Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium . The partially purified enzyme had the following K m values: leucine, 1.1 × 10 −5 m ; adenosine triphosphate, 6.5 × 10 −4 m ; tRNA I Leu , 4.1 × 10 −8 m ; tRNA II Leu , 4.3 × 10 −8 m ; tRNA III Leu , 5.3 × 10 −8 m ; and tRNA IV Leu , 2.9 × 10 −8 m . The tRNA Leu fractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a p H optimum of 8.5 and an activation energy of 10,400 cal per mole, and was inactivated exponentially at 49.5 C with a first-order rate constant of 0.064 min −1 . Strain CV356 ( leuS3 leuABCD702 ara-9 gal-205 ) was isolated as a mutant resistant to dl -4-azaleucine and able to grow at 27 C but not at 37 C. Extracts of strain CV356 had no leucyl-tRNA synthetase activity (charging assay) when assayed at 27 or 37 C. Temperature sensitivity and enzyme deficiency were caused by mutation in the structural gene locus specifying leucyl-tRNA synthetase. A prototrophic derivative of strain CV356 (CV357) excreted branched-chain amino acids and had high pathway-specific enzyme levels when grown at temperatures where its doubling time was near normal. At growth-restricting temperatures, both amino acid excretion and enzyme levels were further elevated. The properties of strain CV357 indicate that there is only a single leucyl-tRNA synthetase in S. typhimurium .

Salmonella typhimurium strain CV123 (ara-9 gal-205 flrBl), isolated as a mutant resistant to trifluoroleucine, has derepressed and constitutive levels of enzymes forming branched-chain amino acids. This strain grows more slowly than the parent at several temperatures, both in minimal medium and nutrient broth. It overproduces and excretes sizeable amounts of leucine, valine, and isoleucine in comparison with the parental strain. Both leuS (coding for leucyl-transfer ribonucleic acid [tRNA]synthetase) and flrB are linked to lip (min 20 to 25) by P1 transduction, whereas only leuS is linked to lip by P22 transduction. Strain CV123 containing an F' lip+ episome from Escherichia coli has repressed levels of leucine-forming enzymes, indicating that flrB+ is dominant to flrB. Leucyl-tRNA synthetase from strain CV123 appears to be identical to the leucyl-tRNA synthetase in the parent. No differences were detected between strain CV123 and the parent with respect to tRNA acceptor activity for a number of amino acids. Furthermore, there was no large difference between the two strains in the patterns of leucine tRNA isoaccepting species after fractionation on several different columns. Several other flrB strains exhibited temperature-sensitive excretion of leucine, i.e., they excreted leucine at 37 C but not 25 C. In one such strain, excretion at 37 C was correlated with derepression of some enzymes specified by ilv and leu. These latter results suggest that flrB codes for a protein. 942 on July 16, 2020 by guest

No abstract