The characterization of mmgE from Bacillus subtilis

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The characterization of mmgE from Bacillus subtilis


Hardesty, Grant Alan



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Bacillus subtilis is a rod shaped gram-positive bacterium that has been extensively studied for decades. The scientific interest lies, in large part, to its use as a model organism for the study of sporulation, which is a simple form of cell differentiation. It is also a very important organism in industry and everyday life as it is used to produce antibiotics, and other important compounds. B. subtilis is also used as a probiotic and as an additive to organic fertilizers and plant treatments because of its antifungal properties.The regulation of the initiation and continuation of sporulation has been well studied, but the metabolic changes the cell must go through are still in question and the subject of current study. One of the metabolic changes that happens in the cell is the activation of the mmg operon by 963;E in the mother cell during sporulation. This operon is composed of six genes that encode a putative fatty acid degradation pathway and the methylcitric acid cycle. This cycle takes propionyl-CoA (a fatty acid degradation metabolite) and condenses it with oxaloacetate into 2-methylcitrate, and through the rest of the cycle produces pyruvate and succinate that can enter the citric acid cycle.One gene in the middle of the putative methylcitric acid cycle is mmgE. This gene has not previously been studied. Its sequence, both genetic and proteomic, are a very close match to 2-methylcitrate dehydratases found in other organisms like Escherichia coli and Salmonella typhimurium.The mmgE gene was cloned into an overexpression strain of E. coli and the protein was isolated for study through Ni-NTA chromatography. Through UV/Vis spectrometry, high performance liquid chromatography (HPLC), and mass spectrometry the activity of this enzyme, on 2-methylcitrate, was studied. It was found that this enzyme does perform 2-methylcitrate dehydratase activity