ACTION OF A HISTIDINE ANALOGUE, 1,2,4-TRIAZOLE-3-ALANINE,IN SALMONELLA TYPHIMURIUM

ALFRED P. LEVIN' AND PHILIP E. HARTMANDepartment of Biology, The Johns Hopkins University, Baltimore, Maryland

Received for publication 3 June 1963

ABSTRACT

LEVIN, ALFRED P. (The Johns Hopkins Uni-versity, Baltimore, Md.), AND PHILIP E. HART-MAN. Action of a histidine analogue, 1,2,4-triazole-3-alanine, in Salmonella typhimurium.J. Bacteriol. 86:820-828. 1963.-The effect ofthe histidine analogue, 1,2,4-triazole-3-alanine(TRA), on growth and enzyme synthesis inhistidine auxotrophs of Salmonella typhimuriumhas been studied. TRA allows an increase ofapproximately 50% in the amount of protein in aculture but does not allow concomitant synthesisof ribonucleic acid and deoxyribonucleic acid.Although the analogue prevents the formationof active bacteriophage and of enzymaticallyactive inosine 5'-phosphate dehydrogenase, itdoes not prevent the formation of enzymaticallyactive L-histidinol phosphate phosphatase or ofimidazoleacetol phosphate transaminase, twoenzymes involved in the biosynthesis of histidine.Of the three known functions of histidine in thecell, TRA mimics two: it is incorporated intoprotein, and it acts as a repressor material forsynthesis of enzymes involved in the formationof histidine. TRA fails to act as a feedback in-hibitor of the first step in the formation of histi-dine.

The metabolism of structural analogues ofamino acids by bacteria, protozoa, yeasts, andmammals has been studied in recent years (seereviews by Shive and Skinner, 1958; Richmond,1962a). These studies have detected the abilityof analogues to mimic various functions of normalmetabolites, such as the ability to be incorporatedinto protein, to act as feedback inhibitors, and toelicit repression. During the process of screeninghistidine analogues for their ability to replacehistidine as a growth factor for histidine auxo-trophs of Salmonella typhimurium, Ames andHartman (1962) noted that 1,2,4-triazole-3-

I Present address: Department of Biochemistry,University of Tennessee, Knoxville.

alanine (TRA) stimulated the growth of oneclass of histidine mutants, namely, mutants ofgene E. We have examined further this findingin relation to the metabolic functions of histidine.It had been reported previously that TRA wasan inhibitor of growth of Escherichia coli eitheralone (Jones and Ainsworth, 1955) or in combina-tion with other metabolic analogues (Wheeler,Schabel, and Skipper, 1956). The results indicatethat TRA mimics L-histidine in its ability to beincorporated into protein and in its ability toelicit repression. A preliminary report of thesefindings has been presented (Levin and Hartman,1963).

\I'ATERIALS AND METHODS

Mutants, media, and chemicals. Most of thehistidine auxotrophs of S. typhimurium strainsLT-2 and LT-7 used in this study have beendescribed (Hartman, Loper, and Serman, 1960b;Hartman, Hartman, and Serman, 1960a). Mutanthis-203-262 is a constitutive mutant describedby Ames, Hartman, and Jacob (1963). MutanthisEa-349 was isolated after growth of the parentLT-2 auxotroph strain (gua-i), with 5-bromo-deoxyuridine and subsequent isolation by meansof the penicillin selection technique (Lederberg,1950).The growth medium was that of Vogel and

Bonner (1956). Growth of cultures at 37 C, intubes with forced aeration or in flasks on a recip-rocating shaker, was followed turbidimetricallyin a Klett colorimeter (54 filter; Klett Mfg. Co.,New York, N.Y.) or in a spectrophotometer(Beckman, DU; Beckman Instruments, Inc.,Fullerton, Calif.) at 650 my. Counts of the num-ber of viable bacteria were made on nutrientagar plates.The L-histidinol phosphate used in the enzy-

matic assays was synthesized by the procedureof Ames and Horecker (1956) as modified byAmes (personal communication). In the modifiedprocedure, the ratio of polyphosphate to L-histidinol dihydrochloride was raised from 3.5:1

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VOL. 86, 1963 EFFECT OF A HISTIDINE ANALOGUE ON S. TYPHIMURIUM

to 12.3:1. After heating the reactants, a smallvolume of water was added, and this mixturewas heated an additional 30 min. The reactionmixture then was applied directly to a Dowex-l-acetate column. After elution with water, thePauly positive material was adjusted to pH 9with ammonia and reapplied to a Dowex-1-acetate column. Elution was started with a lineargradient 0 to 0.2 N acetic acid. After 2.5 columnvolumes, elution was changed to 0.15 N aceticacid, and the L-histidinol phosphate which waseluted between 1 and 2 column volumes waslyophilized to dryness, extracted with ethanol,and dried. The yield of chromatographically purematerial was approximately 50%.

