Vol. 124, No. 3 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Dec. 1975, p. 1366-1373 Copyright 0 1975 American Society for Microbiology
Derepression and Repression of the Histidine Operon: Role of the Feedback Site of the First Enzyme VICTOR M. FERNANDEZ* RAFAEL MARTINDELRIO, ALFREDO R. TEBAR, JOSE M. GUISAN, AND ANTONIO 0. BALLESTEROS Departamento de Catalisis, Consejo Superior de Investigaciones Cientificas, Madrid 6, Spain Received for publication 25 September 1975
Thiazolealanine, a false feedback inhibitor, causes transient repression of the his operon previously derepressed by a severe histidine limitation in strains with a wild-type or feedback-hypersensitive first enzyme but not in feedback-resistant mutants. Since experiments reported here clearly demonstrate that thiazolealanine is not transferred to tRNAH"S, it is proposed that this "transient repression" is effected through the interaction of thiazolealanine with the feedback site of the enzyme. Experiments in the presence of rifampin indicate that this thiazolealanine-mediated effect is exerted at the level of translation. We conclude that histidine (free), in addition to forming co-repressor, also represses the operon at the level of translation through feedback interaction with the first enzyme of the pathway (adenosine 5'-triphosphate phosphoribosyltransferase). Rates of derepression in feedback-resistant strains are roughly half of those observed in controls, suggesting a positive role played by a first enzyme with a normal but unoccupied feedback site. Some feedback-resistant mutants, in contrast to the wild type, were unable to exhibit derepression under histidine limitation caused by aminotriazole. Extensive studies on the regulation of biosynthetic operons in microorganisms have shown that there are two different mechanisms by which end product biosynthesis is regulated. One mechanism, feedback inhibition (fine control), involves inhibition of the first enzyme of the pathway by the end product (43, 48) and determines the internal pool of this metabolite in the cell. The other mechanism, repression and derepression (coarse control), involves changes in the intracellular concentrations of the biosynthetic enzymes (16, 44). The results of several studies suggest that feedback-sensitive first enzyme of a biosynthetic operon is involved in repression (5, 6, 13, 20, 21, 24, 26, 36, 40). In Salmonella typhimurium, the involvement of the first enzyme for histidine biosynthesis (G enzyme) [N-1-(5'-phosphoribosyl) adenosine triphosphate: pyrophosphate phosphoribosyltransferase, EC 2.4.2.171 in the regulation of the operon is well documented (20, 21, 36). In the present work, results of experiments designed to study derepression and repression of the histidine operon lead us to postulate that direct participation of the first enzyme in regulation at the level of translation involves the state of the feedback site of the enzyme.
MATERIALS AND METHODS Bacterial strains. The organisms employed in this study, obtained from the collection of P. E. Hartman (12), were the LT2 (wild type) strain of S. typhimurium and several mutants derived from it (Table 1). Growth conditions and experimental design. Cells were routinely grown in minimal medium (45) with glucose at 0.4%. The cultures were aerated in a rotary shaker at 37 C. The histidine auxotrophs were grown in the presence of a sufficient amount of L-histidine to support growth to an absorbance at 650 nm of approximately 0.4; after depletion of histidine, growth was controlled with L-histidinol (Sigma Chemical Co., final concentration, 15 uM). The growth rate (u = 0.15 generation per h) during derepression was the same in all experiments and did not change after addition of 0.4 to 1.6 mM 2-thiazole-DL-alanine (TA, Cyclo Chemical Co.). Derepression of the prototrophs (LT2 and feedback-resistant mutants) was effected with 3-amino-1,2,4-triazole (AMT, Cyclo Chemical Co., final concentration, 15 mM). Derepression of the hypersensitive bradytroph was effected with 40 mM AMT plus 15 MM histidinol. The design of the kinetic experiments was similar to that of Kovach et al. (18, 19). An MSE sonic oscillator was used to disrupt the cells. The rifampin experiments were similar in design to those of McLellan and Vogel (29), with the following modifications. The wild-type organism, LT2, was grown in the presence of 15 mM AMT plus 15 /M 1366
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TABLE 1. List of mutants Genotype
hisIF135 hisG1102 hisG1104 hisG1108 hisG 109 hisG1102hisIF135 hisG1109hisIF135 hisG1306 hisG52 hisG70 hisG428
Source/reference
Phenotype
Deletion of I and F genes Feedback resistant Feedback resistant Feedback resistant Feedback resistant Double mutant Double mutant Feedback hypersensitive Missense Missense Nonsense
histidinol. This limited amount of histidinol supports growth to an absorbance at 650 nm of approximately 0.7. Since increasing amounts of mRNA accumulate with increasing duration of starvation (29), rifampin (180 jg/ml) was added after 1 h of histidine starvation (i.e., 1 h after cessation of growth); 2 min later additions were made, as indicated below. The removed samples were immediately mixed with ice, chloramphenicol (100 ,g/ml), and NaN, (10 mM) to prevent further protein synthesis before the crude extracts were prepared. Enzyme assays and substrates. Protein was estimated by the method of Lowry et al. (28), with insulin standards. The activities of the G and C (L-histidinolphosphate:2-oxoglutarate aminotransferase, EC 2.6.1.9.) enzymes of the his operon were measured by the procedures of Martin et al. (31). A unit of activity is defined as a change in absorbance at 290 nm of 0.25/5 min (31). Enzyme levels are expressed in units per milligram of protein. The synthesis of histidine biosynthetic enzymes is coordinate (1); therefore, the level of aminotransferase is considered to be representative of the differential expression of the entire operon. Aminoacylation of tRNA to evaluate the his-tRNA synthetase activity was carried out as previouslv described (20). L-Histidinol phosphate and 5phosphoribosyl-1-pyrophosphate were Sigma products. Escherichia coli K-12 tRNA was obtained from General Biochemicals Div. (Mogul Corp.), and 1,2,4triazole-3-DLalanine (TRA) was obtained from Cyclo Chemical Co. Other methods. A simplification of the method of Stieglitz and Calvo (41) was used for testing the ability of TA to be incorporated into protein in vivo. The mutant hisIF135 was grown in the presence of a sufficient amount of L-histidine to support growth to an absorbance at 650 nm of 0.4 and then washed and transferred to three separate flasks with ["CC]valine (final concentration, 0.2 mM). The first flask received L-histidine (final concentration, 0.4 mM); the second flask received TA (final concentration, 0.8 mM); and the third received water. At 20-min intervals, samples of 1 ml were removed, quickly filtered through Millipore membranes, and washed twice with 10 ml of minimal medium containing 0.2 mM valine. The radioactivity retained by the filter, which was measured in a Nuclear-Chicago Mark II scintillation
R. F. Goldberger, 12 R. F. Goldberger, 39 R. F. Goldberger, 39 P. E. Hartman, 39 R. F. Goldberger, 39 R. F. Goldberger, 39 R. F. Goldberger, 39 P. E. Hartman, 37 P. E. Hartman, 12 R. F. Goldberger, 12 R. F. Goldberger, 12
spectrometer using a 0.5% solution of 2-(4-t-butylphenyl)-5(4-biphenyl)-1,2,4-oxidiazole (CIBA) in toluene, reached a maximum that was indicative of the maximal radioactivity incorporated in about 1 h. Periodate oxidation was carried out following the method of Folk and Berg (10) as modified by Lewis and Ames (27). The extracted, aminoacylated tRNA's were treated with periodate, stripped of attached histidine, and tested for their ability to accept L["IC ]histidine. Parallel experiments were run with TA or without additions in the aminoacylation reaction.
