A cis/trans Test of the Effect of the First Enzyme for Histidine Biosynthesis on Regulation of the Histidine Operon

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A cis/trans Test of the Effect of the First Enzyme for Histidine Biosynthesis on Regulation of the Histidine Operon
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  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/18592869 A cis-trans test of the effect of the first enzymefor histidine biosynthesis on regulation of thehistidine operon  Article   in  Journal of Bacteriology · May 1973 Source: PubMed CITATIONS 14 READS 11 5 authors , including:Antonio O. BallesterosSpanish National Research Council 190   PUBLICATIONS   5,649   CITATIONS   SEE PROFILE Marco R SoriaUniversità degli Studi di Salerno 91   PUBLICATIONS   2,689   CITATIONS   SEE PROFILE All content following this page was uploaded by Marco R Soria on 05 March 2014. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  JouRNAL OF BACTERIOLOGY, Apr. 1973, p. 351-356 Copyright   1973 American Society for Microbiology Vol. 114, No. 1 Printed in U.S.A. A cis/trans Test of the Effect of the First Enzyme for Histidine Biosynthesis on Regulation of the Histidine Operon JOHN S. KOVACH, ANTONIO 0 BALLESTEROS,2 MARILYN MEYERS, MARCO SORIA, ND ROBERT F. GOLDBERGER Laboratory of Chemical Biology, National Institutes of Health,Bethesda, Maryland 20014 Previous studies showed that when triazolalanine was added to a derepressed culture of a histidine auxotroph, repression of the histidine operon occurred as though histidine had been added (6). However, when triazolalanine was added to a derepressed culture of a strain with a mutation in the first gene of the histidine operon which renderedthe first enzyme for histidine biosynthesis resistant to inhibition by histidine, repression did not occur. The studies reported here represent a cis/trans test of this effect of mutations to feedback resistance. Using specially constructed merodiploid strains, we were able to show that the wild-type allele is dominant to the mutant (feedback resistant) allele and that the effect operates in trans. We conclude that the enzyme encoded by the first gene of the histidine operon exerts its regulatory effect on the operon not by acting locally at its site of synthesis, but by acting as a freely diffusible protein. Repression of the histidine operon of Sal- monella typhimurium requiresparticipation of aminoacylated histidine transfer ribonucleic acid (histidyl-tRNA; references 1, 7, 12-15, 17). Although the mechanismby which his- tidyl-tRNA acts in repression is unknown, it has been assumed that this molecule fulfills its function in the form of a complex with a specific regulatory protein  2, 3). The histidine analogue, DL-1, 2, 4-triazolo-3- alanine (triazolalanine), is known to cause repression of the histidine operon bybecoming aminoacylated to histidine tRNA  6). We pre- viously reported that this analogue cannotcause repression of the histidine operon when the feedback-sensitive site of the first enzyme for histidine biosynthesis, phosphoribosyl- transferase [N-1-(5'-phosphoribosyl)adenosine triphosphate: pyrophosphate phosphoribosyl- transferase, EC 4.2.1c], is blocked chemically or damaged structurally (5). Because the gene for the first enzyme is adjacent to the operator gene of the histidine operon, we consideredthe  Present address: College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032. 'Presentaddress: Departamento de Catalisis, Consejo Superior de Investigaciones Cientificas, Madrid, Spain.   Present address: Department of Biochemistry and Mo- lecular Biology, Harvard University, Cambridge, Mass. possibility that the effect of the enzyme on *repressibility by triazolalanine wasdue to some action of the enzyme at its site of synthesis. To testthis, we designed experiments to deter- mine whether the effect of a mutation to feedback resistance was cis dominant, asex- pected for an action of the enzyme at its site of synthesis, or whether itis the wild-type allele which is dominantand effective intrans, as expected for an actionof the enzyme as a freely diffusible gene product. like other known regu-latoryproteins. M T RIALS AND METHODS Enzyme assays and substrates. Protein was de- termined by the method of Lowry et al. (8) with insulin standards.Assays for the enzymes specified by the first gene (hisG),third gene (hisC), and seventh gene (hisF) of the histidine operon were performed as previously described (10). Enzyme levels are expressed as the amount of activity per milligram of protein. Substrates were obtained asfollows: L-histidinol phosphateand 5-phosphoribo- syl-lpyrophosphate were purchased from Cyclo Chemical Co.; N'-(5'-phospho-D-ribosylfor-mimino)-5-amino-1-(5 -phosphoribosyl)-4-imidazole- carboxamide was synthesized enzymically from ATP (Sigma Chemical Co.) and 