Vol. 131, No. 1 Printed in U.S.A.

JOURNAL OF BACTERIoLOGY, July 1977, p. 153-162

Copyright ) 1977 American Society for Microbiology

Inactivation and Partial Degradation of Phosphoribosylanthranilate Isomerase-Indoleglycerol Phosphate Synthetase in Nongrowing Cultures of Escherichia coli RAYMOND D. MOSTELLER,* KAORU R. NISHIMOTO, AND RUTH V. GOLDSTEIN Department ofBiochemistry, University of Southern California, School of Medicine, Los Angeles, California 90033 Received for publication 17 January 1977

The stability of tryptophan biosynthetic enzyme activities was examined in cultures of repressor-negative (trpR) strains of Escherichia coli K-12 incubated under conditions of nutrient starvation or chloramphenicol inhibition. The results show that four of the five activities examined are stable under most nongrowing conditions, whereas one activity, indoleglycerol phosphate (InGP) synthetase, carried by the trpC protein, is unstable under most conditions tested. Phosphoribosylanthranilate (PRA) isomerase activity, which is also carried by the trpC protein, is unstable during starvation for ammonium, cysteine, or sulfate but is stable under other nongrowing conditions where InGP synthetase is not. InGP synthetase activity but not PRA isomerase activity is also diminished about twofold in cultures using glycerol as a carbon-energy source. These results indicate that one or both activities of the trpC protein is specifically inactivated under several culture conditions. Experiments with antibodies to the trpC protein show that sulfate-starved and ammonium-starved cultures contain 20 to 40% less immunologically reactive trpC protein than unstarved cultures. This indicates that the trpC protein is probably partially degraded under these conditions. During recovery from sulfate starvation or ammonium starvation, cultures slowly regain normal levels of InGP synthetase and PRA isomerase activities, suggesting that inactivation may be reversible.

The bifunctional enzyme phosphoribosylanthranilate (PRA) isomerase-indoleglycerol phosphate (InGP) synthetase [1-(2'-carboxyphenylamino)-1-deoxyribulose-5-phosphate carboxy-lyase (cyclizing), EC 4.1.1.8] of Escherichia coli catalyzes two sequential reactions in the biosynthesis of tryptophan: phosphoribosylanthranilate -- o-carboxyphenylamino de-

oxyribulose phosphate

--

indoleglycerol phos-

phate (5, 6, 10, 35). This enzyme, which is a monomeric protein (molecular weight, 45,000) with no known cofactors (6), is specified by the third gene, trpC, of the trp operon (34, 42). The kinetic properties of the enzyme demonstrate that the two active centers are distinct and nonoverlapping (5). Genetic and biochemical experiments show that the amino-terminal portion of the protein is primarily responsible for the InGP synthetase activity, and the carboxylterminal portion is principally concerned with the PRA isomerase activity (5, 34, 42). However, experiments with chain termination and deletion mutants indicate that a polypeptide of

near-normal length is needed for both activities (42). The data suggest that this bifunctional enzyme may have resulted from gene fusion, whereas in other organisms the activities are carried by separate proteins (4). While studying the kinetics of derepression of tryptophan biosynthetic enzymes in E. coli, we observed that InGP synthetase activity is not derepressed by the tryptophan analog, 3indolylacrylic acid, although other tryptophan biosynthetic enzymes are derepressed under these conditions (22). This suggests that the trpC protein may be inactivated or degraded in the presence of the analog. In other experiments with repressor-negative (trpR) strains of E. coli, we found that InGP synthetase activity but not other trp enzyme activities is diminished during chloramphenicol or valine inhibition and during growth in glycerol-supplemented medium. These results also suggest that the trpC protein may be inactivated or degraded under some conditions. At this time we had not tested PRA isomerase activity. In