1,2,4-Triazole-3-alanine (TRA) was obtainedfrom R. G. Jones, Eli Lilly & Co., Indianapolis,Ind.

Chemical determinations and methods. The pro-tein contents of bacterial suspensions and ex-tracts were determined by the method of Lowryet al. (1951); ribonucleic acid (RNA) was meas-ured by the orcinol method as described by Morseand Carter (1949); deoxyribonucleic acid (DNA)was determined by the method of Burton (1956).The protein fraction of cells was prepared by

the procedure of Roberts et al. (1955). Hydrolysisof this fraction was performed in 6 N HCI for 15hr at 112 C in sealed ampoules. After hydrolysis,the material was evaporated to dryness in a flashevaporator, dissolved and re-evaporated twicefrom water, and finally dried over silica gel andNaOH pellets in vacuo.

Descending chromatography of the proteinhydrolysates was performed for 24 hr on What-man no. 1 paper with the propanol-ammoniasolvent (solvent I) of Ames and Mitchell (1952).Subsequent descending chromatography of com-ponents eluted from these papers was on What-man no. 1 paper with the butanol-acetic acid-water solvent (solvent II) of Mandelstam (1958);the solvent was allowed to run over the paperfor two 24-hr periods, between which the paperwas dried.Chromatograms were visualized by reaction

with ninhydrin spray (Mandelstam, 1958). TRAreacted to yield a brownish-purple color. Develop-ment was at room temperature.

Preparation of bacterial extracts and enzymeassays. Extracts of bacteria were prepared fromcell suspensions washed and resuspended in 0.02to 0.05 M tris(hydroxymethyl)aminomethanebuffered at pH 7.5 with HCl. Suspensions, at 4 C,were disrupted for 2 min in a sonic oscillator

(MSE, model 60W; Measuring & ScientificEquipment, Ltd., c/o Instrumentation Asso-ciates, New York, N.Y.). Sonic extracts werethen centrifuged at 32,000 X g for 30 min in arefrigerated angle-head centrifuge.Most assays of extracts for imidazoleacetol

phosphate transaminase and for L-histidinolphosphate phosphatase activities were performedunder the conditions described by Ames, Garry,and Herzenberg (1960). However, in obtainingthe results reported in Table 2, assays for L-histidinol phosphate phosphatase were performedin the presence of 10-2 M Mg++. The Mg++ in-creased the activity by approximately 30%(Ames, personal communication). Inosine 5'-phosphate (IMP) dehydrogenase activity wasmeasured by the method of Magasanik, Moyed,and Gehring (1957).

RESULTS

Growth of histidine mutants on TRA. The origi-nal observations by Ames and Hartman (1962),that TRA stimulated growth uniquely of mutantsof gene E, were made on strains which had beenplated on minimal agar and to which crystals ofthe analogue then were applied. Such experi-ments were repeated, and the observations wereconfirmed that E mutants gave a zone of growthwhereas mutants of other genes failed to re-spond. However, this growth behavior contrastedsharply with results obtained in liquid culture.Cultures were grown overnight in minimal me-dium containing excess amounts of histidine(i.e., 20 ,g/ml, an amount in excess of that re-quired for maximal growth) and then inoculatedinto tubes containing limiting amounts of histi-dine (4 to 5 ,ug/ml). The cultures were grown at37 C with forced aeration. Under these condi-tions, TRA, added either at the time of inocula-tion or at the time of exhaustion of histidine,caused similar "growth" responses not only inmutants of gene E but also in mutants of gene G(single-site and multisite) or in multigene "dele-tion" mutants, such as hisE-D-712. Single-sitemutants from genes F, A, H, B, C, and D failedto respond to TRA under these conditions.The "growth" response of a mutant from gene

G (his-203) is depicted in Fig. 1. The opticaldensity of the culture with TRA (30 ,ug/ml) in-creased about 50% over a parallel culture withoutTRA.

