The free amino acid pool contents of Gram-negative bacteria (Aerobacter aerogenes, Erwinia carotovora, Pseudomonas fluorescens) were studied as functions of the growth environment and were compared with those from correspondingly grown cultures of Gram-positive bacteria (Bacillus subtilis var. niger, B. megaterium, B. polymyxa) and the yeast Saccharomyces cere visiae.Although the pools of the Gram-positive bacteria and the yeast contained five to 20 times the concentration of free amino acids present in the pools of Gram-negative bacteria, all pools were similar in containing only a limited range of detectable amino acids. Glutamate invariably predominated and generally accounted for over 50 % of the total amino acid content of the pool.The contents and composition of pools from micro-organisms maintained in steady states in chemostat cultures did not vary with time, but changed significantly with changes in either growth rate or the nature of the growth limitation. However, these pool variations were small compared with those resulting from addition of 2 % (w/v) NaCl to a culture of growing bacteria.With cultures of Gram-negative bacteria, sudden changes in medium salinity effected marked and rapid changes in free glutamate content; with Grampositive bacteria, similar changes occurred, but extremely slowly. Addition of 4 % (w/v) NaCl to growing yeast cultures brought about no observed changes in pool size or composition. These results are discussed with reference to the involvement of free amino acids in synthesis and functioning of microorganisms.
SUMMARYAmmonia-limited Aerobacter aerogenes, Erwinia carotovora, Pseudomonas jluorescens, Bacillus subtilis and B. megaterium synthesized glutamate from NH, and a-oxoglutarate by a process that involved first the synthesis of glutamine and then the reductive transfer of the glutamine amide-nitrogen to the a-position of a-oxoglutarate. The latter step required the recently reported enzyme ' glutamine(amide) : a-oxoglutarate amino-transferase oxido-reductase (NADP) ', some further properties of which are described here. This enzyme, from different organisms, always had a well-defined maximum activity at a pH value between 7.5 and 8.0; it had an apparent K, for 2-oxoglutarate between 0-1 and 2.0 mM and an apparent K, for glutamine between 0-2 and 1.8 mM. Glutamate (the metabolic end-product) and Mg2+ strongly inhibited the enzyme from Gram-negative bacteria but less so that from Grampositive species. Synthesis of glutamate by this enzyme required NADPH, and NADH was inactive ; pyruvate, oxaloacetate, a-oxobutyrate and 2-oxoisovalerate could not substitute for a-oxoglutarate, nor could the requirement for glutamine be met by asparagine, citrulline, arginine or urea. Although conditions that favoured the synthesis of this enzyme generally also favoured synthesis of glutamine synthetase and caused suppression of glutamate dehydrogenase formation, a close correlation between the bacterial contents of these different enzymes was not apparent.
Carbon-limited chemostat cultures of Klebsiella aerogenes NCTC 418 consumed more oxygen per unit of cell synthesis when growing on mannitol or glycerol than when growing on glucose; and since the "maintenance" requirements were similar, this suggested that the extra reducing equivalents present in these compounds were oxidized wastefully. By comparison with carbon-limited cultures, carbon-sufficient cultures that were ammonia-, sulphate- or phosphate-limited generally consumed considerably more oxygen per unit of cell synthesis, particularly at low growth rates. Thus, according to the theory of Pirt, these carbon-sufficient cultures had a greatly increased "maintenance energy" requirement but nevertheless used the remaining energy with a much increased efficiency compared with carbon-limited cultures. This, we suggest, is a false conclusion which stems from the basic assumption that the maintenance requirement does not change with growth rate.
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