153

154

MOSTELLER, NISHIMOTO, AND GOLDSTEIN

light of the interesting nature of this bifunctional enzyme and the possibility of finding proteolytic digestion in vivo of a specific protein, we extended these studies to include other culture conditions and examined the effect of some of these conditions on stability of the trpC protein. MATERIALS AND METHODS Bacteria. All bacterial strains are derivatives of the W3110 strain of E. coli K-12 and contain mutations in the genes for the repressor of the trp operon (trpR2) and for tryptophanase (tna-2). Mutations were derived from strains provided by C. Yanofsky (trpR2, tna-2, trpB9578, thr-1, ilv-1, cysB), J. R. Beckwith (metBI, pyrF502), D. E. Morse [leu277(Am)], T. S. Matney (hisG2743), and W. K. Maas (argAl) and were transferred by bacteriophage P1-mediated transduction (17). A deletion of the his operon (Ahis-8) was obtained by bacteriophage P2-mediated education (32). Mutations pro-3, rif-1, and strAl were derived in this lab. Strains A2/ A2, trpC1117, and RM593 (trpR2, tna-2, AtrpLD102/ F colV, B AtrpLD102) were obtained from C. Yanofsky. Strain IC504 was obtained from I. P. Crawford. Bacterial culture conditions. Bacterial cultures were grown with vigorous aeration at 37°C in minimal-salts medium E (39) supplemented with 0.4% glucose, 50 jg of L-tryptophan per ml, and 40 or 50 ,g of other amino acids per ml when required. Glycerol, sodium succinate, or L-proline was substituted for glucose where indicated. For starvation experiments, cells from logarithmically growing cultures (3 x 108 cells/ml) were washed free of specific nutrients by filtration on membrane filters (Millipore Corp.). In some cases, cultures were allowed to exhaust a limiting amount of substrate (glucose, proline), or centrifugation was used in place of filtration (glucose or histidine starvation). For sulfate starvation, 0.8 mM MgCl2 was substituted for MgSO4 in medium E with citrate omitted (2). Ammonium starvation medium contained 28 mM KH2PO4, 42 mM Na2HPO4, and 0.8 mM MgSO4. For recovery experiments, 0.8 mM Na2SO4 or 16.7 mM NH4Cl was added to the sulfate-starved or ammonium-starved cultures, respectively. For phosphate starvation, 40 mM KCl was substituted for KH2PO4 in single-strength tris(hydroxymethyl)aminomethane (Tris)-minimal medium (15). For inhibition experiments, chloramphenicol (20 ug/ml) or Lvaline (100 jig/ml) was added directly to logarithmically growing cultures. Enzyme and protein assays. Cell extracts were prepared by sonic disruption of washed cells in 0.1 M Tris-hydrochloride buffer, pH 7.8, as described previously (22). InGP synthetase assays were performed in 0.1 M Tris-hydrochloride buffer, pH 7.8, containing 0.2 i.mol of 1-(o-carboxyphenylamino)-1deoxyribulose-5-phosphate in a final volume of 0.5 ml (36). PRA isomerase assays were performed in 0.1 M triethanolamine-HCl buffer, pH 8.6, containing about 0.4 ,mol of PRA and excess InGP synthetase (trpClll 7 mutant extract) in a final volume of 0.5 ml (7). The amount of InGP formed in each assay

J. BACTERIOL.

was determined as indole-3-aldehyde after oxidation with periodate (36). 1-(o-Carboxyphenylamino)-ldeoxyribulose-5-phosphate and PRA were prepared as described previously (7). Where indicated, InGP synthetase activity was followed spectrophotometrically (6). Anthranilate synthetase activity was determined fluorometrically in 0.1 M Tris-hydrochloride buffer containing 10 Atmol of L-glutamine, 4 zmol of magnesium acetate, 3 Amol of mercaptoethanol, and 0.4 ,mol of chorismic acid in a final volume of 2.0 ml (7). Chorismic acid was prepared as described previously (9). Tryptophan synthetase (a or /2) subunit activity was determined in the indole to tryptophan reaction in the presence of 20 U per assay tube of the complementary subunit (36). Where indicated, the InGP-to-indole activity of tryptophan synthetase was determined in 0.1 M KPO4 buffer, pH 7.0, containing 0.2 Amol of InGP and 350 ,umol of salt-free hydroxylamine (8) in a final volume of 0.5 ml (36). InGP was prepared enzymatically from 1-(o-carboxyphenylamino)-1-deoxyribose-5-phosphate (1, 7). Protein concentrations were determined by the method of Lowry et al. (20) using crystalline bovine serum albumin as standard. Anthranilate synthetase is a tetrameric complex composed of two nonidentical polypeptides specified by the operator-proximal E and D genes of the trp operon in E. coli. PRA isomerase-InGP synthetase is specified by trpC, the third gene of the operon. The /2 and a subunits of tryptophan synthetase are specified by the fourth and fifth genes, B and A, respectively. Transcription of the operon is initiated and controlled in the promoter-operator-leader region that precedes trpE (3, 16). Preparation of antibodies and immunological determination of radioactively labeled trpC protein. PRA isomerase-InGP synthetase was purified to homogeneity by the method of Creighton and Yanofsky (6). A white New Zealand rabbit was immunized by periodic injections of the purified enzyme (50 to 100 ,ug/injection) in Freund complete adjuvant. The immunoglobulin G fraction of the immune serum was eluted from diethylaminoethyl-cellulose in 10 mM potassium phosphate, pH 8.0 (32). On immunodiffusion plates the immunoglobulin G fraction gave a single precipitin line with an extract from a derepressed culture of a tryptophan auxotroph (A2/A2). The equivalence point of the immunoglobulin G fraction was determined by precipitation with the purified enzyme. Logarithmically growing cultures (3 x 108 cells/ ml) were pulse-labeled for 10 min with L-[4,53H(N)]leucine (New England Nuclear Corp.). Portions of cultures were harvested immediately or after 3 to 3.5 h of incubation under growing or nongrowing conditions. Cell extracts were prepared as described for enzyme assays. Radioactively labeled trpC protein was precipitated in duplicate as follows. To 300 Al of buffer A (50 mM Tris-hydrochloride [pH 7.5], 2 M KCl, and 2% Triton) were added 200 Al of labeled extract, 10 ,ug of purified PRA isomerase-InGP synthetase, and the equivalent amount of the immunoglobulin G fraction (determined above). Precipitations were done at 0 to 4°C