Protein synthesis in the presence of TRA. Toinvestigate the nature of the constituent(s) re-sponsible for the TRA-stimulated increase in the

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J

cc

H.zJ)1-

0 60 120 180 240

TIME IN MINUTES

FIG. 1. Growth of strain his-203 onhistidine with (A) and without (0) addelalanine (TRA). An overnight culture was iinto medium containing L-histidine - HCliAt 90 min, TRA (30 ,ug/ml, final concewas added to one of two parallel culture&Subsequent growth was followed in both c

3500 _ 2.0 -

Prote,in) 3000 _

D2500

6 I.0 x 090 9-

2000 6iV ,g 7 [

1500 1 6t* No Add

LTRA

oL-histidint-HCI

30 60 90

TIME IN MINUTES

ViableCount

RNA, and DNA, and of the viable number ofcells in a culture of his-46 (gene G) after exhaus-tion of histidine, after exhaustion of an additionalsupplement of histidine, and after growth onTRA (Fig. 2). In the culture to which TRA wasadded, the increase in protein reflected the in-crease in optical density (upper left of Fig. 2).In contrast, the negligible increase in RNA,DNA, and viable count did not correspond to thelarge increase in optical density. The histidinesupplement allowed commensurate increases inprotein, RNA, DNA, and viable counts. Thelarge increase in protein in the culture grown on

300 360 TRA indicated that the analogue or a derivativeof it was being incorporated into protein.

limiting Further confirmation of the above conclusiond triazole- came from determinations on the effect of chlor-inoculated amphenicol on the optical density of the culture(4 ,ug/ml). after addition of TRA. As can be seen in Fig. 3,entration) chloramphenicol completely abolished the growths (arrow). response to the analogue.ultures. Recovery of TRA from protein hydrolysates.

Additional evidence for TRA utilization in pro-tein synthesis was obtained by isolation of theanalogue from the protein of cells grown on TRA.Mutant his-46 was grown on limiting histidine,and TRA was added at the time of exhaustionof the histidine. The culture was grown for anadditional 90 min. The cells then were centri-

O 4.0 -DNA

2 3.0 -

-J

6E2.0J0O

-L I00 30 60 90

TIME IN MINUTES

FIG. 2. Protein, RNA, DNA, and viable countsof Salmonella typhimurium strain his-46 grown onhistidine and on TRA. An overnight culture wasinoculated into medium containing limiting histi-dine. After exhaustion of the histidine, the culturewas divided into three parts: 0 = no additions;A = TRA (final concentration, 15 ,Ag/ml); 0 =L-histidine* HCl (final concentration, 2 ,ug/ml).Zero time is the time of division of the culture intothree portions. The per cent increase in opticaldensity after 90 min with no additions was 8.2,with TRA was 48, and with histidine was 52.

optical density, an experiment, designed similarlyto that shown in Fig. 1, was performed. Deter-minations were made of the amounts of protein,

200

w 160

-J

in 120

I-

z 80

w

-i40Q

120 240 360

TIME IN MINUTES

FIG. 3. Chloramphenicol inhibition of growth ofstrain his-63 on TRA. Growth of parallel cultures onlimiting histidine was followed. After exhaustionof histidine, TRA (final concentration, 16 ug/ml)was added to two cultures (0, OJ) and, after 30 min,chloramphenicol (CAP), at a final concentrationof 50 jug/ml, was added to one of these cultures (O).Nothing was added to the third culture (*).

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fuged, washed several times in minimal medium,and were fractionated as indicated in Materialsand Methods. The hydrolyzed protein fractionwas chromatographed in solvent I and, afterdrying, the TRA area of the chromatogram wascut out, eluted with water, and chromatographedin solvent II. As seen in Fig. 4, a spot appearedin the hydrolysate of the protein derived fromthe his-46 culture which migrated in the samearea as does TRA. No spot was present at thisposition in chromatograms of protein hydroly-sates of wild-type LT-2, but a similar spot didappear in chromatograms of hydrolysates pre-pared by adding TRA to LT-2 protein and hy-drolyzing the mixture. The results indicate thatTRA can be incorporated in vivo into Salmonellaproteins.

