JOURNAL OF BACTERIOLOGY, Apr., 1966Copyright © 1966 American Society for Microbiology

Vol. 91, No. 4Printed in U.S.A.

Distribution and Function of Genes Concerned withAromatic Biosynthesis in Escherichia coli

JAMES PITTARD AND B. J. WALLACE

School of Microbiology, University of Melbourne, Victoria, Australia

Received for publication 6 December 1965

ABSTRACTPITrARD, JAsES (School of Microbiology, University of Melbourne, Victoria,

Australia), AND B. J. WALLACE. Distribution and function of genes concernedwith aromatic biosynthesis in Escherichia coli. J. Bacteriol. 91:1494-1508. 1966.-Anumber of mutant strains of Escherichia coli K-12, which are blocked in the bio-synthesis of the aromatic amino acids, were examined biochemically to determinetheir particular enzymatic deficiencies. The mutations carried by these strainswere mapped by use of the methods of conjugation and transduction. Structuralgenes for five of the enzymes of the common pathway leading to chorismate andfor the two enzymes converting chorismate to phenylpyruvate and p-hydroxy-phenylpyruvate, respectively, were identified. Unlike the genes of the tryptophanoperon. most of these genes are distributed over widely separated regions of thechromosome.

The various enzymatic reactions by means ofwhich the bacterium Escherichia coli convertserythrose-4-phosphate and phosphoenolpyruvateto chorismate are now well established (13, 25).Chorismate is a key intermediate in aromaticbiosynthesis, because it is the last intermediate inthe common pathway and, as such, can be con-verted enzymatically via a number of differentpathways into the end products tyrosine, phenyl-alanine, tryptophan, ubiquinone, vitamin K, andfolic acid (5, 6, 10, 11).Mutant strains blocked in any of the reactions

leading to chorismate show a multiple require-ment for two or more of the aromatic aminoacids and vitamins depending on the complete-ness of the block. Such mutants have been re-ferred to as multiple aromatic auxotrophs as dis-tinct from strains blocked in any of the specificpathways leading from chorismate to tryptophan,phenylalanine, or tyrosine, these latter strainsbeing known as tryptophan auxotrophs or phenyl-alanine auxotrophs, etc.Very little is known about the distribution on

the E. coli chromosome of the structural genes foreach of the enzymes involved in the reactions ofthe common pathway. The mutations carried bysome aromatic mutants have been mapped (24),but, since no biochemical investigations werecarried out, the functions of the mapped geneswere unknown. It is the purpose of this communi-cation to describe the chromosomal location andthe specific function of five of the aro genes con-

cerned with the common pathway and of one ofthe phe and one of the tyr genes concerned withthe phenylalanine and tyrosine pathways, re-spectively. (For a description of symbols used, seeTable 1.) Some of these mutant strains have beenobtained from A. L. Taylor, some from R.Somerville, and the rest have been isolated inthese laboratories.To locate the genes on the chromosome, we

have used both interrupted mating techniquesand phage Plkc-mediated transductions (thisphage will subsequently be referred to as P1).The data from interrupted mating experimentsmake it possible to assign to a gene a definitechromosomal location, and this method has beenused in the construction of most chromosomalmaps. The accuracy of the method, however, doesnot make it possible to distinguish genes that areseparated by less than 1 to 2% of the E. coligenome. Since the E. coli chromosome may con-tain as many as 5,000 genes, mutations separatedby less than 50 genes may not be separable bythis method.Phage P1 is unable to cotransduce two chromo-

somal markers that are separated by more thanapproximately 2% of the E. coli genome. Whentwo markers are cotransducible, however, an esti-mation of the distance between them can be madeby measuring the frequency with which they areseparated by recombination events. This may bemeasured by use of a wild-type donor with a re-cipient that carries both mutant alleles, in which

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case the frequency at which the wild-type allelesare cotransduced will vary inversely with the dis-tance that separates them. This distance may alsobe measured if one mutation is present in thedonor strain and the other in the recipient. In thiscase, the frequency with which wild-type trans-ductants are obtained will vary directly with thedistance that separates the two mutations. Ingeneral, transduction studies involving P22-Sal-monella typhimurium systems and P1-E. coli sys-tems indicate that, if two mutations occur in asingle gene, then the frequency with which wild-type transductants are obtained in crosses be-tween mutants will vary between about 5 and 0%of that obtained with wild-type donor phage, de-pending on the relative positions of the mutationswithin the gene (15, 21, 28).

In some cases in this work, where preliminaryconjugation experiments indicated that a numberof different mutants constituted a single geno-type, only one or two members of the group wereexamined in interrupted mating experiments, andthe group identity of the remainder was verifiedby transduction tests. In those cases where wehave carried out time of entry experiments onmutant strains already mapped by Taylor andThoman (24), our results do not differ signifi-cantly from theirs.We have retained the genotype symbols used by

Taylor and Thoman (24), except in two cases, inwhich we found that mutations to which they havegiven separate gene designations could be shownto be located within a single gene. These findingshave raised certain problems with regard to no-menclature, which we propose to deal with as

PKDH DHQ DHSAldolose Synthease Dehydroquinose Reductose

follows. In the case of mutations which Taylorand Thoman (24) have listed as occurring in twogenes, aro-C and aro-B, and which we found tooccur within a single gene, we have made thefollowing changes. For reasons to be discussedlater, we have retained the aro-C designation forboth mutations and used the aro-B designationfor one of the aromatic genes which had notpreviously been mapped. In the case of the twomutations which Taylor and Thoman (24) haveplaced in the two genes phe-A and phe-B andwhich we found to occur within a single gene, wehave retained the phe-A designation for bothmutations. Since we have not located any othergenes concerned with phenylalanine biosynthesis,phe-B does not occur on our map. The specificreactions by means of which erythrose-4-phos-phate and phosphoenolpyruvate are converted tochorismate and then to phenylalanine and tyro-sine are shown in Fig. 1. The genetic symbolsused to identify the structural genes for each ofthese enzymes are included in the figure.

It is to be noted that the sequence of upper-case letters used to describe different structuralgenes does not necessarily correspond to the se-quence of enzymatic steps. For example, aro A isconcerned with the enzyme carrying out the sixthreaction of the common pathway and not thefirst.

MATERIALS AND METHODS

Organisms. The strains used in this work are allderivatives of E. coli K-12 and are described in Table1. The order of transfer of chromosomal genes by theHfr strains is shown in Table 2.