RESULTS Repression by TA. We first demonstrated the ability of the false feedback inhibitor TA to repress the histidine operon in E. coli (A. R. Tebar, Ph.D. thesis, Universidad Autonoma de Madrid, Madrid, Spain, 1974). We have extended that study to S. typhimurium in which several well-characterized mutants that exhibit altered feedback sensitivity to histidine are available. First, we chose the deletion strain hisIF135 (carrying a wild-type G enzyme), easily derepressible by restricting the supply of exogenous histidinol. Figure 1A shows repression (transient) by TA (0.8 mM) of hisIF135 cells grown with a severe limitation of histidine (doubling time, 6.5 h); the same general pattern was obtained at concentrations of 0.4 or 1.6 mM TA. In contrast, the double mutant hisG1109hisIF135, isogenic with the deletion strain hisIF135 but with an additional mutation (G1109) (39) leading to feedback resistance, was not repressed by identical amounts of TA (Fig. 1B). The same result was obtained in the double mutant hisG1102hisIF135, which carries a different mutation causing feedback resistance. We then studied the effect of the mutation hisG1306, which causes the G enzyme to be 10 times more feedback sensitive than the wildtype enzyme (37). The growth rate was decreased from a generation time of 50 min to 6.5 h with 40 mM AMT and 15 uM histidinol.
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FIG. 1. Kinetics of repression by TA (0.8 mM) of strains previously derepressed with limiting amounts of histidinol. The specific activity of the C enzyme (aminotransferase) is shown. In this and in all other experiments, enzymatic determinations were carried out in triplicate, and protein concentrations were in duplicate. (A) Strain hisIF135 (wild-type G); (B) strain hisG1109hisIF135 (feedback-resistant G); (C) strain hisG1306 (feedback-hypersensitive G).
Figure 1C shows the result of a typical experiment, using 0.8 mM TA. A pattern similar to wild type was observed but the "transient repression" lasted some 25 min more. Derepression rate. In the previously mentioned experiments, derepression was accomplished by severe limitation of histidine, using histidinol to regulate growth rate. In all of these cases, a clear correlation can be seen between the rate of expression and the state of the feedback properties of the G enzyme. The rate of increase in specific activity in the two feedback-resistant strains was approximately half as great as that in the wild type. The rate in the case of the feedback-hypersensitive strain is not well defined but appears to be lower than is obtained with the wild type; possible complications owing to the presence of AMT are unresolved. These experiments have been repeated many times and the results are given in Table 2, which also includes data on three other typical hisG mutants. Derepression by AMT of feedback-resistant mutants. AMT inhibits imidazoleglycerolphosphate dehydratase (EC 4.2.1.19), the seventh step in the pathway for histidine biosynthesis, thus reducing histidine levels and producing derepression of the operon (14). (Because AMT also inhibits purine biosynthesis [14], adenine [50 ug/ml] was added to the culture medium.) Figure 2 shows the results of a parallel experiment with strains LT2 and hisG1102; although after the addition of 15 m M AMT the generation times changed equally in both cases (from 1 h to approximately 10 h), no derepression was observed in the feedbackresistant strain (Fig. 2B). Figure 3 shows an experiment with another feedback-resistant muant, hisG1104. This strain is highly resistant to 15 mM AMT, a concentration that has little effect on the growth rate. Derepression was not elicited, even in the presence of 65 mM AMT (second AMT addition), a concentration that appreciably af-
TABLE 2. Rates of derepression Organism
hisIF135
hisG1102hisIF135 hisG1109hisIF135 hisG428 hisG52 hisG70
Defect in G enzyme
Derepression rate Units of C enzyme! Standard deviation mg of protein/h
None Feedback resistant Feedback resistant Catalytically inactive Catalytically inactive Catalytically inactive
a Numbers in parentheses indicate how many values have been averaged.
9.0 (8)a 4.0 (3) 4.2 (6) 9.1(1) 7.4 (1) 5.4 (1)
± 1.4 ± 0.9
±0.9
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ing in a false co-repressor TAAtRNAH"S. This possibility has been ruled out by three lines of evidence. The first is the transfer to tRNAHIS. I The extent to which a compound can protect tRNA from periodate is a measure of the ability of the compound to be attached to tRNA (4). U A TA did not protect tRNA from periodate oxidation (Table 3). The second line of evidence is the incorporation into protein. The analogue TA IA was not incorporated into protein in place of histidine; the total radioactivity incorporated was no different from the control value (Table I a 3-3A0 -3 S 3 aS (ml TI, iSin 4). Third, TA, up to 2.5 mM (400-fold excess FIG. 2. Derepression with AMT (15 mM) of prot- over histidine), had no effect on the charging otrophic strains. Growth curves are shown at the top. activity of semipurified his-tRNA synthetase. (A) LT2 (wild type); (B) hisG1102 (feedback resistExperiments with rifampin. The design of ant). the experiments with rifampin included accumulation of the potential (presumably formation of mRNA) for histidine biosynthetic enAMT ,_____-O zymes, inhibition of further transcription with I rifampin, and measurement of enzyme forma0.90 tion as a function of the different additions (29). AMT Histidinol, the last precursor of histidine on the I pathway, was used as a source of histidine (2) in 10.60 I all the experiments to translate the accumulated messenger. The control experiment with histidinol (60 gM) alone is shown in Fig. 4A. If histidine (0.8 mM) is also supplied with his0.45 tidinol, translation of the messenger is hindered 0 f~~~~ (Fig. 4D); the same result is found with TA (1.6 5 V mM) instead of histidine (Fig. 4C). The histidine analogue TRA is charged into protein (25) 13 .2 and represses the operon (21; V. M. Fernandez, unpublished data), but it does not inhibit the first enzyme for histidine biosynthesis (2, 30). -20 0 s0 s0 100 20 40 120 (minutes) Now if TRA (0.2 mM) is added along with FIG. 3. Absence of derepression, in the presence of histidinol, an incorporation into histidine enAMT (15 mM and 65 mM), of the feedback-resistant zymes similar to the control is observed (Fig. ANT
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mutant hisG1104. Growth
curve
is shown at the top.
fects the growth rate. Another strain, hisG1108, is totally resistant to growth inhibition. In the case of hisG1109 (experiment not shown), the pattern was like LT2 (Fig. 2A), both in growth inhibition and derepression. The sole known role for AMT is the inhibition of the seventh step in the histidine pathway (14). However, in the present study its effect on two other histidine enzymes has been examined. AMT, up to a concentration of 40 mM, had no effect on the activity of the G enzyme in vitro, either wild type or feedback resistant. AMT, up to 40 mM, had no effect on the charging activity of partially purified his-tRNA
synthetase. Investigation of TA properties. There is a possibility that TA represses the wild-type G strain by being transferred to tRNAH'S, result-
TABLE 3. Protection in vitro of tRNAHIS from periodate oxidation Protected with:
Protection(%)
9.5 ........... None ..... L-Histidine (0.15MM)a ................ 100 12 TA (2mM)a ................ a
Final concentration.
