Production of an Altered Pantothenate-Synthesizing Enzyme by a Temperature-Sensitive Mutant of Escherichia Coli

Abstract

Mutant 99-1 t, a temp.-sensitive pantothenate auxotroph, was isolated by incubating mutant 99-1, which requires pantothenate at any temp., in minimal medium A at 25[degree]C. The temp.-sensitive pantothenate auxotrophs, comprising 5 to 10% of the resulting colonies, were distinguished from the prototrophs by their inability to grow on minimal medium A at 37[degree]C. The pantothenate-synthesizing enzyme was extracted chiefly by grinding fresh cells with glass. Enzyme activity was measured both for the cell-free extracts and for the non-growing cell suspensions. The stability of the enzyme was studied by incubating extracts of 99-1 t and of wild type at various temps. At intervals samples were removed, cooled, and their residual enzyme activity detd. at 15[degree]C, at which temp. the enzyme remained stable. The enzyme in wild-type extract was completely stable for 2 hrs. at 35[degree]C and only slightly inactivated at 47[degree]C, whereas the enzyme from the mutant strain was largely inactivated within 1 hr. at 30[degree]C. Similar results were obtained both in the presence and absence of factors required for enzyme activity. The heat stability of the mutant enzyme varied among different prepns., but these variations were small compared to those between mutant and wild-type. The temp. at which the strains were grown did not affect heat stability of the enzyme, but did affect the yield from the mutant, with aerobic conditions at 25[degree]C or growth at 35[degree]C giving completely inactive prepns. Inactivation rates at 35[degree]C in mixtures of mutant and wild-type extracts were equal to the sum of the activities of the separate controls, indicating that neither extract has any effect on the other and that the differences in thermal stability were due to differences in the enzyme molecules. Further tests indicated a similarity between the behavior of the mutant enzyme and protein denaturations, with respect to activity-time relationships, and protective agents against inactivation. The extracts of wildtype and mutant strains showed slight differences in the Michaelis constant for pantoate, in affintiy for ATP, and in inhibiting effects of urea and acetone. In non-growing cell suspensions the mutant enzyme was less stable than wild-type enzyme, but was more stable in cell suspensions than in extracts. The temp. at which the enzyme becomes unstable in cell suspensions coincides with that at which the cells lose the ability to grow without pantothenate. No enzyme activity was detected in extracts or whole cells of strain 99-1 which requires pantothenate at any temp. The results demonstrate a qualitative change in an enzyme as a result of mutation and provide evidence for a one to one relationship between gene and enzyme.