Vol. 128, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Oct. 1976, p. 202-211 Copyright ) 1976 American Society for Microbiology
Regulation of Histidinol Phosphate Aminotransferase Synthesis by Tryptophan in Bacillus subtilis DOUGLAS. A. WEIGENT' AND EUGENE W. NESTER*
Department of Microbiology and Immunology, School of Medicine, University of Washington, Seattle, Washington 98195
Received for publication 19 July 1976
The effect of tryptophan on the synthesis of histidinol phosphate aminotransferase and prephenate dehydrogenase has been examined. The genes specifying two enzymes for tryptophan biosynthesis (anthranilate synthase and tryptophan synthase-B) were found to be derepressed in a temporal sequence according to their chromosomal location. The genes for histidinol phosphate aminotransferase and prephenate dehydrogenase were derepressed simultaneously approximately 8 min after tryptophan synthase-B. When excess tryptophan was added to a derepressed culture, the pattern of repression of trpE (anthranilate synthase), trpB (tryptophan synthase-B), hisH (histidinol phosphate aminotransferase), and tyrA (prephenate dehydrogenase) was found to be simultaneous. Methyl tryptophan-resistant mutants, which synthesize elevated levels of the tryptophan enzymes, also synthesized elevated levels of histidinol phosphate aminotransferase. Qualitatively similar data were obtained in a temperaturesensitive tryptophanyl-transfer ribonucleic acid synthetase mutant grown at elevated temperatures. The time at which messenger ribonucleic acid was synthesized for anthranilate synthase, tryptophan synthase-B, histidinol phosphate aminotransferase, and prephenate dehydrogenase in the presence of actinomycin D indicated that ordered enzyme synthesis was a result of ordered transcription of the corresponding portion of the genome. The effect of the drug rifampin on enzyme synthesis was also examined. The addition of this drug halted the transcription of anthranilate synthase very rapidly, but later regions of the tryptophan region continued to be transcribed. The transcription of the hisH and tyrA genes was also shut off rapidly after rifampin was added. The significance of these observations to the control of transcription of the hisH gene by tryptophan is discussed. In the past several years, a number of cases have been studied in which starvation for a single amino acid causes elevation of biosynthetic enzymes of several apparently unrelated amino acid pathways (5, 27). This phenomenon, which has been called cross-pathway regulation, can be reciprocal (6). An example of crosspathway regulation includes the histidine, tryptophan, and arginine pathways in the eukaryote Neurospora crassa (7). In this organism, starvation for any one of the three amino acids increases enzyme synthesis in all three pathways (5). Schurch et al. (22) have demonstrated elevation of histidine, tryptophan, and arginine biosynthetic enzymes in histidinestarved Saccharomyces cerevisiae. The molecular basis of these cross-pathway interactions is not known. I Present address: The Hormel Institute, University of Minnesota, Austin, MN 55912.
Cross-pathway regulation has also been reported in the prokaryote Bacillus subtilis. Almost 10 years ago Chapman and Nester (8) isolated mutants resistant to analogues of either histidine or tyrosine. A high proportion of both types of mutants had elevated levels of enzymes of both tyrosine and histidine biosynthesis. Since aminoacyl-transfer ribonucleic acid (tRNA) synthetases and tRNA's have been implicated in the regulation of enzyme synthesis in several well-studied systems (9, 20), these components were examined in both wild-type and mutant cells. No detectable differences could be observed (16). More recently, Roth and Nester (21) presented evidence for a new form of cross-pathway regulation in B. subtilis. The activity of a gene for a histidine biosynthetic enzyme, histidinol phosphate aminotransferase, as well as of a contiguous gene for tyrosine synthesis, prephenate dehydrogenase, was shown to be con-
202
VOL. 128, 1976
HISTIDINOL PHOSPHATE AMINOTRANSFERASE
trolled under certain conditions in parallel with the trp loci. In this situation, unlike the other examples of cross-pathway regulation that have been described, the regulation of histidine synthesis was limited to the single gene for histidine synthesis that was linked to the trp loci. Apparently, the parallel control of synthesis of enzymes of tryptophan, histidine, and tyrosine synthesis does not result from an accumulation of an intermediate in any of these pathways. Mutants blocked in the first enzyme of histidine and aromatic amino acid synthesis still have these three sets of enzymes under parallel control. To account for all of these data, Roth and Nester (21) proposed a read-through mechanism in which RNA synthesis, which is initiated at the beginning of the trp gene cluster, continues uninterrupted into the structural genes for histidinol phosphate aminotransferase and prephenate dehydrogenase. This paper presents data on experiments designed to elucidate further the molecular basis of this unique form of control. MATERIALS AND METHODS Bacterial strains. The strains of B. subtilis used in this investigation are described in Table 1. Growth of bacteria. The minimal salts-glucose medium of Spizizen (23) supplemented with amino acids as indicated in the various experiments was used throughout this study. Derepression of the tryptophan biosynthetic enzymes was achieved either by a limitation in exogenous tryptophan or growth at elevated temperatures for 2 h in the presence of L-tryptophan in the case of the temperaturesensitive tryptophanyl-tRNA synthetase mutant. To derepress the trp operon by limiting tryptophan, the cells were grown in 6 liters of Spizizen (23) minimal glucose medium supplemented with 0.05% casein hydrolysate, excess (50 gg/ml) phenylalanine, tyrosine, tryptophan, and histidine. The cells were grown at 30°C in a New Brunswick fermenter model MF-14 with forced aeration (5 liters/min) and agitation (200 rpm). The exponentially growing culture was harvested and transferred into prewarmed medium that lacked tryptophan. Samples (200 ml) were transferred to cold buffer containing chloramphenicol (100 ,ig/ml) at the times indicated, rapidly
chilled, immediately centrifuged, and stored at -70°C. Growth was monitored with a Klett-Summerson colorimeter (filter no. 42). In the presence of excess tryptophan, the growth of these organisms proceeds with a generation time of approximately 40 min; in the absence of exogenous tryptophan, their growth rate is reduced to a generation time of approximately 8 h. In experiments with mtr strains grown with an excess of the required amino acids, the cells were grown until the late exponential phase and then rapidly chilled and harvested. Preparation of enzyme extracts. Cell extracts were routinely prepared either by lysing the cells with lysozyme (21) or subjecting the thick suspension to sonic disruption (18). For the assay of anthranilate synthase, tryptophan synthase-B and prephenate dehydrogenase, cells were lysed in 0.1 M potassium phosphate buffer, pH 7.5, containing 40% glycerol, 0.1 mM ethylenediaminetetraacetate, 0.05 M glutamine, 0.6 mM pyridoxal phosphate, 200 ,ug of lysozyme per ml, and 10 ug of deoxyribonuclease per ml at 37°C for 20 min. For the assay of histidinol phosphate aminotransferase, cells were disrupted in 0.05 M potassium phosphate buffer, pH 7.5, containing 0.6 mM mercaptoethanol and 20 ;Lg of phenylmethylsulfonylfluoride per ml by sonic treatment at maximum power for 4 min in an MSE ultrasonic disintegrator. Cell debris was sedimented by centrifugation at 25,000 x g for 30 min at 4°C in a Sorvall centrifuge, and the supernatant fluid was used in enzyme assays. For certain experiments, where indicated, the crude cell extract was passed through a column of Sephadex G-25. Enzyme assays. Tryptophan synthase-B was assayed in crude extracts prepared in glycerol buffer as described by Hoch et al. (13). Anthranilate synthase was assayed by measuring the amount of anthranilate synthesized by an increase in fluorescence at 400 nm (activation at 335 nm) in a recording Aminco-Bowman spectrofluorometer (21). Histidinol phosphate aminotransferase activity was assayed as described by Chapman and Nester (8), except that pyridoxal phosphate was used at 0.6 mM.
