Synthesis of glutamate in Aerobacter aerogenes by a hitherto unknown route.

In order to grow in a simple salts medium in which ammonia provides the sole source of nitrogen, micro-organisms must possess some mechanism for the synthesis of amino acids from ammonia and intermediary metabolites. In many bacteria this requirement is fulfilled by the enzyme glutamate dehydrogenase (EC 1.4.1.4), which reductively aminates 2-oxoglutarate to glutamate. In other bacteria (particularly in some species of Bacillus) analogous amino acid dehydrogenases (e.g. alanine dehydrogenase and leucine dehydrogenase) functionally replace glutamate dehydrogenase and glutamate is then formed by an aminotransferase reaction. Available kinetic data indicate that amino acid dehydrogenases generally have high Km values for ammonia (above 5mM; see Sanwal & Zink, 1961; Wiame, Pierard & Ramos, 1962), and one might therefore expect that when bacteria are growing in a simple salts medium the intracellular free ammonia concentration always would be relatively large. When growth takes place in the presence of a large excess of ammonia, the intracellular free ammonia content is difficult to assess without washing the organisms (which is undesirable, since it may cause changes in 'pool' composition; see Britten, 1965). But, when organisms are cultured in a chemostat under conditions where growth is limited by the availability of ammonia, the intracellular free ammonia can be easily determined by extracting the organisms with 0.25% HC104 and applying the extracts to the columns of a Technicon automatic amino acid analyser; washing these organisms is unnecessary since the extracellular free ammonia concentration is negligible (Herbert, 1958). With ammonia-limited chemostat cultures of Aerobacter aerogenes, growing at a dilution rate of 0.3h-1 in the medium of Tempest (1965) at pH6.5 and 35°C, the intracellular free ammonia concentration was found to be less than 0.005% of the bacterial dry weight * Present address: Novo Industri A/S, Fuglebakkevej 115, 2200 Copenhagen N, Denmark. (below 0.5mM), well below theKm value for amnmonia (3-4mM) of A. aerogenes glutamate dehydrogenase. This observation prompted a detailed quantitative study of the relationship between growth condition and bacterial glutamate dehydrogenase activity of