Synergism between TRA and purines. After itwas demonstrated that TRA was incorporatedinto protein, presumably in place of histidine inthese specific histidine auxotrophs, the problem ofwhy so many histidine auxotrophs failed to growon the analogue was puzzling. Hartman (1956)reported that mutants of gene H and othermutants with incomplete genetic blocks wouldrespond to adenine in place of histidine. Shed-lovsky and Magasanik (1962) proposed thatgrowth on adenine was due to the replacementof the internal adenine supply which had beendrained by the excretion of 5-amino-1-ribosyl-4-imidazolecarboxamide (AICAr) into the mediumby these mutants. AICAr is excreted since onlythe N1-C2 unit of an adenine derivative, adeno-sine triphosphate (ATP), is utilized in the bio-synthesis of histidine (Moyed and Magasanik,1960; Ames, Martin, and Garry, 1961). Whenadenine, adenosine, or other purine bases ornucleosides were added together with TRA, allclasses of mutants which had failed to respondto TRA alone now grew. Among histidine mu-tants, the only exceptions were mutants whosesites of mutation were restricted to gene D; theselatter mutants accumulate histidinol. Additionof L-histidinol was found to prevent the TRAresponse in a D-G double mutant.The finding that a purine source together with

TRA could elicit a growth response in mutantsof gene H enabled us to distinguish biologicallybetween histidine and TRA. H mutants wereused to assay the material eluted from the chro-matograms from solvent I (see Fig. 4). Thematerial which was chromatographically identi-fied as containing TRA gave a growth response

only after the addition of adenine. The materialfrom the LT-2 hydrolysate, to which no TRAhad been added, failed to give a response with orwithout adenine. These results substantiate theidentifications denoted in Fig. 4.

Inability of TRA to serve as a feedback inhibitor.As noted above, addition of TRA alone caused agrowth stimulation in mutants blocked in thevery early reactions in the histidine biosyntheticpath (genes G and E), but failed to stimulatemutants blocked in later steps. However, a purinesupplement enabled mutants from this lattergroup to respond to TRA. These results are inkeeping with the idea that TRA fails to mimicL-histidine as a feedback inhibitor of reaction 1.Indeed, Moyed (1961), studying compound IIIformation in extracts of Escherichia coli, foundTRA to be a very weak inhibitor; Ames et al.(1961), using crude extracts of S. typhimurium,and Martin (1963), using a partially purifiedpreparation of phosphoribosyl-ATP pyrophos-phorylase, indicated that TRA failed to act as aninhibitor of this reaction (reaction 1).

Proteins made in the presence of TRA. Since

G46-TRALT2 +TRA

TRATH REO

LT2 ALA

TRA

TH REO

ALA

FIG. 4. Chromatogram of eluates from proteinhydrolysates. TRA areas from chromatograms runin solvent I were eluted and applied to paper fordevelopment by descending chromatography insolvent II, pictured here. G46-TRA is from a proteinhydrolysate of culture hisG-46 grown on TRA. LT-2+ TRA is from a hydrolysate of wild-type proteinto which TRA was added before hydrolysis. LT-2is from a protein hydrolysate of a wild-type culture.Threo = L-threonine; Ala = L-alanine. The natureof the compound reacting with ninhydrin and run-ning behind (above) TRA is unknown.

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:W 1201--z

ioo

0 6

Z 4

U-

z

it 60

Z 40

20

BACTERIOPHAGE H5 -Growth in his-46

* No Add

a TRAo L- histidine - HCI

) _

10 20 30 40 50

TIME IN MINUTES60 70

FIG. 5. Growth of bacteriophage mutant H5 ofP22 on his-46 with and uwthout TRA and histidine.Cells in logarithmic growth were centrifuged, washed,and suspended in minimal medium. Bacteriophagewere added (multiplicity of infection = 0.07).After 6 min for adsorption, the mixture was diluted1:100 into medium containing dextrose and nofurther additions (0), L-histidine* HC1 (0), TRA(A).

