Regulation of the Aroh Operon of Escherichia Coli by the Tryptophan Repressor CINDY L

Home , Derepression, Tryptophan repressor

JOURNAL OF BACTERIOLOGY, May 1987, p. 2158-2164 Vol. 169, No. 5 0021-9193/87/052158-07$02.00/0 Copyright © 1987, American Society for Microbiology Regulation of the aroH Operon of Escherichia coli by the Tryptophan Repressor CINDY L. GROVE' AND ROBERT P. GUNSALUSl 2* Department of Microbiology' and the Molecular Biology Institute,2 University of California, Los Angeles, Los Angeles, California 90024 Received 24 November 1986/Accepted 24 February 1987

Regulation of expression of aroH, the structural gene for the tryptophan-sensitive 3-deoxy-D- arabinoheptulosonic acid-7-phosphate synthetase, by the tryptophan repressor and its corepressor, L- tryptophan, was studied in vivo by using aroH-lacZ fusions. Protein and operon fusions were constructed on multicopy plasmids and subsequently crossed in single copy to the bacterial chromosome via the specialized transducing bacteriophage XRZ5. Analysis of the resulting lysogens demonstrated that aroH-LacZ expression in a trpR mutant strain varied four- to fivefold relative to an isogenic trpR+ strain under fully repressing conditions. In trpR' strains containing either fusion, a modest (ca. 50%) change in activity was seen in response to the addition of L-tryptophan to the culture medium. These data demonstrate that aroH gene expression is only moderately regulated by the tryptophan repressor and that this regulation is at the level of transcription. Addition of L-phenylalanine, L-tyrosine, or Casamino Acids (Difco Laboratories, Detroit, Mich.) to the cell culture medium resulted in a tryptophan repressor-dependent derepression of aroH expression. We believe that this effect is caused by L-tryptophan limitation as a result of repression and feedback inhibition of the tyrosine- and phenylalanine-specffic 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase isoenzymes. Derepres- sion of aroH expression by the L-tryptophan analogs, 3-4-indoleacrylic acid and indole-3-propionic acid, is also documented.

The first step in the biosynthesis of the aromatic amino the genes responsible for subsequent steps in L-phenylal- acids L-tyrosine, L-phenylalanine, and L-tryptophan in Esch- anine biosynthesis (13). erichia coli involves the condensation of the cellular inter- The aroH operon contains a single gene and is located at mediates, phosphoenolpyruvate and erythrose-4-phosphate, 37 min on the E. coli chromosome (O). It has been cloned, to give the seven-carbon sugar, 3-deoxy-D-arabinoheptu- and the DNA sequence has been determined (32). Two sites losonic acid-7-phosphate (DAHP). This reaction is catalyzed for initiation of aroH mRNA transcription have been iden- by three DAHP synthetase isoenzymes encoded by the tified in vitro and are located at +117 (major 5' terminus) and unlinked aroF, aroG, and aroH genes (28). The tyrosine- + 114 (minor 5' terminus) nucleotides prior to the transla- sensitive DAHP synthetase (aroF) accounts for approxi- tional initiation codon. A palindromic nucleotide sequence, mately 20% of the total DAHP synthetase activity, whereas similar in sequence to the tryptophan repressor-specific the phenylalanine-sensitive enzyme (aroG) accounts for operators of the trpR and trpEDCBA operons of E. coli, is approximately 80% of the total activity. The tryptophan- centered approximately 35 base pairs upstream of the major sensitive isoenzyme (aroH) has been reported to account for 5' transcriptional start site within the aroH regulatory re- less than 1% ofthe total DAHP synthetase activity in the cell gion. The tryptophan repressor has been shown to bind at (17). this DNA sequence by in vitro DNA-binding experiments. Control of this first step of aromatic amino acid biosyn- When the tryptophan repressor is prebound at the operator thesis in E. coli occurs both at the level of feedback site, the restriction endonuclease enzyme RsaI is unable to inhibition by the aromatic amino acids and by repression of bind and then cut at its recognition site located within the the genes coding for the DAHP synthetases. Both the aroH operator sequence (32). tyrosine-sensitive and phenylalanine-sensitive enzymes can The tryptophan repressor coordinately regulates three be feedback inhibited up to 95% by the end products of the operons in E. coli involved in L-tryptophan biosynthesis (15, respective branch pathways (17). The L-tryptophan-sensitive 29). When the intracellular L-tryptophan levels are low, the enzyme can be feedback inhibited up to 60% by L- tryptophan repressor exists predominantly in the aporepres- tryptophan (7, 23). sor form (1) that has a very low affinity for operator DNA The three DAHP synthetases are also regulated at the (24). If the intracellular L-tryptophan levels are elevated transcriptional level. The tyrosine repressor, product of the because of increased L-tryptophan biosynthesis or transport, tyrR gene, regulates expression of the unlinked aroF and the tryptophan aporepressor binds its corepressor, L- aroG genes in response to intracellular levels of L-tyrosine, tryptophan, to form the active repressor complex, which L-phenylalanine, and L-tryptophan (6, 9), whereas aroH is subsequently binds the respective operator DNAs to repress regulated by the tryptophan repressor, the product of the trp, trpR, and aroH operon expression. trpR gene (5), and by L-tryptophan. The pheR gene product, To determine the extent of aroH gene regulation by the the phenylalanine repressor, does not appear to have any tryptophan repressor and by its corepressor, L-tryptophan, direct effect on aroG expression, although it does regulate we constructed protein and operon fusions between the aroH and the lacZ genes. Levels of P-galactosidase activity in cells containing these fusions indicate the degree of aroH * Corresponding author. expression at the transcriptional and translational levels in 2158 VOL. 169, 1987 TRANSCRIPTIONAL REGULATION OF THE aroH OPERON 2159