Prephenate dehydrogenase activity was measured as described by Roth and Nester (21), except that quinicrine-hydrochloride was included in the reaction mixture at a final concentration of 0.1 mM. Enzyme activities are expressed as relative specific activities based on a value of 1.0 for wild-type
TABLE 1. Bacillus subtilis strains Strain designation Description Transformable strain of B. subtilis blocked in indoleglycerol168 phosphate synthesis (9) WB 746 Spontaneous revertent of 168 WB 3159 Spontaneous mtr derivative of WB 746 with high constitutive level of the tryptophan biosynthetic enzymes (14) GSY 266 A derivative of 168 and a methionine auxotroph; lacks 3-cystathi-
Source J. Spizizen
Our laboratory Our laboratory W. Steinberg
onase
GSY 1306
203
Strain GSY 266 with a second mutation in tryptophanyl-tRNA synthetase
W. Steinberg
204
WEIGENT AND NESTER
J. BACTERIOL.
cells grown in minimal medium. The specific activity in wild-type cells grown in minimal medium at 30°C in terms of nanomoles of substrate used or product formed each minute per milligram of protein is: tryptophan synthase-B, 1; anthranilate synthase, 0.01; prephenate dehydrogenase, 5; histidinol phosphate aminotransferase, 1.0. Chemicals. Prephenic acid was prepared from chorismic acid as previously described (17). All other chemicals were purchased from commercial sources and used without further purification. Isolation of analogue-resistant mutants. Analogue-resistant mutants were isolated by plating approximately 108 cells of a log-phase culture of strain 746 on Spizizen (23) minimal medium containing 1 mg of 5-methyltryptophan per ml. After incubation at 37°C for 48 to 72 h, 20 resistant colonies were streaked onto fresh plates of the same medium, and colonies from this plate were purified two additional times on minimal medium without the analogue.
containing chloramphenicol. The specific activity of each enzyme was determined in each sample. The relative specific activities were plotted as a function of the sampling time (Fig. 2 and 3). Within the limits of accuracy of the assays, the specific activities of each of these enzymes began to increase in a temporal se-
24 20 -
16 t--
RESULTS Time course of derepression. The readthrough model proposed by Roth and Nester (21) predicts that gene products will be synthesized sequentially with the order corresponding to the sequence on the genetic map (Fig. 1). To determine whether this prediction is fulfilled, we examined the kinetics of derepression of four genes that had been mapped in the proximal (anthranilate synthase), central (tryptophan synthase-B), and distal regions (histidinol phosphate aminotransferase and prephenate dehydrogenase) within the aromatic cluster
a)Q. 12 -
Cr) a)
0 cr a)
8 _
4 _ I-'
0
(19).
Cultures of B. subtilis strain 168 growing exponentially in Spizizen (23) minimal salts medium supplemented with excess amounts of phenylalanine, tryptophan, histidine, and tyrosine were derepressed for synthesis of the tryptophan enzymes by transferring the cells to the identical medium lacking tryptophan. At various times before and after tryptophan depletion, samples were transferred to cold buffer mtr
aroF
arB
10 20 30 40 50 60 70 80 Time in Min.
FIG. 2. Derepression of trpE, trpB, and hisH at 30°C in strain 168. The cells were cultured as described in Materials and Methods. Samples were transferred to cold buffer containing chloramphenicol at the times indicated. Extracts were prepared and passed through Sephadex G-25 to determine the specific activities of anthranilate synthase (a), tryptophan synthase-B (0), and histidinol phosphate aminotransferase (x).
trEtrD
trCtrFtrptrA
hisH trA
aro E
Origin FIG. 1. Genetic map of aromatic amino acid mutations in Bacillus subtilis. The enzymes specified by the genes listed are: mtr, methyltryptophan resistance; aroF, chorismate synthase; aroB, dehydroquinate synthase; trpE, anthranilate synthase; trpD, phosphoribosyl transferase; trpC, indoleglycerol-phosphate synthase; trpF, PRA isomerase; trpB, tryptophan synthase-B; trpA, tryptophan synthase-A; hisH, histidinol phosphate aminotransferase; tyrA, prephenate dehydrogenase; aroE, enolpyruvylshikimate-5-phosphate synthase. '
HISTIDINOL PHOSPHATE AMINOTRANSFERASE
VOL. 128, 1976
20 I 16 [ f--
.)_ .)_
12 H
._
a) 0. a/)
8H
.) 0
0L)
4 0 -z-
n
I
I 0
10 20 30 40 50 60 70 80 TIME IN MIN.