SAKinase

EPSP CASynthetose Syis&tse

PEP b+ F PKDH - DHQ - - DHS - SA - * SAP - EPSP- CA

E-4 -P A--.-- a . , _-

P,epF".,d

N[z"c]- .Phe- yruvic * PhenyInine

CA CAMutasse

AA Tyr-A _ _ DehoeSyrCh#t Prete~~nic IN4FHox- Tyrosine

Aci

Tryptophos

FIG. 1. An outline of the reactions involved in the conversion of erythrose-4-phosphate and phosphoenolpyruvateto chorismate, and ofchorismate to phenylalanine and tyrosine. The genetic symbols for each of the known struc-tural genes are also included. Symbols used: PEP, phosphoenolpyruvate; E-4-P, erythrose-4-phosphate; PKDH,7-phospho-2-keto-3-deoxy-D-arabino-heptonate; DHQ, dehydroquinate; DHS, dehydroshikimate; SA, shikimate;SAP, shikimate-5-phosphate; EPSP, 3-enolpyruvylshikimate-5-phosphate; CA, chorismate; AA, anthranilate;aro B, the structural gene for DHQ synthetase; aro D, the structural gene for dehydroquinase, etc.

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P16IT-ARD AND WALLACE

TABLE 1. List of strains"

Genetic loci relevant to this workbStrain no. Previous no. Sex

aro A aro B aro C aro D aro E phe A tyr A Other loci

AT2022 d + + + + + l c + pro- T6r FAT2092 -d + + + + + 2 + pur C T6r FAB2861 phe l/1, + + + + + 351 + F-AB2859 tyr 2/3e + + + + + + 351 FAT2273 d + + + + + + 352 T6r his- PAT2471 d + + + + + + 4 thi- str-s ci"HfrAB478 t 2 + + + + + + pro- str-s F-AB2860 2 + + + + + + proa str-r FAB2852 358 + + + + + + pro- arg F-AB2858 358 + + + + + + pro- arg- trr FAB2853 359 + + + + + + pro- arg FAB2855 361 + + + + + + pro- arg PAB2851 357 + + + + + + pro- arg str-s 1FAB2856 357 + + + + + + pro- arr str-r PAB2829 10.5e 354 + + + + + + PAB2854 360 + + + + + + pro- arg E-AB2826 10.1 e + 351 + + + + + PAB2847 + 351 + + + + + mat- T6r E-AB347 _d + + 4 + + + + thr- leu- ciHfrAB2830 10.6s + + 355 + + + + F-AB2849 + + 355 + + + + T6r F-AB2850 + + 356 + + + + F-AB477 d + + 1 + + + + F-AB1360 d + + + 362 + + + his- T6r p-AB2827 10.2w + + + 352 + + + F-AB2848 + + + 352 + + + T6r F-AT2472 _d + + + + 24 + + "HfrAB2828 8.5e + + + + 353 + + F-AB2834 + + + + 353 + + T6r mat F-AB313 + + + + + + + thr- leu- thi- T6J a HfrAB311 + + + + + + + thr- leu- thi- T6, c HfrAB259 + + + + + + + thi- str-s d7 Hfr

a The following abbreviations are used: aro, aromatic amino acids and vitamins; pro, proline; pur,purine; thi, thiamine; arg, arginine; his, histidine; thr, threonine; leu, leucine; ilv, isoleucine and valine;phe, phenylalanine; tyr, tyrosine; mal, maltose; xyl, xylose; str, streptomycin; T6, bacteriophage T6;(0), origin.

b For a description of the enzymes affected by mutations in the aro A, aro B, aro C, aro D, aro E,phe A, and tyr A genes, see Fig. 1.

c Numbers refer to allele numbers allotted to mutant strains in this laboratory and in the laboratoriesof E. A. Adelberg and A. L. Taylor.

d These strains or their derivatives are referred to in the paper of Taylor and Thoman (24). StrainAB347 carries the aro 4 allele of strain AB444, and strain AB477 carries the aro I allele of strain AB1320.

6 These strains were provided by R. Somerville.

TABLE 2. Order oftransfer ofchromosomal genes byHfr strains*

Strain no. Order of transfer

AB313 (0) xyl-mal-str-pur C-his-try-pro-leu-ilv-sex factor

AB311 (0) his-try-pro-leu-ilv-xyl-mal-str-purC-sex factor

AB259 (0) leu-pro-try-his-pur C-str-mal-xyl-ilv-sex factor

* For abbreviations, see first footnote inTable 1.

Transducing phage P1 was obtained from N.Schwartz.

Media and culture methods. The media and culturemethods used in this work have been described byAdelberg and Burns (1).

Buffers. The tris(hydroxymethyl)aminomethane(Tris)-HCl, sodium phosphate, and Veronal-HClbuffers which have been used were prepared accord-ing to the methods of Dawson et al. (8).

Mating conditions. The conditions under whichmating was carried out were identical to those de-scribed by Taylor and Thoman (24), except that thediluted cells were incubated in 200-ml bottles insteadof 1-liter Erlenmeyer flasks.

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Nutritional tests. A 0.1-mi amount of a light sus-pension (approximately 106 cells per milliliter) ofcells was added to a soft-agar layer and poured ontoa minimal-medium plate supplemented with all theamino acids required for growth, except the aromaticamino acids. When the layer had set, a sterile filter-paper disc was placed on the surface of the plate, andone drop of a 1 mg/ml solution of the nutrient beingtested was added to the disc. Plates were incubatedfor 48 hr at 37 C, and the results were read.

Interruption of mating. Samples (1.0 ml) wereremoved from the bottles at 2- to 5-min intervalsand transferred to screw-capped tubes containing 1ml of bacteriophage T6 [approximately 1010 plaque-forming units (PFU) per ml]. The cell-phage mixturewas immediately agitated in a Vortex Junior mixerfor 45 sec to disrupt mating pairs, and then incubatedat 37 C for 10 min to allow the phage to adsorb tothe T6-sensitive male parents. Phage T6 was used tokill the male parent in crosses involving the Hfrstrains AB313 and AB311. Any phage-resistantmutants of the male parent were unable to grow onthe selective medium which lacked amino acidsnecessary for their growth. When the streptomycin-sensitive Hfr strain AB259 was used, however, theexconjugant male cells were killed by the strepto-mycin in the medium, and phage T6 was not used.

Transduction techniques. Transduction techniquesused in this work have been described (19).

Syntrophism tests. The method described by Gibsonand Jones (12) was used.

Isolation of mutants. The method described byAdelberg and Meyers (3) was used. In most cases,ultraviolet light was the mutagenic agent, but, insome cases, it was N-methyl-N'-nitro-N-nitrosoguani-dine. When the latter mutagen was used, the proce-dure described by Adelberg, Mandel, and Chen (2)was followed.

Preparation of cell-free extracts. Cells harvestedfrom 18-hr cultures were washed with chilled 0.9%NaCl and then suspended in 4 ml per 1 g (wet weight)of either 0.1 M Tris (pH 7.8) or 0.1 M phosphatebuffer (pH 7.6). Cell breakage was achieved by forcingcells through a French press at a pressure of20,000 psi.

