Summary
The AS-PRT enzyme complex which catalyzes the first two steps in the biosynthesis of tryptophan in S. typhimurium consists of two polypetide subunits: anthranilate synthetase (component I or AS-CoI) and anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT). These polypeptides are the products of the first two structural genes of the trp operon, trpA and trpB respectively. The PRT component has two functions: the aminoterminal 40% of the polypeptide is necessary for glutamine amidotransferase activity (GAT) while the carboxy-terminal 60% carries out the PRT activity proper. The mutant strain SO495 has a mutation, trpA515, which confers a unique phenotype: while the strain is capable of utilizing anthranilic acid (AA) a substrate of PRT, as a growth factor, it can only do so in the presence of the analogue 5-methyltryptophan (MT) normally a potent growth inhibitor. Previous evidence indicates that SO495 may possess a somewhat altered PRT, and that its activity could be inhibited by an altered, enzymatically inactive AS made in this strain under derepression. Some experiments designed to test these possibilities are described in this paper. Various properties of the PRT's of the MT-dependent mutant and several of its MT-independent revertants were examined and compared. These included the determination of their apparent Km's for the substrates anthranilic acid (AA) and phosphoribosyl pyrophosphate (PRPP) and the presence or absence of GAT activity. In addition, the possibility that a complex consisting of PRT and an enzymatically inactive AS-CoI was present in some of the revertant strains only when grown under derepressing conditions was investigated by gel chromatography. The results showed that the MT-dependent strain SO495 and the MT-independent revertants have PRT's which differ from each other as well as from wild type LT7 PRT. In MT-independent revertants which retain the trpPO region and most of trpA, PRT can form a loose aggregate which elutes from Bio-Gel columns as three fast moving peaks. This loose aggregate is absent when the strains are grown under repressing conditions and is always absent in strains which lack most of the trpA gene. These results support the idea that the dependence of strain S0495 on MT for utilization of AA as a growth factor has to do with the inhibition of the altered PRT made in this mutant by an altered AS-CoI polypeptide which is synthesized only under derepression. They also suggest that translation of trpB starts from different points in the wild type, S0495 and the MT-independent revertants.
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References
Balbinder, E., Blume, A., Weber, A., Tamaki, H.: Polar and antipolar mutants in the tryptophan operon of Salmonella typhimurium. J. Bact. 95, 2217–2229 (1968)
Bauerle, R.H., Margolin, P.: A multifunctional enzyme complex in the tryptophan pathway of Salmonella typhimurium: Comparison of polarity and pseudopolarity mutations. Cold Spr. Harb. Symp. quant. Biol. 31, 203–214 (1966)
Blume, A., Balbinder, E.: The tryptophan operon of Salmonella typhimurium. Fine structure analysis by deletion mapping and abortive transduction. Genetics 53, 577–592 (1966)
Callahan, R., Dooley, M.M., Balbinder, E.: A mutation to 5-methyltryptophan-dependence in the trp operon of Salmonella typhimurium-II-Studies of 5-methyltryptophan-dependent mutants and their revertants. Molec. gen. Genet. 165, 129–143 (1978)
Cordaro, J.C., Balbinder, E.: Evidence for the separability of the operator from the first structural gene in the tryptophan operon of Salmonella typhimurium. Genetics 67, 151–169 (1971)
Demerec, M., Adelberg, E.A., Clark, A.J., hartman, P.E.: A proposal for a uniform nomenclature in bacterial genetics. Genetics 54, 61–76, (1966)
Dooley, M.M., Balbinder, E.: Differences between the anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferases of Salmonella typhimurium strains LT2 and LT7. J Gen. Microbiol., in press
Grieshaber, M., Bauerle, R.: Monomeric and dimeric forms of component II of the anthranilate synthetase—anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase complex of Salmonella typhimurium. Implications concerning the mode of assembly of the complex. Biochemistry 13, 373–383 (1974)
Henderson, E.J., Nagano, H., Zalkin, H., Hwang, L.H.: The anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase aggregate. Purification of the aggregate and regulatory properties of anthranilate synthetase. J. biol. Chem. 245, 1416–1423 (1970a)
Henderson, E.J., Zalkin, H., Hwang, L.H.: The anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase aggregate. Catalytic and regulatory properties of aggregated and unaggregated forms of anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase. J. biol. Chem. 245, 1424–1431 (1970b)
Jackson, E.N., Yanofsky, C.: Localization of two functions of phosphoribosyl anthranilate transferase of Escherichia coli to distinct regions of the polypeptide chain. J. Bact. 117, 502–508 (1974)
LaScolea, L.J., Balbinder, E.: Restoration of phosphoribosyltransferase activity by partially deleting the trpB, gene in the tryptophan operon of Salmonella typhimurium. J. Bact. 112, 877–885 (1972)
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 265–275 (1951)
Marcus, S.L., Balbinder, E.: Purification of anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase from Salmonella typhimurium using affinity chromatography: Resolution of monomeric and dimeric forms. Biochem. biophys. Res. Commun. 47, 438–444 (1972)
McPartland, A., Somerville, R.L.: Isolation and characterization of mutations creating high-efficiency transcription initiating signals within the trp operon of Escherichia coli. J. Bact. 128 557–572 (1976)
Smith, O.H., Yanofsky, C.: Enzymes involved in the biosynthesis of tryptophan. In: Methods in enzymology (Colowick, S.P., Kaplan, N.O., eds.), pp. 794–806. New York: Academic Press 1962
Starlinger, P., Saedler, H.: IS-elements in microorganisms. In: Critical reviews in microbiology (Laskin, A.I., Lechevalier, H., eds.). CRC Press. Vol. 6, pp. 112–152 (1977)
St. Pierre, M.L.: Mutations creating a new initiation point for the expression of the histidine operon in Salmonella typhimurium. J. molec. Biol. 35, 71–82 (1968)
Stuttard, C.: Tryptophan biosynthesis in Salmonella typhimurium: location in trpB of a genetic difference between strains LT2 and LT7. J. Bact. 123, 878–887 (1975)
Tanemura, S., Bauerle, R.: Internal reinitiation of translation in polar mutants of the trpB gene of Salmonella typhimurium. Molec. gen. Genet. 153, 135–143 (1977)
Vogel, H.J., Bonner, D.: Acetylornithinase of Escherichia coli: Partial purification and some properties. J. biol. Chem. 218, 97–106 (1956)
Wuesthoff, G., Bauerle, R.H.: Mutations creating internal promoter elements in the tryptophan operon of Salmonella typhimurium. J. molec. Biol. 49, 171–196 (1970)
Yanofsky, C., Horn, V., Bonner, M., Stasiowski, S.: Polarity and enzyme functions in mutants for the first three genes of the tryptophan operon of Escherichia coli. Genetics 69, 409–433 (1971)
Zalkin, H.: Anthranilate synthetase In: Advances in enzymology (Meister, A. ed.) Vol. 38, pp. 1–39. New York: J. Wiley & Sons. 1973
Zalkin, H., Kling, D.: Anthranilate synthetase-purification and properties of component I from Salmonella typhimurium. Biochemistry 7, 3566–3573 (1968)
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LaScolea, L.J., Dooley, M.M., Torget, R. et al. A mutation to 5-methyltryptophan dependence in the trp operon of Salmonella typhimurium . Molec. Gen. Genet. 165, 145–153 (1978). https://doi.org/10.1007/BF00269902
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DOI: https://doi.org/10.1007/BF00269902