FIG. 3. Derepression of trpB, hisH, and tyrA at 30°C in strain 168. The experimental design was identical to that described in the legend to Fig. 2. Samples were transferred to cold buffer containing chloramphenicol at the times indicated and treated as described in Materials and Methods to determine specifwc activities oftryptophan synthase-B (0), histidinol phosphate aminotransferase (0), and prephenate dehydrogenase (x). quence: first anthranilate synthase and then tryptophan synthase-B, followed by the simultaneous appearance of histidinol phosphate aminotransferase and prephenate dehydrogenase. The sequence observed for the four enzymes corresponds with the order of the respective genes on the chromosome. The average lengths of time between the inflection in the growth rate owing to tryptophan limitation and the times at which the specific activities of each of the enzymes began to increase are compiled in Table 2. The intervals between derepression of one gene and the next gene measured are also shown. These data confirm the sequence in the appearance of the four enzyme activities. The time at which histidinol phosphate aminotransferase and prephenate dehydrogenase appeared could not be distinguished even when the cells were grown at a lower temperature (data not shown). Intervals of the same order of magnitude as observed in this study have also been observed for the induction of f3-galactosidase and transacetylase in Escherichia coli (2), as well as for the products of several genes of histidine synthesis in
205
Salmonella typhimurium (10). Effect of tryptophan on enzyme synthesis during derepression. To examine the effect of tryptophan on enzyme synthesis, the derepressed rate of increase in synthesis of the various enzymes was established by the assay of samples at intervals for 30 min under conditions in which growth was limited by tryptophan. Excess L-tryptophan was then added to the culture, and sampling continued for another 30 min. The specific activity of each enzyme was determined in each sample, and their relative specific activities were plotted as a function of the sampling time (Fig. 4). When Lr tryptophan was added to a derepressed culture, further increases in the specific activities of all four of the enzymes examined came to a halt simultaneously. Therefore, the pattern of repression of enzyme synthesis was not in the same temporal sequence as that observed for derepression of enzyme synthesis. Effect of regulatory mutants of tryptophan synthesis on histidine and tyrosine enzyme synthesis. A temperature-sensitive tryptophanyl-tRNA synthetase mutant of B. subtilis was recently isolated by Steinberg and Anagnostopoulos (25). They reported that at elevated temperatures both anthranilate synthase and tryptophan synthase-B were coordinately derepressed (24). The possibility that tryptophanmediated control of histidinol phosphate aminotransferase in these mutants was in some way altered from that resulting from tryptophan limitation was investigated. The level of histidinol phosphate aminotransferase in cells grown at 370C is shown in Table 3. The mutant strain (GSY 1306) was derepressed in both tryptophan and histidine enzyme synthesis as compared with the wild-type parental strain, GSY 266. The level of anthranilate synthase, tryptophan synthase-B, and histidinol phosphate TABLE 2. Summary of derepression of trpE, trpB, hisH, and tyrA in B. subtilis 168 Gene
trpE
Derepression timeP 5
Interval (min)
2.0 10.0
trpB
14 + 3.1
hi&H
22 + 2.8
8.0 1.0 23 2.0 a The data are the average of 10 experiments. Derepression at 3000 took place shortly after an observed reduction in the growth rate. The derepression time is the time between the reduction of growth rate and the time at which an increase in the rate of the enzyme synthesis was observed.
tyrA
206
J. BACTERIOL.
WEIGENT AND NESTER
specifi'c activity of histidinol phosphate aminotransferase (Fig. 5). It was found that the ratio of activity of tryptophan synthase-B and histidinol phosphate aminotransferase, within the limits of accuracy of the assays, remained essentially constant. Although the mutations were not mapped, other evidence suggests that they all occur at one genetic locus, the mtr locus (14). Timing of RNA synthesis for enzymes syn-
TR,p 40 ._
ion
o0
0
10
q) X cv 0
c1
20
3D
40
50
60
TABLz 3. Derepression of trpE, trpB, and hisH in trp Sl and wild-type strains at 30 and 37°C Relative sp act HistidiStrain Athrlfl Trpo nol phosStrain ilate phan syn- phate synthase thase-B transferase 1.0 0.7 GSY 266 (wild 30 0.9 type) 30 1.5 1.3 1.0 GSY 1306 (Si) GSY 266 37 0.5 0.5 1.0 GSY 1306 37 5.0 4.0 2.2
TeC)
4
.4 C)
I
3
I C)
2
U)
| ,~~~~
,
.
.
20
30
40
a)
0
10
50
60
Time in Mn. 20 FIG. 4. The effect oftryptophan on derepression of trpE, trpB, hisH, and tyrA in strain 168. The experimental design was identical to that described in the 16 legend to Fig. 2, except tryptophan (20 uglml) was added at 30 min. Samples were removed, before and after the addition of tryptophan, to cold buffer containing chloramphenicol at the times indicated and r-C treated as described in Materials and Methods to < =0 12 determine specifw activities of anthranilate synthase (@), tryptophan synthase-B (0), histidinol phosphate aminotransferase (A), and prephenate dehydrogen- cn c 8 ase (x).
.
.5
_
0
.
-C
0)
_
0
0
aminotransferase increased five-, four-, and 0 twofold, respectively, above the basal level. Thus, it would appear that at supraoptimal temperatures tryptophanyl-tRNA synthetase mutants have lost control over the synthesis of I I histidinol phosphate aminotransferase as well 2 4 0 1 3 as the tryptophan biosynthetic enzymes. Relative Specific Activity In B. subtilis, Nester et al. (19) described a Histidinol phosphate aminotransferase gene conferring resistance to 5-methyltryptoFIa. 5. Relative specific activities of tryptophan phan. Mutation in this locus results in constitutive synthesis of the tryptophan enzymes (14, synthase-B and histidinol phosphate aminotrans19). The gene lies to the left ofaroF (Fig. 1) (12, ferase in mtr mutants. Enzyme activities were meain extracts prepared fiom cells grown in mini19) and thus is not contiguous to the trp operon. sured mal medium supplemented with L-phenylalanine The level of histidinol phosphate aminotrans- and (50 pg eachlml). Numbers for enferase in 20 independently isolated 5-methyl- zymeL-histidine activities are relative specific activities based on tryptophan-resistant mutants was determined. the value of 1.0 for wild-type cells grown in minimal The relative specific activity of tryptophan syn- medium. Each point is an average determination thase-B was plotted with respect to the relative from two experiments for each mutant. ctl-4O.
0
I
I
HISTIDINOL PHOSPHATE AMINOTRANSFERASE
VOL. 128, 1976
thesized during derepression. If ordered enzyme synthesis resulted from a selective control of messenger RNA (mRNA) synthesis, then that control would be restricted to the time in which the particular cistron was being transcribed. To test whether such a control might be operating, it was necessary to know the times at which mRNA for anthranilate synthase, tryptophan synthase-B, histidinol phosphate aminotransferase, and prephenate dehydrogenase were synthesized, and whether the RNA templates for each of these enzymes had a short half-life. Figure 6 presents the results of an experiment designed to measure the times at which mRNA for anthranilate synthase and tryptophan synthase-B were synthesized as well as 0.1
*,)20
.I6OD 16
Z 0
=0 8
(n CL
w c >
1 II 10
>%
.> I.
I
30
40
50
60
70
20 0
a) 16
_i ~
20
12 0
8
en 0
0., a) =a >
c;s
g4 O
-.
