Inactivation of Aspartic Transcarbamylase in Sporulating Bacillus subtilis : Demonstration of a Requirement for Metabolic Energy

Abstract

The aspartic transcarbamylase (ATCase) activity of Bacillus subtilis cells disappears rapidly from stationary-phase cells prior to sporulation. ATCase activity does not appear in the culture fluid during the stationary phase; hence the enzyme appears to be inactivated in the cells. The enzyme is inactivated normally in two different mutants lacking proteases; the activity is very stable in crude extracts of cells or in the culture fluid. These results suggest that ATCase is not inactivated by the general proteolysis that occurs in sporulating bacteria. The inactivation of ATCase can be completely inhibited after it has begun by oxygen starvation or addition of fluoroacetate. Inhibitors of oxidative phosphorylation and electron transport also interrupt the inactivation of ATCase. The inactivation of ATCase is very slow in two mutant strains that are deficient in enzymes of tricarboxylic acid cycle. Addition of gluconate to stationary cultures of the mutant strains, which is known to restore depleted adenosine 5'-triphosphate pools in these bacteria, also restores inactivation of ATCase. These experiments support the conclusion that the generation of metabolic energy is necessary for the inactivation of ATCase in stationary cells.

Degradation of ornithine transcarbamylase in sporulating Bacillus subtilis cells

“…The second peak of protease activity (at 17 h; see Fig. 1) did not appear after fluoroacetate addition, which showed that the inhibitor did interfere with energy-yielding metabolism, as expected from earlier results (22,23). It is possible that the deprivation of energy induced by fluoroacetate was sufficient to block protease synthesis but not sufficiently extreme to block degradation.…”

“…Unlike the inactivation of aspartate transcarbamylase in B. subtilis (22), the inactivation of OTCase was not inhibited by interfering with the generation of metabolic energy by addition of fluoroacetate. Inactivation was prevented by chloramphenicol, if the antibiotic was added before inactivation began.…”

“…A number of enzyme activities disappear from B. subtilis cells during the stationary phase of growth. Aspartate transcarbamylase (4,9,22), glutamine phosphoribosylpyrophosphate amidotransferase (15,21), and pyrimidine-repressible carbamyl phosphate synthetase (13) all begin to be inactivated immediately after the end of exponential growth. OTCase, on the other hand, is first inactivated 4 to 6 h after the end of exponential growth on glucose-nutrient broth, which is a period of extensive bulk protein turnover (10).…”

“…Previous studies from this laboratory have identified two examples of degradative inactivation in Bacillus subtilis. Aspartate transcarbamylase, the first enzyme of pyrimidine biosynthesis de novo, is rapidly inactivated at the end of exponential growth (4,22). This is also the case for glutamine phosphoribosylpyrophosphate amidotransferase (21).…”

Coordinate synthesis of the enzymes of pyrimidine biosynthesis in Bacillus subtilis.

“…The availability of the strains that produced pyrimidine enzymes constitutively enabled us to examine whether such synthesis was coordinate in cells that were grown under identical conditions in the presence of a constant excess of exogenous uracil and harvested during exponential growth. This was important because interference by inactivation of pyrimidine enzymes in the stationary phase or during pyrimidine starvation (7,11) could be avoided. Simultaneous synthesis and degradation of aspartate transcarbamylase during uracil starvation of B. subtilis cells has been demonstrated (5), and each of the other enzymes is inactivated in stationary cells (7; unpublished data).…”