GENE- RELATIONS OF TRYPTOPHAN MUTANTS IN STREPTOMYCES COELICOLOR A3 (2)

CHARLES M. SMITHERS AND PAULINUS P. ENGEL

Department of Biology, Virginia Polytechnic Institute and State Uniuersity, Blacksburg, Virginia 24061 Manuscript received April 4, 1974 Revised copy received June 25, 1974

ABSTRACT

Mutations in twenty-eight tryptophan mutants of S. coe ;color . 3 (2) were mapped relative to the nearest flanking markers. Mutants lacking single enzymatic activities for phosphoribosyltransferase, phosphoribosylanthranilate , indodeglycerol phosphate synthase, tryptophan synthase A and tryptophan synthase B were identified.

RELIMINARY mapping (ENGEL1973) established that trp mutations in P, codicolor (Figure 1) map on both sides of hisC, between proA and argA. Evidence for the location of twenty-eight trp mutations relative to the nearest flanking markers is summarized in this paper. Enzyme assays using cell-free extracts of twenty-seven of these mutants provide evidence to identify mutants with enzymatic defects. Mutants lacking tryptophan synthase A, tryptophan synthase B and indoleglycerol phosphate synthase activities (Figure 2) map in thc short region between hisC and ammA. Mutants lacking phosphoribosyltrans- ferase and phosphoribiosylanthranilate isomerase activities map in the short

el,e2,e4,5,e5,e6,7,e7, e9,elO,e12,e16, el7, e18,e19,e20.eZl.e23

FIGURE1.-Linkage map of S. coelicolor A3(2) showing the position of markers (Ho~woon et al. 1973) referred to in this paper.

Grnelic\ 78: 799-808 Novemhei, 1974 800

l"d111

FIGURE2.-Pathway of tryptophan biosynthesis. Abbreviations: AS, anthranilate synthase; PRT, phosphoribosyltransferase; PRPP, phosphoribosyl-5-pyrophosphate;PP, inorganic pyro- phosphate; PRA, phosphoribosylanthranilate; PRAI, phosphoribosylanthranilate isomerase; CDRP, 1- (0-carboxyphenylamino) -1-deoxyribulose-5-phosphate; InGP, indoleglycerol phosphate; InGPS, indoleglycerol phosphate synthase; Gly-3-P, glyceraldehyde-3-phosphate; TS, tryptophan synthase; TS-A and TS-B, A and B reactions of tryptophan synthase. region between rifB and thiC. Mutants lacking anthranilate synthase activity have not been identified. Preliminary evidence suggesting the constitutive syn- thesis of indoleglycerol phosphate synthase in S. coelicolor is presented.

MATERIALS AND METHODS

General: S. coelicolor and Aerobacter aerogenes, strain 62-1, were grown at 30". Trp mu- tants of E. coli K12 were grown at 37". The minimal and complete media for S. coelicolor, and concentrations of supplements in these media, were as previously described (HOPWOOD1967; HOPWOODet al. 1973). A. aerogenes was grown in the media described by GIBSON(1964). E. coli was grown in the medium of VOGELand BONNER(1956) supplemented with 0.05% acid-hydro- lyzed casein, 0.5% glucose and 5 pg/ml L-tryptophan. Strains and mutagenesis: A. aerogenes 62-1, isolated by GIBSONand GIBSON(1964), was ob- tained from DR. N. H. GILES.E. coli 9778 (PR mutant), 9830 (PRA isomerase mu- tant), 9941 (InGP synthase mutant) and T8 (mutant in tryptophan synthase a component) were obtained from DR.CHARLES YANOFSKY. S. coelicolor strains (Table 1) were derived from the wild-type strain, A3(2), by mutation or recombination. Multiply marked Trp strains, used in reciprocal crosses, were isolated from crosses of Trp x 876 as previously described (ENGEL1973). DR. D. A. HOPWOODprovided the wild-type strain, mutants Trpl, vl, 2, 5, 7 and 8, and strains bearing markers ProAl, leuAl, rifB37, thiC2, hisC9, ammA.5, argAl, cysC3, strA1 and pheAl. The location of these markers is shown in Figure 1. Gene symbols have conventional meanings (HOPWOODet al. 1973). Mutants Trpl, vl, 2, 5, 7 and 8 were induced with ultraviolet light as described by HOPWOOD and SERMONTI(1962). Mutants Trpe2, e3, e4, e5, e6, e7, e9, e10, ell and e12 were induced with ethyl methane sulfonate (Eastman Organic Chemicals) as previously described ( ENGEL 1973). Trpel, e8, e13, e14, e16, e17, e18, e19, e20, e21, e22 and e23 were induced with N-methyl- N'-nitro-N-nitrosoguanidine (Aldrich Chemical Co.) using the procedure of DEL~C(1970). To preclude the isolation of identical mutants different clones of wild-type cells were used to induce each Trp mutant. The growth response, complementation and preliminary mapping of Trp mu- tants has been described (ENGEL1973). Indole-utilizing mutants include Trpel, e2, 4,e5, 5, &, e7, 7, e8, e9, e10, e12, e16, e17, e18, e19, e20, e21, e22 and e23. TrpS grows poorly on minimal medium supplemented with 50 pg indole/ml. Mutants Trpl, vl, 2, e3, ell, e13 and e14 grow only in the presence of tryptophan. No anthranilate-utilizing mutants have been found. Al- though the location of the trp mutation in Trpel8 was mapped earlier in this investigation, sub- cultures of this strain failed to revive prior to the enzymatic investigation. TRYPTOPHAN MUTANTS IN S. coelicolor 801 TABLE 1 Characteristics of strains