-O
10 20 30 40 50 60 70 Time in Min.
FIG. 6. Timing of RNA synthesis for enzymes synthesized during derepression at 37°C in strain 168. The experimental design was identical to that described in the legend to Fig. 2. At the indicated times, samples were removed and added to: (a) cold phosphate buffer containing chloramphenicol and (0) actinomycin D (3 pg/ml). The actinomycin Dtreated samples were incubated at 37°C until 70 min, at which time enzyme synthesis was arrested by the addition of cold phosphate buffer containing chloramphenicol (100 pg/ml). The cells were collected by centrifugation and prepared for enzyme assays as described in Materials and Methods. (A) Anthranilate synthase; (B) tryptophan synthase-B.
207
the stability of these mRNA molecules. The experiment consisted of starving a culture for tryptophan and at regular intervals transferring samples to warm medium containing actinomycin D. This sample of the culture was incubated in the presence of actinomycin D for 10 min after the last sample was removed. All protein synthesis in the samples treated with actinomycin D was then stopped by the addition of cold buffer containing chloramphenicol. For controls, a second set of samples, removed at the same time as the experimental samples, was added directly to cold buffer containing chloramphenicol. If RNA templates for these enzymes were present at any time after the addition of actinomycin D, the level of enzyme activity at these times should have been the same as in the sample removed at 70 min. This was clearly not the case (Fig. 6). The addition of actinomycin D at any time halted synthesis of anthranilate synthase and tryptophan synthase-B within 2 to 5 min. Thus, the stability of messages for these two enzymes was approximately the same as the stability of pulse-labeled RNA (15). The gene specifying anthranilate synthase was transcribed approximately at the time tryptophan became depleted, and the message from this cistron was translated approximately 3 min later. A similar pattern between transcription and translation of tryptophan synthase-B was observed. A similar experiment was conducted to determine the times at which mRNA for histidinol phosphate aminotransferase and prephenate dehydrogenase were synthesized and to measure the stability of these mRNA templates. The results are shown in Fig. 7. Actinomycin D added at any time shut off histidinol phosphate aminotransferase and prephenate dehydrogenase synthesis within 2 to 5 min. This experiment further demonstrated that the time difference between the appearance of polypeptides of the tryptophan operon and the enzymes coded by the linked genes of histidine and tyrosine synthesis was not a result of a delay in the translation of a previously synthesized message. Therefore, the times at which enzymes appear during derepression appear to be transcriptionally controlled. The data also argue against any assembly of protein subunits accounting for the delays in enzyme appearance. The present study cannot rule out the less likely possibility that an assembly process proceeded during preparation of the extract for enzyme analysis. Intervals of the same order of magnitude between transcription and translation as observed in this study have also been
208
WEIGENT AND NESTER
J. BACTZRIOL.
oE .o4 0) 04-
40/
Q. 0 u.2 a) 4.-
C
o -.
O 0 4-
I
I
10 20 30 40 50 60 70
a)
cn .4= o
o O.
v
plotted as a function of the sampling time. The results of these experiments are shown in Fig. 8. The synthesis of tryptophan synthase-B was shut off approximately 10 min after anthranilate synthase. This is in good agreement with the data on the kinetics of appearance of this enzyme after tryptophan starvation (Fig. 2). These data suggest that RNA synthesis was initiated at the beginning of the tryptophan operon and continued uninterrupted through the tryptophan genes. The specific activities of histidinol phosphate aminotransferase and prephenate dehydrogenase decreased immediately upon the addition of rifampin. The data suggest that these latter two enzytnes are translated from separate monocistronic messages which required new initiations of transcription. An alternative explanation of rifampin inhibition of transcription initiations is that the drug indirectly caused a rapid intracellular accumulation of tryptophan, which then led to
0) >.
V 0)
c.
r.
0
10 20 30 40 50 60 70 Time in Min.
FIG. 7. Timing of RNA synthesis for enzyme synthesized during derepression at 37°C in strain 168. The experimental design was identical to that described in the legend to Fig. 6. Samples were removed at the indicated times and treated as described in Materials and Methods to determine specific activities of histidinol phosphate aminotransferase (A) and prephenate dehydrogenase (B).
observed in the timing of induced a-glucosidase and histidase synthesis during outgrowth of Bacillus cereus spores (26). Effect of rifampin on enzyme synthesis during derepression. Rifampin specifically inhibits the initiation of transcription without influencing the completion of RNA chains being synthesized (11). We used this drug to determine the number and sites at which transcription is initiated during derepression of the loci concerned with tryptophan, histidinol phosphate aminotransferase, and prephenate dehydrogenase activity. For these experiments, strain 168 was grown at 30°C in a medium lacking tryptophan to derepress the tryptophan operon. Rifampin was then added to the culture. At intervals before and after the addition of rifampin, samples were transferred to cold buffer containing chloramphenicol. The specific activity of each enzyme was determined in each sample and the relative specific activities were
0)
c)
0
10 20 30 40 50 60 70
_aL 2 cn ,H 03 _
-A
x
a)
._> I
ILa)
I
0
I
I
I
10 20 30 40 50 60 70 Time in Min.
FIG. 8. The effect of rifampin on derepression of trpE, trpB, hisH, and tyrA in strain 168. The experimental design was identical to that described in the legend to Fig. 4, except rifampin (20 pg/ml) was added at 40 min. The samples were treated as described in Materials and Methods to determine specific activities of anthranilate synthase (O), tryptophan synthase-B (0), histidinol phosphate aminotransferase (A), and prephenate dehydrogenase (x).
HISTIDINOL PHOSPHATE AMINOTRANSFERASE
VOL. 128, 1976
normal repression of the trp operon. To establish that rifampin inhibition did not result solely from tryptophan-mediated repression, we performed an experiment identical to the one described in Fig. 8 with a regulator-constitutive strain, mtr. The results for anthranilate synthase, tryptophan synthase-B, histidinol phosphate aminotransferase, and prephenate dehydrogenase in typical experiments are compiled in Fig. 9. The data indicate that upon the addition of rifampin, the specific activities of anthranilate synthase, histidinol phosphate aminotransferase, and prephenate dehydrogenase decreased immediately, whereas the decrease in specific activity for tryptophan syn-
4>100 80
-o o
',60 a) 0
_~~~~
40
a)
-.20 0 c0 0
5
o~~-o-o
o
F I
I
---x
x
x
x--
41
I* A_
0.