Fnu DII Smo I

9 10 11 12 i 8 thr ala, arg ile IC CCeiGGGG~~A~T C-CC TG AC G CD3 C ATAA GGG'BCCCTAGGG

Fnu DII Smo I PstI Fn uDDl BamHI pDNAI 3.8kb

Kmr

Fnu DI SmaI

ligase Fnu Dll/SmoI 9 10 8 thr ala gly asp pro 5bCT;GC GGAtc TGTACGCCCCCTAGGG FnuDII/SmOI ,' , _ .

FIG. 1. Construction of the aroH'-'lacZ protein fusion plasmid (pCLG3). A 308-base-pair FnuDII fragment, containing the first 10 codons of the aroH gene and its associated regulatory region from pDNA1, was inserted into the SmaI site of pMC1396 to generate pCLG3 as shown. The boxed regions represent the following gene regions: lacZ, cm; lacY, f; lacA, ; aroH, 1. Arrows indicate the direction of transcription. Numbering above the amino acid sequence refers to the position from the amino terminus of the wild-type aroH or lacZ gene product. Abbreviation: kb, kilobases. response to cell culture conditions. The effects of L-tyrosine, Plasmid pDNA1 is a derivative of pAROH924 (33) in L-phenylalanine, Casamino Acids (Difco Laboratories, De- which the 0.9-kilobase BamHI-PstI fragment that contains troit, Mich.), aromatic vitamins, and L-tryptophan analogs the 5' coding portion of the aroH gene, and its corresponding on aroH'-'lacZ expression were also examined in strains regulatory region was ligated into the large BamHI-PstI that are either wild type (trpR+) or defective (trpR) for the fragment of pACYC184 (pDNA1 was obtained from D. N. tryptophan repressor. Arvidson). Plasmid pMC1396 has been described previously (8), and plasmid pRS415 was obtained from R. W. Simons MATERIALS AND METHODS (R. W. Simons, F. Houmon, and N. Kleckner, Gene, in press). The bacteriophage vector, XRZ5, was obtained from Bacterial strains, plasmids, and bacteriophage. All E. coli R. Zagursky. strains used in these experiments were K-12 derivatives. The construction of the aroH'-'lacZ protein fusion plas- P90C [F- A(lac pro) ara thi] (21) and CSH22 [A(lac pro) ara mid, pCLG3, is shown in Fig. 1. The 308-base-pair FnuDII thi trpR F' lacZAM15] (20) were obtained from J. H. Miller. fragment used for this construction contains the first 10 Strain RG42 [A(lac pro) ara thi trpR F+Kan] was derived codons of the aroH gene and its associated 5' regulatory from CSH22 by phenocopy mating with strain S90C [F+Kan] region. The nucleotide sequence at the fusion junction in this (20). Colonies were selected for kanamycin resistance (Kmr) plasmid was confirmed by DNA sequence analysis (data not on kanamycin L plates and were screened for streptomycin shown). Production of a predicted aroH'-'lacZ hybrid ,- sensitivity (Srs) by replica plating on streptomycin L plates. galactosidase protein was confirmed by Western blot analy- Kmr Srs colonies were repurified, and a Lac- Pro- Ara- sis of extracts of cells containing pCLG3 probed with rabbit Thi- TrpR- strain was designated RG42. anti-p-galactosidase antibody (data not shown). 2160 GROVE AND GUNSALUS J. BACTERIOL.

