Aromatic Amino Acid Biosynthesis: Gene-EnzymeRelationships in Bacillus subtilis
DELILL NASSER1 AND E. W. NESTERDepartments ofMicrobiology and Genetics, University of Washington, Seattle, Washington 98105
Received for publication 21 August 1967
Single step mutants of Bacillus subtilis which required either one or all of thearomatic amino acids for growth were isolated. The relevant gene defect was de-termined for each mutant by enzyme assays in vitro. A mutant deficient in eachenzyme step of aromatic amino acid biosynthesis was found with the exceptions ofthe shikimate kinase and the phenylalanine and tyrosine transaminases. Representa-tive mutants carrying the defective genes were mapped by deoxyribonucleic acidmediated transformation by reference to the aromatic amino acid gene (aro) clusterand, alternately, to any of the other unlinked aro genes. The genes coding for dehy-droquinate synthetase, 3-enol pyruvylshikimate 5-phosphate synthetase, one formof chorismate mutase, and prephenate dehydrogenase are linked to the aro cluster.Except for the previously identified linkage between the genes of 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase and one species of chorismate mutase, theother genes involved in this pathway are neither linked to the aro cluster nor to eichother.
The classic studies of Demerec (10) in Sal-monella typhimurium first demonstrated the re-markable clustering of genetic loci concernedwith the same biosynthetic pathway. Since thosestudies, numerous investigators have extendedthe observations to a variety of biosyntheticpathways in a number of microbial species (3).Although the phenomenon has been well sub-stantiated, its physiological significance is notclear. The operon hypothesis ofJacob and Monod(22), which accounts for a coordinate repressioncontrol of linked genes, serves as a reasonableexplanation for some linkage groups. In somesystems, the gene products of closely linked lociare aggregated into stable enzyme complexes(34). However, gene linkage is not an essentialcondition for aggregation (12). Perhaps in somecases gene linkage is solely an evolutionaryremnant (21).The multibranched pathway in Bacillus sub-
tilis (Fig. 1), which results in the synthesis oftyrosine (tyr), tryptophan (trp), phenylalanine(phe), p-hydroxybenzoic acid (POBA), p-amino-benzoic acid (PABA), and a possible third vita-min, appears to be similar in chemical sequenceto that defined in Escherichia coli and Aerobacteraerogenes by Davis (7), Sprinson (43), Rivera
1 Present address: Department of Bacteriology,Division of Biological Sciences, University of Florida,Gainesville 32603.
and Srinivasan (40), and Gibson (16). However,control of enzyme activity and relevant generelationships are quite different (23, 32, 35).
Anagnostopolous and Crawford (5) demon-strated that the segment of deoxyribonucleic acid(DNA) which had previously been shown tocarry genes for indole and histidine biosynthesis(15, 33) carried all of the loci specifically con-cerned with tryptophan biosynthesis. This linkagegroup was later extended by Nester, Schaffer,and Lederberg (35) to include loci concernedwith other steps of aromatic amino acid bio-synthesis. However, mutants were characterizedonly by their growth or lack of growth on eachof the aromatic amino acids, on combinations ofthe three amino acids, or on the intermediatemetabolite, shikimate.
This paper extends that work. We have eitherfound or biochemically defined single step mu-tants for each step in the biosynthetic sequence,with the exceptions of mutants lacking shikimatekinase and the tyrosine and phenylalanine trans-aminases. The gene involved in each biochemicalreaction has been related by DNA-mediatedtransformation to the aro cluster and to the othergenes concerned with aromatic amino acid syn-thesis.
MATERMILS AND METHODSBacterial strains. All of the strains used in this study
were derived from B. subtilis 168 or 23. The mutants
were obtained either after ultraviolet irradiation (35)or by the use of the mutagen N-methyl-N'-nitro-nitrosoguanidine (1, 26).