Q._
_a_
x 1-
.6
a) co
X
10 20 30 40 50 60 70
JOURNAL OF BACTERIOLOGY, Oct. 1976, p. 202-211 Copyright ) 1976 American Society for Microbiology
Regulation of Histidinol Phosphate Aminotransferase Synthesis by Tryptophan in Bacillus subtilis DOUGLAS. A. WEIGENT' AND EUGENE W. NESTER*
Department of Microbiology and Immunology, School of Medicine, University of Washington, Seattle, Washington 98195
Received for publication 19 July 1976
The effect of tryptophan on the synthesis of histidinol phosphate aminotransferase and prephenate dehydrogenase has been examined. The genes specifying two enzymes for tryptophan biosynthesis (anthranilate synthase and tryptophan synthase-B) were found to be derepressed in a temporal sequence according to their chromosomal location. The genes for histidinol phosphate aminotransferase and prephenate dehydrogenase were derepressed simultaneously approximately 8 min after tryptophan synthase-B. When excess tryptophan was added to a derepressed culture, the pattern of repression of trpE (anthranilate synthase), trpB (tryptophan synthase-B), hisH (histidinol phosphate aminotransferase), and tyrA (prephenate dehydrogenase) was found to be simultaneous. Methyl tryptophan-resistant mutants, which synthesize elevated levels of the tryptophan enzymes, also synthesized elevated levels of histidinol phosphate aminotransferase. Qualitatively similar data were obtained in a temperaturesensitive tryptophanyl-transfer ribonucleic acid synthetase mutant grown at elevated temperatures. The time at which messenger ribonucleic acid was synthesized for anthranilate synthase, tryptophan synthase-B, histidinol phosphate aminotransferase, and prephenate dehydrogenase in the presence of actinomycin D indicated that ordered enzyme synthesis was a result of ordered transcription of the corresponding portion of the genome. The effect of the drug rifampin on enzyme synthesis was also examined. The addition of this drug halted the transcription of anthranilate synthase very rapidly, but later regions of the tryptophan region continued to be transcribed. The transcription of the hisH and tyrA genes was also shut off rapidly after rifampin was added. The significance of these observations to the control of transcription of the hisH gene by tryptophan is discussed. In the past several years, a number of cases have been studied in which starvation for a single amino acid causes elevation of biosynthetic enzymes of several apparently unrelated amino acid pathways (5, 27). This phenomenon, which has been called cross-pathway regulation, can be reciprocal (6). An example of crosspathway regulation includes the histidine, tryptophan, and arginine pathways in the eukaryote Neurospora crassa (7). In this organism, starvation for any one of the three amino acids increases enzyme synthesis in all three pathways (5). Schurch et al. (22) have demonstrated elevation of histidine, tryptophan, and arginine biosynthetic enzymes in histidinestarved Saccharomyces cerevisiae. The molecular basis of these cross-pathway interactions is not known. I Present address: The Hormel Institute, University of Minnesota, Austin, MN 55912.
Cross-pathway regulation has also been reported in the prokaryote Bacillus subtilis. Almost 10 years ago Chapman and Nester (8) isolated mutants resistant to analogues of either histidine or tyrosine. A high proportion of both types of mutants had elevated levels of enzymes of both tyrosine and histidine biosynthesis. Since aminoacyl-transfer ribonucleic acid (tRNA) synthetases and tRNA's have been implicated in the regulation of enzyme synthesis in several well-studied systems (9, 20), these components were examined in both wild-type and mutant cells. No detectable differences could be observed (16). More recently, Roth and Nester (21) presented evidence for a new form of cross-pathway regulation in B. subtilis. The activity of a gene for a histidine biosynthetic enzyme, histidinol phosphate aminotransferase, as well as of a contiguous gene for tyrosine synthesis, prephenate dehydrogenase, was shown to be con-
202
VOL. 128, 1976
HISTIDINOL PHOSPHATE AMINOTRANSFERASE
trolled under certain conditions in parallel with the trp loci. In this situation, unlike the other examples of cross-pathway regulation that have been described, the regulation of histidine synthesis was limited to the single gene for histidine synthesis that was linked to the trp loci. Apparently, the parallel control of synthesis of enzymes of tryptophan, histidine, and tyrosine synthesis does not result from an accumulation of an intermediate in any of these pathways. Mutants blocked in the first enzyme of histidine and aromatic amino acid synthesis still have these three sets of enzymes under parallel control. To account for all of these data, Roth and Nester (21) proposed a read-through mechanism in which RNA synthesis, which is initiated at the beginning of the trp gene cluster, continues uninterrupted into the structural genes for histidinol phosphate aminotransferase and prephenate dehydrogenase. This paper presents data on experiments designed to elucidate further the molecular basis of this unique form of control. MATERIALS AND METHODS Bacterial strains. The strains of B. subtilis used in this investigation are described in Table 1. Growth of bacteria. The minimal salts-glucose medium of Spizizen (23) supplemented with amino acids as indicated in the various experiments was used throughout this study. Derepression of the tryptophan biosynthetic enzymes was achieved either by a limitation in exogenous tryptophan or growth at elevated temperatures for 2 h in the presence of L-tryptophan in the case of the temperaturesensitive tryptophanyl-tRNA synthetase mutant. To derepress the trp operon by limiting tryptophan, the cells were grown in 6 liters of Spizizen (23) minimal glucose medium supplemented with 0.05% casein hydrolysate, excess (50 gg/ml) phenylalanine, tyrosine, tryptophan, and histidine. The cells were grown at 30°C in a New Brunswick fermenter model MF-14 with forced aeration (5 liters/min) and agitation (200 rpm). The exponentially growing culture was harvested and transferred into prewarmed medium that lacked tryptophan. Samples (200 ml) were transferred to cold buffer containing chloramphenicol (100 ,ig/ml) at the times indicated, rapidly
chilled, immediately centrifuged, and stored at -70°C. Growth was monitored with a Klett-Summerson colorimeter (filter no. 42). In the presence of excess tryptophan, the growth of these organisms proceeds with a generation time of approximately 40 min; in the absence of exogenous tryptophan, their growth rate is reduced to a generation time of approximately 8 h. In experiments with mtr strains grown with an excess of the required amino acids, the cells were grown until the late exponential phase and then rapidly chilled and harvested. Preparation of enzyme extracts. Cell extracts were routinely prepared either by lysing the cells with lysozyme (21) or subjecting the thick suspension to sonic disruption (18). For the assay of anthranilate synthase, tryptophan synthase-B and prephenate dehydrogenase, cells were lysed in 0.1 M potassium phosphate buffer, pH 7.5, containing 40% glycerol, 0.1 mM ethylenediaminetetraacetate, 0.05 M glutamine, 0.6 mM pyridoxal phosphate, 200 ,ug of lysozyme per ml, and 10 ug of deoxyribonuclease per ml at 37°C for 20 min. For the assay of histidinol phosphate aminotransferase, cells were disrupted in 0.05 M potassium phosphate buffer, pH 7.5, containing 0.6 mM mercaptoethanol and 20 ;Lg of phenylmethylsulfonylfluoride per ml by sonic treatment at maximum power for 4 min in an MSE ultrasonic disintegrator. Cell debris was sedimented by centrifugation at 25,000 x g for 30 min at 4°C in a Sorvall centrifuge, and the supernatant fluid was used in enzyme assays. For certain experiments, where indicated, the crude cell extract was passed through a column of Sephadex G-25. Enzyme assays. Tryptophan synthase-B was assayed in crude extracts prepared in glycerol buffer as described by Hoch et al. (13). Anthranilate synthase was assayed by measuring the amount of anthranilate synthesized by an increase in fluorescence at 400 nm (activation at 335 nm) in a recording Aminco-Bowman spectrofluorometer (21). Histidinol phosphate aminotransferase activity was assayed as described by Chapman and Nester (8), except that pyridoxal phosphate was used at 0.6 mM.