ARZ5 Lac- Aps

pCLG3 Lac+Apr

or1 I

A A Oatt ACLG3 A fxxl______:- CI i s Lac+Apr y Z' I'ro H b/c

4( aroH' '/ccZ )( Hyb)

FIG. 2. Transfer of the aroH'-'lacZ protein fusion from pCLG3 to the chromosome via XRZ5. Cells containing plasmid pCLG3 were infected with XRZ5, and lysates were prepared as described in Materials and Methods. Recombination of the homologous 3' regions of lac and bla regions on the phage and plasmid resulted in transfer of the aroH'-'lacZ fusion from pCLG3 to give XCLG3. The boxed regions represent the following gene regions: lacA, P l; lacY, ; lacZ, _; bla, E1; aroH, EI-. Arrows indicate the direction of transcription. The phenotype of the plasmid- or phage-containing cells is indicated at the right.

The protein fusion was crossed from pCLG3 to the lambda of the manufacturers. T4 DNA ligase was purified by the vector XRZ5 and subsequently to the E. coli chromosome in method of Panet et al. (22). DNA sequence analysis was single copy. The phage XRZ5 contains the 3' regions of the done by the method of Sanger et al. (25). Lambda DNA bla gene (derived from pBR322) and the lacZY+ genes, isolation (10) and phage procedures (26) were done as oriented opposite to one another, and exhibits a Lac- Aps previously described. (ampicillin-sensitive) phenotype (Fig. 2). It also contains Media and culture conditions. Cells were grown in the attP such that integration of the vector or its derivatives into minimal E medium of Vogel-Bonner (27) supplemented with the E. coli chromosome can occur at attB (R. Zagurski, 0.2% glucose, 25 mg of L-proline per liter, and 0.01% personal communication). Cells bearing the plasmid pCLG3 thiamine hydrochloride. L-tryptophan, L-tyrosine, and L- were lytically infected with XRZ5 (26). Homologous recom- phenylalanine were added at final concentrations of 200 or bination between the 3' regions of the lacZ and bla genes on 400 ,uM where indicated. For measurement of P-galactos- the phage and the aroH'-'lacZ fusion contained on pCLG3 idase activities, cells were grown overnight with shaking at resulted in transfer of the 5' aroH'-'lacZ and bla regions 37°C in the medium indicated. A sample of the overnight from the plasmid into the XRZ5 phage to give XCLG3 (Fig. culture was diluted 20-fold into 15 ml of medium in a 125-ml 2). A blue plaque was purified on a lawn of P9OC, phage Delong flask (Bellco Glass, Inc., Vineland, N.J.). Cultures stocks were prepared, and the resulting lysate was used to were vigorously aerated by shaking at 300 rpm. Cells were lysogenize the indicated strains (26; Simons et al., in press). then grown to mid-exponential phase, chilled on ice, and To construct an operon fusion, a 315-base-pair EcoRI- harvested as described below. Luria broth (20) was used for BamHI fragment from pCLG3 was cloned into the operon plasmid and phage construction procedures. Ampicillin was fusion vector, pRS415 (Simons et al., in press). The resulting added at a final concentration of 60 mg/liter (plasmid- plasmid was designated pCLG7. The aroH'-lacZ+ operon containing strains) or 20 mg/liter (lambda lysogens). Kana- fusion on pCLG7 was crossed to XRZ5 to yield the phage mycin and streptomycin (Sigma Chemical Co., St. Louis, designated XCLG7 by using the methods described for the Mo.) were added to culture media at a final concentration of XCLG3 protein fusion. The fusion was then introduced in 30 and 125 mg/liter, respectively. The chromogenic indicator single copy into isogenic E. coli P9OC (trpR+) and RG42 XG (International Biotechnologies) was used at a concentra- (trpR) strains as described for the protein fusion. tion of 40 mg/liter. Bacterial culture media (Casamino Acids, DNA and phage manipulations. Digestion of plasmid DNA Tryptone, yeast extract, and agar) were obtained from Difco and preparation of DNA fragments from polyacrylamide gels Laboratories and used according to Difco instructions. were done as described previouisly (19). Ligation of DNA Enzyme assays. ,B-Galactosidase assays were done as fragments (19), cell transformation (16), and preparation of described by Miller (20), with the following modifications. plasmid DNA (3) were done as described previously. Re- Cells grown to mid-exponential phase were harvested by striction enzymes were obtained from Bethesda Research centrifugation for 5 min (3,000 x g) and suspended in 0.1 Laboratories (Rockville, Md.), International Biotechnolo- volume of PM2 buffer (0.1 M NaPO4 [pH 7.0], 1 mM MgSO4, gies Inc. (New Haven, Conn.), or New England BioLabs 0.2 mM MnSO4). Samples of these cells were then diluted in (Beverly, Mass.) and were used according to the instructions PM2 buffer to the appropriate density for enzyme assay (1 VOL. 169, 1987 TRANSCRIPTIONAL REGULATION OF THE aroH OPERON 2161 ml, final volume). Cells were made permeable by the addi- Previous studies have suggested that aroH expression was tion of toluene (1 drop per tube) and mixed by vortexing. regulated solely by L-tryptophan, via trpR+ (5). To confirm Toluene was removed by evaporation under vacuum before these early observations and to rrmore closely examine aroH the enzyme assay. The samples were brought to 28°C by operon expression in response to L-tyrosine or L-phenyl- placing the tubes in