Transformation procedures. Transformations weredone essentially as described by Nester et al. (35).Donor DNA was extracted from cells grown in TSY[Trypticase Soy Broth (BBL) + 10 g of yeast extractper liter]. In our earlier studies, DNA was preparedby the method described by Marmur (27), but laterDNA preparations were made essentially by themethod of Dubnau, Smith, and Marmur (14) inwhich the cells are broken by lysozyme (WorthingtonBiochemical Corp., Freehold, N.J.) and Pronase(Calbiochem, Los Angeles, Calif.) in a 0.3 M solutionof sucrose-SSC (0.15 M NaCl + 0.015 M Na citrate).Linkage was analyzed with more than one concentra-tion of DNA in order to minimize the uptake ofseparate pieces of DNA, which would lead to anapparent linkage (35). The concentration of DNAused in a particular experiment could be shown to belimiting by the proportionate drop in the transforma-tion frequency upon dilution of the DNA. The linkagevalues reported are those obtained at limiting concen-trations.
Mapping procedures. Genetic linkage was assessedeither by the determination of the cotransfer index(33, 35) or by the recombination index (15, 24). Inthe initial studies, linkage to the aro cluster of geneswas estimated by using either his-2 or trpC (Table 1)as a reference marker. Both of these loci are in the arocluster (35), and the percentage of coincident transfersof the aro marker and the reference marker served asa quantitative estimate of the degree of linkage be-tween the two loci.The genes which were not linked to the aro cluster
were tested for linkage to each other. If the mutantloci could be differentiated nutritionally, repulsioncrosses (i.e., + -, -X, - +) were performed andthe cotransfer index was calculated by assuming thatthe recombinant class which could not be scored wasequivalent to the recombinant class which could bescored. Therefore, in a repulsion cross, the cotransferindex is equal to the number of double transformantsdivided by the sum of the double transformants plustwo times the class of single transformants (33).
H,r;, _ "H CORPH~~PC"2-C-COCH
COOHH IL..,, cH,-c-_ooH
cOO CM1 COO cH tOOC CP 2
JN.cooPI6> c -COOH"
OH \OHIM CM-i-coo
COO" cOm, Co-o-CcmpHP0 cOH o NH
NHM OH OHp-Aoitobe"nzc P-HydHoxynek TrosHe
ocid ocid
acid synthesis in Bacillus subtilis.
If the mutant loci could not be nutritionally differ-entiated, then the recombination index was deter-mined (15, 24). This technique is based on the expec-tation that the number of prototrophic transformantswill be equal whether DNA is isolated from aprototroph or a mutant, provided the mutation in thedonor and recipient are genetically unlinked. If thelocus in the mutant is linked to the locus in the recip-ient, then a crossover must occur to yield a proto-trophic recombinant. Consequently, there will befewer recombinants when DNA isolated from themutant strain is used than when DNA from theprototrophic strain is used. Because of differences inthe transforming efficiencies of donor DNA, fre-quencies were normalized by equalizing the numberof transformations for an unlinked marker with theuse of the two preparations of DNA. Operationally,if loc = the marker being studied, ref = referencemarker, m = mutant DNA, and w = prototrophicDNA, then the recombination index (R.I.) can becalculated from the following formula:
loc mref mloc wref w
Thus, a ratio of 1 indicates nonlinkage. A ratio ofless than 1 would indicate linkage.
Gene designations in this paper have been modifiedfrom preceding papers to conform with the sugges-tions offered by Demerec et al. (11).
Preparation of enzyme extracts. Extracts wereroutinely prepared from cells grown in Spizizen'sminimal medium (42), plus 0.5% glucose, plus therequired supplements. Tyrosine, phenylalanine, tryp-tophan, and the vitamins PABA and POBA wereadded at levels of 20, 20, 5, and 0.1 ,ug/ml, respectively.The cells were harvested, washed once with the bufferto be used in the specific assay, and resuspended in thesame buffer. The extract was then prepared in one oftwo ways. The cells were broken either by sonictreatment in an MSE Ultra Sonic Power Unit for 4min at maximal power or by the addition of 1 mg of
lysozyme per ml (Worthington Biochemical Corp.)and 10 ,ug/ml of deoxyribonuclease (WorthingtonBiochemical Corp.). Cell debris was sedimented bycentrifugation at 25,000 X g for 30 min and thesupernatant fluid was used in enzyme assays withoutfurther purification, unless specifically noted.