TABLE 1. Effect of TRA and L-histidine HC1on IMP dehydrogenase formation in strain

gua-1 hisEa-849*IMP

dehydrogenaseAdditions Amt added at 90 min

(units/mg ofprotein)

JAg/mi(1) None 0.06(2) L-Histidine*HCl 2 0.96(3) TRA 15 0.04(4) L-Histidine HCl 2

8-Azaguanine 20 0.12(5) TRA 15

8-Azaguanine 20 0.06

* A culture was grown on limiting guanine (10,g/ml) and L-histidine HCl (4,ug/ml) until bothsubstrates were exhausted from the medium. Theculture was divided, additions were made as in-dicated in the table, and then each mixture wasincubated for 90 min. Extracts were prepared aridtested. The units/mg of protein at zero time were0.03.

TRA is incorporated into protein, we can examineits effect on the subsequent efficiency of theprotein. We have examined the effect of theanalogue on the ability of cells to produce bac-

)

teriophage P22. Cells of strain his-46 were grownovernight in minimal medium containing histi-dine. These cells then were diluted 1:100 infresh medium of the same composition and weregrown to a cell density of 7 X 108/ml. The cellswere centrifuged, washed, and resuspended inminimal medium. A virulent mutant of bac-teriophage P22 was added to the culture and,after 5 min for adsorption, the mixture wasdiluted 1:100 into minimal medium containingdextrose and into the same medium containingeither histidine or TRA (Fig. 5). The culture wasaerated, and the total number of infectiouscenters was measured at intervals. There was anincrease in the number of infectious centers onlywhen histidine was present. Thus, TRA cannotsubstitute for histidine in the production of P22bacteriophage.TRA also failed to support the production of

active IMP dehydrogenase in nonproliferatingcells suspended under conditions which led tothe rapid formation of enzyme when histidinewas present (Table 1). The inclusion of 8-aza-guanine acted as an additional control, since ithas been shown that in the presence of this com-pound the production of IMP dehydrogenase isrepressed (Levin and Magasanik, 1961).Not all proteins examined after growth of cells

on TRA were inactive. A derivative of strainhis-203, mutant his-203-262, although still re-quiring histidine for growth, has constitutivelevels of enzymes concerned with histidine bio-synthesis which are seven to ten times those inwild-type cells (Ames et al., 1963). These levelsare independent of the histidine content of themedium. When his-203-262 cells are grown onTRA, there is no change in the specific activityof the L-histidinol phosphate phosphatase or ofthe imidazoleacetol phosphate transaminase,despite a considerable increase in protein (Table2). These results indicate, therefore, that bothenzymes made in the presence of TRA are activeenzymatically.TRA and repression. Since active L-histidinol

phosphate phosphatase and imidazoleacetolphosphate transaminase are made in the presenceof TRA, it is possible to assess the role of TRAin the control of the formation of the enzymesinvolved in histidine biosynthesis. Hartman et al.(1960a, b) showed that all of the genes controllingthe synthesis of histidine are closely linked.Ames and Garry (1959) demonstrated that theenzymes in the pathway are coordinately re-

0 -0-.b-T;sE9a;A=Fll

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pressed and derepressed. Using strain his-1l,which is slightly leaky, i.e., will grow slowly inminimal medium, Ames and Garry showed thatafter the exhaustion of histidine from the mediumthe cells became derepressed and enzyme levelsten times those in wild-type cells were reachedin 2 hr. We have repeated this type of experi-ment, using supplements of either histidine or ofTRA. Growth responses to the different condi-tions are indicated at the left of Fig. 6. As seenin the right portion of Fig. 6, the addition ofhistidine causes an inhibition of enzyme forma-tion. This repression is followed by a rapid syn-thesis of the enzymes after the re-exhaustion ofhistidine from the medium at about 90 min. Onthe other hand, TRA completely prevented theincrease in specific activity of the enzymes forthe duration of the experiment (a full hour aftergrowth on TRA ceased). Since active enzymecan be synthesized in the presence of TRA(Table 2), the failure of the specific activity toincrease must be due to a role of TRA in repres-sion.