TABLE 4. Capacity to support valine incorporation Maximal radioactivity Additions
incorporated
(counts/min)
None (water added) TA (0.8 mM) His (0.4 mM)
7,000 7,400 39,300
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FERNANDEZ ET AL.
6
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15, 42, 47). The study of the kinetic pattern of repression in various histidine auxotrophs in the presence of TA (19) and the finding that the histidine analogue TRA cannot cause repression of feed-
2
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back-resistant mutants (21) led Kovach and colleagues (18) to conclude that the feedbacksensitive site of the first enzyme plays a role in regulation of the histidine operon. Later, they presented evidence indicating that the first enzyme must interact with his-tRNA to fulfill
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4
6
Tim. (minutes) FIG. 4. Translation of the messenger accumulated after I h of histidine starvation. At zero time, rifampin was added, and 2 min later the following additions were made: (A) histidinol (60 uM); (B) histidinol (60 AM) plus TRA (0.2 mM); (C) histidinol (60 AM) plus TA (1.6 mM); (D) histidinol (60 MM) plus histidine (0.8 mM). For further details, see text. The specific activity of the C enzyme is shown. The different specific activities in A-D at 2 min reflect the derepression level attained; complications resulting from the presence of AMT during growth could account for this lack of reproducibility.
4B). These results clearly demonstrate that histidine (at 0.8 mM) blocks the expression of accumulated his operon messenger, an effect that is also shared by TA.
DISCUSSION The isolation and characterization of over 2,000 histidine mutants in S. typhimurium by P. E. Hartman and associates (12) has provided the basis for extensive studies on regulation of the histidine operon by Brenner and Ames (8). However, no complete picture of the process has yet emerged. It has been generally accepted that it is the concentration of his-tRNA, and not that of free histidine, that controls repression of this system (3, 27, 35, 38). Aminoacylation of
its regulatory properties (20). It has been demonstrated that TA acts as a false feedback inhibitor (32) of the first enzyme of the histidine biosynthetic pathway (30). In this paper, we present another effect of TA, namely the ability to repress transiently the histidine operon previously derepressed by a drastic limitation in the supply of histidine. That the TA action is exerted at the level of G enzyme is clearly demonstrated by the fact that the double mutants hisG1102hisIF135 and hisG1109hisIF135, which differ from hisIF135 only in an additional G feedback mutation, are not repressed by TA. The above-mentioned effect of TA on the level of enzymes of the histidine operon could be easily explained if TA could be transferred to tRNA and form a false co-repressor that would mimic his-tRNAH"'. The experiments reported here clearly demonstrate that TA cannot protect tRNA from oxidation by periodate (Table 3) and that it cannot be incorporated into protein (Table 4). Moreover, TA neither inhibits the activity of his-tRNA synthetase nor affects the growth rate of a culture suffering a drastic limitation of histidine. All of these data rule out the possibility that TA represses by forming co-repressor. As far as we know, TA behavior is the only case of a false feedback inhibitor that is unable to be incorporated into protein and that represses the operon. This repressing effect can be exerted at the level of transcription or at the level of translation. The experiments with rifampin, carried out to differentiate between them, demonstrate that histidine blocks the expression of present messenger (Fig. 4D), an effect that is also shared by TA (Fig. 4C) but not by TRA (Fig. 4B). Since the primary feature common to histidine and TA is feedback inhibition and TRA allows tormal production of the C enzyme (Fig. 4B), it appears that TA exerts its transitory effect at the level of translation. Control at the level of translation has been
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FEEDBACK SITE IN REGULATION OF THE HIS OPERON
proposed for the trp (23), arg (22, 29, 34), and ilv (46) operons. Interestingly enough, Wasmuth and Umbarger (46) found that free isoleucine (or glycyl-leucine) participates in the postulated translation repression, and not as iletRNA, which is clearly implicated as co-repressor; furthermore, isoleucine is the feedback inhibitor of the first enzyme of the branched pathway, threonine deaminase (43). It is not easy to explain why the repression caused by TA in not permanent. Several explanations would be possible: enzymatic removal from the system, reinitiation of translation on the mRNA at a different site, etc. Here we are dealing with a "false" feedback inhibitor, with a K, 30 times higher than that of histidine (30), and the cell is able to detect it after awhile; the "true" inhibitor histidine does not, of course, produce such a pattern of transient repression. With 0.05 mM TRA, a "false" co-repressor of the histidine operon, transient repression for 30 to 50 min is also obtained (A. 0. Ballesteros and V. M. Fernandez, unpublished data). In addition to this role of the enzyme in repression, the data of Table 2 clearly show the effect of the state of the feedback site on the kinetics of derepression elicited with limiting histidinol and no histidine analogues present. When the feedback site is impaired by mutation, the derepression rate is roughly half of that seen in the wild type. As for the other three G mutants of Table 2, since they have no G activity, it is not possible to ascertain the state of the feedback resistance site, which could be normal, non-existent, or somewhat altered, thus giving normal or intermediate derepression rates. This same effect of lower derepression rate in a feedback-resistant strain was also obtained by Kovach et al. (21). Using the same strains as we used (hisIF135 and hisG1102hisIF135) and following an identical experiment design, they reported derepression rates of 1.7 and 0.9 (arbitrary units), respectively (cf. Fig. 2 and3in [21D. This stimulatory effect of an intact feedback site on the G enzyme in derepression of the operon has been suggested before, on theoretical grounds, by Rothmann-Denes and Martin (36). More recently, Kasai (17) suggested that a positive factor enhancing transcription of the histidine operon binds at the promoter. In yeast, Bollon and Magee (6) found that a normal feedback-sensitive threonine deaminase is necessary for derepression of the ilv pathway, acting possibly as an inducer. Later, they proposed that threonine deaminase acts, in control of the ilv and leu pathways, as a positive
1371
element, determining the amount of leu-tRNA available (7). In E. coli K-12, Levinthal et al. (26) postulated that threonine deaminase is a positive effector in the multivalent control process. The results of the experiments shown in Fig. 2 and 3 reveal more dramatically the effect of the state of the G enzyme feedback site on derepression. We have been unable to elicit derepression of strains hisG1102 and hisG1104 when the histidine limitation is caused by AMT. Loss of feedback inhibition in the mutants leads to an overproduction of histidine, some of the excess being excreted into the medium (39). A large histidine pool in the feedback-resistant mutants hisG1104 and hisG1108 could explain the resistance to growth inhibition by 15 mM AMT. On the other hand, strains hisG1102 and hisG1104 cannot be derepressed, even under conditions of histidine starvation. In strain hisG1102 (Fig. 2), the doubling time of 10 h obtained with 15 mM AMT is indicative of a drastic limitation in the availability of histidine. The same result, absence of derepressibility, was obtained by Kovach et al. (18) in a merodiploid strain with chromosomal hisG deletion and episomal hisG feedback resistance, when also inhibited with 15 mM AMT, a concentration that severely affected growth rate. Patthy and Denes (33) have reported a feedback-insensitive mutant of E. coli K-12 able to derepress only when reaching a high histidine deprivation. All of this means that, under conditions of scarcity of histRNAHis, the state of the feedback site determines whether or not the mechanism of derepression can operate. Other feedback-resistant strains, like hisG1109, can be derepressed, indicating that, as far as regulation is concerned, possibly no two such mutants are alike. Because of the general nature of the process of feedback inhibition, this new kind of repression at the level of mRNA translation by the feedback inhibitors of the first enzyme of the pathway could be easily extended to other biosynthetic operons. Since the feedback inhibition of allosteric first enzymes is commonly accompanied by changes in the quaternary structure, it is tempting to speculate that this translational repression is caused by such changes. ACKNOWLEDGMENTS A.O.B. thanks R. F. Goldberger, who introduced him to the histidine system, and P. E. Hartman for constant advice and help, as well as B. N. Ames, F. Blasi, R. G. Martin, J. R. Roth, M. Levinthal, and H. Whitfield for their comments. We are indebted to A. Albert and co-workers for laboratory facilities; to R. F. Goldberger, A. Sols, and G. Ailhaud for
1372
FERNANDEZ ET AL.