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155

for 16 to 24 h. The resulting precipitates were growing cultures. Several isogenic or nearly washed twice with buffer B (50 mM Tris-hydrochlo- isogenic repressor-negative (trpR) strains of E. ride [pH 7.5], 1.2 M KCl, and 1.2% Triton) and once coli W3110 containing various auxotrophic muwith buffer C (50 mM Tris-hydrochloride [pH 7.5] tations were tested for trp enzyme activities and 0.1 M NaCl). Each of the washed precipitates during nutrient starvation or chloramphenicol was dissolved in 100 ,ul of 40 mM Tris-acetate (pH 6.1), 1% sodium dodecyl sulfate, 40 mM dithiothrei- inhibition (Table 1). Four of the five activities tol, and 1 mM ethylenediaminetetraacetic acid, are stable under most conditions tested. The heated for 15 min at 95°C, and subjected to electro- fifth, InGP synthetase, is reduced under all phoresis on 6% polyacrylamide gels for 2.5 h. Each nongrowing conditions except glucose starvagel was cut into 60 to 65 1-mm slices and digested in tion. PRA isomerase activity is diminished by 0.2 ml of 30% H202-1% NH40H for 2 h at 70°C or for starvation for ammonium, cysteine, or sulfate 12 to 14 h at 37°C. The digested slices were neutral- but is not significantly altered by other condiized with acetic acid and suspended in 4 ml of Aqua- tions. The results in Table 1 indicate that the sol-2 (New England Nuclear Corp.). Radioactivity loss of InGP synthetase and PRA isomerase was determined in a Beckman LS-245 liquid scintillation system. Radioactivity in total protein was activities exhibits at least three patterns: (i) determined in material precipitated with 5% trichlo- InGP synthetase activity was diminished about twofold, and PRA isomerase activity was stable roacetic acid. (e.g., chloramphenicol inhibition and proline RESULTS starvation); (ii) InGP synthetase activity was Stability of trp enzyme activities in non- diminished 80 to 90%, and PRA isomerase acTABLE 1. Effect of various nongrowing conditions on activities of tryptophan biosynthetic enzymes Relative enzyme activitiesc (%) Bacterial

strain

Relevant geno-

tRea

. .Tryptophan syntheCulture conditionsb AnthraniPRA InGP syn- 3 Sub tase a Sub synlate thetase

isomerase

thetase

lSuunit 138 god 107

aub

unit Glucose starvation 119 78 148 83 trpB9578 Glucose starvation 108 94 115 119 Chloramphenicol inhi92 94 48 92 bition (4 h) RM312 Chloramphenicol inhi69 94 34 NTe 91 bition (24 h) RM382 trpB9578 Chloramphenicol inhi89 NT 47 108d 102 bition RM362 pro-3 Proline starvation 94 113 39 125 112 RM605 Threonine starvation 85 88 45 92 102 thr-I RM312 144 NT 82 Valine inhibition 53 95 RM634 94 ilv-l Isoleucine and valine 82 86 13 109 starvation RM623 84 121 107 71 16 leu-277(Am) Leucine starvation 84 90 132 RM597 metBI Methionine starvation 88 15 RM312 117 11 93 110 145 Phosphate starvation 86 119 RM607 124 130 10 Uridine starvation pyrF502 RM386 Ahis-8 7 115 103 Histidine starvation 134 134 10 71 89 RM596 hisG2743 75 65 Histidine starvation 96 103 87 RM576 130 9 argAl Arginine starvation 118 127 7 RM312 Ammonium starvation 83 81 4 8 67 85 111 RM309 cysBI Cysteine starvation 117 108 28 7 30 RM312 Sulfate starvation a All strains are trpR2 tna-2. b Cultures were treated as indicated for 4 h except where noted. Experiments are listed in approximate order of decreasing InGP synthetase activity. c Specific activities are expressed relative to those of an untreated (growing) culture of the same strain. Typical absolute specific activities (units per milligram of protein) for untreated cultures are 2.0 for anthranilate synthetase, 14 for PRA isomerase, 3.0 for InGP synthetase, 15 for tryptophan synthetase A subunit, and 19 for tryptophan synthetase a subunit. One unit of enzyme activity in each case is the amount of activity that catalyzes the conversion of 0.1 ,umol of substrate to product in 20 min. d Activity determined in the InGP to indole reaction in the presence of 20 U of added a subunit. NT, Not tested.