In some cases, whole cells were assayed for shi-kimate kinase activity. The cells were grown in 10 mlof TSY at 37 C with aeration. The cells werepacked by centrifugation, washed once, and resus-pended in the appropriate buffer at one-tenth theoriginal concentration; 10 ,ug of CETAB (cetyl tri-methyl ammonium bromide; Eastman) per ml wasthen added (13). After mixing, the suspension wasshaken for 10 min at 37 C. The suspension was usedwithout further treatment in the assays.DNA determinations. DNA concentration was
determined by the method of Burton (6) with deoxyadenylic acid as a standard.
Assays for DAHP (3-deoxy-D-arabino heptulosonicacid 7-phosphate) synthetase. DAHP synthetase wasdetermined by the method of Srinivasan and Sprinson(45) as modified by Jensen and Nester (23).
Assay for DHQ (dehydroquinate) synthetase. DHQsynthetase activity was determined by the method ofSrinivasan, Rothschild, and Sprinson (44) as modi-fied by Jensen and Nester (23). The substrate (DAHP)for this assay was isolated and purified from cells ofSB167 by R. A. Jensen of this laboratory. This mu-tant (SB167), which accumulates DAHP as a resultof a lesion in dehydroquinase activity, was grownovernight at 37 C with aeration in Spizizen's minimalmedium supplemented with 0.5% glucose and 25 ggof shikimic acid per ml. The cells were harvested bycentrifugation, washed once, and resuspended in3 to 5 ml of Spizizen's minimal medium. The cellsuspension was heated for 10 min at 80 C, after whichthe cells were disrupted by sonic vibration for 5 minin an MSE sonic oscillator. All succeeding steps wereperformed at 4 C. The sonic extract was added to acolumn (1 X 16 cm) of Dowex 1, chloride form. TheDAHP was eluted with 0.1 M (NH4)2CO3, pH 8.1.Volumes of 10 ml were collected in each tube, andthe flow rate was 20 to 25 ml per hr. The tubes con-taining DAHP were pooled, the pH was adjusted to5 with formic acid, and the solution was lyophilized.The residue was dissolved in about 5 ml of water andexcess Ba acetate was added. The pH was adjusted to8.0 with NH4OH and 2 volumes of cold ethyl alcoholwere added. The precipitate was collected by centrifu-gation and washed once with cold ethyl alcohol.The precipitate was resuspended with vigorous
shaking in 3 to 5 ml of water. The suspension wascentrifuged; the supernatant fluid was retained andthe residue was subjected to the same process. Afterrepeating the process several times, the supernatantfluids, which contained the DAHP, were pooled, andexcess 2 M (NH4)2CO3 was added to remove Ba. Theprecipitate was removed by centrifugation; the pHof the supematant fluid was adjusted to 5 with formicacid and then lyophilized. The dried material wasused as the substrate for DHQ synthetase withoutfurther purification.
Assay for dehydroquinase. The assay procedure of
Mitsuhashi and Davis (30) was employed, with theculture filtrate of E. coli mutant strain 170-27 used asthe source of DHQ. This strain was grown under theconditions specified by Weiss, Davis, and Mingioli(47) for the accumulation of DHQ.
Assay for DHS (dehydroshikimate) reductase. DHSreductase activity was determined by the method ofYaniv and Gilvarg (48).
Assay for shikimate kinase. Shikimate kinase ac-tivity was determined by the method described byNester, Lorence, and Nasser (34). In essence, theassay consists of the conversion of l4C-shikimate(uniformly labeled) to 14C-shikimate 5-phosphate.