Cell-free extracts were obtained after centrifugationat 21,600 X g for 20 min, and were stored at-20 C.Column chromatography of enzymes. This was

carried out by use of the method described by Cottonand Gibson (5).

Assay of dehydroquinate (DHQ) synthetase. Themethod described by Srinivasan and Sprinson (23)was used.

Assay of dehydroquinase. The method describedby Mitsuhashi and Davis (17) was used. In theseassays, since no dehydroquinic acid was available,the substrate for the reaction was the filtered super-natant fluid containing the accumulation products ofE. coli 83-1 (see 26).

Assay of dehydroshikimate (DHS) reductase. Themethod described by Yaniv and Gilvarg (27) wasused. Cell-free extracts were assayed for their abilityto convert shikimate to dehydroshikimate. Dehydro-shikimate formed was estimated by measuring opticalabsorbance at 234 mu in a Unicam SP500 spectro-photometer.

Assay of shikimate kinase. The following incuba-tion mixture was used: shikimic acid, 2 Asmoles;adenosine triphosphate (ATP), 4 /Amoles; MgC12, 5jAmoles; cell-free extract, 1 to 2 mg of protein; 0.05M Veronal buffer (pH 9.0) to a total volume of 2.0 ml.

Kinase activity was determined by measuring thedisappearance of shikimate according to the methodof Gaitonde and Gordon (9).

Estimation of shikimate-5-phosphate. This wasmeasured by conversion to shikimate with alkalinephosphatase according to the method of Morgan,Gibson, and Gibson (18).

Assay of 3 - enolpyruvyl - shikimate - 5 - phosphate(EPSP) synthetase and chorismate synthetase. SinceEPSP was not available for use as a substrate, cell-free extracts were examined for their ability to carryout the overall conversion of shikimate to anthranilate.The general conditions used for this assay have al-ready been described by Morgan et al. (18). Shikimatewas used rather than shikimate-5-phosphate as it wasreadily available, and all the strains being examinedwere tested for their ability to convert shikimate toshikimate-5-phosphate. Anthranilate was measuredrather than chorismate because of the ease and sensi-tivity of its fluorimetric estimation. Although someof the chorismate formed from shikimate can be con-verted to prephenate by these extracts, this amountwas kept to a minimum by preparing cell-free ex-tracts from cells in which anthranilate synthetasewas derepressed. This was achieved by growing cellsin a minimal medium containing excess tyrosine andphenylalanine (2 X 10-4 M), p-aminobenzoic andp-hydroxybenzoic acids (2 X 10-6 M), and limitingindole at a concentration of 1.5 X 10-5 M.

Complementation tests. Cell-free extracts unable tocarry out the overall conversion of shikimate to an-thranilate were mixed in various combinations andtested for their ability to complement each other inthe overall reaction. As a result of these tests, cell-free extracts could be directed into two groups(groups I and II), significant conversion of shikimateto anthranilate only being observed when extractsfrom both groups were present.

Identification ofenzyme activities present in cell-freeextracts of groups I and II. To establish which enzy-matic activities were missing from extracts of groupsI and II, complementation tests were carried out inwhich different extracts were added sequentially. Inthese tests, 0.2 ml of a cell-free extract from onegroup was added to the reaction mixture and incu-bated at 37 C for 40 min. The incubation mixture wasthen heated at 100 C for 5 min and then cooled to37 C (step 1). Then 0.2 ml of an extract from theother group was added, and the reaction mixture wasagain incubated at 37 C for 40 min (step 2). Theanthranilate formed was measured as described foranthranilate synthetase. Anthranilate will only beformed when the extract added first is able to convertshikimate to EPSP (i.e., possessing full activity forEPSP synthetase), and when the extract added lastpossesses full activity for chorismate synthetase.

Assay of anthranilate synthetase. A modification ofthe method described by Morgan et al. (18) wasused. Chorismic acid (2.0 ,umoles), MgCl2 (5 ,Amoles),glutamine (5 ,Amoles), and Tris buffer, pH 8.2 (50

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jAmoles), were added per milliliter of incubationmixture.

Assay ofchorismate mutase, prephenate dehydratase,and prephenate dehydrogenase. The method describedby Cotton and Gibson (5) was used for all of theseassays.

Protein estimation. Protein was estimated accordingto the method of Lowry et al. (16).

Specific activities. The specific activity of eachenzyme preparation is expressed as the number of0.1 ,umole of substrate used or product formed per20 min per mg of protein at 37 C.

RESULTS

Mutant strains were originally screened fortheir growth responses to shikimate, and to one

or more of the end products of aromatic biosyn-thesis. The results of these tests are shown inTable 3.

Biochemical analysis of mutant strains blockedbefore shikimate. Those strains showing a com-

plete or partial response to shikimate were testedfor their ability to cross-feed each other (Table 4).

These strains were then examined for their abilityto carry out the second, third, and fourth reactionsof the common pathway. The results of theseenzyme assays are shown in Table 5. As can beseen, the assays clearly indicate three differentgroups of mutants, each blocked in a separatereaction. The existence of three different pheno-types amongst these mutants had already beensuggested by the cross-feeding data. It is of inter-est that the group whose aromatic requirementscan only be partially satisfied by shikimic acid,and which cross-feeds the other two groups, lacksDHS reductase activity. Similar inability ofstrains blocked between DHS and shikimate togrow on shikimic acid alone has already been re-

ported by Davis (7).Genetic analysis ofmutant strains blocked before

shikimate. Somerville (personal communication)has already shown that the mutations affectingstrains AB2826, AB2827, and AB2828 occurredin genes which were carried in different transduc-ing fragments by phage P1. We determined the

TABLE 3. Results ofgrowth responses ofmutant strains to shikimic acid and to the endproductsof aromatic biosynthesisa

Strain PHE, TYR, TRY, PHETYR, SA SA, PHE, PFE, TYR PHlE TYRPAB,POB TRY TYR

AB2847 +b ++ ++ _ - _AB2827 ++ ++ ++ ++ + - _AB1360 ++ ++ ++ ++ _ _ _AB2834 + _ -c + _ - _AT2472 + + -c ++AB2829 + + _ _ _ _ -AB478 ++ ++ _ _ _ _ -AB2851 ++ ++ _ ++ ++ _ +AB2852 ++ ++ - ++ ++ - +AB2853 ++ ++ - ++ ++ _ +AB2854 ++ ++ _ ++ ++ - +AB2855 ++ ++ _ + + _ -AB2830 + _ _ _ _ - -AB477 ++ ++ _ ++ + - _AB2850 ++ ++ _ ++ ++ - _AB347 ++ ++ _ ++ ++ + +AT2022 ++ ++ _ ++ ++ ++ -AT2273 ++ ++ _ ++ ++ - ++AT2471 ++ ++ _ ++ ++ - ++AB2859 ++ ++ _ ++ ++ - ++AT2092 ++ ++ _ ++ ++ ++ -AB2861 ++ ++ +d ++ ++ ++ +d

a Abbreviations used: PHE, phenylalanine; TYR, tyrosine; TRY, tryptophan; PAB, p-aminobenzoicacid; POB, p-hydroxybenzoic acid; SA, shikimic acid; -, no response; +, weak response; ++, stronggrowth response.

b A number of strains grow poorly on media supplemented with these five compounds. Yeast extractand, in some cases, shikimic acid will satisfy their extra requirements.

c Although unable to grow on shikimic acid alone, or on a mixture of phenylalanine and tyrosine,these strains grow well on shikimic acid, phenylalanine, and tyrosine.

d Although phenylalanine markedly stimulates growth of this organism, it grows slowly in the absenceof phenylalanine.