critical reading of the manuscript; and to Laboratorios a gift of rifampin. J. VegaLeal and M. C. Ceinos contributed skilled technical assist-
Lepetit, Sociedad An6nima, for ance.
LITERATURE CITED 1. Ames, B. N., and B. Garry. 1959. Coordinate repression of
J. BACTERIOL. 19. Kovach, J. S., M. A. Berberich, P. Venetianer, and R. F. Goldberger. 1969. Repression of the histidine operon: effect of the first enzyme on the kinetics of repression. J. Bacteriol. 97:1283-1290. 20. Kovach, J. S., J. M. Phang, F. Blasi, R. W. Barton, A. 0.
Ballesteros, and R. F. Goldberger. 1970. Interaction between histidyl transfer ribonucleic acid and the first enzyme for histidine biosynthesis of Salmonella typhimurium. J. Bacteriol. 104:787-792. 21. Kovach, J. S., J. M. Phang, M. Ference, and R. F. Goldberger. 1969. Studies on repression of the histidine operon. II. The role of the first enzyme in control of the histidine system. Proc. Natl. Acad. Sci. U.S.A. 63:481-488. 22. Lavalle, R. 1970. Regulation at the level of translation in the arginine pathway of Escherichia coli K12. J. Mol. Biol. 51:449-451. 23. Lavalle, R., and G. De Hauwer. 1970. Tryptophan messenger translation in Escherichia coli K12. J. Mol. Biol.
the synthesis of four histidine biosynthetic enzymes by histidine. Proc. Natl. Acad. Sci. U.S.A. 45:1453-1461. 2. Ames, B. N., R. G. Martin, and B. J. Garry. 1961. The first step of histidine biosynthesis. J. Biol. Chem. 236:2019-2026. 3. Ant6n, D. N. 1968. Histidine regulatory mutants in Salmonella typhimurium. V. Two new classes of histidine regulatory mutants. J. Mol Biol. 33:533-546. 4. Berg, P., U. Lagerkvist, and M. Dieckmann. 1962. Enzymic synthesis of amino acyl derivatives of ribonucleic acid. VI. Nucleotide sequences adjacent to the ..pCpCpA end groups of isoleucine- and leucine51:435-447. specific chains. J. Mol. Biol. 5:159-171. 5. Bollon, A. P. 1974. Fine structure analysis of a eukaryotic 24. Leisinger, T., R. H. Vogel, and H. J. Vogel. 1969. multifunctional gene. Nature (London) 250:630-634. Repression-dependent alteration of an arginine enzyme in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 6. Bollon, A. P., and P. T. Magee. 1971. Involvement of 64:686-692. threonine deaminase in multivalent repression of the isoleucine-valine pathway in Saccharomyces 25. Levin, A. P., and P. E. Hartman. 1963. Action of a histidine analogue, 1,2,4-triazole-3-alanine, in Salmocerevisiae. Proc. Natl. Acad. Sci. U.S.A. 68:2169-2172. 7. Bollon, A. P., and P. T. Magee. 1973. Involvement of nella typhimurium. J. Bacteriol. 86:820-828. threonine deaminase in repression of the isoleucine- 26. Levinthal, M., L. S. Williams, M. Levinthal, and H. E. valine and leucine pathways in Saccharomyces Umbarger. 1973. Role of threonine deaminase in the cerevisiae. J. Bacteriol. 113:1333-1344. regulation of isoleucine and valine biosynthesis. Nature 8. Brenner, M., and B. N. Ames. 1971. The histidine operon (London) New Biol. 246:65-68. and its regulation, p. 349-387. In H. J. Vogel (ed.), 27. Lewis, J. A., and B. N. Ames. 1972. Histidine regulation in Salmonella typhimurium. XI. The percentage of Metabolic pathways, vol. 5. Academic Press Inc., New York. transfer RNA"H' charged in vivo and its relation to the 9. Eidlic, L., and F. C. Neidhardt. 1965. Role of valyl-sRNA repression of the histidine operon. J. Mol.Biol. synthetase in enzyme repression. Proc. Natl. Acad. Sci. 66:131-142. U.S.A. 53:539-543. 28. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. 10. Folk, W. R., and P. Berg. 1970. Characterization of Randall. 1951. Protein measurement with the Folin altered forms of glycyl transfer ribonucleic acid synthephenol reagent. J. Biol. Chem. 193:265-275. tase and the effects of such alterations on aminoacyl 29. McLellan, W. L., and H. J. Vogel. 1970. Translational repression in the arginine system of Escherichia coli. transfer ribonucleic acid synthesis in vivo. J. Bacteriol. 102:204-212. Proc. Natl. Acad. Sci. U.S.A. 67:1703-1709. 11. Freundlich, M. 1967. Valyl-transfer RNA: role in repres- 30. Martin, R. G. 1963. The first enzyme in histidine biosynsion of the isoleucine-valine enzymes in Escherichia thesis: the nature of feedback inhibition by histidine. J. coli. Science 157:823-825. Biol. Chem. 238:257-268. 12. Hartman, P. E., Z. Hartman, R. C. Stahl, and B. N. 31. Martin, R. G., M. A. Berberich, B. N. Ames, W. W. Ames. 1971. Classification and mapping of spontaneDavis, R. F. Goldberger, and J. D. Yourno. 1971. ous and induced mutations in the histidine operon of Enzymes and intermediates of histidine biosynthesis in Salmonella, p. 1-34. In E. W. Caspari (ed.), Advances Salmonella typhimurium, p. 3-44. In H. Tabor and C. in genetics, vol. 16. Academic Press Inc., New York. W. Tabor (ed.), Methods in enzymology, vol. 17B. 13. Hatfield, G. W., and R. 0. Burns. 1970. Specific binding Academic Press Inc., New York. of leucyl transfer RNA to an immature form of L-threo- 32. Moyed, J. S. 1961. Interference with the feed-back control nine deaminase: its implications in repression. Proc. of histidine biosynthesis. J. Biol. Chem. 236:2261-2267. Natl. Acad. Sci. U.S.A. 66:1027-1035. 33. Patthy, L., and G. Denes. 1970. Altered repression 14. Hilton, J. L., P. C. Kearney, and B. N. Ames. 1965. Mode behaviour in a feedback insensitive mutant of Escheof action of the herbicide 3-amino-1,2,4-triazole (amirichia coli K12. Acta Biochim. Biophys. Acad. Sci. inhibition of trole): an enzyme of histidine biosyntheHung. 5:147-157. sis. Arch. Biochem. Biophys. 112:544-547. 34. Rogers, P., R. Krzyzek, T. M. Kaden, and E. Arfman. 15. Ito, K., S. Hiraga, and T. Yura. 1969. Tryptophanyl 1971. Effect of arginine and cannavanine on arginine transfer RNA synthetase and expression of the tryptomessenger RNA synthesis. Biochem. Biophys. Res. phan operon in the trpS mutants of Escherichia coli. Commun. 