ABSTRACT AUERNIK, KATHRYNE SHERLOCK. Functional Genomics Analysis of Metal Mobilization by the Extremely Thermoacidophilic Archaeon Metallosphaera sedula. (Under the direction of Dr. Robert Kelly.) Biomining processes recovering base, strategic and precious metals have predominantly utilized mesophilic bacteria, but relatively low yields have impacted wider application of this biotechnology. However, the use of high temperature microorganisms offers great potential to increase metal mobilization rates. Metallosphaera sedula (Mse) is an extremely thermoacidophilic archaeon with bioleaching capabilities, although little is known about the physiology of this microorganism. To better characterize Mse, its genome was sequenced and a whole genome oligonucleotide microarray was constructed for transcriptional response analysis. The physiological and bioenergetic complexities of Mse bioleaching were studied focusing on iron oxidation, sulfur oxidation, and growth modes (heterotrophy, autotrophy, and mixotrophy). The transcriptomes corresponding to each of these elements were examined for clues to the mechanisms by which Mse oxidizes inorganic energy sources (i.e. metal sulfides) and fixes CO2. Quinol/terminal oxidases important for maintaining intracellular pH and contributing to ATP generation via proton pumping were stimulated by different energy sources. The soxABCDD’L genome locus (Msed_0285-Msed_0291) was stimulated in the presence of reduced inorganic sulfur compounds (RISCs) and H2, while the soxNL-CbsABA cluster (Msed_0500-Msed_0504) was induced by Fe(II). Two similar copies of the SoxB/CoxI-like cytochrome oxidase subunit, foxAA’ (Msed_0484/Msed_0485) were implicated in fox cluster oxidation of Fe(II), as well as other energy sources. The doxBCE locus (Msed_2030-Msed2032) did not respond uniformly to either Fe(II) or RISCs, but was up-regulated in the presence of chalcopyrite (CuFeS2). A similar response was also observed for a putative rusticyanin (Msed_0966, rus), thiosulfate: quinone oxidoreductase (Msed_0363/Msed_0364, doxDA), and a putative sulfide:quinone oxidoreductase (Msed_1039, sqr), all three of which are candidates to serve as primary electron acceptors from inorganic substrates. Putative proteins implicated in the generation of reducing equivalents were identified (Dms/Sre-like reductase and Hdr-like reductases). Mixotrophy in Mse was defined as a strong preference for organic carbon combined with concomitant use of multiple inorganic (and organic) energy sources, if available. This growth mode was observed during CuFeS2 bioleaching, with organic carbon most likely obtained via recycling of lysed cell material. Functional Genomics Analysis of Metal Mobilization by the Extremely Thermoacidophilic Archaeon Metallosphaera sedula by Kathryne Sherlock Auernik A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Chemical and Biomolecular Engineering Raleigh, North Carolina 2009 APPROVED BY: _________________________ ___________________________ David F. Ollis Jason M. Haugh __________________________ ___________________________ Amy M. Grunden Robert M. Kelly Chair of Advisory Committee BIOGRAPHY Kathryne Sherlock Auernik grew up in a small town outside of Rochester, NY. She attended the University of Notre Dame (Notre Dame, IN) and received a Bachelor of Science degree in chemical engineering and a certificate in materials science in January 2001. After spending 3.5 years providing technical support for the manufacture of viral vaccines (Merck & Co., Inc., West Point, PA), she matriculated to the graduate program in the department of chemical and biomolecular engineering at North Carolina State University in search of more formalized biotechnology-related coursework and an opportunity to conduct academic research. She obtained a non-thesis Master of Science in chemical engineering in 2006. Following completion of her doctoral degree in chemical and biomolecular engineering and minor in biotechnology in 2009, she plans to return to work providing technical support for the manufacture of viral vaccines (Merck & Co., Inc., Durham, NC). ii TABLE OF CONTENTS LIST OF TABLES................................................................................................................ v LIST OF FIGURES ............................................................................................................. viii LIFE IN HOT ACID: PATHWAY ANALYSES IN EXTREMELY THERMOACIDOPHILIC ARCHAEA ................................................................................... 1 Abstract................................................................................................................... 2 Introduction ............................................................................................................. 2 Extremely thermoacidophilic archaea and their physiological characteristics......... 3 Mechanisms of resistance to and survival in hot acid ............................................. 4 Molecular genetics of extreme thermoacidophiles .................................................. 8 Bioleaching.............................................................................................................. 10 Other developments................................................................................................ 13 Summary................................................................................................................. 15 Acknowledgements ................................................................................................. 15 References.............................................................................................................. 15 THE GENOME SEQUENCE OF THE METAL-MOBILIZING, EXTREMELY THERMOACIDOPHILIC ARCHAEON METALLOSPHAERA SEDULA PROVIDES INSIGHTS INTO BIOLEACHING-ASSOCIATED METABOLISM....................................... 29 Abstract................................................................................................................... 30 Introduction ............................................................................................................. 31 Materials and Methods............................................................................................ 35 Results and Discussion........................................................................................... 37 Organization and general features of the genome.................................................. 37 Iron oxidation........................................................................................................... 38 Sulfur oxidation....................................................................................................... 41 Carbon metabolism................................................................................................. 42 Metal Tolerance...................................................................................................... 44 Adhesion ................................................................................................................. 45 Transcriptional response of M. sedula to FeSO4..................................................... 47 Summary................................................................................................................. 49 Acknowledgements ................................................................................................. 50 References.............................................................................................................. 50 Appendix: Supplementary Materials ....................................................................... 65 COMPONENTS OF ELECTRON TRANSPORT CHAINS IN THE EXTREMELY THERMOACIDOPHILIC CRENARCHAEON METALLOSPHAERA SEDULA IDENTIFIED THROUGH IRON AND SULFUR COMPOUND OXIDATION TRANSCRIPTOMES .......................................................................................................... 70 Abstract................................................................................................................... 71 Introduction ............................................................................................................. 72 Materials and Methods............................................................................................ 74 Results .................................................................................................................... 76 Quinone reduction from organic electron donors .................................................... 76 Quinone reduction via inorganic electron donors.................................................... 77 iii TABLE OF CONTENTS (continued) New components of Fe(II)-induced electron transport chains.............................. 78 New components of RISC-induced electron transport chains.............................. 78 Quinone oxidation via terminal oxidase complexes ............................................. 81 Fe(II) and RISC-induced hypothetical proteins .................................................... 84 Discussion............................................................................................................ 85 Summary.............................................................................................................. 90 Acknowledgements .............................................................................................. 91 References..........................................................................................................