Assay for EPSP (3-enolpyruvyl shikimate 5-phos-phate) synthetase and CHA (chorismate) synthetase.Substrates for the specific assays of these enzymeswere unavailable. Therefore, an in vitro complementa-tion method was used. An extract of a mutant of A.aerogenes, lacking either the EPSP synthetase or theCHA synthetase was mixed with an extract made fromB. subtilis. This mixture of extracts was then testedfor the ability to synthesize anthranilate from thesubstrates shikimate and ATP (adenosine triphos-phate). Mutants of A. aerogenes known to be deficientin these activities were obtained from B. D. Davisof Harvard Medical School. Strain A170-40 lacksEPSP synthetase and A170-44 CHA synthetase (9,17, 18). These strains were grown on minimal medium(8), supplemented with (ug/ml): trp, 5; tyr, 15; phe,15; and PABA plus POBA, 5; plus 0.2% glucose.Extracts were made by treatment of the cells with aBranson Sonifier at maximal power at a setting of 5for 4 to 5 min. The debris was removed by centrifuga-tion at 25,000 X g for 30 min. The reaction mixturewas as described by Morgan, Gibson, and Gibson(31), except that shikimate and ATP (1 pmole/mleach) were the initial substrates rather than shikimate5-phosphate. The B. subtilis extracts were preparedfrom cells grown in Spizizen's minimal mediumsupplemented with limiting (,ug/ml: trp, 5; tyr, 15;phe, 15; PABA and POBA, 0.1) amounts of the endproducts. The cells were washed once with 0.1M tris(hydroxymethyl)aminomethane (Tris) chloridebuffer (pH 8.2) and broken in the same bufferby sonic disruption. The amount of anthrani-late produced by the mixture of B. subtilis extractand A. aerogenes extract in the reaction mixtureafter incubation for 30 min at 37 C was propor-tional to the enzyme concentration of the B. subtilisextract. Anthranilate was determined by a modifiedBratton-Marshall assay (4).
Sucrose density gradient. The method of Martinand Ames (28) was used with slight modification. A4.6-ml linear gradient from 10 to 30% sucrose in 0.1M Tris chloride (pH 8.2) + 0.01 M MgC92, 10-4 Methylenediaminetetraacetate (EDTA), and 6 X 10-3 Mmercaptoethanol was prepared. An 0.5-ml amount ofthe extract prepared in the same buffer was layeredon top. The extract was centrifuged in a SW 39Lswinging-bucket rotor in a model L Spinco centrifugeat 150,000 X g for approximately 16 hr at 4 C. Eachtube was punctured with a small-gauge needle and10-drop fractions were collected in a total of 41 to 43tubes. An internal standard of human hemoglobin
(Pentex, Inc., Kanhakee, Ill.) was used in each grad-ient. The hemoglobin concentration was measured byits adsorbancy at 410 m,.
Diethylaminoethyl (DEAE) cellulose chromatog-raphy. DEAE cellulose (Carl Schleicher & SchuellCo., Keene, N.H.) was processed and a column(2 X 20 cm) was packed as described by Peterson andSober (37). Extracts were always treated with prota-mine sulfate prior to chromatography. Approximately0.1 ml of a 2% solution of protamine sulfate was
added for every 10 mg of protein in the crude extract.The mixture was allowed to stand for 15 min at 0 C,and the precipitate was removed by centrifugation at25,000 X g for 30 min. A maximum of approximately40 mg of protein was applied to the column which hadbeen previously equilibrated with the starting buffer.A linear gradient was formed by eluting with 125 mlof 0.1 M Tris chloride (pH 8.2) containing 0.01 M
MgC12, 10-4 M EDTA, 6 X 10-3 M mercaptoethanol,and 0.1 M NaCl in the mixing chamber, and the same
volume of Tris buffer containing 0.5 M NaCl in thereservoir. Approximately 1.5-ml fractions were
collected. The flow rate was 25 ml/hr. Columns were
run at 4 C, and the fractions were assayed for en-
zyme activity as soon as possible.