40:1220-1226. Genetics 61:521-538. 35. Roth, J. R., D. F. Silbert, G. R. Fink, M. J. Voll, D. 16. Jacob, F., and J. Monod. 1961. Genetic regulatory mechAnt6n, P. E. Hartman, and B. N. Ames. 1966. Transfer anisms in the synthesis of proteins. J. Mol Biol. RNA and the control of the histidine operon. Cold 3:318-356. Spring Harbor Symp. Quant. Biol. 31:383-392. 17. Kasai, T. 1974. Regulation of the expression of the 36. Rothman-Denes, L., and R. G. Martin. 1971. Two mutahistidine operon in Salmonella typhimurium. Nature tions in the first gene of the histidine operon of (London) 249:523-527. Salmonella typhimurium affecting control. J. Bacte18. Kovach, J. S., A. 0. Ballesteros, M. Meyers, M. Soria, riol. 106:227-237. and R. F. Goldberger. 1973. A cis/trans test of the effect 37. St. Pierre, M. L. 1968. Mutations creating a new initiaof the first enzyme for histidine biosynthesis on regulation point for expression of the histidine operon in tion of the histidine operon. J. Bacteriol. 114:351-356. Salmonella typhimurium. J. Mol. Biol 35:71-82.
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38. Schlesinger, S., and B. Magasanik. 1964. Effect of amethylhistidine on the control of histidine synthesis. J. Mol. Biol. 9:670-682. 39. Sheppard, D. W. 1964. Mutants of Salmonella typhimurium resistant to feedback inhibition by L-histidine. Genetics 50:611-623. 40. Somerville, R. L., and C. Yanosfky. 1965. Studies on the regulation of tryptophan biosynthesis in Escherichia coli. J. Mol. Biol. 11:747-759. 41. Stieglitz, B., and J. M. Calvo. 1971. Effect of 4-azaleucine upon leucine metabolism in Salmonella typhimurium. J. Bacteriol. 108:95-104. 42. Szentirmai, A., M. Szentirmai, and H. E. Umbarger. 1968. Isoleucine and valine metabolism of Escherichia coli. J. Bacteriol. 95:1672-1679. 43. Umbarger, H. E. 1956. Evidence for a negative-feedback mechanism in the biosynthesis of isoleucine. Science 123:848-848.
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44. Vogel, H. J. 1957. Repression and induction as control mechanisms of enzyme biogenesis: the "adaptive" formation of acetylornithinase, p. 276-289. In N. P. McElroy and B. Glass (ed.), The chemical basis of
heredity. The Johns Hopkins Press, Baltimore. 45. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218:97-106. 46. Wasmuth, J. J., and H. E. Umbarger. 1974. Role of free
isoleucine or glycyl-leucine in the repression of threonine deaminase in Escherichia coli. J. Bacteriol. 117:29-39. 47. Yaniv, M., F. Jacob, and F. Gros. 1965. Mutations thermosensibles des systemes activant la valine chez E. coli. Bull. Soc. Chim. Biol. 47:1609-1626. 48. Yates, R. A., and A. B. Pardee. 1956. Control of pyridine biosynthesis in Escherichia coli by feedback mechanism. J. Biol. Chem. 221:757-770.
JOURNAL OF BACTERIOLOGY, Dec. 1975, p. 1366-1373 Copyright 0 1975 American Society for Microbiology
Derepression and Repression of the Histidine Operon: Role of the Feedback Site of the First Enzyme VICTOR M. FERNANDEZ* RAFAEL MARTINDELRIO, ALFREDO R. TEBAR, JOSE M. GUISAN, AND ANTONIO 0. BALLESTEROS Departamento de Catalisis, Consejo Superior de Investigaciones Cientificas, Madrid 6, Spain Received for publication 25 September 1975
Thiazolealanine, a false feedback inhibitor, causes transient repression of the his operon previously derepressed by a severe histidine limitation in strains with a wild-type or feedback-hypersensitive first enzyme but not in feedback-resistant mutants. Since experiments reported here clearly demonstrate that thiazolealanine is not transferred to tRNAH"S, it is proposed that this "transient repression" is effected through the interaction of thiazolealanine with the feedback site of the enzyme. Experiments in the presence of rifampin indicate that this thiazolealanine-mediated effect is exerted at the level of translation. We conclude that histidine (free), in addition to forming co-repressor, also represses the operon at the level of translation through feedback interaction with the first enzyme of the pathway (adenosine 5'-triphosphate phosphoribosyltransferase). Rates of derepression in feedback-resistant strains are roughly half of those observed in controls, suggesting a positive role played by a first enzyme with a normal but unoccupied feedback site. Some feedback-resistant mutants, in contrast to the wild type, were unable to exhibit derepression under histidine limitation caused by aminotriazole. Extensive studies on the regulation of biosynthetic operons in microorganisms have shown that there are two different mechanisms by which end product biosynthesis is regulated. One mechanism, feedback inhibition (fine control), involves inhibition of the first enzyme of the pathway by the end product (43, 48) and determines the internal pool of this metabolite in the cell. The other mechanism, repression and derepression (coarse control), involves changes in the intracellular concentrations of the biosynthetic enzymes (16, 44). The results of several studies suggest that feedback-sensitive first enzyme of a biosynthetic operon is involved in repression (5, 6, 13, 20, 21, 24, 26, 36, 40). In Salmonella typhimurium, the involvement of the first enzyme for histidine biosynthesis (G enzyme) [N-1-(5'-phosphoribosyl) adenosine triphosphate: pyrophosphate phosphoribosyltransferase, EC 2.4.2.171 in the regulation of the operon is well documented (20, 21, 36). In the present work, results of experiments designed to study derepression and repression of the histidine operon lead us to postulate that direct participation of the first enzyme in regulation at the level of translation involves the state of the feedback site of the enzyme.