5-phosphoribosyl-1-pyro- phosphate (10). Bacterial strains. The strains employed in this study are listed in Table 1. The following strains were 351  352 KOVACH TABLE 1. Strains employed Strain Histidine no.a Genotype require- ment TA 830 hisOGDCBHAF644 Yes hisIF135b hisIF135 Yes TA929C hisG1102 hisIF135 Yes SB900d hisOG1302 his W1824 Yes TG5718d hisOG1302hisW1824 pur-804 YesTG5720c,F 80hisG1929 hisB+/ No hisOG1302 his W1824 pur-804 TG5722b, C F 80hisG1929 hisB+/hisIF135 No TR75° F 80hisB2405/hisDCBHAFIE712 Yes arg-501 ser-821 TR59b F 80hisC2389/hisDCBHAFIE712 Yes arg-501 ser-821 TG769b, C F'80hisC2389/hisG1102 hisIF135 No TG5705b.C F 80hisC2389/hisOGDCBHAF644 Yes TG5719b, F 80hisB2405/hisOG1302 No his W1824 pur-804 TG5721c d F 80hisG1929 hisB+/ No hisOGDCBHAF644 aAll strains designated TG were constructed as part of the present study (see materials and meth- ods). bProduces normal phosphoribosyltransferase. c Produces feedback-resistant phosphoribosyl- transferase. d Produces no phosphoribosyltransferase. obtainedfrom the collectionofP. E. Hartman: hisOGDCBHAF644, hisIF135, TA929, SB900, TR75,andTR59. The mutation, hisG1102 (in strains TA929 and TG5769), is a missense mutationwhich results in the production of a catalytically active feedback- resistant phosphoribosyltransferase (16); the muta- tion his W1824 (in strains SB900,TG5718,and TG5720) is not linked to the histidine operon and resultsinconstitutive expression of the operon (1). Strain TG5769 wasprepared by transferof the episomefrom strain TR59 intostrain TA929, select- ing for histidine prototrophy; strain TG5705 wasprepared by transfer of the episome from strain TR59 into strain TA830, selecting for growthon histidinol. In both cases, the donor, strain TR59, was counter- selected by omission of arginine and serine from the mating plates; strain TG5718 was derived from strain SB900by penicillin selection for a spontaneously arising adeninerequirement (pur-804). Isolation of a strain carrying anepisome with a mutation to feedback resistance was accomplished by the followingprocedure, which is a modification ofthe method of Sheppard (16). Strain TG5719 was prepared by transferring the episome of strain TR75 into strain TG5718. This transfer was identified by selecting for histidine prototrophy. The merodiploid strain wasused for selection of feedback-resistant mutations. Since it contained no chromosomal hisG gene, only episomal mutationscould be obtained by selection for feedback resistance. It had been shown ET AL. J. BACrERIOL. by Sheppard (16) that a mutation to feedback resistance causes the organism to excrete histidine. This excretionofhistidine can be detected bygrowth on minimal plates of a histidine auxotroph in the immediate vicinityof a colony of the feedback-resist- ant strain. We found, in keeping withthe findings of J. R. Roth (unpublished data), that the presence of an additional(constitutive) mutation causing dere- pression of the histidine operonenhances this  feed- ing by a feedback-resistant strain. Therefore, we startedselection for a feedback-resistance episome by mutagenesis of strain TG5719 which contains the chromosomal constitutive mutation, hisW1824. Mu- tagenesis was carried out by the addition ofa few crystals of N-methyl-N'-nitro-N-nitrosoguanidine (Aldrich Chemical Co.) to each of 20 mid-log-phase cultures, each in 10 ml of medium E of Vogel andBonner (18), supplemented with glucose(0.5 ) and adenine (20 pg/ml). After the cultures had been shaken at 37 C for 10 min, the cells were centrifuged, washed, and suspended in fresh medium containing D,L-2-thiazolalanine (thiazolalanine, 10 mM; Cyclo Chemical Co.). Thiaz6lalanine is an analogue of histidine that inhibits phosphoribosyltransferase by the same mechanism as does histidine (9, 11). By inhibiting the biosynthesis ofhistidine, it inhibits growth of all strains prototrophic for histidine except thosethatcontaina mutation affecting the abilityof phosphoribosyltransferase to be inhibited (feedback- resistant mutant) or a mutation affecting the abilityof the cell to transport the analogue(permease mutant). Thus,growth of the mutagenized cultures in thepresence of thiazolalanine enrichedthe cul-tures for such mutants. Feedback-resistant mutants could thenbe distinguished frompermeasemutants by their abilityto  feed. After growth to saturation in thiazolalanine, approximately 10' mutagenized cells were mixed with approximately 10' cells of the histidine auxotroph, strain TA830, in 2 ml of 0.6 melted agar (45 C) containing thiazolalanine (50 mM) and adenine (15 ug/ml). Thismixture was poured onto a medium E plate. The presence ofthiazolalanine in the pour plate further enhanced selection for feedback-resistant mutants and per- mease mutants. After incubating the plates for 48 h at 37 C, feeder colonies were easily recognized and picked. Twenty-five such colonies were purified by repeated