a circulating water bath for 5 min, and alanine supplementation or both, we monitored aroH'-'lacZ 3.5 [lI of P-mercaptoethanol was then added. The reaction expression in an E. coli wild-type strain for aroF+, aroG+, was initiated by the addition of 0.25 ml of o-nitrophenyl-p- and aroH+. P9OC (trpR+) lysogens grown in a minimal D-galactopyranoside substrate (4 mg/ml in 0.25 M NaPO4, medium containing L-phenylalanine exhibited 1.4-fold higher pH 7.0). When sufficient color had developed (A420 of 0.2 to levels of the hybrid AroH'-p-galactosidase protein than did 0.6), the reaction was stopped by the addition of 2 ml of 1 M cells grown in minimal medium alone (Table 1). However, Na2CO3, and the elapsed time was noted. Protein concen- the addition of L-tyrosine to the culture medium did not tration was estimated by measuring the density of the cell result in a similar derepression of aroH'-'lacZ expression in cultures at 600 nm (Uvikon 810 spectrophotometer; Kontron P9OC relative to expression in cells grown in medium with- AG, Zurich, Switzerland). For the cells used, an A600 of 1.4 out L-tyrosine. When both L-tyrosine and L-phenylalanine is equivalent to 109 cells per ml of 150 ,ug of protein per ml were added to the culture medium (in the absence of (20). One unit of P-galactosidase is defined as 1 nmol of L-tryptophan), aroH'-'lacZ expression was further dere- o-nitrophenol-p-D-galactopyranoside hydrolyzed per min pressed relative to that seen in those cells grown with per mg of protein. Under the conditions used, the molar L-phenylalanine alone (Table 1; 1.4- to 2-fold). The observed extinction coefficient of o-nitrophenol is 0.0045. derepression of aroH'-'lacZ expression is clearly dependent on the tryptophan repressor present in P9OC (trpR+), as RESULTS indicated by the uniformly high levels of aroH'-'lacZ expres- Construction of aroH'-'lacZ protein fusion and aroH-lac+ sion in a trpR strain (RG42) grown under identical condi- operon fusion plasmids. Direct assay of the L-tryptophan- tions. Addition of all three aromatic amino acids (L-tyrosine, specific DAHP (aroH) synthetase is difficult in wild-type E. L-phenylalanine, and L-tryptophan; 200 puM) to the culture coli cells because of the presence of the tyrosine-sensitive medium of P9OC(XCLG3) cells resulted in an intermediate (aroF) and phenylalanine-sensitive (aroG) DAHP synthetase level of aroH'-'lacZ expression when compared with that for activities. As a result, it has not been possible to quantita- cells grown in minimal medium plus tryptophan alone. tively evaluate the contribution of either the tryptophan Increasing the concentration of L-tryptophan in the culture repressor or the L-tryptophan levels on aroH operon expres- medium to 400 puM reduced P-galactosidase levels to the sion. To indirectly examine the regulation of aroH expres- same as that seen for minimal medium plus 200 puM L- sion, we constructed an aroH'-'lacZ protein fusion plasmid tryptophan. This suggests that the derepression of aroH'- that produces a hybrid AroH'-3-galactosidase protein 'lacZ expression by phenylalanine and tyrosine is a result of (pCLG3) and an aroH'-lacZ+ operon fusion (pCLG7) which a reduced intracellular tryptophan concentration. We also produces a wild-type ,-galactosidase protein (see Materials tested the effect of the aromatic vitamins p-aminobenzoic and Methods; Fig. 1). The fusions were subsequently acid (10 pum), p-hydroxybenzoic acid (40 pum), and 2,3- crossed to the chromosome by using specialized phage XRZ5 dihydroxybenzoic acid (500 ,um) on aroH'-'lacZ gene ex- to create XCLG7 and XCLG3 (Fig. 2). Assays of hybrid pression. No change in activity was observed, whether these ,-galactosidase levels in fusion-bearing cells grown under vitamins were present or absent under all of the culture alternative culture conditions indicate the degree of tran- conditions tested in Table 1 (data not shown). scriptional and translational regulation of the aroH gene Effect of Casamino Acids on aroH'-'IacZ expression. We expression. also tested the effect of Casamino Acids on aroH'-'lacZ Expression of the aroH'-'lacZ protein fusion in trpR+ and expression in trpR+ (P9OC) and trpR (RG42) strains. trpR strains. To determine the extent that aroH'-'lacZ ex- Casamino Acids, an acid hydrolysate of casein, is composed pression is regulated by the tryptophan repressor, isogenic of a mixture of amino acids minus L-tryptophan, which is E. coli P90C (trpR+) and RG42 (trpR) strains lysogenic for destroyed during the hydrolysis. When P9OC(XCLG3) cells XCLG3 were grown in minimal medium in the presence or were grown in medium containing 1% Casamino Acids, absence of L-tryptophan (200 jxM). Cells were harvested, hybrid P-galactosidase levels were twofold higher than in and levels of hybrid ,B-galactosidase activity were determined as described in Materials and Methods. Levels of TABLE 1. Expression of an aroH'-lacZ protein fusion in strains P-galactosidase activity in these strains varied four- to P9OC (trpR+) and RG42 (trpR) lysogenic for XCLG3 fivefold overall (Table 1). Omission of L-tryptophan from the culture medium re- Additions to culture P-Galactosidase activityb for strain: sulted in levels of P-galactosidase activity in trpR