MATERIALS AND METHODS Bacterial strains. The organisms employed in this study, obtained from the collection of P. E. Hartman (12), were the LT2 (wild type) strain of S. typhimurium and several mutants derived from it (Table 1). Growth conditions and experimental design. Cells were routinely grown in minimal medium (45) with glucose at 0.4%. The cultures were aerated in a rotary shaker at 37 C. The histidine auxotrophs were grown in the presence of a sufficient amount of L-histidine to support growth to an absorbance at 650 nm of approximately 0.4; after depletion of histidine, growth was controlled with L-histidinol (Sigma Chemical Co., final concentration, 15 uM). The growth rate (u = 0.15 generation per h) during derepression was the same in all experiments and did not change after addition of 0.4 to 1.6 mM 2-thiazole-DL-alanine (TA, Cyclo Chemical Co.). Derepression of the prototrophs (LT2 and feedback-resistant mutants) was effected with 3-amino-1,2,4-triazole (AMT, Cyclo Chemical Co., final concentration, 15 mM). Derepression of the hypersensitive bradytroph was effected with 40 mM AMT plus 15 MM histidinol. The design of the kinetic experiments was similar to that of Kovach et al. (18, 19). An MSE sonic oscillator was used to disrupt the cells. The rifampin experiments were similar in design to those of McLellan and Vogel (29), with the following modifications. The wild-type organism, LT2, was grown in the presence of 15 mM AMT plus 15 /M 1366
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TABLE 1. List of mutants Genotype
hisIF135 hisG1102 hisG1104 hisG1108 hisG 109 hisG1102hisIF135 hisG1109hisIF135 hisG1306 hisG52 hisG70 hisG428
Source/reference
Phenotype
Deletion of I and F genes Feedback resistant Feedback resistant Feedback resistant Feedback resistant Double mutant Double mutant Feedback hypersensitive Missense Missense Nonsense
histidinol. This limited amount of histidinol supports growth to an absorbance at 650 nm of approximately 0.7. Since increasing amounts of mRNA accumulate with increasing duration of starvation (29), rifampin (180 jg/ml) was added after 1 h of histidine starvation (i.e., 1 h after cessation of growth); 2 min later additions were made, as indicated below. The removed samples were immediately mixed with ice, chloramphenicol (100 ,g/ml), and NaN, (10 mM) to prevent further protein synthesis before the crude extracts were prepared. Enzyme assays and substrates. Protein was estimated by the method of Lowry et al. (28), with insulin standards. The activities of the G and C (L-histidinolphosphate:2-oxoglutarate aminotransferase, EC 2.6.1.9.) enzymes of the his operon were measured by the procedures of Martin et al. (31). A unit of activity is defined as a change in absorbance at 290 nm of 0.25/5 min (31). Enzyme levels are expressed in units per milligram of protein. The synthesis of histidine biosynthetic enzymes is coordinate (1); therefore, the level of aminotransferase is considered to be representative of the differential expression of the entire operon. Aminoacylation of tRNA to evaluate the his-tRNA synthetase activity was carried out as previouslv described (20). L-Histidinol phosphate and 5phosphoribosyl-1-pyrophosphate were Sigma products. Escherichia coli K-12 tRNA was obtained from General Biochemicals Div. (Mogul Corp.), and 1,2,4triazole-3-DLalanine (TRA) was obtained from Cyclo Chemical Co. Other methods. A simplification of the method of Stieglitz and Calvo (41) was used for testing the ability of TA to be incorporated into protein in vivo. The mutant hisIF135 was grown in the presence of a sufficient amount of L-histidine to support growth to an absorbance at 650 nm of 0.4 and then washed and transferred to three separate flasks with ["CC]valine (final concentration, 0.2 mM). The first flask received L-histidine (final concentration, 0.4 mM); the second flask received TA (final concentration, 0.8 mM); and the third received water. At 20-min intervals, samples of 1 ml were removed, quickly filtered through Millipore membranes, and washed twice with 10 ml of minimal medium containing 0.2 mM valine. The radioactivity retained by the filter, which was measured in a Nuclear-Chicago Mark II scintillation
R. F. Goldberger, 12 R. F. Goldberger, 39 R. F. Goldberger, 39 P. E. Hartman, 39 R. F. Goldberger, 39 R. F. Goldberger, 39 R. F. Goldberger, 39 P. E. Hartman, 37 P. E. Hartman, 12 R. F. Goldberger, 12 R. F. Goldberger, 12
spectrometer using a 0.5% solution of 2-(4-t-butylphenyl)-5(4-biphenyl)-1,2,4-oxidiazole (CIBA) in toluene, reached a maximum that was indicative of the maximal radioactivity incorporated in about 1 h. Periodate oxidation was carried out following the method of Folk and Berg (10) as modified by Lewis and Ames (27). The extracted, aminoacylated tRNA's were treated with periodate, stripped of attached histidine, and tested for their ability to accept L["IC ]histidine. Parallel experiments were run with TA or without additions in the aminoacylation reaction.
RESULTS Repression by TA. We first demonstrated the ability of the false feedback inhibitor TA to repress the histidine operon in E. coli (A. R. Tebar, Ph.D. thesis, Universidad Autonoma de Madrid, Madrid, Spain, 1974). We have extended that study to S. typhimurium in which several well-characterized mutants that exhibit altered feedback sensitivity to histidine are available. First, we chose the deletion strain hisIF135 (carrying a wild-type G enzyme), easily derepressible by restricting the supply of exogenous histidinol. Figure 1A shows repression (transient) by TA (0.8 mM) of hisIF135 cells grown with a severe limitation of histidine (doubling time, 6.5 h); the same general pattern was obtained at concentrations of 0.4 or 1.6 mM TA. In contrast, the double mutant hisG1109hisIF135, isogenic with the deletion strain hisIF135 but with an additional mutation (G1109) (39) leading to feedback resistance, was not repressed by identical amounts of TA (Fig. 1B). The same result was obtained in the double mutant hisG1102hisIF135, which carries a different mutation causing feedback resistance. We then studied the effect of the mutation hisG1306, which causes the G enzyme to be 10 times more feedback sensitive than the wildtype enzyme (37). The growth rate was decreased from a generation time of 50 min to 6.5 h with 40 mM AMT and 15 uM histidinol.
