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α-Isopropylmalate synthase and β-isopropylmalate dehydrogenase activities were detected in extracts of the following organisms: ChromatiumD, Rhodopseudomonas spheroides, HydrogenomonasH16, Pseudomonas aeruginosa, Pseudomonas fluorescens, Vibrio extorquens, Rhizobium japonicum, Alcaligenes viscolactis, Escherichia coliB, Proteus vulgaris, Aerobacter aerogenes, Salmonella typhimurium, Micrococcussp., Micrococcus lysodeikticus, Bacillus polymyxa, Bacillus subtilis, and Nocardia opaca. The α-isopropylmalate synthase activity in these extracts was inhibited by low concentrations of l-leucine. Taken together with other data, these results suggest that the isopropylmalate pathway is widespread among organisms that can synthesize leucine.

dl-4-Azaleucine (5 × 3−3m) added to exponentially growing cells of Salmonella typhimuriumresulted in an abrupt cessation of growth lasting 4 to 8 hr followed by a resumption of division. The transitory nature of inhibition was not due to the instability or modification of the analogue or to a derepression of leucine-forming enzymes. Of many compounds tested, leucine served most efficiently to reverse 4-azaleucine-induced inhibition. Inhibition of growth can be explained by the fact that 4-azaleucine inhibits α-isopropylmalate synthase, the first enzyme unique to leucine biosynthesis. The analogue was a poor inhibitor of both the transamination of α-ketoisocaproate to leucine and the charging of leucine to transfer ribonucleic acid. With a leucine auxotroph starved for leucine, the analogue was incorporated into protein specifically in place of leucine. Such incorporation was accompanied by the death of almost all of the cells.

Cell suspensions of pseudomonad C, a bacterium capable of growth on methanol as sole carbon source, were able to oxidize methanol, formaldehyde, and formate, although the rates of oxidation for the latter two compounds were much slower. The latter compounds also could not serve as sole carbon sources. Through the use of labeled compounds, it was shown that in the presence of methanol, formaldehyde, formate, and bicarbonate were incorporated into trichloroacetic acid-precipitable material. Hexose phosphate synthetase activity was found, indicating the assimilation of methanol via an allulose pathway. No hydroxypyruvate reductase activity was found, nor was any complex membrane structure observed. Such a combination of characteristics has been observed in an obligate methylotroph (PseudomonasW1), but pseudomonad C can utilize a variety of non-methyl substrates.

Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium. The partially purified enzyme had the following Kmvalues: leucine, 1.1 × 10−5m; adenosine triphosphate, 6.5 × 10−4m; tRNAILeu, 4.1 × 10−8m; tRNAIILeu, 4.3 × 10−8m; tRNAIIILeu, 5.3 × 10−8m; and tRNAIVLeu, 2.9 × 10−8m. The tRNALeufractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a pH 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.