SUMMARYTryptophan auxotrophs were isolated from Staphylococcus aureus strain 655 following mutagenesis with ultraviolet radiation, N-methyl-N'-nitro-Nnitrosoguanidine or ethyl methanesulphonate. The mutants were placed in five mutant classes on the basis of auxotrophic response to and accumulations of tryptophan intermediates or derivatives. The tryptophan loci were mapped by comparing relative distances of each locus from two reference loci, trpB and trpE. Relative distances of loci were determined by ratio test analysis of cotransduction data. The tryptophan loci were shown to be contained on a cluster on the S. aureus genome; the mapping data suggested the gene order: trpE, trp(DF), trpC, trp(DF), trpB, trpA. The genetically distinct trp(DF) regions presumably control two enzymic functions in the conversion of anthranilic acid to I-(0-carboxypheny1amino)-I-deoxyribulose 5-phosphate.
Mutants blocked before indole-3-glycerol phosphate formation in the tryptophan biosynthetic pathway of P. putida ("early-blocked" mutants) are unable to use indole as a source of tryptophan for growth on. minimal medium. The uninduced level oftryptophan synthase [EC 4.2.1.20; L-serine hydro-lyase (adding indole)] in such mutants was thought to be responsible for this property. We have shown that levels of indole higher than those previously tested will support growth of these mutants. In addition, the growth rate of these mutants on a given indole concentration was shown to be proportional to the synthase level induced under the same conditions. This apparent induction of tryptophan synthase by indole in "early-blocked" mutants was shown to be caused by formation of the normal effector molecule, indole-3-glycerol-P, from indole.Secondary mutations occur in "early-blocked" trp strains, which enable them to grow on low concentrations of indole. One type of "indole-utilization" mutation occurs in the trpA gene, inactivating its product. Tryptophan synthase is readily induced by low concentrations of indole in these mutants, even though they are unable to convert indole to indole-3-glycerol-P. We propose that the a-chain of the synthase has an autogenous regulatory function, serving as the repressor or the indole-3-glycerol-P recognition component of the repressor of the trpAB operon (synthase a-and j3-chains). Our hypothesis holds that the trpA type of "indole-utilization" mutation alters the repressor (synthase a-chain) so that indole as well as indole-3-glycerol-P serves as an effector molecule for tryptophan synthase induction.Various modifications and elaborations of the basic operon model of gene regulation (1) have been necessary to accommodate details of diverse regulatory systems which have been studied since its introduction. A recent review by Goldberger (2) served to underscore compelling evidence from many different laboratories of yet another variation of the basic operon model-autogenous regulation. The principle of autogenous regulation-that the protein encoded by a cistron serves as a modulator of the activity of that cistron or of the operon that encompasses it-appears to be a basic one, which is manifested by many diverse biological regulatory systems (2). This concept in various forms has been expounded for many years (2) and has also been referred to as "self-regulation" of finished enzymes (3), and more recently as "autoregulation" (4). Another review of this subject is in press (D. W. Calhoun and G. W. Hatfield, to appear in Annual Reiews of Microbiology, 1975). In this paper, we present evidence that the tryptophan synthase (synthase) [EC 4.2 Structural genes for the enzymes of the tryptophan synthetic pathway are distributed in three locations on the chromosome of P. putida (5) (Fig. 1). Each trp gene cluster has a different mode of regulation (6, 7). The trpEDC cluster resembles the equivalent, operator-proximal portion of the trp operon in iEcherichia coli (8), Salmonella typhimuri...
Tryptophan biosynthetic enzymes were assayed in various tryptophan mutants of Staphylococcus aureus strain 655 and the wild-type parent. All mutants, except trpB mutants, lacked only the activity corresponding to the particular biosynthetic block, as suggested previously by analysis of accumulated intermediates and auxonography. Tryptophan synthetase A was not detected in extracts of either trpA or trpB mutants but appeared normal in other mutants. Mutants in certain other classes exhibited partial loss of another particular tryptophan enzyme activity. Tryptophan synthetase B activity was not detected in cell extract preparations but was detected in whole cells. The original map order proposed for the S. aureus tryptophan gene cluster was clarified by the definition of trpD (phosphoribosyl transferase − ) and trpF (phosphoribosyl anthranilate isomerase − ) mutants. These mutants were previously unresolved and designated as trp ( DF ) mutants (anthranilate accumulators). Phosphoribosyl anthranilate isomerase and indole-3-glycerol phosphate synthetase enzymes were separable by molecular sieve chromatography, suggesting that these functions are coded by separate loci. Molecular sieve chromatography failed to reveal aggregates involving anthranilate synthetase, phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, and indole-3-glycerol phosphate synthetase, and this procedure provided an estimate of the molecular weights of these enzymes. Tryptophan was shown to repress synthesis of all six tryptophan biosynthetic enzymes, and derepression of all six activities was incident upon tryptophan starvation. Tryptophan inhibited the activity of anthranilate synthetase, the first enzyme of the pathway.
Studies of a trpA mutant constitutive for tryptophan synthase production support the hypothesis of autogenous regulation (R. F. Goldberger, 1974; A. R. Proctor and I. P. Crawford, 1975) of the Pseudomonas putida trpAB loci.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.