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TA
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-
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12
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TA 8
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-20
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FIG. 1. Kinetics of repression by TA (0.8 mM) of strains previously derepressed with limiting amounts of histidinol. The specific activity of the C enzyme (aminotransferase) is shown. In this and in all other experiments, enzymatic determinations were carried out in triplicate, and protein concentrations were in duplicate. (A) Strain hisIF135 (wild-type G); (B) strain hisG1109hisIF135 (feedback-resistant G); (C) strain hisG1306 (feedback-hypersensitive G).
Figure 1C shows the result of a typical experiment, using 0.8 mM TA. A pattern similar to wild type was observed but the "transient repression" lasted some 25 min more. Derepression rate. In the previously mentioned experiments, derepression was accomplished by severe limitation of histidine, using histidinol to regulate growth rate. In all of these cases, a clear correlation can be seen between the rate of expression and the state of the feedback properties of the G enzyme. The rate of increase in specific activity in the two feedback-resistant strains was approximately half as great as that in the wild type. The rate in the case of the feedback-hypersensitive strain is not well defined but appears to be lower than is obtained with the wild type; possible complications owing to the presence of AMT are unresolved. These experiments have been repeated many times and the results are given in Table 2, which also includes data on three other typical hisG mutants. Derepression by AMT of feedback-resistant mutants. AMT inhibits imidazoleglycerolphosphate dehydratase (EC 4.2.1.19), the seventh step in the pathway for histidine biosynthesis, thus reducing histidine levels and producing derepression of the operon (14). (Because AMT also inhibits purine biosynthesis [14], adenine [50 ug/ml] was added to the culture medium.) Figure 2 shows the results of a parallel experiment with strains LT2 and hisG1102; although after the addition of 15 m M AMT the generation times changed equally in both cases (from 1 h to approximately 10 h), no derepression was observed in the feedbackresistant strain (Fig. 2B). Figure 3 shows an experiment with another feedback-resistant muant, hisG1104. This strain is highly resistant to 15 mM AMT, a concentration that has little effect on the growth rate. Derepression was not elicited, even in the presence of 65 mM AMT (second AMT addition), a concentration that appreciably af-
TABLE 2. Rates of derepression Organism
hisIF135
hisG1102hisIF135 hisG1109hisIF135 hisG428 hisG52 hisG70
Defect in G enzyme
Derepression rate Units of C enzyme! Standard deviation mg of protein/h
None Feedback resistant Feedback resistant Catalytically inactive Catalytically inactive Catalytically inactive
a Numbers in parentheses indicate how many values have been averaged.
9.0 (8)a 4.0 (3) 4.2 (6) 9.1(1) 7.4 (1) 5.4 (1)
± 1.4 ± 0.9
±0.9
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ing in a false co-repressor TAAtRNAH"S. This possibility has been ruled out by three lines of evidence. The first is the transfer to tRNAHIS. I The extent to which a compound can protect tRNA from periodate is a measure of the ability of the compound to be attached to tRNA (4). U A TA did not protect tRNA from periodate oxidation (Table 3). The second line of evidence is the incorporation into protein. The analogue TA IA was not incorporated into protein in place of histidine; the total radioactivity incorporated was no different from the control value (Table I a 3-3A0 -3 S 3 aS (ml TI, iSin 4). Third, TA, up to 2.5 mM (400-fold excess FIG. 2. Derepression with AMT (15 mM) of prot- over histidine), had no effect on the charging otrophic strains. Growth curves are shown at the top. activity of semipurified his-tRNA synthetase. (A) LT2 (wild type); (B) hisG1102 (feedback resistExperiments with rifampin. The design of ant). the experiments with rifampin included accumulation of the potential (presumably formation of mRNA) for histidine biosynthetic enAMT ,_____-O zymes, inhibition of further transcription with I rifampin, and measurement of enzyme forma0.90 tion as a function of the different additions (29). AMT Histidinol, the last precursor of histidine on the I pathway, was used as a source of histidine (2) in 10.60 I all the experiments to translate the accumulated messenger. The control experiment with histidinol (60 gM) alone is shown in Fig. 4A. If histidine (0.8 mM) is also supplied with his0.45 tidinol, translation of the messenger is hindered 0 f~~~~ (Fig. 4D); the same result is found with TA (1.6 5 V mM) instead of histidine (Fig. 4C). The histidine analogue TRA is charged into protein (25) 13 .2 and represses the operon (21; V. M. Fernandez, unpublished data), but it does not inhibit the first enzyme for histidine biosynthesis (2, 30). -20 0 s0 s0 100 20 40 120 (minutes) Now if TRA (0.2 mM) is added along with FIG. 3. Absence of derepression, in the presence of histidinol, an incorporation into histidine enAMT (15 mM and 65 mM), of the feedback-resistant zymes similar to the control is observed (Fig. ANT
ANT
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0
0
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mutant hisG1104. Growth
curve
is shown at the top.
fects the growth rate. Another strain, hisG1108, is totally resistant to growth inhibition. In the case of hisG1109 (experiment not shown), the pattern was like LT2 (Fig. 2A), both in growth inhibition and derepression. The sole known role for AMT is the inhibition of the seventh step in the histidine pathway (14). However, in the present study its effect on two other histidine enzymes has been examined. AMT, up to a concentration of 40 mM, had no effect on the activity of the G enzyme in vitro, either wild type or feedback resistant. AMT, up to 40 mM, had no effect on the charging activity of partially purified his-tRNA
synthetase. Investigation of TA properties. There is a possibility that TA represses the wild-type G strain by being transferred to tRNAH'S, result-
TABLE 3. Protection in vitro of tRNAHIS from periodate oxidation Protected with:
Protection(%)
9.5 ........... None ..... L-Histidine (0.15MM)a ................ 100 12 TA (2mM)a ................ a
Final concentration.
TABLE 4. Capacity to support valine incorporation Maximal radioactivity Additions
incorporated
(counts/min)
None (water added) TA (0.8 mM) His (0.4 mM)
7,000 7,400 39,300
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FERNANDEZ ET AL.
6
Additions
J. BACTERIOL.
j
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0
15, 42, 47). The study of the kinetic pattern of repression in various histidine auxotrophs in the presence of TA (19) and the finding that the histidine analogue TRA cannot cause repression of feed-
2
¢
back-resistant mutants (21) led Kovach and colleagues (18) to conclude that the feedbacksensitive site of the first enzyme plays a role in regulation of the histidine operon. Later, they presented evidence indicating that the first enzyme must interact with his-tRNA to fulfill
II
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4
6
Tim. (minutes) FIG. 4. Translation of the messenger accumulated after I h of histidine starvation. At zero time, rifampin was added, and 2 min later the following additions were made: (A) histidinol (60 uM); (B) histidinol (60 AM) plus TRA (0.2 mM); (C) histidinol (60 AM) plus TA (1.6 mM); (D) histidinol (60 MM) plus histidine (0.8 mM). For further details, see text. The specific activity of the C enzyme is shown. The different specific activities in A-D at 2 min reflect the derepression level attained; complications resulting from the presence of AMT during growth could account for this lack of reproducibility.
4B). These results clearly demonstrate that histidine (at 0.8 mM) blocks the expression of accumulated his operon messenger, an effect that is also shared by TA.
