Analysis of a possible multienzyme complex encoded by mother cell metabolic gene (MMG) operon of Bacillus subtilis strain 168.

摘要

Bacillus subtilis is a rod shaped, aerobic, endospore forming gram-positive bacterium. B. subtilis is used as a model organism to study cell differentiation during sporulation in prokaryotes. When there is inadequate supply of carbon resources, B. subtilis undergoes a process of sporulation.The mother cell metabolic gene (mmg) operon is expressed at early stages of sporulation. It has six open reading frames mmgABCDE and yqiQ. The products of these genes mmg A, B, C, D, E and yqiQ in mmg operon share sequence homology with enzymes involved in fatty acid metabolism and methyl citric acid cycle. The genes mmgABCD were successfully cloned, over expressed and purified. The fatty acid degradation enzymes exist, as a complex in E.coli and our hypothesis is that the fatty acid degradation proteins may function as a complex as well. We tested this hypothesis by cloning and coexpressing all four mmg ABCD in a single strain of E.coli. This was accomplished by cloning mmgA with a His-tag for Ni-NTA affinity chromatography. If the four proteins formed a complex, the remaining mmgBCD would co-purify with the His-tagged mmgA. We observed a ~26 kDa protein coeluting with mmgA (which may be mmgB) and also 40-50 kDa protein.(mmgC - 40710 Da and mmgD- 41946 Da)