Investigating Manganese Oxidation by Aurantimonas manganoxydans SI85-9A1 By Nicholas Caputo B.S. in Chemistry, Colorado School of Mines A THESIS Presented to the Institute of Environmental Health, Division of Environmental and Biomolecular Systems and the Oregon Health & Science University School of Medicine in partial fulfillment of the requirements for the degree of Master of Science June 2011 Division of Environmental & Biomolecular Systems School of Medicine Oregon Health & Science University ________________________________________________ Certificate of Approval ________________________________________________ This is certify that the Master’s thesis of Nicholas Caputo has been approved ______________________________________ Dr. Bradley Tebo Thesis Research Advisor Professor, OHSU ______________________________________ Dr. James W. Whittaker Thesis Committee Chair Professor, OHSU ______________________________________ Dr. Arthur Glasfeld Department of Chemistry Professor, Reed College ii Table of Contents Certificate of Approval ................................................................................................. ii Table of Contents ......................................................................................................... iii List of Tables .................................................................................................................v List of Figures ................................................................................................................ i List of Equations ........................................................................................................... ii Acknowledgments ........................................................................................................ iii Abstract ..........................................................................................................................v Chapter 1: Introduction of the Biogeochemistry of Manganese ..................................1 Importance of manganese in the environment.......................................................................... 1 Mn(II) oxidation by microorganisms ....................................................................................... 3 Purpose of this thesis .............................................................................................................. 7 Chapter 2: Characterization of Mn(II) oxidizing Peroxidase A ..................................9 Introduction ............................................................................................................................ 9 Methods and Materials.......................................................................................................... 11 Results and Discussion ......................................................................................................... 18 Summary .............................................................................................................................. 36 Chapter 3: Development of a genetic system for A. manganoxydans SI85-9A1 ........ 38 Introduction .......................................................................................................................... 38 Methods and Materials.......................................................................................................... 40 Results and Discussion ......................................................................................................... 48 Summary .............................................................................................................................. 61 Chapter 4: The Mechanism of Mn(II) oxidation in vivo and in vitro ......................... 63 Introduction .......................................................................................................................... 63 Methods and Materials.......................................................................................................... 68 Results and Discussion ......................................................................................................... 72 Summary .............................................................................................................................. 80 Chapter 5: Summary and Future Directions .............................................................. 83 References .................................................................................................................... 86 Appendix A: Expression of Mn(II) oxidizing Proteins ............................................... 93 Introduction .......................................................................................................................... 93 Methods and Materials.......................................................................................................... 93 Results and Discussion ....................................................................................................... 100 Summary ............................................................................................................................ 103 Appendix B: Tandem MS Data ................................................................................. 105 iii Introduction ........................................................................................................................ 105 Results................................................................................................................................ 105 Biographical Sketch ................................................................................................... 120 iv List of Tables Table 1: Complexity of proteins in each stage of purification ......................................... 21 Table 2: Top four proteins by most assigned spectra per group ...................................... 23 Table 3: Purification table for MopA purification .......................................................... 24 Table 4: Amino Acid composition of Type 1 secretion signal ........................................ 29 Table 5: Gram negative bacteria with RTX peroxidases ................................................. 32 Table 6: Strains, plasmids, and primers used in Chapter 3 .............................................. 41 Table 7: PCR reaction conditions. .................................................................................. 47 Table 8: Antibiotic MIC for A. manganoxydans SI85-9A1 ............................................. 49 Table 9: Properties of proteins found near MopA and other accessory proteins .............. 58 Table 10: Qualitative Mn(II) oxidation and cell density of EMS mutants ....................... 60 Table 11: Strains, plasmids and primers used in Appendix A ......................................... 94 Table 12: PCR conditions used in Appendix A .............................................................. 96 Table 13: Variables tested for elution of Mop from chitin resin .................................... 102 Table 14: Proteins identified after each stage of purification ........................................ 106 Table 15: Peptide sequences top five identified proteins from LBOM protein matrix ... 108 Table 16: Peptide sequences top five identified proteins after IEX purification stage ... 111 Table 17: Peptide sequences top five identified proteins after HIC purification stage ... 114 Table 18: Peptide sequences top five identified proteins after SEC purification stage... 116 v List of Figures Figure 1: SDS-PAGE gel of MopA purification showing enrichment of protein bands ... 19 Figure 2: Localization of proteins identified using MS/MS ............................................ 21 Figure 3: Mn(II) oxidation inhibition by cyanide ........................................................... 26 Figure 4: Native PAGE activity correlation gel .............................................................. 27 Figure 5: MopA predicted 3o structure based on 1o sequence ......................................... 28 Figure 6: Growth of A. manganoxydans SI85-9A1 in M and K-media............................ 50 Figure 7: Restreak plate of pJH1 conjugants and contaminant ........................................ 53 Figure 8: Plasmid DNA isolated from the six conjugants ............................................... 55 Figure 9: PCR screen to prove the identity of conjugants. .............................................. 55 Figure 10: Putative ‘Moperon’ ....................................................................................... 56 Figure 11: In vitro Mn(IIIV) oxidation disruption ....................................................... 73 Figure 12: In vitro Mn(IIIII) oxidation disruption ....................................................... 74 Figure 13: In vitro Mn(IIIIV) oxidation disruption ..................................................... 75 Figure 14: In vitro Mn(IIIIV) oxidation disruption; 10x [Cat] over Figure 13 ............. 76 Figure 15: In vitro Mn(IIIV) oxidation disruption ....................................................... 77 Figure 16: Time course of Mn(II) oxidation in vivo........................................................ 78 Figure 17: Effect of xanthine concentration on in vivo Mn(II) oxidation ........................ 80 Figure 18: Purification of MoxA using immobilized metal affinity chromatography .... 101 Figure 19: Western blot of purified MoxA ................................................................... 102 List of Equations