Microbes interact with their surroundings through a variety of mechanisms, ranging from extracellular machineries like flagella, pili, and surface layer proteins to protein complexes embedded in the cell membrane. In this dissertation, we used a variety of techniques to characterize these mechanisms of interaction, with a focus on exploiting recent advances in electron microscopy to better understand these systems. The dissertation opens with an overview of the history and development of electron microscopy (EM) with a focus on its historical contributions to microbial interactions in the literature, its recent technical developments in electron microscopy, and helical reconstruction of protein filaments by cryo electron microscopy (cryoEM). EM has developed into an indispensable tool for studying all aspects of microbial interactions from the gross cellular level to protein structures at atomic resolution.
The following chapter of the thesis focuses on characterizing the interaction between two bacteria in the oral microbiome by utilizing scanning electron microscopy (SEM) in conjunction with light microscopy and genetic experiments. The relationship between the newly described obligately parasitic bacterium TM7x (Candidatus Saccharimonas formerly Candidate Phylum TM7) and its host Actinomyces odontolyticus species XH001 is described. Evidence from qPCR experiments, light microscopy, and SEM show that TM7x causes stress to its host XH001 and that this stress is additive with other stress factors in the environment. This demonstrates that the relationship between the two is actively harmful to XH001 whereas it was previously unclearly if XH001 host was impacted by the growth of TM7x. Light microscopy and SEM were also used to demonstrate that TM7x divides by budding and that no flagella or pili are visible on either cell. This data suggests that TM7x adheres to the host cell in a directional manner using cell surface or membrane proteins.
In chapter three, the cell envelope of the bacterium Syntrophamonus wolfei was characterized using biochemical assays and transmission electron microscopy (TEM). S.wolfei is a syntroph which must live in symbiotic relationships or consortium withother prokaryotes that consume the syntrophic metabolic products. A method wasdevised to separate the membrane portion of the cells from the soluble cell contents toproduce cell ghosts. These cell ghosts were analyzed via mass spectroscopy to identifythe three major protein components. One of these proteins, Swol_0141 has domainswhich identify it as a potential surface (S) layer protein domains. Transmission electronmicroscopy (TEM) of the cell ghosts revealed paracrystalline array of P4 symmetry,consistent with the production of a proteinaceous S-layer. CryoEM of the cells showsthe protein arrangement of the cell envelope.
Finally, I present an atomic structure of the archaeal flagellum from Methanospirillum hungatei strain JF-1, obtained with cryo electron microscopy, helical reconstruction, and de novo model building. The archaeal flagellum is a nanomachine which rotates to drive cell motility and adheres to other cells and surfaces. The thin filament of the flagellum is only 10 nm in diameter, but can extend to be several times longer than the cell length. This structure is the first complete atomic resolution model of an archaeal flagellin, and it describes the intermolecular interactions which allow for the stability of the flagellar filament under rotational stress. The cryoEM structure of the native protein also revealed eight sites of post-translational modification. To conclude, a comparison with the bacterial flagella and type IV pili shows that the archaeal flagellum is a structurally distinct cell motility and adhesion apparatus.
In summary, these thesis projects demonstrate the breadth of utility electron microscopy has for studying microbes and their environmental interactions. These include the cell to cell interactions of an oral parasitic bacteria and host bacteria, the definitions of an undescribed single cell envelope, and the atomic resolution protein structure of a flagellar nanomachine. The depth of information which can be explored using electron microscopy to solve complex microbial cell and protein structures continues to expand.