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VOL. 91, 1966 AROMATIC BIOSYNTHESIS IN E. COLI

TABLE 4. Results of cross-feeding tests betweenmutant strains showing a growth

response to shikimic acid

Strains being fedStrains feeding

AB2834 AT2472 AB2827 AB1360 AB2847

AB2834 _* _ + + +AT2472 _ - + + +AB2827 _ _ _ - +AB1360 _ - _ - +AB2847 _ _

* Symbols: -, no cross-feeding; +, cross-feeding occurs.

TABLE 5. Presence of DHQ synthetase, dehydro-quinase, and DHS reductase in cell-freeextracts of mutant showing a growth

response to shikimic acid

Cell-free extract Specific Specific Specificprepared from activity of activity of activity of

strain DHQ'¶~a dehydro- DHSssynthetase quinase reductase

AB2834 1.16 18 <0. 31AT2472 0.74 26 0.23AB2827 1.10 1.8 13AB1360 0.915 2.2 13AB2847 0.025 20 12

approximate positions of these genes on thechromosome by mating these aro- recipients with anumber of different Hfr strains. A more accuratemap location was then found by carrying outinterrupted mating experiments between the re-

cipients and the particular Hfr strain that trans-ferred the aro+ allele at the highest frequency. Inthe case of two of these strains, AB2826 andAB2828, the genes aro B (the structural gene forDHQ synthetase) and aro E (the structural genefor DHS reductase) both showed a fairly closelinkage to mal. Consequently, mal- derivatives ofthese strains were prepared (AB2847 andAB2834). Figures 2 and 3 show the time of entryof aro B and aro E genes relative to mal, with theHfr strain AB313 as donor. The close linkage ofaro B and mal was confirmed by transductiontests, and these results are expressed in Table 6.To confirm that both AT2472 and AB2828

carried mutations in the same gene (aro E),transductions were carried out between thesestrains. The results of Table 7 show that, in trans-duction between P1 prepared on AB2828 and therecipient AT2472, aro E+ transductants areformed at approximately 1.7% of wild-type fre-quency. In the reciprocal transduction, when therecipient was AB2828 and the donor AT2472,

Time of sampling in minutes

FIG. 2. Kinetics of zygote formation wvheni the HfrAB313 is mated with the female AB2847.

the transduction frequency was reduced to 8% ofthe wild-type frequency. Both these results arecompatible with the idea that the mutations ofstrains AB2828 and AT2472 occur within thesame gene (aro E). This conclusion is furthersupported by the biochemical data.The aro D gene (the structural gene for dehy-

droquinase) is transferred distally to his by HfrAB313, and hence as an early marker by the HfrAB311 (see Table 2). In interrupted mating ex-periments, with the male strain AB311 as donorand either AB1360 or AB2848 as recipients, thearo D gene was transferred at 14 min. Since thetime of entry for his in these experiments was 8min, these results confirmed the earlier observa-tions of Taylor and Thoman (24). The results oftransduction tests confirming the close linkageof mutations affecting AB2848 and AB1360 aregiven in Table 8.

Biochemical analysis ofmutant strains unable torespond to shikimic acid but requiring two or morearomatic amino acids for growth. Extracts of thesestrains were initially examined for their ability to

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i 20

10

mCI aro B

oL0

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1500

60

30

50X 40

Q

x

0-o

4,

00

z

101

PIITARD AN]

malaro E

10 20 30

Time of sampling in minutes

FIG. 3. Kinetics of zygote formation when the HfrAB313 is mated with the female AB2834.

carry out the conversion of shikimate to anthrani-late as described in Materials and Methods. Theresults of these assays are shown in Table 9. Thespecific activities for anthranilate synthetase areincluded to indicate those strains which could notbe fully derepressed for this activity. Extractsprepared from fully derepressed cells (AB1360)and from wild-type cells (AB1171) are includedas controls. In every case, extracts were preparedfrom cells grown in media containing 1.5 X 10-5M indole as described in Materials and Methods.

Conversion of shikimate to shikimate-S-phos-phate. All of the extracts unable to convert shiki-mate to anthranilate could convert shikimate toshikimate-5-phosphate. Extracts of only onestrain, AB347, showed a reduced activity for thisreaction, and we will return to this strain later.

D WALLACE J. BACTERIOL.

The results of these shikimate kinase assays areincluded in Table 10.

Complementation between extracts. Since thesestrains could be blocked in either EPSP synthe-tase or in chorismate synthetase, different extractswere mixed together to determine whether pairs

TABLE 6. Testing for the cotransduction of genesaro B, aro E and mal

No. of No. of No. of No. of aro+Recint* mal+ aro+ aro+mal+ transductantsRecipient* trans- trans- trans- that are mal+

ductants ductants ductants unselected

AB2847 aro B 215 372 88 9/40351 mal 354

AB2834 aro E 267 200 0 0/40353 mal 352

* In both cases, the P1 phage used had beenprepared on a mal+ aro B+ aro E+ donor.

TABLE 7. Identification of aro E mutants bytransduction*

No. of aro+Donor Recipient trans-

ductants

AB2826 aro C+ aro E+ AB2830 aro C 355 ca. 8,000AB2826 aro C+ aro E+ AT2472 aro E 24 ca. 8,000

AB2828 aro E 353 AB2830 aro C 355 ca. 3, 600AB2828 arc E 353 AT2472 aro E 24 60

AT2472 aro E 24 AB2830 aro C 355 ca. 2, 500AT2472 aro E 24 AB2828 aro E 353 200

* Pairs of figures represent two experiments using the sameamount of donor phage (plaque-forming units per milliliter)with two different recipients. In this and subsequent tables,the numbers of aro+ transductants represent the total number oftransductant colonies obtained on three plates. In those caseswhere the numbers obtained were very high, i.e., 8,000, numberswere estimated only to the nearest 100. Since for each testtransduction a control with an unrelated recipient was alwayscarried out, no attempt has been made to normalize thesenumbers to a fixed amount of plaque-forming units of inputphage.