Prephenate dehydrogenase activity was measured as described by Roth and Nester (21), except that quinicrine-hydrochloride was included in the reaction mixture at a final concentration of 0.1 mM. Enzyme activities are expressed as relative specific activities based on a value of 1.0 for wild-type
TABLE 1. Bacillus subtilis strains Strain designation Description Transformable strain of B. subtilis blocked in indoleglycerol168 phosphate synthesis (9) WB 746 Spontaneous revertent of 168 WB 3159 Spontaneous mtr derivative of WB 746 with high constitutive level of the tryptophan biosynthetic enzymes (14) GSY 266 A derivative of 168 and a methionine auxotroph; lacks 3-cystathi-
Source J. Spizizen
Our laboratory Our laboratory W. Steinberg
onase
GSY 1306
203
Strain GSY 266 with a second mutation in tryptophanyl-tRNA synthetase
W. Steinberg
204
WEIGENT AND NESTER
J. BACTERIOL.
cells grown in minimal medium. The specific activity in wild-type cells grown in minimal medium at 30°C in terms of nanomoles of substrate used or product formed each minute per milligram of protein is: tryptophan synthase-B, 1; anthranilate synthase, 0.01; prephenate dehydrogenase, 5; histidinol phosphate aminotransferase, 1.0. Chemicals. Prephenic acid was prepared from chorismic acid as previously described (17). All other chemicals were purchased from commercial sources and used without further purification. Isolation of analogue-resistant mutants. Analogue-resistant mutants were isolated by plating approximately 108 cells of a log-phase culture of strain 746 on Spizizen (23) minimal medium containing 1 mg of 5-methyltryptophan per ml. After incubation at 37°C for 48 to 72 h, 20 resistant colonies were streaked onto fresh plates of the same medium, and colonies from this plate were purified two additional times on minimal medium without the analogue.
containing chloramphenicol. The specific activity of each enzyme was determined in each sample. The relative specific activities were plotted as a function of the sampling time (Fig. 2 and 3). Within the limits of accuracy of the assays, the specific activities of each of these enzymes began to increase in a temporal se-
24 20 -
16 t--
RESULTS Time course of derepression. The readthrough model proposed by Roth and Nester (21) predicts that gene products will be synthesized sequentially with the order corresponding to the sequence on the genetic map (Fig. 1). To determine whether this prediction is fulfilled, we examined the kinetics of derepression of four genes that had been mapped in the proximal (anthranilate synthase), central (tryptophan synthase-B), and distal regions (histidinol phosphate aminotransferase and prephenate dehydrogenase) within the aromatic cluster
a)Q. 12 -
Cr) a)
0 cr a)
8 _
4 _ I-'
0
(19).
Cultures of B. subtilis strain 168 growing exponentially in Spizizen (23) minimal salts medium supplemented with excess amounts of phenylalanine, tryptophan, histidine, and tyrosine were derepressed for synthesis of the tryptophan enzymes by transferring the cells to the identical medium lacking tryptophan. At various times before and after tryptophan depletion, samples were transferred to cold buffer mtr
aroF
arB
10 20 30 40 50 60 70 80 Time in Min.
FIG. 2. Derepression of trpE, trpB, and hisH at 30°C in strain 168. The cells were cultured as described in Materials and Methods. Samples were transferred to cold buffer containing chloramphenicol at the times indicated. Extracts were prepared and passed through Sephadex G-25 to determine the specific activities of anthranilate synthase (a), tryptophan synthase-B (0), and histidinol phosphate aminotransferase (x).
trEtrD
trCtrFtrptrA
hisH trA
aro E
Origin FIG. 1. Genetic map of aromatic amino acid mutations in Bacillus subtilis. The enzymes specified by the genes listed are: mtr, methyltryptophan resistance; aroF, chorismate synthase; aroB, dehydroquinate synthase; trpE, anthranilate synthase; trpD, phosphoribosyl transferase; trpC, indoleglycerol-phosphate synthase; trpF, PRA isomerase; trpB, tryptophan synthase-B; trpA, tryptophan synthase-A; hisH, histidinol phosphate aminotransferase; tyrA, prephenate dehydrogenase; aroE, enolpyruvylshikimate-5-phosphate synthase. '
HISTIDINOL PHOSPHATE AMINOTRANSFERASE
VOL. 128, 1976
20 I 16 [ f--
.)_ .)_
12 H
._
a) 0. a/)
8H
.) 0
0L)
4 0 -z-
n
I
I 0
10 20 30 40 50 60 70 80 TIME IN MIN.