Strain Genotype' A3 (2) wild type P4 pheAl proAI hisC9 ammA5 cysC3 strAl P8 pheAl hisC9 ammA5 argAl cysC3 strAl P22 trpell pheAl argAl cysC3 strA1 P38 trpel4 pheAl proAI cysC3 strAl P59 trpeb pheAl argAl cysC3 strAl P67 trpeb proAl cysC3 strAl PI43 trpe8 pheAl proAI strA1 P144 trpe8 pheAl argA1 strAl PI47 trpel0 proAl strAl PI51 irp8 pheAl argAl cysC3 strAl PI59 trpe22 pheAl argAl strA1 PI60 trpe22 pheAl proAl strAl PI 78 trp8 pheAl proAl strA1 PI83 trpel0 argAl strAl P189 proAI leuAl rifB37 thiC2 strAl PI90 pheAl 1euAl rifB37 thiC2 argA1 strAl 876 proAI hisC9 argAl cysC3 pheA1 strAl

* Gene symbols (HOPWOODet al. 1973) have conventional meanings.

Chemicals: Chorismate was isolated as the free acid from culture filtrates of A. aerogenes 62-1 by the method of EDWARDSand JACKMAN (1965). Chorismate concentration was estimated after enzymatic conversion to anthranilate in the presence of E. coli 9778 extract. Anthranilate concentration was determined from the fluorescence of standard anthranilate solutions. The method of CREIGHTONand YANOFSKY(1970) was used to synthesize 1-(0-carboxyphenylamino) - I-deoxyribulose-5-phosphate (CDRP) . CDRP concentration was estimated by enzymatic con- version to indoleglycerol phosphate (InGP) with E. coli T8 extract. InGP was prepared enzy- matically from CDRP by the method of WEGMANand CRAWFORD(1968) using E. coli T8 extract. InGP concentration was determined by metaperiodate oxidation (YANOFSKY1956) and enzy- matically by conversion to indole in the presence of E coli 9941 extract. Indole was measured by the method of YANOFSKY(1955). All other chemicals were obtained from commercial sources. Crosses: Crosses were done and analyzed as described by HOPWOOD(1967). The rationale of reciprocal crosses, designed to determine the location of trp mutations relative to the nearest flanking markers, is illustrated in Tables 2 and 3. Preparation of S. coelicolor extracts for enzyme assays: Complete medium, 50 ml in 250-ml flasks, containing 50 pg L-tryptophan/ml and 1% glucose was inoculated from slants and in- cubated three days while standing. Two-litter flasks containing 390 ml complete medium, sup plemented with 10 pg L-tryptophan/ml and 1% glucose, were inoculated with 10 ml of cells from the three-day-old static culture. L-tryptophan was not added to cultures of wild-type cells. These cultures were incubated on a shaker for 16 hours (New Brunswick G25, 300 rpm). Cells were harvested by centrifugation, washed in extraction buffer (0.1 M potassium phos. phate buffer, pH 7.8, containing 0.8 M sucrose, 0.1 mM EDTA, 0.5 M KCl, 6 mM 2-mercapto- ethanol) and centrifuged. The pellet was suspended in twice its volume of extraction buffer, sonicated for 30 seconds (Virsonic Cell Disruptor, operating at 150 watts) and centrifuged. All centrifugations were done at 16,3(Eo x g, 4", U) minutes. These extracts were used for enzyme assays without further purification or concentration of activity. Extracts lacking a particular activity were also assayed for that activity after dialysis of 1 ml of extract in 150 ml extraction buffer for 4 hours. 802 C. M. SMITHERS AND P. P. ENGEL