TABLE 8. Identification of aro D mutants bytransduction*

No. ofDonor Recipient tanos

ductants

AB1360 aro D 362 AB2826 aro B 351 183AB1360 aro D 362 AB2848 aro D 352 0AB1360 aro D 362 AB2828 aro E 353 207

* The same amount of donor phage was usedin each cross.

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TABLE 9. Ability of cell-free extracts to2convertshikimate to anthranilate

Cell-free extract Mficromoles (X l04) of Specific activity ofprepared from anthranilic acid formed antlhranliate

stan per 20 min per mg of sytetestr.un ~~protein snhts

AB1360* 370 4.6AB2829 3 3.9AB478 2 3.4AB2851 57 2.0AB2852 44 1.6AB2853 85 1.3AB2854 27 0.5AB2855 25 1.1AB2830 <1 8.4AB477 12 2.6AB2850 15 0.33AB347 <1 1.0AB2858 42 10.7AB171 t 60 0.17

* AB1360, which is unable to convert DHQ toDHS, has been used as a wild-type control for theconversion of shikimate to anthranilate. Becausethis strain requires all the aromatic amino acidsincluding tryptophan for growth, it was possibleto derepress its anthranilate synthetase.

t Strain ABl171 has no aromatic requirementsand is included as a wild-type control for thosemutants in which it was not possible to fully de-repress anthranilate synthetase.

of extracts could complement each other in theoverall reaction. As can be seen from the resultsof Table 11, these tests allowed us to divide themutants into two groups (groups I and II). Anyextract from one group was able to complementan extract from the other in the overall reaction,but no complementations were observed betweenmembers of the same group. The identification ofthe specific reaction that was missing in the caseof either group I and II mutants was determinedby sequential mixing experiments as described inMaterials and Methods. Complementations onlyoccurred when group I extracts were added firstin step 1, and group II extract was added forstep 2 (Table 12). From these results, we con-cluded that group I mutants possess activeEPSP synthetase and inactive chorismate synthe-tase, whereas extracts of group II possess activechorismate synthetase and inactive EPSP syn-thetase.

Reduced kinase activity of extracts of strainAB347. Even though extracts of this strain possessonly one-fourth the kinase activity of wild-typestrains, the fact that extracts of AB347 can com-plement group II extracts in sequential mixingexperixents suggested that this reduction of ac-tivity does not drastically affect the formation of

EPSP. That this reduced kinase activity was notdue to the presence of an inhibitor in extracts ofAB347 was demonstrated by mixing an extract ofAB347 with an extract of strain AB2858 andmeasuring the combined kinase activity. Thespecific activity of the mixture was found to equalthe mean of the two separate specific activities(Table 10).As no kinase mutants have yet been reported in

the literature, it was decided to try to determinewhether the reduced kinase activity of strainAB347 was associated with its loss of chorismatesynthetase activity. It seemed possible that, if thekinase gene and the chorismate synthetase genewere contiguous, either a polarity mutation inone gene or a deletion extending into both mighthave caused the loss of the chorismate synthetaseand the reduction of the kinase activity. To testthis notion the aro+ allele from a wild-type strainwas introduced into strain AB347 by transduc-tion, recombinants being selected on minimalmedia not supplemented with the aromatic aminoacids. A similar transduction was carried outwith strain AB2830 as a recipient. This strain isalso a group I mutant deficient in chorismatesynthetase, but possessing full kinase activity.Two aro+ transductants in each case were puri-fied, and then examined for their kinase activity.The extracts prepared from the aro+ transductantsof AB347 had only one-fourth the kinase activityof the aro+ transductants prepared from strainAB2830. From this, then, we inferred that thekinase gene and the chorismate synthetase genesare not contiguous and that the reduced kinaselevels in extracts of AB347 can not be explained

TABLE 10. Ability of cell-free extracts to convertshikimate into shikimate-5-phosphate

Cell-free extract prepared from strain Specific activity

AB1360* 1.9AB2829 2.4AB478 2.9AB2851 3.2AB2852 1.5AB2853 1.6AB2854 1.2AB2855 1.2AB2830 1.8AB477 2.9AB2850 2.3AB347 0.7AB2858 1.8

AB2858 and AB347 1.2

* Strain AB1360 is unable to convert DHQ intoDHS, but has normal activity for the conversionof shikimate to chorismate. It has been used as awild-type control for the shikimate kinase assay.

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TABLE 11 Ability ofcell-free extracts ofstrains in group I andgroup ll to convert shikimate to anthranilatein mixing experiments

Micromoles (X 104) of anthranilate formed inoverall reaction per 20 min per mg of mixed protein

Cell-free extracts prepared from strains Complemen-Valuexpecte if no tation

Value obtained in mixing Value epected if noexperiment occurs*

AB2830 (I) and AB2829 (Il) 106 4 YesAB2830 (I) and AB478 (II) >107 3 YesAB477 (I) and AB478 (II) 175 14 YesAB2850 (I) and AB2829 (II) >100 18 YesAB2850 (I) and AB2851 (II) 160 72 YesAB2850 (I) and AB478 (II) 156 17 YesAB347 (I) and AB478 (II) 186 3 YesAB2830 (I) and AB2851 (II) 272 58 YesAB2830 (I) and AB2852 (II) 226 45 YesAB2830 (I) and AB2853 (II) 255 86 YesAB2830 (I) and AB2854 (II) 194 28 YesAB2830 (I) and AB2855 (II) 356 26 Yes

AB2830 (I) and AB477 (I) 5 13 NoAB2830 (I) and AB2850 (I) 5 16 NoAB347 (I) and AB2850 (1) 1 16 NoAB347 (I) and AB2830 (I) 4 2 No

AB2851 (II) and AB478 (Il) 34 59 NoAB2829 (II) and AB478 (II) 1 5 NoAB2852 (II) and AB478 (II) 24 46 NoAB2853 (II) and AB478 (II) 27 87 NoAB2854 (II) and AB478 (II) 18 29 NoAB2855 (II) and AB478 (II) 14 27 NoAB2852 (II) and AB2853 (Il) 32 129 NoAB2853 (Il) and AB2854 (Il) 8 112 NoAB2852 (II) and AB2851 (Il) 41 101 No

* This value is determined by adding valuesTable 9).

