Magnetotactic Bacteria: Isolation, Imaging, and Biomineralization

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 (Fe3S 4) 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 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) microm long and 0.62 (+/- 0.1) microm 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) microm long 0.34 (+/- 0.02) microm 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. 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.