cells that mediuma RG42 (trpR) P9OC (trpR+) were nearly identical to the levels in L-tryptophan- None 437 156 supplemented cells. However, when P90C (trpR+) cells that Trp 470 104 are wild type for the tryptophan repressor were grown under Tyr 437 147 the same conditions, enzyme levels varied by about 1.5-fold Phe 430 220 in response to L-tryptophan (Table 1). Increasing the L- Tyr, Phe 460 318 tryptophan levels in the culture medium from 200 to 400 ,uM Trp, Tyr 466 110 did not lead to lower levels in P90C(ACLG3), Trp, Phe 477 122 P-galactosidase Phe 490 151 indicating that aroH'-'lacZ expression is maximally re- Trp, Tyr, pressed under these conditions (data not shown). These a Cultures were grown as described in Materials and Methods. Each amino results establish that aroH expression, like trpR expression acid was added at a final concentration of 200 ,uM: Trp, L-tryptophan; Tyr, L- tyrosine; Phe, L-phenylalanine. (18), does not vary dramatically in response to levels of b P-Galactosidase units are given as nanomoles of o-nitrophenyl-3-D- L-tryptophan in the cell culture medium compared with galactopyranoside hydrolyzed per minute per milligram of protein. Each value levels of trp operon expression in E. coli (31). is the average of at least three individual determinations. 2162 GROVE AND GUNSALUS J. BACTERIOL. cells grown in minimal medium alone (319 U versus 156 U). (trpR) upon the addition of either analog under identical This is similar to what is seen when cells are grown with growth conditions. L-phenylalanine and L-tyrosine (Table 1). Addition of both Calculation of the number of molecules per cell. If the L-tryptophan (200 ,um) and 1% Casamino Acids to the specific activity of P-galactosidase is 400,000 U/mg (12), and medium resulted in partial derepression of aroH'-'lacZ ex- we assume 150 jig of protein per 109 cells (20), then pression (i.e., 20%), when compared with levels of I- (6.02 x 1023 molecules/mol) galactosidase in cells grown in the presence of L-tryptophan x alone. These effects were not seen in the trpR strain (RG42) (1.16 x 108 mg/mol)(400,000 U/mg) when grown under identical culture conditions. 0.15 mg of protein 156 U Expression of an aroH'-lacZ' operon fusion in isogenic x = 304 molecules/cell trpR+ and trpR strains. The regulation of aroH'-lacZ+ ex- 109 cells mg of protein pression by the tryptophan repressor and its corepressor, L-tryptophan, was evaluated in P9OC (trpR+) and RG42 Therefore, cells growing in minimal medium contain approx- (trpR) strains lysogenic for XCLG7 (Materials and Methods). imately 300 molecules of DAHP synthetase (trp) per cell. The production of ,3-galactosidase in these operon fusion strains is under control of the lacZ translational initiation DISCUSSION signals, whereas transcription occurs from the aroH promoter-operator element. Levels of ,-galactosidase in the corre- We examined the contribution of the tryptophan repres- sponding trpR+ and trpR strains, when grown in the pressor, its corepressor L-tryptophan, and the other two aro- ence of L-tryptophan, varied about fourfold (Table 2). Thus, matic amino acids (L-phenylalanine and L-tyrosine) on aroH the maximum range of aroH'-lacZ+ expression in wild-type regulation. We also tested the effect of the L-tryptophan (trpR+) strains versus defective (trpR) strains is similar to analogs, 3-p-indoleacrylic acid and indole-3-propionic acid, that seen for the protein fusion strains. The maximum range on aroH expression. Comparison of hybrid AroH'-13- of aroH'-lacZ+ expression in a trpR+ strain (P9OC) wild type galactosidase activities in the lysogenic E. coli strains for tryptophan repressor was 1.5-fold in response to cell RG42(ACLG3) and P9OC(XCLG3) reveals that the maximum growth in the presence or absence of L-tryptophan. In- range of aroH'-'lacZ expression from fully repressed condi- creased L-tryptophan in the culture medium (final concen- tions [P9OC (trpR+) cells grown in minimal medium plus tration, 400 ,uM) did not result in further repression of L-tryptophan] to nonrepressed conditions [RG42 cells (trpR) aroH'-lacZ+ expression, indicating that aroH expression grown minus L-tryptophan] are four- to fivefold (Table 1). varies by only about 50o in response to added L-tryptophan, These results are in disagreement with previous suggestions as seen for the protein fusion strain P9OC(XCLG3), under that aroH expression was regulated 10- to 20-fold by the similar culture conditions. However, when compared on an tryptophan repressor (30), although they are in accord with absolute basis, the operon fusion strains have about a 20-fold results obtained from mRNA measurements (32). higher level of ,-galactosidase activity than the protein The effect of L-tryptophan supplementation on aroH ex- fusion strains under corresponding cell growth conditions pression was determined by using the aroH'-'lacZ protein (Table 2 versus Table 1). and operon fusion strains. Levels of j-galactosidase in trpR+ Effect of L-tryptophan analogs on aroH'-'lacZ expression. cells grown in the absence of L-tryptophan were 50% higher Previous studies demonstrated that the addition of the L- than those in cells grown in presence of this amino acid tryptophan analogs, 3-,-indole acrylic