TABLE 12. Ability of cell-free extracts of strains ingroup I and group IH to convert shikimate toanthranilate in sequential mixing experiments

Micromoles (X 104) ofanthranilate formed inCell-free extract Cell-free extract overall reaction per

added in step 1* added in step 2 20 min perme ofmixed protein

AB478 (II) AB2850 (I) 2AB2850 (I) AB478 (II) 30

AB478 (II) AB347 (I) 1AB347 (I) AB478 (II) 69

AB478 (II) AB2830 (I) 1AB2830 (I) AB478 (II) 122

AB2851 (II) AB2830 (I) 20AB2830 (I) AB2851 (II) 97

* For a description of steps 1 and 2, see text.

obtained when each extract is incubated single (see

in terms of the mutation which caused the loss ofactivity of chorismate synthetase. The aro+ trans-ductants of AB347 and AB2830 were also ex-amined for their growth rates in liquid and onsolid minimal media, with and without the aro-matic amino acids. Stimulation of growth rate bythe aromatic amino acids was the same in bothcases, indicating that the reduced level of shiki-mate kinase observed in cell-free extracts does notmean that the cells are starved for the aromaticamino acids. Nevertheless, experiments are inprogress to determine the location of the mutationin AB347 causing this decreased specific activityfor shikimate kinase.

Genetic analysis of mutant strains unable to re-spond to shikimic acid but requiring two or morearomatic amino acids for growth. On the basis oftransduction tests, these mutant strains could bedivided into two groups which were identical tothe groups formed on the basis of the enzymatic

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studies. Transduction between members of agroup gave aro+ transductants at low frequencies(approximately 1 % of wild-type frequency),whereas transduction between representatives ofdifferent groups gave aro+ transductants at wild-type frequencies.

Group I mutants. The results of an interruptedmating experiment between one member of groupI, AB2849, and the Hfr AB313 are shown in Fig.4. This gene affecting chorismate synthetase ac-tivity we have termed aro C. Transduction ex-periments demonstrating that all members ofgroup I carry mutations in the aro C gene areshown in Table 13. On the basis of interruptedmating experiments, Taylor and Thoman (24)placed the mutations affecting AB347 and AB477,which are present in their strains AB444 andAB1320, approximately 3.5 min apart on thechromosome, and designated them aro C andaro B, respectively. However, in this study, aro+transductants were formed at less than 1 % wild-type frequency in crosses between these mutants(Table 13). Furthermore, in other transductionexperiments, we have been able to show that thearo Cl allele of AB477 and the aro C4 allele ofAB347 are both cotransduced with pur C at fre-quencies of 38 and 36%, respectively. Because ofthis latter result we have retained the aro C desig-nation rather than the aro B for these mutations.Group II mutants. The mutations carried by

two group II mutants, AB478 and AB2856, andaffecting EPSP synthetase were mapped in in-terrupted mating experiments using the male

TABLE 13. Identification of aro C mutants bytransduction*

No. of aro+Donor Recipient trans-

ductants

AB2826 aro C+ aro A+ AB347 aro C 4 ca. 6,000AB2826 aro C+ aro A+ AB2829 aro A 354 ca. 6,000

AB2830 aro C 355 AB347 aro C 4 155AB2830 aro C 355 AB2829 aro A 354 ca. 7,000

AB2850 aro C 356 AB347 aro C 4 73AB2850 aro C 356 AB2829 aro A 354 ca. 7,000

AB477 aro C 1 AB347 aro C 4 30AB477 aro C 1 AB2829 aro A 354 ca. 11,000

* Pairs of figures represent two experiments in which thesame amount of donor phage (plaque-forming units per milli-liter) was used with two different recipients.

strain AB259. In both cases, the time of entry ofthis gene, aro A, was 30 min, which is in agree-ment with the results of Taylor and Thoman (24).Transduction experiments demonstrating that allmembers of group II carry mutations in the aro Agene are given in Tables 14 and 15.

Biochemical analysis of mutant strains blockedbetween chorismate and phenylalanine. Three mu-tants of this class were examined. Of these,AT2022 and AT2092 were both obtained fromDr. Taylor, and AB2861, from Dr. Somerville.Cotton and Gibson (5) have shown in Aerobacteraerogenes and in E. coli W that there are twochorismate mutase enzymes that are separablewhen cell-free extracts are chromatographed ondiethylaminoethyl (DEAE)-cellulose columns.They have also shown that prephenate dehy-drogenase activity is associated with one peakand prephenate dehydratase activity with theother. We found a similar situation in E. coliK-12, and the chromatographic separation ofthese enzymes from cell-free extracts of a wild-type strain is shown in Fig. 5. Figure 6 shows asimilar experiment involving extracts from thephenylalanine auxotroph AT2022. It can be seenthat one of the chorismate mutase peaks and itscorresponding dehydratase activity are missingfrom extracts of the phenylalanine auxotroph.Extracts of the phenylalanine auxotroph strainAT2092 show a similar loss of prephenate dehy-dratase and its corresponding chorismate mutasepeak (Cotton, personal communication). On theother hand, extracts of mutant AB2861, althoughhaving no prephenate dehydratase activity, stillpossess two peaks of chorismate mutase activity.

Genetic analysis of mutant strains blocked be-tween chorismate and phenylalanine. Again on the

Time of sompling in minutes

FIG. 4. Kinetics of zygote formation when the HfrAB313 is mated with the female AB2849.

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basis of interrupted mating data, Taylor andThoman (24) have referred to the mutation ofAT2022 as occurring in the phe B gene, and thatof AT2092 as occurring in the phe A gene; theyhave placed these genes approximately 6.5 minapart on the E. coli chromosome. We found that,in transduction tests between these two mutants,phe+ transductants were formed at less than0.25% of the frequency with a wild-type donor.In these tests, we used AT2022 as the recipient inall crosses and compared the frequency of pro+and phe+ transductants. On the basis of theseresults, we concluded that the mutations can not

TABLE 14. Identification of aro A mutants bytransduction*

No. of aro+Donor Recipient trans-

ductants

AT2471 aro D+ aro A+ AB1360 aro D 362 980AT2471 aro D+ aro A+ AB478 aro A 2 1,350

AB2852 aro A 358 AB1360 aro D 362 530AB2852 aro A 358 AB478 aro A 2 7

AB2853 aro A 359 AB1360 aro D 362 167AB2853 aro A '359 AB478 aro A 2 12

AB1360 aro A+ aro C+ AB2830 aro C 355 2,150AB1360 aro A+ aro C+ AB2852 aro A 358 1,760

AB2855 aro A 361 AB2830 aro C 355 690AB2855 aro A 361 AB478 aro A 2 1

AB2851 aro A 357 AB2830 aro C 355 1,300AB2851 aro A 357 AB478 aro A 2 4

AB2854 aro A 360 AB2830 aro C 355 Ca. 3,600AB2854 aro 4 360 AB478 aro A 2 4

AB2829 aro A 354 AB2830 aro C 355 Ca. 8,700AB2829 aro A 354 AB478 aro A 2 186

AB478 aro A 2 AB2830 aro C 355 ca. 3,200AB478 aro A 2 AB2852 aro A 358 0

* Pairs of figures represent two experiments in which thesame amount of donor phage (plaque-forming units per milli-liter) was used with two different recipients. In every pair ofcrosses, one cross, in which either AB1360 or AB2830 is therecipient, serves as a control to measure the transducing abilityof the donor phage.