single-colony isolation on minimal medium.Each of these strains was found to contain a phos-phoribosyltransferasehighly resistant to inhibition by histidine. One of them, strain TG5720, was chosen for further studies. The phosphoribosyltransferase ofthis strain was not inhibited at all by histidine at aconcentration (5 x 10-1 M), 1,000-fold higher than the Kg of the wild-type enzyme. Transfer of the episome tostrain hisOGDCBHAF644 yielded strain TG5721 which, as expected, was highly feedback resistant butwhich, in addition, was prototrophic,indicating that a reversion of the episomal hisB missense mutation had occurred. This episome was also transferred to strain hisIF135 to yield strain TG5722. Growth conditions. Cells were routinely grown in 2 liters of medium E of Vogel andBonner (18), with  HISTIDINE BIOSYNTHESIS glucose at 0.5 , in a 4-literflask. The cultures were aerated vigorously in a New Brunswick rotary shaker at 37 C. The histidine auxotrophs were grown in the presence of a sufficient amount of L-histidine to supportgrowth to an optical density at 700 nm of approximately 0.35 (3.4 x 101 cells/ml).  fter deple- tion ofhistidine from the growth medium, growth was supported by L-histidinol (2.5 x 10-i M). Dere- pressionof prototrophic merodiploid strains was producedbyadding 3-amino-1,2,4-triazole (amino- triazole, 15 mM; Aldrich Chemical Co.). Amino- triazole inhibits imidazole glycerol phosphatedehy- dratase, the enzyme catalyzing the seventh step of the histidine pathway.Because aminotriazole also inhibits a step of purine biosynthesis'(4), adenine (50 gg/ml; Cyclo Chemical Co.) was added to the culture medium. Experimental design. In a typical experiment, 2 liters ofculture wasgrown in a rotary shaker in a 4-liter flask. Samples (80-100 ml) were withdrawn periodically,so that six samples were collected dur- ingderepression and six samples were collected after the addition ofhistidine (1.6 x 10-3 M) or triazolala- nine (3.2 x 10-i M; Cyclo Chemical Co.). Each sample wasimmediately mixed with ice and excesshistidine. The samples werethen centrifuged, and the pelleted cells were washed with 0.05 M tris(hy- droxymethyl)aminomethane buffer, pH 8.0, and re- centrifuged. The cells were then suspended in3.5 ml of the same buffer. Extracts were prepared from the suspensions with a French pressure cell (American Instrument Co.) at 6,000 lb/in2 (ca. 4,218, 600 kg/mi, clarified by centrifugation, and assayed. RESULTS The studies reportedhere concern the rela- tionship between wild-type andmutant alleles of the hisG genewith respect to their effects on repressibility of the histidine operon by triazol- alanine. The experimental design was essen-tially the same as that employed in aprevious study (5) which showed that triazolalanine does not cause repression in feedback-resistant mutants. In that study we showed that, when triazolalanine was added to a derepressed cul- ture ofa histidine auxotroph, repression of the histidine enzymes occurred as though histidine had been added. Repression failed to occur, however, when triazolalanine was added to a derepressed culture of an isogenic strain which had, in addition, a mutation in the hisG gene which rendered thephosphoribosyltransferase resistantto feedback inhibition (5). In the first series of experiments, we ex- amined thequestion of whether the response to triazolalanine in a feedback-resistant strain would be altered if an episome,bearing a wild-type hisG gene, were present in the cyto- plasm (Fig. 1). Figure 1A shows the results of a control experiment in which the episome was in the cytoplasm of a strain fromwhich the histidine operon had been deleted. The histi- dineoperon of the episomewas derepressed by limiting the amount of histidine available in the culture medium and was repressed by the addition of triazolalanine. Figure 1B shows the results of another control experiment, in which derepression and repression were studied in the feedback-resistant strainalone. Asshown pre- viously (5) and confirmed here, when triazolal- anine was added to the derepressed culture, repression of the histidine operon did not occur, despite the fact that the growth rate returned to that characteristic of repressed cells. Figure 1C shows the results obtained in the merodip- loid strain: episomewith a wild-type hisG gene; chromosome with a feedback-resistance mutation in the hisG gene. The presence of a mutation in the hisC gene of the episome allowed us to use C-enzyme (aminotransferase) activity to assess chromosomal expression; the presence of a deletion of the hisI and hisF genes of the chromosome allowed us to use I- and F-enzyme activities toassess episomal expres- sion. The addition of triazolalanine to a dere- pressed culture ofthis merodiploid strain caused repression of the histidine operons of both chromosome and episome. The same re- sults were obtained in