Table 2

Location of 9 mutations in indolelrtilirin8 strains relative

to nonselected markers 1euN. rifB37 and U.

Cr.082 Cross*

-+ +Ifp+ + rif + +hi + _-- -__--- pro leu ------__------+ a + + + a+=

Frequency of Frequency of

progeny (X) proaeny (Z)

P190 x 110. Trp+ Trp Trp' P189 x lo. Trp+ Trp Trp+

Trp strain progeny Leu+ Rif Thl+ TIP strain progeny Leu+ Wf Thi+

e6 (P67) 138 1 2 5 e6 (P59) 141 68 0.7 <0.7 e10 (P147) 134 2 s 8 e10 (~183) 123 63

e22 (P160) 130 C0.7 7 <0.7 e22 (P154) 125 81 1 6

* selected markers were proAl+ and U+;Rif+ and Pif denote the

rifanpin-sensitive and rifampin-reslstant phenotypes, respectively.

p'0Ce"Y (Z)

P4 I NO. Pro' His' Trp'

TIP .tr.in prOReay rrg+ ~ ...' 8 (P151) 751" 2.3 0.7 144' 36 e8 (P144) 101* 4 (1 49 50

el4 (P22) 411. 1.5 1.3 196* 29

e22 (P159) 101' 5.9 (1 49 43

Preparation of E. coli extracts: E. coli was grown in two-liter flasks containing 400 ml of the medium described above. Cells were harvested by centrifugation after 16 hours of shaking (New Brunswick, G25, 300 rpm). Cells were washed in extraction buffer (0.1 M potassium phosphate buffer, pH 7.0, 1 mM EDTA and 1 mM dithioerythritol) (CREIGHTONand YANOFSKY1970). The pellet was suspended in two volumes of extraction buffer and sonicated for 15 seconds and cen- trifuged. All centrifugations were done at 16,300 x g, 4". 20 minutes. Extracts were used without further purification. TRYPTOPHAN MUTANTS IN S. coelicolor 803

Enzyme assays: All assays were done at 37". Change in fluorescence was monitored con- tinuously in the anthranilate synthase and PR transferas3 assays using a model A4 Farrand fluorometer (Farrand Optical Co., Inc., Mt. Vernon, N.Y.). The primary filter was a 313 nm interference filter (Farrand Optical Co.). The secondary filter, CSO-51, (Corning Glass Works, Corning, N.Y.) gives maximal transmission of light at 393 nm. The reaction mixture for the anthraniliate synthase assay contained 50 pmoles tris (hy- droxymethyl) aminomethane-HC1, pH 7.5, 10 pmoles MgCl,, 23 ,pmoles L-, 100 nmoles chorismic acid and extract in a final volume of 1 ml. The reaction rate was linear for at least five minutes. PR transferase was assayed as described by KANE and JENSEN (1970). The reaction rate was linear for at least two minutes. PRA isomerase was assayed by measuring the formation of indoleglycerol phosphate from anthranilate. This was a coupled assay in which E. coli 9830 extract, a PRA isomerase mutant, supplied excess PR transferase and InGP synthase activities. The reaction mixture contained 150 nmoles anthranilate, 300 nmoles PRPP, 2 pmoles MgSO,, 30 pmoles potassium phosphate buffer, pH 7.8, 120 pmoles sucrose, E. coli 9830 and S. coelicolw extracts in a final volume of 0.5 ml. Indoleglycerol phosphate was measured by metaperiodate oxidation (YANOFSKY1956) after 20 minutes incubation. InGP synthase was assayed in a 0.5 ml reaction mixture containing 30 pmoles potassium phosphate buffer, pH 7.8, 120 pmoles sucrose, 700 nmoles CDRP and extract. Indoleglycerol phosphate was measured by metaperiod ate oxidation (YANOFSKY1956) after 20 minutes incuba- tion. Tryptophan synthase A activity was assayed in a reaction mixture containing 100 pmoles potassium phosphate buffer, pH 7.8, 120 pmoles sucrose, 250 pmoles NH,OH-HC1, pH 7.0, 200 nmoles indoleglycerol phosphate and extract in a final volume of 0.5 ml. Indole formation was determined (YANOFSKY1955) after 20 minutes' incubation. Tryptophan synthase B activity was measured in a 0.5 ml reaction mixture containing 50 pmoles potassium phosphate buffer, pH 7.8, 200 nmoles indole, 120 umoles sucrose, 15 pmoles L-serine, 20 nmoles pyridoxal phosphate and extract. The disappearance of indole was deter- mined (YANOFSKY1955) after 20 minutes' incubation. Protein concentrations were adjusted in the discontinuous assays for PRA isomerase, InGP synthase, tryptophan synthase A and B to obtain linear reaction rates over 20 minutes. Specific activities are expressed as nmoles of formed or utilized per minute per mil- ligram protein. Protein was determined by the method of LOWRYet al. (1951) using bovine serum albumin as a standard.