acid and indole-3-pro- (Tables 1 and 2). Thus, the aroH operator-tryptophan re- pionic acid, to cell culture medium resulted in derepression pressor regulatory element responds only marginally to high of trp and trpR operon expression (11, 14). To establish and low levels of L-tryptophan. This response is less than whether these two analogs have a similar effect on aroH'- that reported for the other two operons under tryptophan 'lacZ expression, P9OC (trpR+) cells were grown in the repressor control. Under similar cell culture conditions, the presence of various amounts of the analogs. Addition of autoregulated trpR operon expression varied approximately 3-,-indoleacrylic acid (20 p,M) to the minimal cell culture 2.5- to 3-fold in response to L-tryptophan supplementation as medium resulted in complete derepression of aroH'-'lacZ measured by radioimmunoassay (14) or by gene fusion expression to levels seen in strain RG42 (trpR). Addition of methods (18). The trp operon however, appears to be indole-3-propionic acid to the cell culture medium at a final regulated approximately 13-fold (in the absence of attenua- concentration of 100 ,uM also resulted in depression of tion) under similar culture conditions (31). aroH'-'1acZ expression, although the maximum levels of We examined the effect of the other aromatic amino acids, hybrid 3-galactosidase were about one-half of that observed L-tyrosine and L-phenylalanine, on aroH'-'lacZ protein fu- for cells grown in the presence of 3-p-indoleacrylic acid. No sion expression the P9OC(XCLG3) (trpR+) and change in aroH'-'lacZ expression was observed in RG42 RG42(XCLG3) (trpR) lysogens (Table 1). Addition of L- phenylalanine to the culture medium resulted in derepression of aroH'-'lacZ expression in P90C. Addition of L- TABLE 2. Expression of an aroH'-lacZ+ operon fusion in strains tyrosine to the cell culture medium did not result in derepres- P9OC (trpR+) and RG42 (trpR) lysogenic for XCLG7 sion of aroH expression under analogous culture conditions. However, when both L-tyrosine and L-phenylalanine were Additions to 1-Galactosidase activityb for strain: present, there appeared to be a synergistic effect resulting in culture mediuma RG42 (trpR) P90C (trpR+) greater derepression of aroH'-'lacZ expression, compared None 7,587 3,254 with cell growth in amino acid-free medium (Table 1). That Trp 7,964 2,388 this effect is dependent on the presence of functional tryptophan repressor is evident from the uniformly high a Cultures were grown as described in Materials and Methods. Trp, L- tryptophan (200 ,uM). ,-galactosidase levels in trpR cells grown under correspond- b P-Galactosidase units expressed as nanomoles of o-nitrophenyl-1-D- ing conditions. We suggest that this effect is caused by galactopyranoside hydrolyzed per minute per milligram of protein. L-tryptophan limitation as a result of repression and feed- VOL. 169, 1987 TRANSCRIPTIONAL REGULATION OF THE aroH OPERON 2163 back inhibition of the tyrosine- and phenylalanine-specific GM-29456 from the National Institutes of Health and by a UCLA DAHP synthetase isoenzymes, which causes a reduction in Biomedical Research grant to R.P.G. the levels of chorismic acid, a precursor in L-tryptophan biosynthesis (5). LITERATURE CITED The observed derepression of aroH'-'lacZ expression in 1. Arvidson, D. N., C. Bruce, and R. P. Gunsalus. 1986. Interaction the Escherichia coli Trp aporepressor with its ligand, L- XCLG3 lysogens in response to Casamino Acids can also be of described tryptophan. J. Biol. Chem. 261:238-243. explained by the aromatic amino acid effect 2. Bachmann, B. J. 1983. Linkage map of Escherichia coli K-12, above. In cell culture medium containing 1% Casamino edition 7. Microbiol. Rev. 47:180-230. Acids (wt/vol), the concentration of L-phenylalanine is ap- 3. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction proximately 1.2 mM and the concentration of L-tyrosine is procedure for screening recombinant plasmid DNA. Nucleic approximately 0.5 mM, whereas L-tryptophan is absent Acids Res. 7:1513-1523. (product analysis from Difco). These concentrations are far 4. Bogosian, G., and R. S. Somerville. 1984. Analysis in vivo of higher (sixfold and threefold, respectively) than those used factors affecting the control of transcription initiation at promot- in experiments described in Table 1 to obtain maximum ers containing target sites for trp repressor. Mol. Gen. Genet. of the aroH'-'lacZ protein fusion expression. 193:110-118. derepression 5. Brown, K. D. 1968. Regulation of amino acid biosynthesis in Therefore, we suggest that the derepression of aroH'-'lacZ Escherichia coli K-12. Genetics 60:31-48. expression seen when trpR+ cells grown in Casamino Acids- 6. Camakaris, H., and J. Pittard. 1973. Regulation of tyrosine and containing medium (lacking L-tryptophan) is caused by the phenylalanine biosynthesis in Escherichia coli K-12: properties high content of L-tyrosine and L-phenylalanine. As would be of the tyrR gene product. J. Bacteriol. 115:1135-1144. predicted from the above considerations, the derepression of 7. Camakaris, J., and J. Pittard. 