Magnetotactic Bacteria: Isolation, Imaging, and Biomineralization Dissertation Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Zachery Walter John Oestreicher Graduate Program in Geological Sciences. The Ohio State University 2012 Committee: Steven K. Lower, Advisor Wendy Panero Olli Tuovinen Brian H. Lower Copyright by Zachery Walter John Oestreicher 2012 Abstract Magnetotactic bacteria (MTB) are a specialized group of bacteria that produce very small magnets inside their cells. There are a number of reasons that I decided to study these particular microorganisms. MTB are universally found in aquatic environments and they can be isolated with a simple magnet. These bacteria have the distinct ability to synthesize nanometer-scale crystals of magnetite (Fe3O4) or greigite (Fe3S4) inside their cells. This type of biomineralization serves as a model for mineral formation in more complex organisms such as birds, bees, and fish. The magnetite from MTB can be used as a biomarker, called magnetofossils, for past life on earth as well as possible extraterrestrial life forms (e.g., putative magnetofossils in Martian meteorites such as the Allan Hills meteorite). Magnetofossils are novel biomarkers because the magnetite from MTB has a specific crystal shape, narrow size range, and flawless chemical composition, which make them easily identified as biological origin. These same crystallographic attributes could also be exploited in biomimicry. For example, in vitro synthesis of magnetic crystals could have applications in medicine, electronic storage devices, and even environmental remediation. The work in this dissertation touches on all of these concepts. For the environmental isolation of MTB, I collected water samples from two field sites, an arsenic-rich hot spring in Oregon (Mickey Hot Spring) and a freshwater, microbialite-containing lake in British Columbia, Canada (Pavilion Lake). These sites were selected because MTB have never been isolated from these locations, and these two ii sites are often used as proxies for conditions on the early Earth or extraterrestrial bodies. To isolate MTB from these two samples, I used a relatively simple method that takes advantage of a bar magnet and capillary racetrack created using a cotton-plugged glass pipette. The MTB from Mickey Hot Spring in Oregon were rod to vibrioid-shaped cells that were 2.2 (± 0.6) µm long and 0.62 (± 0.1) µm wide. The magnetosomes were composed of bullet-shaped crystals of magnetite that were 84 (± 17) nm long and 39 (± 8) nm wide. These magnetosomes from the Mickey Hot Spring specimens were usually arranged in a single chain. The 16S rRNA gene sequence analysis identified the Mickey Hot Spring specimens as part of the Nitrospirae phylum. MTB isolated from Lake Pavilion in British Columbia were spirillum-shaped cells that were 2.9 (± 0.6) µm long 0.34 (± 0.02) µm wide (n = 7). Their magnetite crystals were 47 (± 5) nm long and 44 (± 5) nm wide. The magnetosomes were arranged in a single chain. The 16S rRNA analysis showed that the Lake Pavilion cells were from the Alphaproteobacteria phylum. After isolating MTB from two different environments, I turned my attention to the biomineralization of magnetite within MTB. For this portion of my dissertation, I examined a protein called Mms-6, which has recently been shown to play a key role in the nucleation and/or growth of magnetite. I used high-resolution transmission electron microscopy (TEM) to examine gold-conjugated, immunolabeled Mms-6 in thin sections of Magnetospirillum magneticum AMB-1. I found that the Mms-6 proteins are not located on the cell membrane or within the cytoplasm, but are only clustered on the magnetosome membrane. This was confirmed by using confocal laser scanning microscopy on Mms-6 proteins labeled with green fluorescence proteins in cells of M. iii magneticum AMB-1. These studies constrain the spatial and temporal function of Mms-6 proteins during the mineralization of magnetite by MTB. Mms6 is confined to the magnetosome membrane after invagination from the cell membrane. The last portion of my dissertation included the high-resolution analysis of two different types of magnetotactic bacteria: M. magneticum AMB-1 and M. gryphiswaldense MSR-1 using atomic force microscopy (AFM) and TEM. In these experiments I examined the ultrastructure and magnetosomes from both species as well as Mms6 proteins and determined the advantages of both techniques to examining MTB. The main advantage that AFM has over the TEM is that cells or biomolecules can be examined under physiological conditions. This allows direct observation of proteins interacting with magnetite in vitro. However, AFM could not be used to visualize structural details within