RESULTS Location of trp mutations relative to leuA, rifB and thiC: The location of mutations in the twenty indole-utilizing mutants relative to leuA, rifB and thiC was determined from progeny of reciprocal crosses. Trp8 grows so poorly on minimal medium containing indole that it is considered unable to utilize indole. Although progeny from forty crosses of indole-utilizing mutants were analyzed, data from only eight of these crosses are summarized in Table 2. Eighteen, among them trpeb and e10, of the twenty mutations in indole-utilizing mutants map between rifB and thiC. The other two mutations in indole-utilizing strains Trpe8 and e22 may between hisC and ammA. Trp+ Leu+ progeny were at least 30-fold more frequent from crosses of P189 x Trp Arg, for all twenty indole-utilizing mutants, than in reciprocal crosses (Table 2). These mutations therefore map to the right of ZeuA. Thi+ Trp+ prog- eny were at least four times more frequent in crosses of P189 x Trpe8 Arg and 804 C. M. SMITHERS AND P. P. ENGEL P189 x Trpe22 Arg than in reciprocal crosses (Table 2). This indicates that mutations trpe8 and e22 map to the right of thiC. By contrast, the remaining eighteen mutations in indole-utilizing strains, among them trpe6 and e10 (Table 2), map to the left of thiC. Among these eighteen mutations that map to the left of thiC, sixteen gave at least 4-fold higher frequencies of Thi+ Trp+ progeny in crosses of P190 x Pro Trp than in reciprocal crosses. The other two indole-utiliz- ing mutants gave between two and three times more Thi+ Trp+ progeny from P190 x Pro Trp crosses than from reciprocal crosses. Not considering the location of trpe8 and e22 relative to rifB since these muta- tions map to the right of thiC, Trp Rif progeny (Table 2) from P190 x Pro Trp, for fourteen of the eighteen remaining indole utilizers, were at least four times more frequent than in reciprocal crosses. Four of the eighteen mutants gave between two and three times more Trp Rif progeny from P190 x Pro Trp crosses than from reciprocal crosses. Although unusually low, the difference in fre- quencies of Trp Rif progeny in P190 x Pro Trpe6 (Table 2) compared with P189 x Pro Trpe6 is consistent with data from other mutants and supports the conclusion that mutation trpe6 is located to the right of rifB. Mutations in eight- een indole-utilizing strains, excluding mutations trpe8 and e22, therefore map between rifB and thiC. Efforts were made to compare the sequence of mutations based on the Pro+ Arg+ selection (Table 2) with the order based on selection of Trp+ Thi+ progeny. Sufficient thiamine was carried over from the complete (crossing) medium, even after repeated washing of cells, to make selection of Thi+ Trp+ progeny from the confluent background growth impossible. Selection of Trp+ Rif progeny was considered impractical because this requires that all crosses be repeated using Trp Rif strains as one parent, after isolation of all the appropriate Trp Rif strains. Location of trp mutations relative to hisC and ammA: Mutations in the eight strains that do not utilize indole (Trpl, vl, 2, e3, 