1974. Purification and properties aroH'-'lacZ expression caused by Casamino Acids is re- of 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase lieved when high amounts of L-tryptophan (>200 ,uM, final (trp) from Escherichia coli. J. Bacteriol. 120:590-597. concentration) is added to the cell culture medium. 8. Casadaban, M. J., J. Chou, and S. N. Cohen. 1980. In vitro gene levels of aroH-lacZ expression in trpR+ fusions that join an enzymatically active P-galactosidase seg- The relative ment to amino-terminal fragments of exogenous proteins: Esch- lysogens containing the protein and operon fusions were erichia coli plasmid vectors for the detection and cloning of compared. It was observed that P-galactosidase levels were translational initiation signals. J. Bacteriol. 143:971-980. 20-fold higher in the operon fusion strains (XCLG7) than in 9. Cornish, E. C., V. P. Argyropoulos, J. Pittard, and B. E. Davidson. the protein fusion strains (XCLG3) under all conditions 1986. Structure of the Escherichia coli K-12 regulatory gene tyrR. tested. Since the pattern of regulation is qualitatively the J. Biol. Chem. 261:403-410. same, we conclude that aroH regulation by L-tryptophan is 10. Davis, R. W., D. Botstein, and J. R. Roth. 1980. A manual for occurring primarily at the transcriptional level. genetic engineering: advanced bacterial genetics, p. 109. Cold The range of aroH operon expression under similar cell Spring Laboratory, Cold Spring Harbor, N.Y. C. Yanofsky. 1968. Mutants of Escherichia in response to the tryptophan repressor is 11. Doolittle, W. F., and culture conditions coli with an altered tryptophanyl-transfer ribonucleic acid syn- almost identical to that observed for trpR (four- to fivefold; thetase. J. Bacteriol. 95:1283-1294. 18). When the trp operon expression data are evaluated 12. Fowler, A. V., and I. Zabin. 1983. Purification, structure and independently of the attenuator element, trp operon expres- properties of hybrid P-galactosidase proteins. J. Biol. Chem. sion varies approximately 70-fold in response to the 258:14354-14358. tryptophan repressor (4, 31). Thus, the maximum range in 13. Gowrishankar, J., and J. Pittard. 1982. Regulation of phenylal- operon expression seen for trp is more than 10 times that anine biosynthesis in Escherichia coli K-12: control of transcrip- seen for aroH and trpR. The range of expression observed in tion of the pheA operon. J. Bacteriol. 150:1130-1137. 1986. lysogens in response to the addition of L-tryptophan to 14. Gunsalus, R. P., A. G. Miguel, and G. L. Gunsalus. trpR+ in Escherichia coli. J. Bacte- among the three operons. Intracellular Trp repressor levels the medium varies significantly riol. 167:272-278. Expression of the trp operon is decreased 13-fold in response 15. Gunsalus, R. P., and C. Yanofsky. 1980. Nucleotide sequence to added tryptophan (31) and trpR is decreased 3-fold (14, and expression of Escherichia coli trpR, the structural gene for 18), whereas aroH expression was decreased by only the trp aporepressor. Proc. Natl. Acad. Sci. USA 77:7117-7121. 50%. Thus, E. coli has evolved a system such that a single 16. Gunsalus, R. P., G. Zurawski, and C. Yanofsky. 1979. Structural regulatory protein can finely tune the expression of at least and functional analysis of cloned deoxyribonucleic acid contain- three different operons involved in the regulation and bio- ing the trpR-thr region of the Escherichia coli chromosome. J. synthesis of L-tryptophan in response to a variety of growth Bacteriol. 140:106-113. 17. Herrmann, K. M. 1983. The common aromatic biosynthetic conditions. R. L. to the overall rates of trp, pathway, p. 301-322. In K. M. Herrmann and Somerville Two major factors contributing (ed.), Amino acids: biosynthesis and genetic regulation. trpR, and aroH operon transcription are the promoter Addison-Wesley Publishing Co., Reading, Mass. strengths and the relative affinities of the tryptophan repres- 18. Kelley, R. L., and C. Yanofsky. 1982. trp aporepressor produc- sor for the respective operators of the three operons. Quan- tion is controlled by autogenous regulation and inefficient trans- titative measurements of these interactions are under way lation. Proc. Natl. Acad. Sci. USA 79:3120-3124. and should yield information regarding the contributions of 19. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular each to the coordinate regulation of aroH, trpR, and cloning: a laboratory manual, p. 504. Cold Spring Harbor trpEDCBA operon expression. Laboratory, Cold Spring Harbor, N.Y. 20. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. ACKNOWLEDGMENTS 21. Miller, J. H., D. Ganem, P. Lu, and A. Schmitz. 1977. Genetic studies of the lac repressor. I. Correlation of mutational sites We thank J. H. Miller for providing strains P9OC and CSH22, with specific amino acid residues: construction of a colinear R. W. Simons and D. N. Arvidson for plasmids pRS415 and gene-protein map. J. Mol. Biol. 109:275-301. pDNA1, respectively, R. Zagursky for the bacteriophage vector, 22. Panet, A., J. H. van de Sande, P. C. Loewen, H. G. Khorana, XRZ5, and K. Whittaker for DNA sequence analysis. A. J. Raae, J. R. Lillehaug, and K. Kleppe. 1973. Physical This work was supported by Public Health Service grant characterization and simultaneous purification of bacteriophage 2164 GROVE AND GUNSALUS J. BACTERIOL.