RM312 RM382 RM312

156

tivity was stable or moderately diminished (e.g., uridine starvation and ammonium starvation); and (iii) both activities were markedly diminished (cysteine starvation and sulfate starvation). These observations suggest that more than one mechanism may be responsible for the loss of these activities. The enzyme activities were also examined at various times during starvation or inhibition. Representative data for chloramphenicol inhibition, ammonium starvation, and sulfate starvation are shown in Fig. 1. It should be noted that InGP synthetase activity in chloramphenicol-inhibited cells does not decrease below about 35% of control even after 24 h of inhibition (Table 1) and that PRA isomerase activity does not decrease below about 50 to 80% of control (Fig. 1B). Thus, the data indicate differences in both the rate (Fig. 1) and extent (Table 10 "Z

9 :tl

t;

0

uzu

J. BACTERIOL.

MOSTELLER, NISHIMOTO, AND GOLDSTEIN

530

K 4A di

16 0

1-

1.IJI

0

2

4 0

2

.I 4 0

2

4

time, hrs

FIG. 1. Effect of chloramphenicol inhibition, ammonium starvation, or sulfate starvation on PRA isomerase and InGP synthetase activities. Inhibition or starvation of cultures was initiated at time zero. Samples were harvested at times indicated and assayed for PRA isomerase (A) and InGP synthetase (0) activities. Specific activities are expressed relative to those of the uninhibited or unstarved culture. (A) Chloramphenicol inhibition of strain RM312 (trpR2 tna-2). (B) Ammonium starvation of strain RM805 (trpR2 tna-2 AtrpLD102). (C) Sulfate starvation of strain RM805.

1) of loss of activities. The fact that both activities are diminished under some conditions suggests that the trpC protein may be degraded or partially degraded under these conditions. Anthranilate synthetase activity is diminished by cysteine or sulfate starvation but is not significantly altered by the other conditions tested. Tryptophan synthetase a and 12 subunit activities are stable under all conditions tested. These results indicate that the four polypeptides responsible for these activities (see Materials and Methods) are stable under most nongrowing conditions. Effect of carbon-energy source on trp enzyme activities. The trp enzyme activities were also examined in cultures of strain RM312 (trpR2 tna-2) grown in minimal-salts medium supplemented with glucose, glycerol, succinate, or proline as carbon and energy source. The activities examined are not markedly altered by the different carbon-energy sources, except that InGP synthetase activity is consistently two- to threefold lower in glycerol-grown cultures (Table 2). The mechanism by which glycerol effects this diminished activity is not known. The fact that InGP synthetase activity is not diminished in cultures supplemented with succinate or proline (Table 2) shows that slow growth is not a sufflcient condition for the lower activity. Effect of nongrowing conditions on immunologically reactive trpC protein. The effect of sulfate starvation on the amount of immunologically reactive trpC protein was examined using two methods. In one case, the ability of extracts from sulfate-starved cultures to block antibody neutralization of InGP synthetase activity was determined. For these experiments, strain IC504 (trpR2 tna-2 AtrpLD102 trpB8IF colV,B AtrpLD102 trpB8) containing elevated levels of PRA isomerase-InGP synthetase was used. The elevated enzyme results from a deletion, AtrpLD102, of the trp leader region (3,

TABLE 2. Effect of carbon-energy source on activities of tryptophan biosynthetic enzymes Relative enzyme activitiesb (%) Carbon-energy sourcea

Doubling time of culture (mi) Anthranilate synthetase

PRA

isomerase

InGP synthetase

Tryptophan synthetase a subunit

100 (18.4) 100 (2.7) 100 (16.4) 100 (2.6) Glucose 75 112 31 96 81 130 Glycerol 111 144 96 76 Succinate 180 160 131 151 78 240 Proline a Cultures of strain RM312 (trpR2 tna-2) were grown for about 10 generations in single-strength minimal medium E with citrate omitted (2) and supplemented with the indicated carbon-energy source (0.4% glucose or glycerol, 1% sodium succinate, and 0.3% L-proline). b All specific activities are expressed relative to those of the glucose-supplemented culture. The absolute specific activities (units per milligram of protein) for the glucose-supplemented culture are given in parentheses.