RESULTS
Table 1 lists the mutants with their phenotypesand genotypes used in this study. These mutantswere initially classified nutritionally on the basisof their ability to grow on shikimate and theirgrowth response to one or all of the aromaticamino acids. This procedure divided the mutantsinto one of several possible categories. Thosestrains capable of growing on medium supple-mented with shikimate were presumed to lack one
of the enzymatic activities prior to shikimatekinase. Those requiring tyrosine, phenylalanine,
TABLE 1. Strains of Bacillus subtilis used in the mapping experiments
WB698b High levels of activity for CHA mutase,shikimate kinase, and DAHP synthetase
WB834 Prototroph
a The SB prefix indicates those strains which were isolated at Stanford University. The WB prefixindicates strains isolated at the University of Washington.
b B. subtilis 168 and all strains with its genetic background lack the aroH gene. In this respect, there-fore, they can be considered to be mutant for this enzyme. For further details concerning this gene, seeLorence and Nester (27).
and tryptophan were presumed to be mutant inone of the enzymatic activities between shikimateand the branch point compound prephenate.Those requiring only a single amino acid forgrowth were presumed to be defective in theterminal portion of the pathway specific for thatend product.
Strains were considered to be mutant for a spe-cific enzyme if there was no measurable activityor only a few per cent of the level of wild-typeactivity. However, in all cases, spontaneous singlestep prototrophic revertants were isolated andenzymatic activity was determined to ensure thatenzyme activity was regained with the con-comitant regain of prototrophy. Mutants lackingshikimate kinase and the final transaminases fortyrosine and phenylalanine were not found.The studies of Meister on E. coli (29) suggested
that a single enzyme functioned in the trans-aminase reactions of tyrosine and phenylalaninesynthesis. Assays of extracts from strains whichrequire both tyrosine and phenylalanine as wellas those which require only one or the otheramino acid for growth have not yielded anyphenylalanine or tyrosine transaminase mutants.
Shikimate kinase. A mutant of B. subtilis de-ficient in shikimate kinase activity has not yetbeen found, although cell extracts or CETAB-treated whole cell suspensions of approximately100 multiple aromatic amino acid-requiring mu-tants have been assayed for this activity. This en-zyme activity was studied more closely to deter-mine why such a mutant was not found.
Extracts from WB 698b, a genetically dere-pressed prototroph, were made by sonically dis-rupting cells grown in minimal medium. In someexperiments, the extract was freed from smallmolecules by passing through a Sephadex G 25column or, alternatively, untreated extracts wereused. The results with both procedures were com-parable. In one set of experiments, extracts werecentrifuged through a sucrose gradient, and thecollected fractions were assayed for shikimatekinase activity. Figure 2 shows the profile ob-tained. There are two molecular species of thisenzyme. Resolution of two species of enzymeswas also obtained when an extract of WB 698bwas chromatographed on DEAE cellulose (Fig.3).The simplest explanation for our lack of suc-
cess in finding a shikimate kinase mutant is thatthe two molecular forms are the result of twoseparate genes. Thus, the probability of finding amutant lacking both activities would be veryslight.
Linkage of genes to the tryptophan cluster. Acotransfer index relating each gene concerned
_ 800-'-z 700-<, 600-
q) 500-" 400-Z 300-'6 200-- 00-6-
5 10 15 20 25 30 35Tube Fraction
40 45
FIG. 2. Sucrose gradient profile of shikimate kinaseactivity ofan extract of 698b.
S
* 2000
>. 1600
S 1200q)
Z 800
.1 400
25 30 35 40 45 50 55 60Tube Fraction
FIG. 3. DEAE cellulose profile of shikimate kinaseactivity of an extract of 698b.
with a specific enzyme in this pathway to the arocluster was determined.
In most cases, two independent mutants defi-cient in the same enzyme activity were used forthe mapping experiments. In all mapping experi-ments, three concentrations of DNA were em-ployed. The representative data shown in Table 2indicate that the genes concerned with DHQsynthetase, EPSP synthetase, one form of theCHA mutase (aroH), and the prephenate de-hydrogenase are linked to the aro cluster. Thesegenes were ordered in relation to the other lociin the linkage group concerned with aromaticamino acid synthesis by Nester et al. (35) priorto the identification of their enzyme lesion. Table3 summarizes the linkage relationships of thegenes encoding for the enzymes of this pathwayas determined from the experiments described inthis and other papers from this laboratory (26,34, 35).