8, ell, e13 and e14) and in two indole-utilizing mutants, Trpe8 and e22, are located between hisC and ammA (Table 3). Am+Trp+ progeny from crosses of P8 X Trp Pro, for nine mutants, were at least four times more frequent than in reciprocal crosses (Table 3). Recip- rocal crosses involving Trpe8 were exceptional since the difference in frequencies of Amm+ Trp+ progeny may be two- to threefold (Table 3). Comparing the order of trp relative to amm based on Pro+ Arg+ selection with the order based on Trp+ Amm+ selection was not possible since the leakiness of Amm precludes selection of A”+progeny. The data in Table 3 support the conclusion that trp maps to the left of amm. His+ Trp+ progeny (Table 3) in crosses of P4 x Trp Arg are more than twice as frequent as in reciprocal crosses. Furthermore, selection of His+ Trp+ progeny results in at least a twofold higher frequency of Pro+ Arg+ progeny in P4 x Trp Arg crosses than in reciprocal crosses. The data therefore indicate that trp muta- tions in the eight mutants that do not utilize indole, among them Trp8 and e14, and in two indole-utilizing mutants, Trpe8 and e22, map to the right of hisC. TRYPTOPHAN MUTANTS IN S. coelicolor 805 Enzymatic activities in Trp mutants: Experiments with Trpe8 and e14 ex- tracts from cells grown in different concentrations of tryptophan indicated that optimal specific activities for tryptophan , except for InGP synthase, were attained in cultures supplemented with 10 pg L-tryptophan/ml and aerated for 16 hours in two-liter flasks containing 400 ml complete medium. Specific activities for InGP synthase do not change in Trpe14 extracts from cells in- cubated for 16 hours with aeration in 400 ml complete medium supplemented with 10 to 100 pgJml L-tryptophan. Extracts from Trpe8 grown in 25 pg 1,-tryptaphan/ml for 16 hours with aeration have one-half the specific activity €or Trp enzymes (InGP synthase is lacking in this mutant) that is found in extracts from cells grown in 10 pg/ml tryptophan. Except for InGP synthase, enzyme activity is virtually undetectable in extracts from cells pm in 50 pg tryptophan/ml. Whether diminished enzyme activity in extracts from cells grown in higher tryptophan concentrations is caused by inhibition of activity or repression of enzyme synthesis has not been determined. Trpel4, a mutant that does not utilize indole, has extremely weak tryptophan synthase B activity (Table 4). Tryptophan synthase B activity is either extremely low or undetectable in extracts of mutants TRPI, vl, 2, e3, ell and e13. Trp8 lacks tryptophan synthase A activity and has relatively weak tryptophan syn- thase B activity (Table 4). The latter observation agrees with the fact that Trp8 grows poorly in the presence of indole. Extracts of indole-utilizing mutants Trpe8 and e22 lack InGP synthase activity (Table 4). Trp7 lacks PRA isomerase activ- ity (Table 4). PR transferase activity was not found in extracts of indole-utilizing mutants Trpe6 and e10 (Table 4). Extracts of indole-utilizing mutants Trpel, e2,e4, e5, 5, e7, e9, e10, e12, e16, e17, e19, e20, e21 and e23 also lacks PR trans-