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PRA ISOMERASE-InGP SYNTHETASE OF E. COLI

16). Antibody neutralization of InGP synthetase activity in extracts from unstarved and sulfate-starved cells is shown in Fig. 2A. The amount of antibody used in these experiments neutralizes a maximum of about 1 U of activity in the extract from unstarved cells. When extract from sulfate-starved cells is reacted with antibody before adding the antibody to the extract from unstarved cells, increased enzyme activity is observed (Fig. 2B). The relative amount of immunologically reactive trpC protein in the two extracts is indicated by the ratio of the amounts of extracts required to give the same increase in activity in the presence of antibody. The results of these experiments (Table 3) show that extracts from sulfate-starved cultures contain 20 to 30% less immunologically reactive trpC protein than extracts from un-

157

Similar results were obtained with strain RM312 (trpR2 tna-2) using immunoprecipitation of the trpC protein followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (experiment 1, Table 4). Using this method, a single major band of radioactivity that co-migrates with purified trpC protein is observed (Fig. 3). The identity of the radioactive material as trpC protein was confirmed by double-antibody precipitation and competition with unlabeled purified trpC protein (data not shown). The effect of chloramphenicol inhibition, glucose starvation, or ammonium starvation was also examined by this method (experiment 2, Table 4). The results indicate that there is a significant decrease in the amount of immunologically reactive trpC protein during ammonium starvation but not under the other two starved cultures. conditions. These findings suggest that the trpC protein is partially degraded during starvation for sulfate or ammonium. Alternatively, the protein may be partially denatured under these conditions such that its antigenicity is diminished. However, the latter possibility does not seem probable, since a denatured protein should not be stable in the cell (see Discussion). The fact that the radioactive trpC protein from each sample co-migrates with the purified enzyme indicates that the molecular weight of the remaining protein is not significantly altered. Inactivation of the trpC protein in vivo. InGP synthetase and PRA isomerase activities in extracts from sulfate-starved or ammoniumstarved cells are usually less than that expected Fg of protein FIG. 2. Tests for immunologically reactive trpC from the loss of immunologically reactive proprotein in extracts from sulfate-starved cells using tein, indicating that some portion of the trpC antibody neutralization of InGP synthetase activity. protein remains chemically intact but is enzy(A) Various amounts of extracts (indicated on the matically inactive. This could result from inacabscissa) from unstarved and sulfate-starved (4 h) tivation ofthe enzyme in vivo during starvation cultures of strain IC504 (trpR2 tna-2 AtrpLD102 or in vitro after sonic disruption of the cells. trpB8lAtrpLD102 trpB8) were assayed for InGP syn- However, using the spectrophotometric assay thetase activity in the presence or absence of 25 of (6), we found that extracts from sulfate-starved antibody preparation. Twenty-milliliter cultures cells (strain IC504) contain diminished InGP were harvested and sonically treated in 1.0 ml of when determined as soon as buffer. Protein concentrations of extracts are 2.1 mg/ synthetase activityafter disruption of the cells. ml (unstarved) and 1.3 mg/ml (starved). Symbols: possible (10 min) Extract from unstarved cells (0); extract from un- Further, the half-life of InGP synthetase activstarved cells plus antibody (a); extract from starved ity is about the same (2 h) in extracts from cells (A); extract from starved cells plus antibody unstarved and starved cells. Therefore, we con(A). (B) Various amounts of extract from the un- clude that inactivation of InGP synthetase acstarved culture of strain IC504 were assayed in the tivity probably occurs in vivo. presence of 25 p1 of antibody preparation that had Restoration of trpC enzyme activities durbeen incubated for 15 min at 0°C with 20 pl (26 pg of ing recovery from sulfate or ammonium starprotein) or 40 pi (51 pg ofprotein) of extract from the vation. InGP synthetase, PRA isomerase, and sulfate-starved culture. Symbols: Extract from un- tryptophan synthetase a subunit activities starved cells plus antibody (0; replotted from Fig. 2A); extract from unstarved cells plus antibody were determined for cultures during recovery treated with 20 p1 of extract from starved cells (0); from sulfate starvation or ammonium starvaextract from unstarved cells plus antibody treated tion (Table 5). The data show that both activities of the trpC protein increase slowly during with 40 pi of extract from starved cells (O). 0.