numerous experiments. In all cases, repression of the histidine operon by triazolalanine could beobtained in a feed- back-resistant strain only when an episome bearing a wild-type hisG gene was present in the cytoplasm. Thus, the normal phos- phoribosyltransferase,specified by the wild- type episomal hisG gene, was able torestore repressibility to the chromosome, demonstrat- ing a trans effect of the wild-type allele. Because morethan one copy of an episome is present in the cytoplasm, in order to pursuethe question of dominance, we carried outa series of experiments which are reciprocal to those described above. For these experiments we studied the effect of a wild-type chromosomal hisG gene on repressibilityof an episome bear- ing a feedback-resistance mutation. It was first necessary to isolate a feedback-resistance epi- some. A procedure was developed by which very highly feedback-resistant episomal mu- tants could be selected with the same basic principles srcinally used by Sheppard (16) to select feedback-resistant chromosomal mu- tants. Assays of extracts of strains carryingthese episomes (but with the chromosomal histidine operon deleted) showed that the phos- phoribosyltransferase they produced was veryhighly resistantto inhibition by histidine. 353 OL. 114, 1973  2  600 TR T o TRA TR R o ~0.45 B o T 0 401   4_e0 o ~~~ 30 0 00 20 20 2 0 2 F IG. 1. Effect of a wild-type hisG gene carried by an episome in the cytoplalsm of a strain with a chromo- somalmutation to feedback resistance. For each part, the growth curve is shown at the top. During the first portion of the experiments, the organisms were g'rown on limiting histidine which caused derepression of the histidine operon. When triazolalanine was added to the derepressed cultulres (arrows), the growth rates im- mediately increased to that characteristic of repressed cells. The specificactivity of the C enzyme or F en- zyme at various times during the experiments is shown at the bottom. (A) F-enzyme activity of -strain TG5705 (episome with a mutation in the hisC gene; chromosome with a deletion of almost the entire histidine operon). T'he data show that the episomal histidine operon is derepressible by histidinelimitation and repressible by triazolalanine. (B) C-enzyme'activity of strain TA929 (haploid strain in which there is a hisGmutation to feed- back resistance and a deletion of the hisI and hisF genes). The data show that the (chromosomab[ operon of this feedback-resistant strain is derepressible by histidine limitation but cannot be repressed by triazolalanine. (C) C-enzyme Iactivity of strain TG5769 (episome with a mutation in the hisC gene; chromosome with a muta- tion to feedback resistance in the hisG gene and a deletion of the hisI and hisF genes). ThDe data show that the episome (carrying a wild-type hisG gene)confers upon thehistidine operon of the chromosome the ability to be repressed by triazolalanine. The effect of triazolalanine on repressibility was tested by using a feedback-resistance epi- some in a strain in which the histidine operon was deleted from the chromosome. Surpris- ingly, when the growth of this merodiploid strain was limited by histidine deprivation, the histidine operon failed to become dere- pressed at all. An experiment which illustrates this phenomenon is shown in Fig. 2A. Although repressibility of the episome alone could not be studied, the question of whether or not a wild-type hisG geneon the chromosome would confer upon the episome the ability to become derepressed and repressedcould be studied.Therefore, we introduced the feedback-re- sistance episome into a strain with a wild-type chromosomal hisG gene butwith a deletion of the hisI and hisF genes. Because of the chro- mosomal deletion inthis strain, we could use I- and F-enzyme activities to assess episomal expression. When the growth of this merodi- ploid strain was limited by histidine depriva- tion, the histidine operon of the episome did become derepressed; when triazolalanine was then added to the derepressed culture, the histidine operon of the episome became re- pressed. These findings are illustrated by the experiment shown in Fig.2B. Numerous sim- ilar experiments gave the same results. Thus, it is clear thatthe episomal operon, which is by itself unresponsive to the availability of his- tidine, becomes responsive andbecomes re- pressible by triazolalanine when it is in the cytoplasm of a strain which carries a wild-type chromosomalhisG gene. These findings dem- onstrate again the trans effect of the wild-type allele. They also demonstrate that the feed- back-resistance mutation does not show a position effect due to gene dosage. Table 2 summarizes the results of morethan 50experiments, each involving duplicate as- says for several enzymesand for protein in at 354 KOVACH ET AL. J. BACTEIOL.
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