A Regulatory Gene of Phenylalanine Biosynthesis 4-Fluorophenylalanine-resistant mutants of Salmonella typhimurium were isolated in which synthesis of chorismate mutase P-prephenate dehydratase ( specified by pheA ) was highly elevated .
Transduction analysis showed that the mutation affecting pheA activity was not linked to pheA , and conjugation and merodiploid analysis indicated that it was in the 95-to 100-min region of the Salmonella chromosome .
Evidence is presented for the hypothesis that the mutation responsible for constitutivity of chorismate mutase P-prephenate dehydratase occurred in pheR , a gene specifying a cytoplasmic product that affected pheA .
pheR mutants were found to carry a second mutation , tyrO .
The tyrO mutation acts cis to cause increased levels of the tyrosine biosynthetic enzymes 3-deoxy-D-arabinoheptulosonate 7-phosphate synthetase ( tyr ) and pre-phenate dehydrogenase , but it has no effect on regulation of pheA .
pression of chorismate mutase P-prephenate dehydratase .
The mutation affecting the latter enzyme is not a pheA operator mutation since it is not linked to pheA .
Evidence is presented suggesting that this mutation has resulted in alteration of a diffusible product specified by pheR .
Apparently , only regulation of pheA is affected ; regulation of aroG , the structural gene for DAHP synthesis ( phe ) , is not affected .
pheR is located in the 95-to 100-min region of the Salmonella chromosome .
MATERIALS AND METHODS Materials .
DL-4-Fluorophenylalanine and 4-hydroxyphenylpyruvic acid were purchased from commercial sources .
Monocyclohexylammonium enol-pyruvate-P was prepared by the method of Clark and Kirby ( 4 ) ; erythrose-4-P was prepared by the method of Ballou and MacDonald ( 1 ) ; barium prephenafe was prepared by the method of Dayan and Sprinson ( 8 ) ; and chorismic acid was prepared by the method of Gibson ( 11 ) .
The minimal-medium used was previously described ( 14 ) .
Supplements were added as indicated in tables .
Difco nutrient broth and nutrient agar served as complete media for preparation of phage lysates and for transductions .
Penassay Broth ( Difco ) was used to grow donors and recipients for conjugational crosses and episome transfers .
Salmonella strains were derivatives of Salmonella typhimurium LT-2 .
Hfr strains ( with the exception of SC19 ) and strain serA13 were kindly supplied by K. E. Sanderson : strain SC19 was YLALANINE BIOSYNTHESIS 1 TABLE 4 .
Specific activities of prephenate dehydratase and chorismate mutase in merodiploidsa Prephenate Chorismate Mating type dehydrataseb mutaseb SG300 F-5.7 6.0 SG302 F-4.2 NTc F111/SG302 F ' 4.4 NT SG303 F-5.0 6.5 F116/SG303 F ' 0.30 0.49-F-3.7 5.1 Segregant SG351 F-3.5 5.1 F116/SG351 F ' 0.15 0.35 SerA F-0.14 NT F116/SerA F ' 0.41 NT LT-2 F-0.37 0.61 a Methods and definition of specific activity are given in Materials and Methods .
Activities are average values from assays of at least two independently prepared cell-free extracts .
Merodiploids were grown in minimal-medium to avoid growth of segregants .
Haploid auxotrophic strains were supplemented with 0.3 mM thymine or required amino-acid .
` Activities of cells grown on minimal-medium .
Similar results obtained with cells grown on 0.5 mM phenylalanine .
c NT , not tested .
SG304 , thy-and tyrA-derivatives , respectively , of strain SG300 .
Chorismate mutase-prephenate dehydratase was not repressed in the resulting merodiploids ( Table 5 ) .
Hence , pheR is not in the lysine-thymine region of F116 , and must be located between 95 and 100 min on the Salmonella chromosome .
In a mero-diploid prepared from F143 and strain SG351 , which contained a normal tyrosine operon , prephenate dehydratase was also highly elevated ( Table 5 ) , thus lending further support to the conclusion that the mutation affecting the tyrosine operon in strain SG300 was not related to the pheR mutation .
Chorismate mutase-prephenate dehydratase activities in the merodiploids were elevated four-to fivefold over the high constitutive levels present in the parent strain , thus resulting in derepression of over fiftyfold relative to wild type ( Table 5 ) .
The presence of multiple copies of the episome in the merodiploid is unlikely , since enzyme levels were not excessively elevated in the control diploid F143/tyrA - .
The unexpectedly high enzyme levels suggest that the mutant repressor may recognize an E. coli operator even less effectively than a Salmonella operator .
As discussed above , the second mutation that occurred in strain SG300 and that was responsi strain SG300 showed the same high constitutive levels of chorismate mutase P-prephenate dehydratase , although they were no longer as resist-ant to fluorophenylalanine as strain SG300 .
Hence , the tyrO mutation was responsible for the selection of these strains on the analogue .
