Abstract
The levels of two aspartokinase isozymes, a lysine-sensitive enzyme and an aspartokinase that is inhibited synergistically by lysine plus threonine, differ strikingly in different strains of Bacillus subtilis. In derivatives of B. subtilis 168 growing in minimal medium, the predominant isozyme is the lysine-sensitive aspartokinase. In B. subtilis ATCC 6051, the Marburg strain, the level of the lysine-sensitive aspartokinase is much lower during growth in minimal medium, and the major aspartokinase activity is the lysine-plus-threonine-sensitive isozyme. Molecular cloning and nucleotide sequence determination of the genes for the lysine-sensitive isozymes from the two B. subtilis strains and their upstream control regions showed these genes to be identical. Evidence that the lysine-sensitive aspartokinase, referred to as aspartokinase II, is distinct from the threonine-plus-lysine-sensitive aspartokinase comes from the observation that disruption of the aspartokinase II gene by recombinational insertion had no effect on the latter. Mutants were obtained from the aspartokinase II-negative strain that also lacked the threonine-plus-lysine-sensitive aspartokinase, which will be referred to as aspartokinase III. Aspartokinase II could be selectively restored to these mutants by transformation with plasmids carrying the aspartokinase II gene. Study of the growth properties of the various mutant strains showed that the loss of either aspartokinase II or aspartokinase III had no effect on growth in minimal medium but that the loss of both enzymes interfered with growth unless the medium was supplemented with the three major end products of the aspartate pathway. It appears, therefore, that aspartokinase I alone cannot provide adequate supplies of precursors for the synthesis of lysine, threonine, and methionine by exponentially growing cells.
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