DISCUSSION The isolation and characterization of over 2,000 histidine mutants in S. typhimurium by P. E. Hartman and associates (12) has provided the basis for extensive studies on regulation of the histidine operon by Brenner and Ames (8). However, no complete picture of the process has yet emerged. It has been generally accepted that it is the concentration of his-tRNA, and not that of free histidine, that controls repression of this system (3, 27, 35, 38). Aminoacylation of
its regulatory properties (20). It has been demonstrated that TA acts as a false feedback inhibitor (32) of the first enzyme of the histidine biosynthetic pathway (30). In this paper, we present another effect of TA, namely the ability to repress transiently the histidine operon previously derepressed by a drastic limitation in the supply of histidine. That the TA action is exerted at the level of G enzyme is clearly demonstrated by the fact that the double mutants hisG1102hisIF135 and hisG1109hisIF135, which differ from hisIF135 only in an additional G feedback mutation, are not repressed by TA. The above-mentioned effect of TA on the level of enzymes of the histidine operon could be easily explained if TA could be transferred to tRNA and form a false co-repressor that would mimic his-tRNAH"'. The experiments reported here clearly demonstrate that TA cannot protect tRNA from oxidation by periodate (Table 3) and that it cannot be incorporated into protein (Table 4). Moreover, TA neither inhibits the activity of his-tRNA synthetase nor affects the growth rate of a culture suffering a drastic limitation of histidine. All of these data rule out the possibility that TA represses by forming co-repressor. As far as we know, TA behavior is the only case of a false feedback inhibitor that is unable to be incorporated into protein and that represses the operon. This repressing effect can be exerted at the level of transcription or at the level of translation. The experiments with rifampin, carried out to differentiate between them, demonstrate that histidine blocks the expression of present messenger (Fig. 4D), an effect that is also shared by TA (Fig. 4C) but not by TRA (Fig. 4B). Since the primary feature common to histidine and TA is feedback inhibition and TRA allows tormal production of the C enzyme (Fig. 4B), it appears that TA exerts its transitory effect at the level of translation. Control at the level of translation has been
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FEEDBACK SITE IN REGULATION OF THE HIS OPERON
proposed for the trp (23), arg (22, 29, 34), and ilv (46) operons. Interestingly enough, Wasmuth and Umbarger (46) found that free isoleucine (or glycyl-leucine) participates in the postulated translation repression, and not as iletRNA, which is clearly implicated as co-repressor; furthermore, isoleucine is the feedback inhibitor of the first enzyme of the branched pathway, threonine deaminase (43). It is not easy to explain why the repression caused by TA in not permanent. Several explanations would be possible: enzymatic removal from the system, reinitiation of translation on the mRNA at a different site, etc. Here we are dealing with a "false" feedback inhibitor, with a K, 30 times higher than that of histidine (30), and the cell is able to detect it after awhile; the "true" inhibitor histidine does not, of course, produce such a pattern of transient repression. With 0.05 mM TRA, a "false" co-repressor of the histidine operon, transient repression for 30 to 50 min is also obtained (A. 0. Ballesteros and V. M. Fernandez, unpublished data). In addition to this role of the enzyme in repression, the data of Table 2 clearly show the effect of the state of the feedback site on the kinetics of derepression elicited with limiting histidinol and no histidine analogues present. When the feedback site is impaired by mutation, the derepression rate is roughly half of that seen in the wild type. As for the other three G mutants of Table 2, since they have no G activity, it is not possible to ascertain the state of the feedback resistance site, which could be normal, non-existent, or somewhat altered, thus giving normal or intermediate derepression rates. This same effect of lower derepression rate in a feedback-resistant strain was also obtained by Kovach et al. (21). Using the same strains as we used (hisIF135 and hisG1102hisIF135) and following an identical experiment design, they reported derepression rates of 1.7 and 0.9 (arbitrary units), respectively (cf. Fig. 2 and3in [21D. This stimulatory effect of an intact feedback site on the G enzyme in derepression of the operon has been suggested before, on theoretical grounds, by Rothmann-Denes and Martin (36). More recently, Kasai (17) suggested that a positive factor enhancing transcription of the histidine operon binds at the promoter. In yeast, Bollon and Magee (6) found that a normal feedback-sensitive threonine deaminase is necessary for derepression of the ilv pathway, acting possibly as an inducer. Later, they proposed that threonine deaminase acts, in control of the ilv and leu pathways, as a positive
1371
element, determining the amount of leu-tRNA available (7). In E. coli K-12, Levinthal et al. (26) postulated that threonine deaminase is a positive effector in the multivalent control process. The results of the experiments shown in Fig. 2 and 3 reveal more dramatically the effect of the state of the G enzyme feedback site on derepression. We have been unable to elicit derepression of strains hisG1102 and hisG1104 when the histidine limitation is caused by AMT. Loss of feedback inhibition in the mutants leads to an overproduction of histidine, some of the excess being excreted into the medium (39). A large histidine pool in the feedback-resistant mutants hisG1104 and hisG1108 could explain the resistance to growth inhibition by 15 mM AMT. On the other hand, strains hisG1102 and hisG1104 cannot be derepressed, even under conditions of histidine starvation. In strain hisG1102 (Fig. 2), the doubling time of 10 h obtained with 15 mM AMT is indicative of a drastic limitation in the availability of histidine. The same result, absence of derepressibility, was obtained by Kovach et al. (18) in a merodiploid strain with chromosomal hisG deletion and episomal hisG feedback resistance, when also inhibited with 15 mM AMT, a concentration that severely affected growth rate. Patthy and Denes (33) have reported a feedback-insensitive mutant of E. coli K-12 able to derepress only when reaching a high histidine deprivation. All of this means that, under conditions of scarcity of histRNAHis, the state of the feedback site determines whether or not the mechanism of derepression can operate. Other feedback-resistant strains, like hisG1109, can be derepressed, indicating that, as far as regulation is concerned, possibly no two such mutants are alike. Because of the general nature of the process of feedback inhibition, this new kind of repression at the level of mRNA translation by the feedback inhibitors of the first enzyme of the pathway could be easily extended to other biosynthetic operons. Since the feedback inhibition of allosteric first enzymes is commonly accompanied by changes in the quaternary structure, it is tempting to speculate that this translational repression is caused by such changes. ACKNOWLEDGMENTS A.O.B. thanks R. F. Goldberger, who introduced him to the histidine system, and P. E. Hartman for constant advice and help, as well as B. N. Ames, F. Blasi, R. G. Martin, J. R. Roth, M. Levinthal, and H. Whitfield for their comments. We are indebted to A. Albert and co-workers for laboratory facilities; to R. F. Goldberger, A. Sols, and G. Ailhaud for
1372
FERNANDEZ ET AL.
critical reading of the manuscript; and to Laboratorios a gift of rifampin. J. VegaLeal and M. C. Ceinos contributed skilled technical assist-
Lepetit, Sociedad An6nima, for ance.
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