be separated by 6.5 min but, in fact, occur withinthe same gene. The results of these experimentsand of a transduction involving the phenylalanineauxotroph AB2861 are shown in Table 16. Therelatively high ratio of pro+ to phe+ obtained inthe wild-type cross was unexpected. Phenylala-nine auxotrophs, however, grow slowly in theabsence of phenylalanine because of the chemicalconversion of prephenate to phenylpyruvate. It ispossible that the background growth of the phe-recipient prevented some of the phe+ transduc-tants from forming colonies.On the basis of the transduction data and the

enzymatic analyses, we concluded that the phenyl-alanine auxotrophs AT2092, AT2022, andAB2861 possess mutations affecting the same

gene. We propose, therefore, that the phe B locus

of Taylor and Thoman (24) should be deletedfrom the map until another gene concerned withphenylalanine biosynthesis is discovered. In in-terrupted mating experiments with the maleAB313 and the female AT2092, the phe A gene

was transferred at 30 min and his at 40 min,confirming the results of Taylor and Thoman(24).

Biochemical analysis of mutant strains blockedbetween chorismate and tyrosine. We examinedonly three strains blocked in tyrosine biosynthe-sis: AT2471, AT2273, and AB2859. When ex-tracts of AT2471 were chromatographed as al-ready described for the phenylalanine auxo-trophs, the chorismate mutase peak with itscorresponding prephenate dehydrogenase activitywas found to be missing (Fig. 7). We obtained thesame results with extracts of strain AT2273 andof strain AB2859.

Genetic analysis of mutant strains blocked be-tween chorismate and tyrosine. The results oftransduction tests between these three tyr- strainsare presented in Table 17. Interrupted matingexperiments which were carried out between themale AB313 and the female AT2273 showed thatthe tyr A gene was transferred at 30 min and hisat 40 min, in agreement with the results of Taylorand Thoman (24).

Since interrupted mating experiments indicated

TABLE 15. Identification of aro A mutants by transduction

Recipient ~~~No of aro+ No. of arg'Donor Recipient transductants transductants

AT2472 aro A+ arge AB2852 aro A 358 arg 3 1,030 1,540AB478 aro A 2 arg+ AB2852 aro A 358 arg 3 10 ca. 2,400AB478 aro A 2 arg+ AB2855 aro A 361 arg 3 15 ca. 2,370AB478 aro A 2 arg+ AB2851 aro A 357 arg 3 0 ca. 2,500AB478 aro A 2 arge AB2853 aro A 359 arg 3 4 ca. 2,500AB478 aro A 2 arg+ AB2854 aro A 360 arg 3 0 ca. 2,500

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.11~~wfraction No.

FIG. 5. Chromatography of a cell-free extract ofstrain AB1360 on a DEAE cellulose column. (A)Chorismate mutase activity (0), protein (0); (B)prephenate dehydratase activity; (C) prephenate de-hydrogenase activity.

that the phe A and tyr A genes were relativelyclose together on the chromosome, transductionexperiments were carried out to see whether thetwo genes were cotransducible. Phage P1 was pre-pared on the tyrosine auxotroph AT2273 andused to transduce the phe+ gene into strainAT2092. Transductants were selected on minimalmedia supplemented with tyrosine but no phenyl-alanine. The phe+ transductants were thenstreaked to minimal media with and without tyro-sine to see whether any had inherited the tvr-allele. Approximately 50% ofthese"phe+ transduc-tants failed to grow in the absence of tyrosine. Inthe reciprocal cross in which AT2092 was thedonor and AT2273 the recipient, 40% of the tyr+

Fraction No.

FIG. 6. Chromatography ofa cell-free extract of thephenylalanine auxotroph AT2022 on a DEAE cellulosecolumn. (A) Chorismate mutase activity (@), protein(0); (B) prephenate dehydratase activity; (C) pre-phenate dehydrogenase activity.

transductants inherited the phe allele and wereunable to grow in the absence of phenylalanine.We conclude therefore that the tyr A and thephe A genes are cotransducible by phage Pl.

DISCUSSION

These results indicate that most of the genesconcerned with aromatic biosynthesis are widelyseparated on the E. coli chromosome, unlikethose concerned with the specific pathway oftryptophan biosynthesis which form a singleoperon. In some cases, the data that we have ob-tained from transduction tests lead us to differentconclusions from those of Taylor and Thoman(24), and we would like to propose the followingexplanation for the different results.

Taylor and Thoman mated the Hfr strainAB313 with two different females, AT2022 and

TABLE 16. Identification of phe A mutants by transduction

Donor Recipient No. of phe+ No. of pro+transductants transductants

Wild-type pro+ phe A+ AT2022 pro 2 phe Al 400 ca. 4,000AT2092 pro+ phe A2 AT2022 pro 2 phe Al 0 ca. 4,000AB2861 pro+ phe A 351 AT2022 pro 2 phe Al 5 1,010l~~~~~~~~~~~~~~ ~~~ ~ ~~~ ~ ~~~ ~ ~~~ ~ ~~~ ~ ~~~ ~ ~~~ ~~~~ ~~~~ ~~~~ ~~~ ~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

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AT2092. Botir of these recipient strains carriedmutant alleles for genes concerned with histidineand phenylalanine biosynthesis. Strain AT2092carried the his I and phe A alleles, and strainAT2022 carried the his 4 and phe B alleles. In anumber of interrupted mating experiments in-volving AB313 and .AT2092, the wild-type allelescorresponding to his I and phe A (his 1+ andphe A+) were transferred at 40.5 and 29 min,respectively. In a number of experiments involv-ing the same male strain and the recipientAT2022, the wild-type alleles corresponding tohis 4 and phe B were transferred at 46 and 35min, respectively. Because the difference of 6.5min-between the times of entry of the his 1+ andhis 4+ alleles was unexpected, these results weretested by crossing both recipient strains withanother Hfr, strain AB311. The order in which

oC

:0

CZ

_

-i

A ,

ct

c-0'D o

02 1-58

-t 'y 1-0c4, w

a g0 -

. -V

, z" 0,5I Iw 't,ct

v TO 2u 30 40 50

Froction No.40 70

FIG. 7. Chromatography ofa cell-free extract of thetyrosine auxotroph AT2411 on a DEAE cellulosecolumn. (A) Chorismate mutase activity (@), protein(0); (B) prephenate dehydratase activity; (C) pre-

phenate dehydrogenase activity.