DISCUSSION

Apparent incorporation of amino acid ana-logues into cellular components has been observedmany times (see reviews by Cohen and Gros,1960; Richmond, 1962a). The finding that TRAis incorporated into protein extends this list andconfirms such a proposal by Moyed (1961) forthe action of the analogue on cultures of E. coli.In addition, we have shown that TRA elicitsrepression of the histidine biosynthetic enzymesjust as does L-histidine.The effect of TRA in apparently replacing

histidine in protein is similar to those effectsobserved for other analogues such as tryptazan(Brawerman and Ycas, 1957; Pardee and Pres-tidge, 1958) or 7-azatryptophan (Pardee andPrestidge, 1958) for tryptophan; norleucine(Munier and Cohen, 1959) for methionine;canavanine (Richmond, 1959) for arginine; ando-, m-, and p-fluorophenylalanine (Munier, 1959;Munier and Cohen, 1959) and fl-2-thienylalanine(Munier and Cohen, 1959) for phenylalanine.In contrast, the effect of TRA on nucleic acidformation differed when compared with tryp-tazan, p - fluorphenylalanine, or ,B -2- thienyl-alanine. All three of these analogues allowed anincrease in nucleic acid formation, the first twoincreasing the nucleic acids in proportion to theprotein increase. The last compound allowed

TABLE 2. Effect of TRA and L-histidine HC1on enzyme formation in strain his3-203-262*

Enzyme formed(units/mg of protein)

Relative Relative LHsAddition optical protein tidinol Imida-protein phos zo~leacetol

pht ph~osphate

phatase aminase

None 0.59 0.57 4.31.0 1.0 4.3 2.121.09 1.08 4.7 2.11

L-Histidine HCl 1.40 1.33 4.7(2 qg/ml) 1.43 1.36 4.8 2.23

TRA (15 ,ug/ml) 1.50 1.31 4.31.53 1.49 4.5 2.23

* A culture was grown on limiting histidine and,at the time of exhaustion of the histidine (relativeoptical density = 1.0), the culture was dividedinto three parts. One part received histidine, thesecond received TRA, and the third received noadditions. The final concentrations of the supple-ments are indicated in the table. Protein and en-zymatic analyses were made at the indicated rela-tive optical densities.

60 1201M80TIME IN MINUTES

IMIDAZOLEACETOLPHOSPHATE r

TRANSAMINASE /

Z HISTIOINOLPHOSPHATE

0° 1.0 PHOSPHATASE /F

E .

o 60 120 180

TIME IN MiNUTES

2.5

2.0

1.5 (A

.0oZv

FIG. 6. Effect of TRA as a repressor of enzymesynthesis in strain his-1l of Salmonella typhi-murium. After exhaustion of a limiting amount ofhistidine, TRA (final concentration, 15,6g/ml) andL-histidine HC1 (final concentration, 2 ,ug/ml) wereadded to two portions of the culture, respectively,and nothing was added to the third portion. Growthand L-histidinol phosphate phosphatase and imida-zoleacetol phosphate transaminase values werefollowed with time. (0) = no addition; (0) =L-histidine*HC1; (A) = TRA.

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much more synthesis of DNA than of either RNAor protein (Pardee and Prestidge, 1956). TRAblocked the formation of both types of nucleicacids while allowing protein synthesis. This mayreflect the more critical role of amino acids suchas histidine, present in low amounts in the cell,plays in cellular metabolism compared withphenylalanine.

Replacement of a normal amino acid (e.g.,methionine) by an analogue (e.g., selenomethio-nine) may be complete and yet allow logarithmicgrowth for long periods of time (Cowie andCohen, 1957). Replacement seems to be equallyprobable for any site occupied by the aminoacid (Cowie et al., 1959; Yoshida and Yamaski,1959; Richmond, 1963). The degree of replace-ment varies (Munier and Cohen, 1959; Rich-mond, 1962b) and apparently is dependent onthe concentration of the analogue in the medium(Richmond, 1962b). The fact that two enzymesinvolved in histidine biosynthesis when made inthe presence of TRA are functionally normal mayindicate that: (i) replacement for histidine doesnot affect these enzymes, similar to the effect ofp-fluorophenylalanine in alkaline phosphatase(Richmond, 1962b); (ii) histidine or TRA is notat the active site or at a critical folding site; or(iii) there is no histidine in these enzymes. Thefact that enzymatically active IMP dehydro-genase is not made when TRA substitutes forhistidine may be of value for the study of thestructure of this enzyme. The effect of the ana-logue here may be similar to the effect of p-fluorophenylalanine on exopenicillinase of Bacilluscereus (Richmond, 1960).Although TRA, like 7-azatryptophan, did not