Similarly pheO mutants in E. coli were selected on fluorophenylalanine as doubly mutated strains , the second mutation having resulted in a feedback resistant DAHP synthetase ( phe ) ( 19 ) .
Genetic investigation indicated that the regulatory mutation affecting pheA was not an operator mutation , and provided evidence for the presence of a regulatory gene pheR located at some distance from pheA .
The presence of the wild-type allele on episome F116 and its absence on F143 established the location of pheR at approximately 95 to 100 min on the Salmonella chromosome .
Precise mapping of its location will depend on availability of a more direct selection procedure than enzyme assay , since pheR-mutants can not be readily selected on fluorophenylalanine .
Scarcity of known markers in the region of pheR may further contribute to the difficulty of mapping .
Dominance of pheR + over pheR-suggested that a diffusible , cytoplasmic product was involved in regulating pheA activity .
The simplest explanation of our results , therefore , would be that pheR specifies an aporepressor which with phenylalanine as corepressor acts on the pheA operator according to the model of Jacob and Monod ( 20 ) .
It is assumed that phenylalanine , and not charged phenylalanyl transfer ribonucleic acid ( tRNA ) , is the true corepressor for chorismate mutase P-prephen-ate dehydratase .
Although aminoacylated tRNAs apparently act as corepressors for regu 5 .
Cohen , G. , and F. Jacob .
Sur la repression de la synthese des enzymes intervenant dans la formation du tryptophane chez Escherichia coli .
Cotton , R. G. H. , and F. Gibson .
The biosynthesis of phenylalanine and tyrosine : enzymes converting ehorismic acid into prephenic acid and their relationship to prephenate dehydrogenase .
Dayan , J. , and D. B. Sprinson .
Determination of prephenic dehydrogenase activity , p. 562 .
In H. Tabor and C. W. Tabor ( ed .
) , Methods in enzymology , vol .
Academic Press Inc. , New York .
Dayan , J. , and D. B. Sprinson .
Preparation of prephenic acid , p. 559 .
In H. Tabor and C. W. Tabor ( ed .
) , Methods in enzymology , vol .
Academic Press Inc. , New York .
Dayan , J. , and D. B. Sprinson .
Enzymes alterations in tyrosine and phenylalanine auxotrophs of Sal-monella typhimurium .
DeLeo , A. B. , J. Dayan , and D. B. Sprinson .
Puri-fication and kinetics of tyrosine sensitive 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthetase from Salmonella .
Fink , G. R. , and J. R. Roth .
Histidine regulatory mutants in Salmonella typhimurium .
Gollub , E. , and D. B. Sprinson .
A regulatory mutation in tyrosine biosynthesis .
Gollub , E. , and D. B. Sprinson .
A regulatory mutation in tyrosine biosynthesis .
Gollub , E. , H. Zalkin , and D. B. Sprinson .
Correlation of genes and enzymes and studies on regulation of the aromatic pathway in Salmonella .
Gollub , E. , H. Zalkin , and D. B. Sprinson .
Assay for 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthetase , p. 349 .
In H. Tabor and C. W. Tabor ( ed .
Academic Press Inc. , New York .
Hartman , P. E. , J. C. Loper , and D. Serman .
Fine structure mapping by complete transduction between histidine requiring Salmonella mutants .
Heinonen , J. , S. W. Artz , and H. Zalkin .
Regulation of the tyrosine biosynthetic enzymes in Salmonella typhimurium : analysis of the involvement of tyrosyltransfer ribonucleic acid and tyrosyl-transfer ribonucleic acid synthetase .
Im , S. W. K. , H. Davidson , and J. Pittard .
Phenylalanine and tyrosine biosynthesis in Escherichia coli K-12 : mutants derepressed for 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthetase ( phe ) , 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthetase ( tyr ) , chorismate mutase T-prephenate dehydrogenase and transaminase A. J. Bacteriol .
Im , S. W. K. , and J. Pittard .
Phenylalanine biosynthesis in Escherichia coli K-12 : mutants derepressed for chorismate mutase P-prephenate dehydra-tase .
Jacob , F. , and F. Monod .
Genetic regulatory mechanisms in the synthesis of proteins .
H. , N. J. Rosebrough , A. L. Farr , and R. J. Randall .
Protein measurement with the Folin phenol reagent .
Mattern , I. , and J. Pittard .
Current linkage map of Sal-monella typhimurium .
Sanderson , K. E. , and M. Demerec .
The linkage map of Salmonella typhimurium .
Schlesinger , S. , and E. W. Nester .
Mutants of Escherichia coli with an altered tyrosyl-transfer ribo-nucleic acid synthetase .
Smith , H. O. , and M. Levine .
A phage P22 gene controlling integration of prophage .
Tryptophan operon of Escherichia coli : Regulatory behavior in Salmonella typhimurium cytoplasm .
Wallace , B. J. , and J. Pittard .
Regulator gene controlling enzymes concerned in tyrosine biosynthesis in Escherichia coli .