158

MOSTELLER, NISHIMOTO, AND GOLDSTEIN

J. BACTERIOL.

TABLE 3. Relative amounts of trpC protein in unstarved and sulfate-starved cells determined by antibody neutralization tests" InGP synthetase activity" (U/ mg of protein) in: Expt no.

Unstarved cells

1 2

SO4-starved

37 31

cells 7 3

Equivalent Extract added froct added amount of extract from starved from unstarved cellsc (gg of (pog cellse of pro-

tein)

27 26 51

21 18 32

Relative amounts of trpC proteind

(starved/unstarved)

0.78 0.69 0.63

a Twenty-milliliter cultures of strain IC504 (trpR2 tna-2 AtrpLD102 trpB8/F'AtrpLD102 trpB8) were harvested during logarithmic growth (unstarved) or after 4 h of sulfate starvation and sonically treated in 1.0 ml of buffer. Protein concentrations of the extracts are 2.0 mg/ml (unstarved) and 1.4 mg/ml (starved) for experiment 1 and 2.1 mg/ml (unstarved) and 1.3 mg/ml (starved) for experiment 2. b Specific activities in the absence of antibodies. e Several volumes of extract from the unstarved cells were assayed for InGP synthetase activity in the presence of 25 1AI of antibody preparation or in the presence of 25 ,ul of antibody preparation that had been incubated for 15 min at 0°C with the indicated amount of extract from the starved cells. The data from experiment 2 are shown in Fig. 2. The equivalent amount of extract from unstarved cells is represented by the horizontal difference between the parallel portions of curves when the data are plotted as depicted in Fig. 2B. d The relative amount of trpC protein is the ratio of the two previous columns: equivalent amount of extract from unstarved cells/extract added from starved cells.

TABLE 4. Effect of various culture conditions on trpC protein determined by immunoprecipitation Expt no.no. Expt

Conditions" Condition8a

Total

pro~~~~tein (pAg)

Total radio-

activity x 106) (cpm,

trpC protein cpm/lO cM1 cpm

12 1.5 Pulse-labeled 2,426 2,022 133 1.6 159 Chased (3.5 h) 2,120 14 1.2 Sulfate starved (3 h) 1,534 1,096 2.2 252 86 2 Pulse-labeled 2,170 85 1.8 263 2,236 Chloramphenicol inhibited (3.5 h) 79 1.6 244 Glucose starved (3.5 h) 1,930 168 Ammonium starved (3.5 h) 70 1.0 1,178 a In experiment 1, a 9-ml culture of strain RM312 (trpR2 tna) was labeled for 10 min with 109 ng of L[3H]leucine per ml (50 pACi/ml, 60 Ci/mmol). One 3-ml portion was harvested immediately, and 3-ml portions were chased with 100 ,ug of non-radioactive Lxleucine per ml or starved for sulfate in the presence of 100 Ag of non-radioactive L-leucine per ml and then harvested. Cells from each subculture were sonically treated in 0.6 ml of buffer, and 35-,ul portions of each extract were used for immunoprecipitation. Total protein and radioactivity are given for 35 ,ul of extract. In experiment 2, a 25-ml culture of strain RM312 was labeled for 10 min with 22 ng of L-[3H]leucine per ml (10 ,uCi/ml, 60 Ci/mmol). One 5-ml portion was harvested immediately, and 5-ml portions were treated as indicated in the presence of 100 ,ug of non-radioactive L-leucine per ml and then harvested. Cells from each subculture were sonically treated in 1 ml of buffer, and 200-jil portions of each extract were used for immunoprecipitation. Total protein and radioactivity are given for 200 ,ul of extract. b Radioactivity in trpC protein per 10 ,ug of total protein.

1

recovery, reaching nearly normal levels after 2 h and, as expected, tryptophan synthetase a subunit activity remains fairly constant. The fact that the activities do not resume normal levels in a short period of time (e.g., 30 min) indicates that inactivation is not readily reversible. However, the fact that total cellular protein increases only 2.5- to 3-fold during 2 h of recovery suggests that new synthesis alone cannot account for the increased activities, and

thus some reactivation must occur. Alternatively, the results obtained (Table 5) could be explained by new synthesis if the rate of trpC protein synthesis were approximately 1.5 times greater than the average rate of other cellular proteins. However, the latter possibility does not seem probable, since strain RM805 (trpR2 tna-2 AtrpLD102) used in these studies has maximal rates of enzyme synthesis specified by the operator-distal trp genes (trpC, trpB, and

explained on the same basis, since both extracts contain approximately the same amount of tryptophan synthetase activity. When an extract from a sulfate-starved culture of strain RM593 was passed through a Sephadex G-25 gel filtration column in 0.1 M Tris-hydrochloride, pH 7.8, the specific activity of InGP synthetase in the column eluate was the same (2 U/mg of protein) as in the initial extract, and no inhibitory activity was detected in later eluting fractions. Therefore, we conclude that reduced InGP synthetase activity in extracts from sulfate-starved cells is not due to a low-molecular-weight inhibitor that is loosely bound to the enzyme molecule.

