the structural gene for the DAHP synthetase (phe). Since feedback inhibition of anthranilate synthetase by L-tryptophan (or 7MT) is competitive with chorismic acid, alterations in growth conditions (added tyrosine) or in a mutant (MAR 13) which increase the amount of chorismic acid available to the tryptophan pathway result in resistance to 7MT derepression. Owing to this competitive nature of tryp- tophan feedback inhibition of anthranilate synthetase by chorismic acid, the early pathway apparently serves to exert a regulatory influence on tryptophan biosynthesis. In Escherichia coli, the level of intermediates of anthranilate synthetase. Unlike other methyl- in aromatic biosynthesis is regulated by several tryptophan analogues, 7MT does not appear to different allosteric interactions. The end products function as an active corepressor. of the pathway (principally phenylalanine, tyro- In this report, we present evidence that ana- sine, and tryptophan) inhibit three isoenzymatic logue-induced derepression is antagonized by the forms of the first enzyme of the pathway, 3-deoxy- inclusion of tyrosine in the growth medium of D-arabinoheptulosonic acid-7-phosphate (DAHP) wild-type cells, and that mutants with a feedback- synthetase, and further interact at the branch resistant DAHP synthetase (phe) are also re- point with the enzymes metabolizing chorismic factory to this derepression. The most likely acid along the various specific biosynthetic routes explanation for these observations is that both (1, 5, 6, 9, 15, 19, 20). Thus, alterations in the alterations increase the amount of chorismic acid levels of the allosteric effectors and the activities available to the tryptophan pathway, thereby of the allosteric enzymes would be expected to reversing the inhibitory effect of 7MT on anthran- demonstrate the complex regulatory inter-rela- ilate synthetase. Thus, the early pathway involv- tionships in the aromatic pathway. ing the production of chorismic acid apparently In the preceding paper (11), evidence was pre- serves to exert a regulatory influence on trypto- sented showing that compounds capable of being phan biosynthesis. metabolized to 7-methyltryptophan (7MT), i.e., 3-methylanthranilic acid (3MA) and 7-methylin- MATERIALS AND METHODS dole, inhibit growth and cause derepression of Organisms. All mutants described were derived the trp operon in wild-type strains of E. coli. Pre- from W1485 tna (a mutant lacking tryptophanase sumably, both phenomena are manifestations ofa isolated in this laboratory) except aroG (a mutant lacking the phenylalanine-inhibitable DAHP synthe- tryptophan limitation caused by 7MT inhibition tase), obtained from K. D. Brown. The parent strain I Taken in part from a thesis submitted to Marquette University of aroG is W1485. The mutants were obtained by by W.A.H. in partial fulfillment of the requirements for the Ph.D. ultraviolet or nitrosoquanidine treatment and plating degree. on appropriate selective media. 3MA-resistant 202 VOL. 101,p 19701REGULATION OF E. COLI TRYPTOPHAN OPERON 203 mutants were obtained by plating mutagen-treated TABLE 1. Cation activation of DAHP synthetase cells on minimal agar plates containing 6.7 X 10-4 M 3MA. Cationa DAHPrelativesynthetaseactivityb Transduction procedures. Plkc transducing lysates of the mutants were prepared by the confluent lysis None....100 technique (13). Control lysates from wild-type strains Co+2.... 182 and controls for sterility were done routinely. Mg+2 ..... 104 Growth media. All strains and mutants were grown Mn+2 .... 158 in the minimal medium of Vogel and Bonner (18) Zn+2..... 65 containing 0.5% glucose and other supplements as Fe+2... 69 desired. The cultures were agitated on a rotary shaker Fe+3... 