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HARDESTY, GRANT ALAN, MS the Characterization of Mmge

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HARDESTY, GRANT ALAN, MS the Characterization of Mmge HARDESTY, GRANT ALAN, M.S. The Characterization of MmgE from Bacillus subtilis. (2012) Directed by Dr. Jason J. Reddick. 56 pp. 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 σE 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. THE CHARACTERIZATION OF MMGE FROM BACILLUS SUBTILIS by Grant Alan Hardesty A Thesis Submitted to the Faculty of The Graduate School at The University of North Carolina at Greensboro in Partial Fulfillment of the Requirements for the Degree Master of Science Greensboro 2012 Approved by ________________________________ Committee Chair APPROVAL PAGE This thesis has been approved by the following committee of the Faculty of The Graduate School at The University of North Carolina at Greensboro. Committee Chair___________________________________ Committee Members___________________________________ ___________________________________ __________________________ Date of Acceptance by Committee _____________________________ Date of Final Oral Examination ii TABLE OF CONTENTS Page LIST OF FIGURES .............................................................................................................v CHAPTER I. BACKGROUND .................................................................................................. 1 I. A Sporulation ............................................................................................ 1 I. B The mmg Operon ................................................................................... 5 I. C Methylcitric Acid Cycle ........................................................................ 6 I. D Introduction to MmgE ......................................................................... 10 I. E ClustalW Alignment ............................................................................ 13 II. EXPERIMENTAL .............................................................................................. 16 II. A Project Overview and Goals ............................................................... 16 II. B Expected Significance ......................................................................... 18 II. C Cloning of mmgE ............................................................................... .19 II. D Overexpression of the MmgE Protein ................................................. 20 II. E Purification of MmgE .......................................................................... 21 II. F Agarose Gel Protocol ........................................................................... 22 II. G SDS-PAGE Protocol ........................................................................... 23 II. H MmgD Purification and Analysis (28) ................................................ 24 II. I MmgE Activity Assay With MmgD Product ....................................... 24 II. J MmgE Activity Assay with Commercial 2-Methylcitrate ................... 25 II. K HPLC Analysis ................................................................................... 26 II. L Mass Spectrometry .............................................................................. 26 II. M CitB Expression .................................................................................. 27 II. N CitB Purification ................................................................................. 28 II. O Concentration of Protein Samples ....................................................... 29 II. P CitB Activity Assay ............................................................................. 29 III. RESULTS AND DISCUSSION ......................................................................... 30 III. A Cloning of mmgE ................................................................................ 30 III. B Purification of MmgE .......................................................................... 31 III. C MmgD ................................................................................................. 33 III. D HPLC .................................................................................................. 34 III. E Phenomenex Synergi Column ............................................................. 35 III. F Direct Infusion Mass Spectrometry ..................................................... 40 III. G LC-MS ................................................................................................ 43 iii III. H CitB ..................................................................................................... 49 III. I Conclusion ............................................................................................ 53 REFERENCES ................................................................................................................. 54 iv LIST OF FIGURES Page Figure 1. The mmg operon ...................................................................................................6 Figure 2. The methylcitric acid cycle ................................................................................10 Figure 3. The reaction catalyzed by MmgD to form 2-methylcitrate, the substrate for MmgE (24) ...........................................................................12 Figure 4. The expected reaction catalyzed by MmgE ........................................................12 Figure 5. ClustalW alignment of prpD from Salmonella typhimurium, E.coli strain K-12, and Bacillus subtilis strain 168 (24) (25) (26) (27) ....................13 Figure 6. Agarose gel confirming the presence of mmgE ................................................31 Figure 7. SDS-PAGE of MmgE ........................................................................................32 Figure 8. SDS-PAGE of MmgD .......................................................................................33 Figure 9. HPLC chromatogram of 1mM citrate (isocratic 100% phosphate buffer) .........36 Figure 10. 1mM isocitrate in phosphate buffer pH 2.9 (isocratic 100% phosphate buffer) ...............................................................36 Figure 11. 1mM Trans-aconitate in phosphate buffer pH 2.9 (isocratic 100% phosphate buffer) ...............................................................37 Figure 12. HPLC chromatogram of 1mM 2-methylcitrate ...............................................37 Figure 13. MmgE enzyme reaction at 15 minutes .............................................................38 Figure 14. MmgE enzyme reaction at 30 minutes .............................................................38 Figure 15. MmgE enzyme reaction at 45 minutes .............................................................39 Figure 16. MmgE enzyme reaction at 60 minutes .............................................................39 Figure 17. Direct infusion lacking MmgE ........................................................................42 Figure 18. Direct infusion of 2-methylcitrate ....................................................................42 v Figure 19. Direct infusion lacking 2-methylcitrate ...........................................................42 Figure 20. Direct infusion of the full reaction ..................................................................43 Figure 21. Control experiment lacking 2-methylcitrate .....................................................44 Figure 22. Control experiment lacking MmgE ..................................................................45 Figure 23. LC-MS analysis of MmgE reaction .................................................................45 Figure 24. A broad spectrum (200-400nm) scan of the MmgE reaction ..........................47 Figure 25. UV/Vis time-course of the mmgE enzymatic reaction at 240nm ....................48 Figure 26. SDS-PAGE of CitB Protein ..............................................................................50
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