15

101_ o

x E 0. u

5

_

0SXI 0

159

PRA ISOMERASE-InGP SYNTHETASE OF E. COLI

VOL. 131, 1977

40 20 slice number

io

FIG. 3. Sodium dodecyl sulfate-acrylamide go,el electrophoresis of immunoprecipitated trpC proteiin labeled in vivo. A culture of strain RM312 (trpR?2 tna-2) was labeled for 10 min with L-[3H]leucine aas described in the footnote to Table 4 (experiment 2)..A 200- portion of the cell extract was used for immiunoprecipitation, and the resulting precipitate was arnalyzed on sodium dodecyl sulfate-acrylamide go,el electrophoresis as described in Materials and Metlhods. Migration is from left to right. The position of purified trpC protein is indicated by an arrow.

DISCUSSION The results of our experiments show that the bifunctional enzyme PRA isomerase-InGP synthetase specified by the third gene (trpC) of the tryptophan (trp) operon is inactivated in nongrowing cultures of E. coli. Under most conditions, only the synthetase activity is diminished, whereas under some conditions (ammonium, cysteine, or sulfate starvation) both activities

are

diminished. We have also demon-

strated that the amount of immunologically reactive trpC protein is diminished in ammonium-starved or sulfate-starved cultures. The TABLE 5. Enzyme activities during recovery from sulfate or ammonium starvation Relative enzyme activitiesb

trpA) due to genetic lesions in trpR and trpl11. Further, the cultures were grown, starved, anid allowed to recover in the presence of exce.i 38 tryptophan (40 ,ug/ml) to avoid any effects of varying tryptophan concentrations. Tests for an inhibitor in cell extracts fro im sulfate-starved cultures. When extracts froxIn unstarved and sulfate-starved cultures of straiin RM593 (trpR2 tna-2 AtrpLDl02/F colV, ,B AtrpLD102) are mixed and assayed for InG 1P synthetase, the activity observed is signif cantly less (20 to 40%) than the sum of activiti es of the separate extracts. This suggests that afinm1l'inhibitor of InGP synthetase may be present iin one of the extracts. However, the apparent ixnhibition is due to large amounts of tryptophain synthetase activity in these extracts which nvert InGP to indole or tryptophan (10). ThulS, when strains containing mutationally altereed tryptophan synthetase are used (e.g., strai,in IC504, Fig. 2 and Table 3), the apparent inhibAtion is not observed. The difference in InGJP synthetase activity found in extracts from uinstarved and sulfate-starved cultures cannot Ibe coi

Culture condi-

tiona

Unstarved Sulfate starvation Recovery from sulfate starvation

Unstarved Ammonium starvation Recovery from ammonium starvation

(%)

R~eovery Trypto-

period

(min)

PRA InGP syn- phan synisomerase thetase thetase a subunit 100 (195) 100 (24) 100 (92)

0

45

6

92

15 30 60 120

60 67 82 97

11 28 45 106

93 94 118 139

100 (17) 15

100 (93) 103

0

15 30 60 120

100 (122) 49

38 39 88 153

20 31 61

95

110 108 109 134

a Cultures of strain RM805 (trpR2 tna-2 AtrpLD102) were starved for sulfate or ammonium for 3 h and then allowed to recover in the presence of Na2SO. or NH4Cl,

respectively. Samples were harvested at the times indicated and used to prepare cell extracts for the enzyme assays. b All specific activities are expressed relative to those of the respective unstarved cultures. Absolute specific activities (units per milligram of protein) for the unstarved cultures are given in parentheses.