T4 induced polynucleotide kinase, polynucleotide ligase and 218:97-106. deoxyribonucleotide acid polymerase. Biochemistry 12:5045- 28. Wallace, B. J., and J. Pittard. 1967. Genetic and biochemical 5050. analysis of the isoenzymes concerned in the first reaction of 23. Pittard, J., L. Camakaris, and B. J. Wallace. 1969. Inhibition of aromatic biosynthesis in Escherichia coli. J. Bacteriol. 93: 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase 237-244. (trp) in Escherichia coli. J. Bacteriol. 97:1242-1247. 29. Yanofsky, C. 1971. Tryptophan biosynthesis in Escherichia coli. 24. Rose, J. K., C. L. Squires, C. Yanofsky, H. L. Yang, and G. J. Am. Med. Assoc. 218:1026-1035. Zubay. 1973. Regulation of in vitro transcription of the 30. Yanofsky, C. 1984. Comparison of regulatory and structural tryptophan operon by purified RNA polymerase in the presence regions of genes of tryptophan metabolism. Mol. Biol. Evol. of partially purified repressor and tryptophan. Nature (London) 1:143-161. New Biol. 245:133-137. 31. Yanofsky, C., R. L. Kelley, and V. Horn. 1984. Repression is 25. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc- relieved before attenuation in the trp operon of Escherichia coli ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. as tryptophan starvation becomes increasingly severe. J. Bac- USA 74:5463-5467. teriol. 158:1018-1024. 26. Silhavy, T. J., M. L. Berman, and L. W. Enquist. 1984. Exper- 32. Zurawski, G., R. P. Gunsalus, K. D. Brown, and C. Yanofsky. iments with gene fusions. Cold Spring Harbor Laboratory, Cold 1981. Structure and regulation of aroH, the structural gene for Spring Harbor, N.Y. the tryptophan-repressible 3-deoxy-D-arabino-heptulosonic ac- 27. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinease of E. id-7-phosphate synthetase of Escherichia coli. J. Mol. Biol. coli: partial purification and some properties. J. Biol. Chem. 145:47-73.