support the production of bacteriophage, it didnot lead to a loss in the number of infectiouscenters as occurred in the B. co1i-T2r system(Pardee and Prestidge, 1958). We have no in-formation as to what stages of bacteriophageformation actually were prevented.There have been several reports that amino

acid analogues can mimic the role of the naturalcompound in repression of enzyme formation(Lester and Yanofsky, 1961; Rowbury andWoods, 1961; Maas, 1961). In some cases, theanalogue is not substantially incorporated intoprotein (e.g., 6-methyltryptophan), while inothers (e.g., canavanine, norleucine, ethionine,norvaline), as well as in the present study, theanalogue is incorporated. There is some indirect

evidence indicating that the repressor substancesare proteins (Jacob, Sussman, and Monod, 1962).An obvious, although completely speculative,possibility for protein repressors of enzymesinvolved in amino acid biosynthesis are the aminoacid-activating enzymes. To be incorporatedinto proteins, amino acid analogues must beactivated, and several, such as TRA, are activein repression. Those amino acid analogues, whichare not incorporated but nevertheless are effectivein repression, also are known to react with aminoacid-activating enzymes (Sharon and Lipmann,1957; Nisman and Hirsch, 1958). Recent isolationof a mutant with an altered activating enzymeby Neidhardt and Fangman (1963) and, in thislaboratory, of derepressed TRA-resistant mutants(J. Roth, personal communication) provide mate-rial by which this possibility can be investigatedexperimentally.

ACKNOWLEDGMENTSThis work was supported in part by research

grant E1650 and training grant 2G-504 from theNational Institute of Allergy and InfectiousDiseases, U.S. Public Health Service.

LITERATURE CITEDAMES, B. N., AND B. GARRY. 1959. Coordinate

repression of the synthesis of four histidinebiosynthetic enzymes by histidine. Proc.Natl. Acad. Sci. U.S. 45:1453-1461.

AMES, B. N., B. GARRY, AND L. A. HERZENBERG.1960. The genetic control of the enzymes ofhistidine biosynthesis in Salmonella typhi-murium. J. Gen. Microbiol. 22:369-378.

AMES, B. N., AND P. E. HARTMAN. 1962. Genes,enzymes, and control mechanisms in histidinebiosynthesis, p. 322-345. In Molecular basisof neoplasia. University of Texas Press,Austin.

AMES, B. N., P. E. HARTMAN, AND F. JACOB. 1963.Chromosomal alterations affecting the regula-tion of histidine biosynthetic enzymes inSalmonella. J. Mol. Biol. 7:23-42.

AMES, B. N., AND B. L. HORECKER. 1956. Thebiosynthesis of histidine: imidazole acetolphosphate transaminase. J. Biol. Chem.220:113-128.

AMES, B. N., R. G. MARTIN, AND B. GARRY. 1961.The' first step of histidine biosynthesis. J.Biol. Chem. 236:2019-2026.

AMES, B. N., AND H. K. MITCHELL. 1952. Thepaper chromatography of imidazoles. J. Am.Chem. Soc. 74:252-253.

BRAWERMAN, G., AND M. YCAS. 1957. Incorpora-

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tion of the amino acid analog tryptazan intothe protein of Escherichia coli. Arch. Biochem.Biophys. 68:112-117.

BURTON, K. 1956. A study of the conditions andmechanism of the diphenylamine reaction forthe colorimetric estimnation of deoxyribonu-cleic acid. Biochem. J. 62:315-323.

COHEN, G. N., AND F. GROS. 1960. Protein bio-synthesis. Ann. Rev. Biochem. 29:525-546.

COWIE, D. B., AND G. N. COHEN. 1957. Biosyn-thesis by Escherichia coli of active alteredprotein containing selenium instead of sulfur.Biochim. Biophys. Acta 26:252-261.

COWIE, D. B., G. N. COHEN, E. T. BOLTON, ANDH. DERoBICHON-SZULMASTER. 1959. Aminoacid analog incorporation into bacterialproteins. Biochim. Biophys. Acta 34:39-46.

HARTMAN, P. E. 1956. Linked loci in the control ofconsecutive steps in the primary pathway ofhistidine synthesis in Salmonella typhimurium,p. 35-62. In Genetic studies with bacteria.Carnegie Inst. Wash. Publ. 612.

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