41 at 37C. Growth curves were obtained by growing EDTA... 16 50-ml cultures in a 500-ml side-arm flask and esti- mating growth at various times by turbidity at 660 nm. a Cations were added to DAHP synthetase Chemicals. D-Erythrose-4-phosphate dimethylacetal reaction mixture at a concentration of 1I- M. dicyclohexylammonium salt was obtained from Calbio- b A crude extract of W1485 tna dialyzed over- chem, Los Angeles, Calif., and converted to free night in buffer containing 10- M EDTA was used erythrose-4-phosphate by the method of Ballou, as a source of enzyme. Fischer, and MacDonald (2) before use. Chorismic acid was prepared as the free acid according to the 10-3 M EDTA. It is apparent that Co+2 and Mn+2 procedure of Gibson (10). All other chemicals were significantly stimulate DAHP synthetase activity and obtained commercially. that EDTA almost completely inhibits the reaction. Preparation of extracts. Cells were harvested by Maximal activation of DAHP synthetase occurs at centrifugation, washed once in saline, and resuspended 10-' M Co+2, and this concentration was used in all in 0.1 M K2HPO4, pH 7.0, containing 10-3 M 2-mer- subsequent experiments. captoethanol and 10-3 M ethylenediaminetetraacetate Diethylaminoethyl (DEAE)-Sephadex chromatog- (EDTA). The cells were broken by sonic oscillation, raphy. DEAE-Sephadex chromatography was carried and cell debris was removed by centrifugation at out on a column of 2.5 X 32 cm by use of a linear 30,000 X g for 30 min. For assay of DAHP synthe- gradient elution starting with 350 ml of 10-2 M K2- tase, extracts were prepared in 5 X 10-2 M K2HPO4, HPO4, pH 7.0, plus 0.1 M KCI in the mixing bottle pH 7.0, containing 10-3 M 2-mercaptoethanol. and 350 ml of 10-2 M K2HPO4, pH 7.0, plus 0.6 M Enzyme assays. The a subunit of tryptophan KCL in the inlet bottle. Eluting buffers contained synthetase (A protein) was assayed according to 10-' M 2-mercaptoethanol. Nucleic acids were not Smith and Yanofsky (16), and anthranilate synthetase removed before crude extracts were placed on the was assayed as described in the preceding paper (11). column. At the end of the fractionation, a small DAHP synthetase was assayed according to the amount of 10-2 M K2HPO4, pH 7.0, containing 1.0 M procedure of Doy and Brown (7), except that the KCl was washed through the column to insure that reaction mixture contained 10-3 M Co2+ (see below). all DAHP synthetase activity had been eluted. Re- After heating with thiobarbituric acid, the reaction covery of DAHP synthetase varied between 32 and tubes were cooled briefly and extracted with 4.0 ml 58%. of cyclohexanone to avoid the turbidity usually en- countered in the assay. The concentration of DAHP RESULTS in the cyclohexanone was determined spectrophoto- metrically at 549 nm by use of a molar extinction Since the analogue-induced trp operon dere- coefficient of 6.0 X 104. pression results from tryptophan limitation, it A unit of enzyme activity is equivalent to 1 ,umole should be reversed by added tryptophan or pre- of substrate consumed or product formed per minute cursors such as anthranilic acid and indole. Table at 37 C. Specific activity is given as units per milligram 2 shows that these compounds did repress cul- of protein. The protein was determined by the method tures to the and that an of Lowry et al. (14). In some cases (relative activity), exposed analogues early the specific activity relative to that found in W1485 intermediate, shikimic acid, had only a slight tna grown in minimal medium was used. A relative effect in decreasing the enzyme levels. Shikimate activity of 1.0 corresponds to 0.0027 units of anthranil- is a precursor of chorismic acid and, in light of ate synthetase per mg of protein and 0.0152 units of the arguments presented below, we have no tryptophan synthetase A protein per mg of protein. ready explanation for its relative ineffectiveness. Co+2 activation of DAHP synthetase. A recent Table 2 further shows that tyrosine, but not report indicated that the DAHP synthetase (trp) is phenylalanine or a mixture of the two, effectively stimulated by Co+2 (15). We have found that Co+2 reversed the derepression. We felt that, as inhibi- appears to activate DAHP synthetase in crude extracts tion of anthranilate synthetase by tryptophan or partially purified preparations of W1485 tna which (or 7-methyltryptophan) is competitive with contain predominantly the DAHP synthetase (phe). acid reversal of 3MA Table 1 shows the effect of various cations on the total chorismic (1, 11), tyrosine DAHP synthetase activity in crude extracts of W1485 and 7-methylindole derepression might be due to tna which was dialyzed overnight in buffer containing tyrosine causing increased chorismic acid avail- 204 HELD AND SMITH J.