Table 4

Specific activities of tryptophan biorynthetlc enzymes in

extracts of S. coelicolor.

Specific activity. -_-_Strain AS PRT PRAI -InCPS E G(2) 0.1 0.4 U.24 1.6 0.11 0.02

iPe6 15.0 t 0.24 1.9 0.46 4.1

lrpelo 13.2 + 0.43 1.s 0.7: 7.5

Trpl 6.3 2.5 t 1.3 0.61 9.2

lrp8 9.5 1.7 1.35 t 1.20 10.5 lrpe22 8.7 2.3 1.25 - 0.79 s.2

Irp3 16.4 4.1 0.98 :.2 + 1.8

rrie14 ij,3 3.i 0.31 2,; 1.40 0.36

* speclr,c activities, defined in \:aterials and

Liethoas, are average values from two assa>s: Ahre-

Ylatloni for enzymes are qiven zn the legen+ to

Figure 2.

+ r.0 detectable aCtlvltY. 806 C. M. SMITHERS AND P. P. ENGEL ferase activity. All twenty-seven mutants have anthranilate synthase activity. It is not likely that failure to detect enzyme activities resulted from an inhibitor in the extracts for the following reasons: (i) missing activities were not restored following dialysis of 1 ml extract in 150 ml extraction buffer for 4 hours; (ii) no inhibition of enzyme activity was observed when mutant and wild-type extracts were mixed. InGP synthase activities vary slightly in wild-type and mutant extracts (Table 4). This observation and the fact that InGP synthase specific activity is not affected by tryptophan concentration in the culture medium suggest constitutive synthesis of this enzyme. Specific activities for the other enzyme vary consider- ably among the different mutants. Tryptophan may be more limiting in some cultures than in others, since growth rates of mutants vary considerably, possibly leading to variation either in rates of enzyme synthesis or in degree of inhibition of activity.

DISCUSSION Synthesis of InGP synthase is repressed by tryptophan in E. coli (ITOand CRAWFORD1965), Salmonella typhimurium (BAUERLEand MARGOLIN1966), Bacillus subtilis (HOCH,ANAGNOSTPOULOS and CRAWFORD 1969), Pseudomonas putida (CRAWFORDand GUNSALUS1966) and Staphyloccus aureus (PROCTORand KLOOS1973). Assuming the evidence suggesting constitutive synthesis of InGP synthase is valid, the regulation of this enzyme is different in S. coelicolor than in the above-mentioned bacteria. Anthranilate synthase is the only repressib!e tryp- tophan biosynthetic enzyme in Acinetobacter calcoaceticus (TWAROGand LIG- GINS 1970). Perhaps the regulation of InGP synthase synthesis in S. coelicolor and A.calcoaceticus is similar. Assuming the trp mutations in S. coelicolor that are described in this paper affect structural genes, it appears that genes for InGP synthase and the compon- ents of tryptophan synthase are near each other, perhaps even contiguous, in the chromosome. The genes controlling anthranilate synthase, InGP synthase and the tryptophan synthase components are closely linked in Micrococcus luteus and separate from the genes for PR transferase and PRA isomerase (KLOOSand ROSE1970). Evidence for similarity between S. coelicolor and M. hteus relative to arrangement OI trp loci might be confirmed when mutations affecting anthran- ilate synthase are identified in S. coelicolor. The location, in S. coelicolor, of mutations affecting InGP synthase and the tryptophan synthase components in one cluster and PR transferase and PRA isomerase in a second cluster indicates that the organization of trp genes in the chromosome of S. coelicolor is different from that found in E. coli (YANOFSKYand LENNOX1959), Salmonella typhinu- rium (BLUME and BALBINDER1966), Bacillus subtilis (CARLTONand WH TT 1969; KANE,HOLMES and JENSEN1972), Staphylococcus aureus (PROCTORand KLOOS1970), Pseudomonas putida (GUNSALUSet al. 1968) and Acinetobacter calcoaceticus (SAWULAand CRAWFORD1972).

This research was supported in part by NSF grant GB-6765. TRYPTOPHAN MUTANTS IN S. coelicolor 807