the cells even when the AFM tip was used as a micro-scalpel to open the outer cell wall of the bacteria. The advantage of TEM is its superior ability to visualize ultrafine, intracellular detail. An obvious disadvantage of TEM is that the bacteria are not living as they are in the AFM. Used together, AFM and TEM offer complementary information for the analysis of biominerals within MTB. iv Dedication This document is dedicated to Chiharu Yaginuma. v Acknowledgments I have many people to acknowledge and I may have left some people out, but after 75 months of doing my PhD it is hard to remember everyone. Steven Lower and Brian Lower for advising me, providing me with support and giving me the opportunity to do my PhD, Olli Tuovinen, Wendy Panero, Richard Dick and Linda Dick for using their lab, Chiharu Yaginuma for a lot of things, Uncle Doctor Steve Goldsmith for all the BBQs, Charles Park for giving me somewhere to write my dissertation, Eric Taylor, Lumarie Perez-Guzman for help with my dissertation and for the wonderful food, Lijun Chen, Nadia Casillas-Ituarte, Alyssa Bancroft for being a good friend, Sara Cole and Richard Montione at CMIF for putting up with all my requests, Todd Matulnik at the fermentation lab, Henk Colijn at CEOF, Nanotech West, Azuma Taoka and Yoshihiro Fukumori at Kanazawa University, Japan Society for the Promotion of Science and the National Science Foundation East Asia and Pacific Summer Institutes for providing me with the opportunity to go to Japan, Marit Nilsen-Hamilton and Pierre Palo for sending me the plasmid containing the mms6 gene and for helping me with the protein purification, Dennis Bazylinski and Chris Lefevre for helping me culture AMB-1, Wei Lin, Jinhua Li and Yongxin Pan at the Chinese Academy of Science the Institute of Geology and Geophysics for showing me how to isolate MTB, the National Science vi Foundation East Asia and Pacific Summer Institutes for giving me another chance to do research in Asia, The Ohio State University for giving me a one year fellowship and for giving me a grant through the Alumni Grants for Graduate Research and Scholarship, the Geological Society of America for their grant which allowed me to collect all the samples for chapter 3, the Friends of Orton Hall for providing me with money to present at M&M, Carmen Valverde-Tercedor for her help with my research and dissertation, Brent Curtiss for all the computer assistance, Angeletha Rogers for over six years of help in the Geology Department, Olivia Amerendes for helping me order countless supplies, Michael Carter for helping me centrifuge and lyse my bacteria, Aurelie Snyder and Scott Wetzel for all of their confocal knowledge, Jim Pawley who allowed me to participate five amazing times in his live cell microscopy workshop in Vancouver, Peter Styger for giving me the chance to learn confocal microscopy, Takatoshi Karasawa, Amanda Davey for helping out in Senegal, the Microscopy Society of America, Eric Rees at Research and Testing for answering all of questions, PNNL and EMSL for giving me the chance to do research at their facility several times, JoAnn Donohue at the Ohio State Department of Speech and Hearing, Sander Flaum for all of his support and inspiration, my parents Bill and Candy McHoes, Tonya and Todd Elmer, Juliana Lam, Sherry Cady for inspiring me to do field work and microscopy and getting me started in geomicrobiology, and of course Dr. John Dash for teaching me how to use the TEM way back in September of 1996, for inspiring me to pursue microscopy, and for many other things. vii Vita 1997................................................................B.S. Biology, Portland State University 2004................................................................M.S. Geology, Portland State University 2006-7………………………………….. ......The Ohio State University Graduate School Fellowship 2007-2011 ......................................................Graduate Teaching Associate, School of Earth Sciences, The Ohio State University 2007................................................................Summer Research Institute Fellow, Pacific Northwest National Laboratory 2008…............................................................Summer Research Fellowship in Kanazawa, Japan, National Science Foundation East Asia and Pacific Summer Institutes 2010…............................................................Summer Research Fellowship in Beijing, China, National Science Foundation East Asia and Pacific Summer Institutes Publications Lower, B. H., Lins, R. D., Oestreicher, Z., Straatsma, T. P., Hochella Jr, M. F., Shi, L., and Lower, S. K., 2008, In vitro evolution of a peptide with a hematite binding motif that may viii constitute a natural