Additional information concerning linked genes.In a previous publication, Nester et al. (35), whiledesignating aro2 and aro3 as two separate geneson the linkage map, suggested that they could beblocked in the same enzyme step and that the
TABLE 2. Linkage analysis of aro genes to the his-2 or trpC loci (aro cluster)a
His+ or Trp+ His± or Trp+ His+ or CotransferEnzyme Cross Aro±b Aro+ Aro+ Trp+ index
DAHP synthetase ... WB834-XSB155 1.0 X 104 9.2 X 103 2 X 101 0.001DHQ synthetase........... WB834-XSB138 7.1 X 101 5.4 X 102 4.1 X 102 0.49Dehydroquinase............ WB834-XSB122 1.3 X 104 1.1 X 104 1.4 X 102 0.006DHS reductase............. WB834-XSB120 3.5 X 104 5.1 X 104 5.0 X 101 0.0006Prephenate dehydratase. WB834-XSB112 2.0 X 105 2.5 X 105 1.1 X 103 0.003
Plate supplementations used were (jAg/ml): trp, 20; phe, 50; tyr, 50; shikimic acid, 50; POBA andPABA, 1; his, 50. In all experiments, the medium was supplemented for all requirements of the donoras well as the recipient cells, so that the only selected marker was that which is indicated. Cotransferindices were calculated as described by Nester and Lederberg (33). Total cell count was 108 to 2 X 10'cells/ml.
b The symbol 1 refers to the fact that the medium on which these selections were made contained thissupplementation and consequently there was no selection for this marker.
TABLE 3. Gene designations for the described enzymelesions and their linkage relationships
ase................. tyrA103 aro clusterPrephenate dehydratase. pheA12 None
a Linkage data for the two loci encoding for thechorismate mutases are presented by Lorence andNester (26) and Nester et al. (34).
observed nutritional differences between the twostrains might reflect only a difference in the com-pleteness of the metabolic block. Enzymaticanalysis has shown this to be the case. Conse-quently, aro,3 has been eliminated from the linkagemap as a separate gene. AroB (aro2) has been re-tained to designate the gene coding for dehydro-quinase.The locus designated inh has been retained, al-
though recent biochemical studies indicate a veryclose relationship to the locus coding for pre-phenate dehydrogenase (tyrA). Previous workcharacterized the SB419 strain (inh-419) as grow-ing on minimal, but not on histidine-supple-mented medium. SB419 does not have detectablelevels of prephenate dehydrogenase in vitro. Theenzymological basis for this phenotype will bethe subject of a future publication (Nester, inpreparation).
Genetic relationship of genes unlinked to thetryptophan cluster. The possibility was tested
that the aromatic loci which were not linked tothe tryptophan cluster were linked to each other.Tables 4 through 6 show the data which indicatenonlinkage of genes not in the aro cluster to eachother.The only genes concerned with this pathway
which have been found to be linked to each other,except for those in the aro cluster, are involvedwith DAHP synthetase and one form of theCHA-mutase (aroG). The data concerned withthe genetic and biochemical relationships of theseenzymes are presented in other papers from thislaboratory (26, 34). Figure 4 summarizes ourdata on the genetic relationships of loci concernedwith the biosynthesis of the aromatic amino acidsin B. subtilis.