various markers are transferred by both theseHfr strains can be seen in Table 2. Strain AB311transferred the his 1+ allele and the his 4+ alleleat 6 min; i.e., the 6.5-min difference between thesemarkers had now disappeared. Unfortunately,strain AB311 transfers the phe A+ and phe B+allele as distal markers, and it was not possibleto check the difference in times of entry observedfor phe A+ and phe B+. On the basis of these re-sults, Taylor and Thoman have proposed that (i)the his 1+ and his 4+ alleles are normally less than1 min apart, but in Hfr AB313 the his 4 allele hasundergone a transposition, and (ii) that the twomutations affecting phenylalanine biosynthesisoccur in two distinct genes separated by approxi-mately 6 min on the chromosome.As we have already shown, transduction tests

demonstrate that the phe A and the phe B muta-tions occur, in fact, in the same gene. In otherwords, the 6-min difference in the times of entry ofphe A+ and phe B+ found in the crosses withAB313 has to be accounted for by a hypothesiswhich will also explain the observed difference inthe times of entry of his J+ and his 4+ alleles. Inthe crosses involving AT2092 and AT2022, selec-tion was also made for the early markers xyl+ ormal+, and the times of entry obtained for thesemarkers indicated that each one was enteringeither recipient at approximately the same time.Therefore, we cannot explain the 6-min differenceby postulating that the initation of chromosometransfer is delayed by 6 min when strain AT2092is the recipient. What we do propose, however, isthat recipient strains are able to affect the rate atwhich the chromosome is transferred from themale donor, and that in matings involving therecipient AT2022 this rate is depressed. To ex-plain the identical time of entry for early markersin these crosses, it is necessary to postulate thatthis alteration of rates of transfer affects distalmarkers much more than it does proximal ones.This hypothesis will also explain the differencesin times of entry for the alleles that we have re-ferred fo as aro Cl+ and aro C4+. We have atpresent no direct evidence in support of this hy-pothesis, but, in view of our results, it seemsmore acceptable than the transposition postu-lated by Taylor and Thoman.We have not examined any of our mutant

TABLE 17. Identification of tyr A mutants by transduction

Donor Recipient No. of tyr+ No. of his+transductants transductants

AB2826 tyr+ his+ AT2273 tyr A 352 his- Ca. 2,500 Ca. 5,000AB2859 tyr A 351 his+ AT2273 tyr A 352 his- 420 Ca. 7,500AT2471 tyr A his+ AT2273 tyr A 352 his- 3 2,820

(A)

O~~~~~~~~~ .

__.~~~~~~~~~~~V

(C)

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AROMATIC BIOSYNTHESIS IN E. COLI

FIG. 8. Distribution of aromatic genes on the chromosome of Salmonella typhimurium and Escherichia coliK-12. The map ofS. typhimurium is based on the publication ofSanderson and Demerec (20). The functions of thedifferent aromatic genes have not been described, and the gene symbols used, e.g., aro A etc., need not correspondto similar gene designations on the E. coli map. The parentheses around a symbol signify that the position is onlyknown approximately. The map ofE. coli K-12 is constructed from our own data and that of Taylor and Thoman(24). A description of the functions of the various genes is to be founid in Fig. I and in the text. The relative orderofphe A and tyr A has not yet been determined.

strains for their ability to carry out the first reac-tion of the common pathway. Since it has beenshown in E. coli W and in E. coli K-12 that thereare probably three isoenzymes able to carry outthis reaction (4, 14, 22), the isolation of mutantsblocked in the first reaction would not be ex-pected after the selection technique that was used.

Sanderson and Demerec (20) have recentlypublished a genetic map of Salmonella typhi-murium, in which they listed five aromatic loci:aro A, aro B, aro C, aro D, and aro E. Since theypresent neither genetic nor biochemical evidencein support of separate gene designations for aro Aand aro E on the one hand and aro B and aro Con the other (in each case the pair of genes beingseparated by less than 1 min on the chromosome),it is not possible to examine their claim that thedistribution and function of the aromatic loci onthe chromosomes of both E. coli and S. typhi-murium are probably both the same. If eachgenetic locus that they refer to controls the struc-ture of a different enzyme, then it appears asthough divergence may exist between the twoorganisms as far as these loci are concerned. Acomparison of the S. typhimurium map and theE. coli map is shown in Fig. 8.

ADDENDUM IN PROOF

We have recently isolated a mutant strain which,although able to grow on minimal media alone, has astrict requirement for tyrosine in the presence ofphenylalanine and tryptophan. Either dehydroquinicacid or shikimic acid can replace tyrosine as a growthfactor. It is probable that this mutant has lost thetyrosine sensitive isoenzyme which converts erythrose-4-PO4 and phosphoenolpyruvate to PKDH.

This new gene, for which we propose the designa-tion aro-F, is cotransducible with the phe A gene at afrequency of 50% and with the tyr A gene at a fre-quency of 60%. Hence, two genes controlling two ofthe first specific reactions after the branch point andone of the genes involved in the first specific reactionof the common pathway are situated very close to-gether on the chromosome. The significance of thisarrangement for the regulation of this pathway iscurrently under investigation.

ACKNOWLEDGMENTSWe would like to thank Laurence Mason-Jones for

excellent technical assistance and Michael Edwardsfor the chorismic acid. We are also indebted to D. B.Sprinson for a gift of 7-phospho-2-keto-3-deoxy-D-arabino-heptonate.

This investigation was supported by a grant fromtne Australian National Health and Medical ResearchCouncil, and by Public Health Service grant AM/4632from the Institute of Arthritis and Metabolic Diseases.

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3. ADELBERG, E. A., AND J. W. MEYERS. 1953.Modification ot' the penicillin technique forthe selection of auxotrophic bacteria. J.Bacteriol. 65:348-353.

4. BROWN, K. D., AND C. H. Doy. 1963. End prod-uct regulation of the general aromatic path-way in Escherichia coli W. Biochim. Biophys.Acta 77:170-172.

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6. Cox, G. B., AND F. GIBSON. 1964. Biosynthesis ofvitamin K and ubiquinone. Relation to theshikimic acid pathway in Escherichia coli.Biochim. Biophys. Acta 93:204-206.

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14. LIM, P. G., AND R. I. MATBES. 1964. Tryptophan-and indole-excreting prototrophic mutant ofEscherichia coli. J. Bacteriol. 87:1051-1055.

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16. LowRY, 0. H., N. J. RosEBROUGH, A. L. FARR,AND R. J. RANDALL. 1951. Protein measure-ment with the Folin phenol reagent. J. Biol.Chem. 193:265-275.

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18. MORGAN, P. N., M. I. GIBSON, AND F. GIBSON.1963. The conversion of shikimic acid intocertain aromatic compounds by cell-free ex-tracts of Aerobacter aerogenes and Escherichiacoli. Biochem. J. 89:229-239.

19. PrrrARD, J. 1965. Effect of integrated sex factor ontransduction of chromosomal genes in Escheri-chia coli. J. Bacteriol. 89:680-686.

20. SANDERSON, K. E., AND M. DEMEREC. 1965. Thelinkage map of Salmonella typhimurium.Genetics 51:897-913.

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