The Regulation of Aspartokinase During Growth and Sporulation of Bacillus Cereus

Abstract

The problem. Diaminopimelic acid (DAP) and dipicolinic acid (DPA) are thought to be important chemical constituents in the formation of heat-resistant spores in baccilli. They are formed through a multistep biosynthetic pathway in which aspartokinase is the first enzyme. The aspartokinas activity in Bacillus cereus has been shown to be repressed by lysine and yet still be able to form heat-resistant spores with near normal amounts of DPA. This study was undertaken to attempt to elucidate how the products formed through the functioning of aspartokinase in B. cereus could be present when the enzyme had been repressed to low levels of lysine. Procedure. Cells of B. cereus T were grown by batch method in lysine assay medium (LAM) with or without lysine added. The cells were harvested and broken by sonication or a French press and the cell-free extracts assayed for aspartokinase activity in various buffers at different times along the growth curve. For inhibition studies, the inhibitors were added to the assay mixture. Findings. A high ionic strength buffer containing 4M NaCl that was tested revealed the presence of aspartokinase activity that was not repressed or inhibited by lysine but was sensitive to feedback inhibition by DAP. Conclusions. B. Cereus is found to have two isozymes of aspartokinase. Aspartokinase I (AI I) is repressed and inhibited by lysine. It is the predominant enzyme functioning during vegetative growth of B. cereus cells. AI II is sensitive to feedback inhibition by diaminopimelate but insensitive to repression or inhibition by lysine. AI II functions during sporulation to ensure channeling of carbon compounds to DPA and DAP for sporogenesis. AK II is rapidly inactivated in most standard buffers; however, a 4M NaCl buffer was developed to stabilize the enzyme for assay.