LITERATURE CITED

BAUERLE,R. H. and P. MARGOLIN,1966 The functional organization of the tryptophan gene cluster in Salmonella typhimurium. Proc. Xatl. Acad. Sci. U.S. 56: 111-118. BLUME,A. J. and E. BALBINDER,1966 The tryptophan operon of Salmonella typhimurium: fine structure analysis by deletion mapping and abortive transduction. Genetics 53 : 577-592. CARLTON,B. C. and D. D. Whitt, 1969 The isolation and genetic characterization of mutants of the tryptophan system of Baci!lus subtilis. Genetics 62: 4.45-460. CRAWFORD,I. P. and I. C. GUNSALUS,1966 Inducibility of tryptophan synthetase in Pseudo- monas pulida. Proc. Natl. Acad. Sci. U.S. 56: 717-724. CREIGHTON,T. E. and C. YANOFSKY,1970 Chorismate to tryptophan (Escherichia coli)- anthranilate synthetase, PR transferase, PRA isomerase, InGP synthetase, tryptophan syn- thetase. pp. 365-380. In: Methods in Enzymology. Vol. XVIIA. Edited by H. TABORand C. W. TABER.Academic Press, N.Y. DEL~C,V.. D. A. HOPWOODand E. J. FRIEND,1970 Mutagenesis by N-methyl-"-nitro-N- nitroguanidine (NTG) in Streptomyces coelicolor. Mutation Res. 9: 167-182. EDWARDS,J. M. and L. M. JACKMAN,1965 Chorismic acid: a branch point intermediate in aromatic biosynthesis. Australian J. Chem. 18 : 1227-1239. ENGEL,P. P., 1973 Genetic control of tryptophan biosynthesis in Streptomyces coelicolor. pp. 125-147. In: Genetics of Industrial Microorganisms: Actinomycetes and Fungi. Vol. 11. Edited by Z. VANEK,Z. HOSTALEKand J. CUDL~N.Academia, Publishing House of the Czechoslovak Academy of Sciences, Prague. GIBSON,F., 1964 Chorismic acid: purification and some chemical and physical studies. Biochem. J. 90: 256-261. GIBSON,M. I. and F. GIBSON,1964 Preliminary studies on the isolation and metabolism of an intermediate in aromatic biosynthesis: chorismic acid. Biochem. J. 90: 248-256. GUNSALUS,I. C., C. F. GUNSALUS,A. M. CHAKRABARTY,S. SIKES and I. P. CRAWFORD,1968 Fine structure mapping of the tryptophan genes in Pseudomonas putida. Genetics 60: 419-435. HOCH,S. O., C. ANAGNOSTOPOULOSand I. P. CRAWFORD,1969 Enzymes of the tryptophan operon of Bacillus subtilis. Biochem. Biophys. Res. Commun. 35: 838-844. HOPWOOD,D. A., 1967 Genetic analysis and genome structure in Streptomyces coelicolor. Bac- teriol. Rev. 31 : 373-403. HOPWOOD,D. A. and G. SERMONTI,1962 The genetics of Streptomyces coelicolor. Advan. Genet. 11: 273-342. HOPWOOD,D. A., K. F. CHATER,J. E. Dowding and A. VIVIAN,1973 Advances in Streptomyces coelicolor genetics. Bacteriol. Rev. 37: 371-405. ITO,J. and I. P. CRAWFORD,1965 Regulation of the enzymes of the tryptophan pathway in Escherichia coli. Genetics 52: 1303-1316. KANE,J. F. and R. A. JENSEN,1970 Metabolic interlock: the influence of histidine on tryptophan biosynthesis in Bacillus subtilis. J. Biol. Chem. 245: 2384-2390. KANE,J. F., W. M. HOLMESand R. A. JENSEN,1972 Metabolic interlock: the dual function of a folate pathway gene as an extraoperonic gene of tryptophan synthesis. J. Biol. Chem. 247: 1587-1596. KLOOS,W. E. and N. E. ROSE, 1973 Transformation mapping of tryptophan loci in Micrococcus luteus. Genetics 6'6: 595-605.

LOWRY,0. H., N. J. ROSEBROUGH,A. L. FARRand R. J. R4NDALL, 1951 Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. 808 C. M. SMITHERS AND P. P. ENGEL PROCTOR,A. R. and W. E. KLoos, 1970 The tryptophan gene cluster of Staphylococcus aureus. J. Gen. Microbiol. 64: 319-327. --, 1973 Tryptophan biosynthetic enzymes of Staphylococcus aureus. J. Bacteriol. 114: 169-177. SAWULA,R. V. and I. P. CRAWFORD,1972 Mapping of the tryptophan genes of Acinetobacter calcoaceticus by transformation. J. Bacteriol. 112 : 797-805. TWAROG,R. and G. L. LIGGINS,1970 Enzymes of the tryptophan pathway in Acinetobacter calco-aceticus. J. Bacteriol. 104: 254-263. VOGEL,H. J. and D. M. BONNER,1956 Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218: 97-106. WEGMAN,J. and I. P. CRAWFORD,1968 Tryptophan synthetic pathway and its regulation in Chromobacterium uiolaceum. J. Bacteriol. 95 : 2325-2335. YANOFSKY,C., 1955 Tryptophan synthetase from Neurospora crassa. pp. 233-238. In: Methods in Enzymology. Vol. 11. Edited by S. P. COLOWICKand N. 0. KAPLAN.Academic Press, N. Y. -, 1956 The enzymatic conversion of anthranilic acid to indole. J. Biol. Chem. 223: 171-184. YANOFSKY,C. and E. S. LENNOX,1959 Transduction and recombination study of linkage rela- tionships among the genes controlling tryptophan synthesis in Escherichia coli. Virology % : 425-447. Corresponding editrr: R. H. DAVIS