FIG. 3. Derepression of trpB, hisH, and tyrA at 30°C in strain 168. The experimental design was identical to that described in the legend to Fig. 2. Samples were transferred to cold buffer containing chloramphenicol at the times indicated and treated as described in Materials and Methods to determine specifwc activities oftryptophan synthase-B (0), histidinol phosphate aminotransferase (0), and prephenate dehydrogenase (x). quence: first anthranilate synthase and then tryptophan synthase-B, followed by the simultaneous appearance of histidinol phosphate aminotransferase and prephenate dehydrogenase. The sequence observed for the four enzymes corresponds with the order of the respective genes on the chromosome. The average lengths of time between the inflection in the growth rate owing to tryptophan limitation and the times at which the specific activities of each of the enzymes began to increase are compiled in Table 2. The intervals between derepression of one gene and the next gene measured are also shown. These data confirm the sequence in the appearance of the four enzyme activities. The time at which histidinol phosphate aminotransferase and prephenate dehydrogenase appeared could not be distinguished even when the cells were grown at a lower temperature (data not shown). Intervals of the same order of magnitude as observed in this study have also been observed for the induction of f3-galactosidase and transacetylase in Escherichia coli (2), as well as for the products of several genes of histidine synthesis in
205
Salmonella typhimurium (10). Effect of tryptophan on enzyme synthesis during derepression. To examine the effect of tryptophan on enzyme synthesis, the derepressed rate of increase in synthesis of the various enzymes was established by the assay of samples at intervals for 30 min under conditions in which growth was limited by tryptophan. Excess L-tryptophan was then added to the culture, and sampling continued for another 30 min. The specific activity of each enzyme was determined in each sample, and their relative specific activities were plotted as a function of the sampling time (Fig. 4). When Lr tryptophan was added to a derepressed culture, further increases in the specific activities of all four of the enzymes examined came to a halt simultaneously. Therefore, the pattern of repression of enzyme synthesis was not in the same temporal sequence as that observed for derepression of enzyme synthesis. Effect of regulatory mutants of tryptophan synthesis on histidine and tyrosine enzyme synthesis. A temperature-sensitive tryptophanyl-tRNA synthetase mutant of B. subtilis was recently isolated by Steinberg and Anagnostopoulos (25). They reported that at elevated temperatures both anthranilate synthase and tryptophan synthase-B were coordinately derepressed (24). The possibility that tryptophanmediated control of histidinol phosphate aminotransferase in these mutants was in some way altered from that resulting from tryptophan limitation was investigated. The level of histidinol phosphate aminotransferase in cells grown at 370C is shown in Table 3. The mutant strain (GSY 1306) was derepressed in both tryptophan and histidine enzyme synthesis as compared with the wild-type parental strain, GSY 266. The level of anthranilate synthase, tryptophan synthase-B, and histidinol phosphate TABLE 2. Summary of derepression of trpE, trpB, hisH, and tyrA in B. subtilis 168 Gene
trpE
Derepression timeP 5
Interval (min)
2.0 10.0
trpB
14 + 3.1
hi&H
22 + 2.8
8.0 1.0 23 2.0 a The data are the average of 10 experiments. Derepression at 3000 took place shortly after an observed reduction in the growth rate. The derepression time is the time between the reduction of growth rate and the time at which an increase in the rate of the enzyme synthesis was observed.
tyrA
206
J. BACTERIOL.
WEIGENT AND NESTER
specifi'c activity of histidinol phosphate aminotransferase (Fig. 5). It was found that the ratio of activity of tryptophan synthase-B and histidinol phosphate aminotransferase, within the limits of accuracy of the assays, remained essentially constant. Although the mutations were not mapped, other evidence suggests that they all occur at one genetic locus, the mtr locus (14). Timing of RNA synthesis for enzymes syn-
TR,p 40 ._
ion
o0
0
10
q) X cv 0
c1
20
3D
40
50
60
TABLz 3. Derepression of trpE, trpB, and hisH in trp Sl and wild-type strains at 30 and 37°C Relative sp act HistidiStrain Athrlfl Trpo nol phosStrain ilate phan syn- phate synthase thase-B transferase 1.0 0.7 GSY 266 (wild 30 0.9 type) 30 1.5 1.3 1.0 GSY 1306 (Si) GSY 266 37 0.5 0.5 1.0 GSY 1306 37 5.0 4.0 2.2
TeC)
4
.4 C)
I
3
I C)
2
U)
| ,~~~~
,
.
.
20
30
40
a)
0
10
50
60
Time in Mn. 20 FIG. 4. The effect oftryptophan on derepression of trpE, trpB, hisH, and tyrA in strain 168. The experimental design was identical to that described in the 16 legend to Fig. 2, except tryptophan (20 uglml) was added at 30 min. Samples were removed, before and after the addition of tryptophan, to cold buffer containing chloramphenicol at the times indicated and r-C treated as described in Materials and Methods to < =0 12 determine specifw activities of anthranilate synthase (@), tryptophan synthase-B (0), histidinol phosphate aminotransferase (A), and prephenate dehydrogen- cn c 8 ase (x).
.
.5
_
0
.
-C
0)
_
0
0
aminotransferase increased five-, four-, and 0 twofold, respectively, above the basal level. Thus, it would appear that at supraoptimal temperatures tryptophanyl-tRNA synthetase mutants have lost control over the synthesis of I I histidinol phosphate aminotransferase as well 2 4 0 1 3 as the tryptophan biosynthetic enzymes. Relative Specific Activity In B. subtilis, Nester et al. (19) described a Histidinol phosphate aminotransferase gene conferring resistance to 5-methyltryptoFIa. 5. Relative specific activities of tryptophan phan. Mutation in this locus results in constitutive synthesis of the tryptophan enzymes (14, synthase-B and histidinol phosphate aminotrans19). The gene lies to the left ofaroF (Fig. 1) (12, ferase in mtr mutants. Enzyme activities were meain extracts prepared fiom cells grown in mini19) and thus is not contiguous to the trp operon. sured mal medium supplemented with L-phenylalanine The level of histidinol phosphate aminotrans- and (50 pg eachlml). Numbers for enferase in 20 independently isolated 5-methyl- zymeL-histidine activities are relative specific activities based on tryptophan-resistant mutants was determined. the value of 1.0 for wild-type cells grown in minimal The relative specific activity of tryptophan syn- medium. Each point is an average determination thase-B was plotted with respect to the relative from two experiments for each mutant. ctl-4O.
0
I
I
HISTIDINOL PHOSPHATE AMINOTRANSFERASE
VOL. 128, 1976
thesized during derepression. If ordered enzyme synthesis resulted from a selective control of messenger RNA (mRNA) synthesis, then that control would be restricted to the time in which the particular cistron was being transcribed. To test whether such a control might be operating, it was necessary to know the times at which mRNA for anthranilate synthase, tryptophan synthase-B, histidinol phosphate aminotransferase, and prephenate dehydrogenase were synthesized, and whether the RNA templates for each of these enzymes had a short half-life. Figure 6 presents the results of an experiment designed to measure the times at which mRNA for anthranilate synthase and tryptophan synthase-B were synthesized as well as 0.1
*,)20
.I6OD 16
Z 0
=0 8
(n CL
w c >
1 II 10
>%
.> I.
I
30
40
50
60
70
20 0
a) 16
_i ~
20
12 0
8
en 0
0., a) =a >
c;s
g4 O
-.