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other enzyme activities specified by the trp operon (anthranilate synthetase and the tryptophan synthetase a and 82 subunits) are stable under all conditions tested (Table 1), except that anthranilate synthetase activity is moderately diminished during starvation for cysteine or sulfate. We conclude that the proteins responsible for these activities are stable under these conditions. The mechanism(s) of inactivation of PRA isomerase and InGP synthetase is not known. The rate and extent of inactivation observed under different conditions (Table 1 and Fig. 1) suggest that more than one mechanism may be involved. The fact that PRA isomerase activity is diminished only under conditions where InGP synthetase activity is markedly reduced (Table 1) suggests that loss of the synthetase activity may be a prerequisite for loss of the isomerase activity. For example, denaturation of the amino terminal portion of the trpC protein resulting in loss of InGP synthetase activity could lead to proteolysis of the polypeptide chain and concomitant loss of the PRA isomerase activity. Other investigators have reported the loss of various enzyme activities during nutrient starvation (40, 41) or in stationary-phase cells (24, 31, 33; E. M. Tecson and E. W. Westhead, Fed. Proc. 33: 1345, 1974) of E. coli, but the mechanism or mechanisms of inactivation have not been elucidated. In two cases, the loss of activity is correlated with the loss of the immunologically reactive protein (protein synthesis initiation factor IF-3, reference 32; aspartokinase III, E. M. Tecson and E. W. Westhead, Fed. Proc. 33:1345, 1974). During induction of bacteriophage X lysogens with ultraviolet-light irradiation or mitomycin C treatment, the bacteriophage repressor protein is inactivated by proteolytic cleavage (30). We have found that InGP synthetase is not inactivated under similar conditions (ultraviolet irradiation to 10% survival of cells followed by incubation in the dark for 30 or 60 min; Mosteller and Nishimoto, unpublished observations). Goldberg et al. (11, 12, 29) have shown that abnormal proteins containing amino acid analogs are degraded more rapidly in vivo than normal proteins and that these proteins are more susceptible to proteolytic digestion in vitro than normal proteins. Other investigators have shown that nonsense or deletion fragments of f-galactosidase (14, 19, 38) or lac operon repressor (28) are degraded more rapidly than the normal proteins. Recently, Zipser and Bhavsar (43) demonstrated that some missense mutations in lacZ also result in degradation of

J. BACTERIOL.

,3-galactosidase. The reason that abnormal or, mutant proteins are degraded faster than normal proteins is not known, although it has been suggested that the conformation of a protein may determine its stability (13). The loss of immunologically reactive trpC protein during ammonium or sulfate starvation (Tables 3 and 4) indicates that the protein is either partially degraded or that its tertiary structure is unfolded (denatured) such that its antigenicity is diminished. Our data do not distinguish these possibilities. However, since proteins with abnormal structure are known to be degraded in E. coli (see above), we believe that a trpC protein with altered tertiary structure would be unstable in the cell, and we therefore favor the idea of at least partial degradation of the protein. The fact that radioactive trpC protein obtained by immunoprecipitation from extracts of starved cells exhibits normal molecular weight during sodium dodecyl sulfate-acrylamide gel electrophoresis is consistent with both possibilities, although this observation suggests that degradation, if it occurs, does not result solely from cleavage of a fragment from the terminus of each protein molecule. On the other hand, the results are consistent with the possibility that a portion (20 to 40%) of the polypeptide chains are degraded and that the remainder are left chemically intact. Nutrient starvation is known to increase the release of radioactive amino acids from previously labeled bacterial proteins (13, 23, 25-27, 40). We have found that 3 h of sulfate starvation of strain RM312 (see Table 1) increases the release of L-[3H]leucine (10-min labeling period, 10 ,uCi/ml, 60 Ci/mmol) from 15% (unstarved) to 27% (starved) of the total radioactivity incorporated. Assuming that PRA isomerase-InGP synthetase comprises 0. 1% of the total cellular protein (see Table 4), complete digestion of 30% of the enzyme molecules would account for only about 2.5% of the increased release of radioactive leucine. In a normal (trpR+) strain, this protein comprises at least 10-fold less of the total protein, depending on culture conditions, and thus would contribute proportionately less to the released amino acids. We have found that InGP synthetase activity in derepressed cultures of Salmonella typhimurium strain LT-2 is unstable during ammonium or sulfate starvation (Mosteller and Nishimoto, unpublished observations). These observations show that instability of the enzyme activity is not strain specific. However, we have also found that InGP synthetase activity in S. typhimurium but not in E. coli is derepressed by the

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PRA ISOMERASE-InGP SYNTHETASE OF E. COLI

tryptophan analog 3-indolylacrylic acid (22), suggesting that the E. coli but not the S. typhimurium enzyme is unstable under these conditions. Differences in stability of the activities could be due to differences in the primary sequence of the two proteins (18). ACKNOWLEDGMENTS We are grateful to Douglas Stevenson for advice regarding immunological procedures and to Varian Hagglund for typing the manuscript. These studies were supported by Public Health Service grant CA14089 from the National Cancer Institute and grant GM23292 from the National Institute of General Medical Sciences.

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