DIscussIoNB. subtilis is unique among microorganisms
thus far studied in having at least four of thegenes concerned with the biosynthesis of thearomatic amino acids linked to the trp cluster ofgenes. While, on the one hand, Neurospora crassahas a gene cluster concerned with the secondthrough the sixth enzymes of this pathway (19),in S. typhimurium (36) and E. coli (38), all arogenes are separate on the chromosome with theexception of those coding for prephenate dehy-drogenase and prephenate dehydratase in E. coli(38). However, N. crassa has trp genes distributedamong the seven chromosomes (2) in contrast toS. typhimurium (41), E. coli (46), and B. subtilis(5), all of which have the trp genes in a singlecluster. Pseudomonas putida is somewhat differentin that the trp genes are distributed into threeseparate clusters (20).The physiological significance of this gene
grouping in B. subtilis is not at all clear. The en-zymes specified by linked genes, except for the trpcluster, are not sequential in the biochemical se-quence, and our initial efforts to demonstrate
TABLE 4. Linkage determination of genes unlinkedto the aro cluster
Genes Recombinants/mlCross tested Cotrans-foi fer index
linkage Trp+ Aro±a Trp:k Aro+
SB155-XSB120. aroA 2.4 X 103 1.5 X 103 1.1WB834-
XSB120..... aroD 1.1 X 104 6.5 X 103
SB155- XSB121. aroA 1.0 X 106 1.0 X 106 1.7WB834-
XSB121.. ... aroC 1. 3 X 106 7.5 X 105
SB719- XSB121. aroD 8.6 X 106 7.2 X 106 1.5WB834-
XSB121.. ... aroC 1. 2 X 106 1.6 X 106
a The selection for Trp+ Aro± strains was made by supple-mentation of the plates with shikimic acid rather than the fullcomplement of aromatic amino acids. The concentrations ofthe supplements were as described in Table 2. Total cell countsin this series of experiments were 108 to 2 X 108 cells/ml.
TABLE 5. Linkage determination of genes unlinkedto the aro cluster to pheAa
CosGenes
Cross tested for Aro+ Phe± Aro+ Phe indexlinkage
SB112-XSB155 aroA 242 2 0.004pheA
SB112-XSB121 aroC 107 0 0pheA
SB112-XSB120 aroD 262 0 0pheA
SB112-XSBl16 aroF 143 0 0pheA
a The indicated number of Aro+ Phe* recom-binants was streaked onto nutrient agar platesand the number of cells with the Aro+ Phe- pheno-type was determined by replica plating.
physical relationships among the enzymes havebeen unsuccessful. Tryptophan synthetase andanthranilate synthetase can be separated in asucrose gradient (unpublished data) and the CHAmutases (aroH and aroG) are easily separatedfrom prephenate dehydrogenase and dehydrataseby DEAE cellulose chromotography (26). Pre-liminary evidence indicates that the enzymescoded for by these genes, if repressible, are notcoordinate in their regulation. Thus, it appearsunlikely that this linkage group represents asingle operon.
ACKNOWLEDGMENTSThis investigation was supported by Public Health
Service grants GM 00511 and GM 09848, and by the
TABLE 6. Linkage determination of genes unlinkedto aro clusters
Recombinantstested
Genes tested CotransferCross for linkage indexAro+ Aro+
Aro(s)±b Aro(s)-
SB121-XSB116 aroC 133 1 0.004aroF
SB120-XSB116 aroD 142 1 0.003aroF
SB155-XSB116 aroA 195 3 0.007aroF
a The indicated number of Aro+ Aro(s)+ recom-binants was streaked onto nutrient agar plates andthe number of cells with the Aro+ Aro(s)- pheno-type was determined by replica plating.
b The Aro (s) designation indicates that selectionfor these classes was made on shikimic acid-sup-plemented medium.
mtr oroB trp cluster his-2 tyrA inh-491 oroE
yIIr 1 1 1sI
aroA aroG aroH amt
FIG. 4. Linkage groups of Bacillus subtilis con-cerned with aromatic amino acid biosynthesis: aroHhas not been ordered in the aro cluster; aroG and aroAhave not been ordered since other genes in this vicinityare not known. The amt (3-amino tyrosine resistance)locus was shown to be linked to the aro cluster byPolsinelli (39). It has not been ordered in the linkagegroup. The symbol mtr = 5-methyltryptophan resistance.
University of Washington Initiative 171 for biologicaland medical research.
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