-O
10 20 30 40 50 60 70 Time in Min.
FIG. 6. Timing of RNA synthesis for enzymes synthesized during derepression at 37°C in strain 168. The experimental design was identical to that described in the legend to Fig. 2. At the indicated times, samples were removed and added to: (a) cold phosphate buffer containing chloramphenicol and (0) actinomycin D (3 pg/ml). The actinomycin Dtreated samples were incubated at 37°C until 70 min, at which time enzyme synthesis was arrested by the addition of cold phosphate buffer containing chloramphenicol (100 pg/ml). The cells were collected by centrifugation and prepared for enzyme assays as described in Materials and Methods. (A) Anthranilate synthase; (B) tryptophan synthase-B.
207
the stability of these mRNA molecules. The experiment consisted of starving a culture for tryptophan and at regular intervals transferring samples to warm medium containing actinomycin D. This sample of the culture was incubated in the presence of actinomycin D for 10 min after the last sample was removed. All protein synthesis in the samples treated with actinomycin D was then stopped by the addition of cold buffer containing chloramphenicol. For controls, a second set of samples, removed at the same time as the experimental samples, was added directly to cold buffer containing chloramphenicol. If RNA templates for these enzymes were present at any time after the addition of actinomycin D, the level of enzyme activity at these times should have been the same as in the sample removed at 70 min. This was clearly not the case (Fig. 6). The addition of actinomycin D at any time halted synthesis of anthranilate synthase and tryptophan synthase-B within 2 to 5 min. Thus, the stability of messages for these two enzymes was approximately the same as the stability of pulse-labeled RNA (15). The gene specifying anthranilate synthase was transcribed approximately at the time tryptophan became depleted, and the message from this cistron was translated approximately 3 min later. A similar pattern between transcription and translation of tryptophan synthase-B was observed. A similar experiment was conducted to determine the times at which mRNA for histidinol phosphate aminotransferase and prephenate dehydrogenase were synthesized and to measure the stability of these mRNA templates. The results are shown in Fig. 7. Actinomycin D added at any time shut off histidinol phosphate aminotransferase and prephenate dehydrogenase synthesis within 2 to 5 min. This experiment further demonstrated that the time difference between the appearance of polypeptides of the tryptophan operon and the enzymes coded by the linked genes of histidine and tyrosine synthesis was not a result of a delay in the translation of a previously synthesized message. Therefore, the times at which enzymes appear during derepression appear to be transcriptionally controlled. The data also argue against any assembly of protein subunits accounting for the delays in enzyme appearance. The present study cannot rule out the less likely possibility that an assembly process proceeded during preparation of the extract for enzyme analysis. Intervals of the same order of magnitude between transcription and translation as observed in this study have also been
208
WEIGENT AND NESTER
J. BACTZRIOL.
oE .o4 0) 04-
40/
Q. 0 u.2 a) 4.-
C
o -.
O 0 4-
I
I
10 20 30 40 50 60 70
a)
cn .4= o
o O.
v
plotted as a function of the sampling time. The results of these experiments are shown in Fig. 8. The synthesis of tryptophan synthase-B was shut off approximately 10 min after anthranilate synthase. This is in good agreement with the data on the kinetics of appearance of this enzyme after tryptophan starvation (Fig. 2). These data suggest that RNA synthesis was initiated at the beginning of the tryptophan operon and continued uninterrupted through the tryptophan genes. The specific activities of histidinol phosphate aminotransferase and prephenate dehydrogenase decreased immediately upon the addition of rifampin. The data suggest that these latter two enzytnes are translated from separate monocistronic messages which required new initiations of transcription. An alternative explanation of rifampin inhibition of transcription initiations is that the drug indirectly caused a rapid intracellular accumulation of tryptophan, which then led to
0) >.
V 0)
c.
r.
0
10 20 30 40 50 60 70 Time in Min.
FIG. 7. Timing of RNA synthesis for enzyme synthesized during derepression at 37°C in strain 168. The experimental design was identical to that described in the legend to Fig. 6. Samples were removed at the indicated times and treated as described in Materials and Methods to determine specific activities of histidinol phosphate aminotransferase (A) and prephenate dehydrogenase (B).
observed in the timing of induced a-glucosidase and histidase synthesis during outgrowth of Bacillus cereus spores (26). Effect of rifampin on enzyme synthesis during derepression. Rifampin specifically inhibits the initiation of transcription without influencing the completion of RNA chains being synthesized (11). We used this drug to determine the number and sites at which transcription is initiated during derepression of the loci concerned with tryptophan, histidinol phosphate aminotransferase, and prephenate dehydrogenase activity. For these experiments, strain 168 was grown at 30°C in a medium lacking tryptophan to derepress the tryptophan operon. Rifampin was then added to the culture. At intervals before and after the addition of rifampin, samples were transferred to cold buffer containing chloramphenicol. The specific activity of each enzyme was determined in each sample and the relative specific activities were
0)
c)
0
10 20 30 40 50 60 70
_aL 2 cn ,H 03 _
-A
x
a)
._> I
ILa)
I
0
I
I
I
10 20 30 40 50 60 70 Time in Min.
FIG. 8. The effect of rifampin on derepression of trpE, trpB, hisH, and tyrA in strain 168. The experimental design was identical to that described in the legend to Fig. 4, except rifampin (20 pg/ml) was added at 40 min. The samples were treated as described in Materials and Methods to determine specific activities of anthranilate synthase (O), tryptophan synthase-B (0), histidinol phosphate aminotransferase (A), and prephenate dehydrogenase (x).
HISTIDINOL PHOSPHATE AMINOTRANSFERASE
VOL. 128, 1976
normal repression of the trp operon. To establish that rifampin inhibition did not result solely from tryptophan-mediated repression, we performed an experiment identical to the one described in Fig. 8 with a regulator-constitutive strain, mtr. The results for anthranilate synthase, tryptophan synthase-B, histidinol phosphate aminotransferase, and prephenate dehydrogenase in typical experiments are compiled in Fig. 9. The data indicate that upon the addition of rifampin, the specific activities of anthranilate synthase, histidinol phosphate aminotransferase, and prephenate dehydrogenase decreased immediately, whereas the decrease in specific activity for tryptophan syn-
4>100 80
-o o
',60 a) 0
_~~~~
40
a)
-.20 0 c0 0
5
o~~-o-o
o
F I
I
---x
x
x
x--
41
I* A_
0.
Q._
_a_
x 1-
.6
a) co
X
10 20 30 40 50 60 70
