Phenotypic and Physiological Characterization of the Epibiotic Interaction Between TM7x and Its Basibiont Actinomyces

Despite many examples of obligate epibiotic symbiosis (one organism living on the surface of another) in nature, such an interaction has rarely been observed between two bacteria. Here, we further characterize a newly reported interaction between a human oral obligate parasitic bacterium TM7x (cultivated member of Candidatus Saccharimonas formerly Candidate Phylum TM7), and its basibiont Actinomyces odontolyticus species (XH001), providing a model system to study epiparasitic symbiosis in the domain Bacteria. Detailed microscopic studies indicate that both partners display extensive morphological changes during symbiotic growth. XH001 cells manifested as short rods in monoculture, but displayed elongated and hyphal morphology when physically associated with TM7x. Interestingly, these dramatic morphological changes in XH001 were also induced in oxygen-depleted conditions, even in the absence of TM7x. Targeted quantitative real-time PCR (qRT-PCR) analyses revealed that both the physical association with TM7x as well as oxygen depletion triggered up-regulation of key stress response genes in XH001, and in combination, these conditions act in an additive manner. TM7x and XH001 co-exist with relatively uniform cell morphologies under nutrient-replete conditions. However, upon nutrient depletion, TM7x-associated XH001 displayed a variety of cell morphologies, including swollen cell body, clubbed-ends, and even cell lysis, and a large portion of TM7x cells transformed from ultrasmall cocci into elongated cells. Our study demonstrates a highly dynamic interaction between epibiont TM7x and its basibiont XH001 in response to physical association or environmental cues such as oxygen level and nutritional status, as reflected by their morphological and physiological changes during symbiotic growth

Using Advances in Electron Microscopy to Study Microbial Interactions

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

Using Advances in Electron Microscopy to Study Microbial Interactions

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

Resting state structure of the hyperdepolarization activated two-pore channel 3

Voltage-gated ion channels endow membranes with excitability and the means to propagate action potentials that form the basis of all neuronal signaling. We determined the structure of a voltage-gated sodium channel, two-pore channel 3 (TPC3), which generates ultralong action potentials. TPC3 is distinguished by activation only at extreme membrane depolarization (V50 ∼ +75 mV), in contrast to other TPCs and NaV channels that activate between -20 and 0 mV. We present electrophysiological evidence that TPC3 voltage activation depends only on voltage sensing domain 2 (VSD2) and that each of the three gating arginines in VSD2 reduces the activation threshold. The structure presents a chemical basis for sodium selectivity, and a constricted gate suggests a closed pore consistent with extreme voltage dependence. The structure, confirmed by our electrophysiology, illustrates the configuration of a bona fide resting state voltage sensor, observed without the need for any inhibitory ligand, and independent of any chemical or mutagenic alteration

Erratum: Corrigendum: CryoEM structure of the Methanospirillum hungatei archaellum reveals structural features distinct from the bacterial flagellum and type IV pilus

Archaea use flagella known as archaella-distinct both in protein composition and structure from bacterial flagella-to drive cell motility, but the structural basis of this function is unknown. Here, we report an atomic model of the archaella, based on the cryo electron microscopy (cryoEM) structure of the Methanospirillum hungatei archaellum at 3.4 Å resolution. Each archaellum contains ∼61,500 archaellin subunits organized into a curved helix with a diameter of 10 nm and average length of 10,000 nm. The tadpole-shaped archaellin monomer has two domains, a β-barrel domain and a long, mildly kinked α-helix tail. Our structure reveals multiple post-translational modifications to the archaella, including six O-linked glycans and an unusual N-linked modification. The extensive interactions among neighbouring archaellins explain how the long but thin archaellum maintains the structural integrity required for motility-driving rotation. These extensive inter-subunit interactions and the absence of a central pore in the archaellum distinguish it from both the bacterial flagellum and type IV pili

CryoEM structure of the Methanospirillum hungatei archaellum reveals structural features distinct from the bacterial flagellum and type IV pilus.

Archaea use flagella known as archaella-distinct both in protein composition and structure from bacterial flagella-to drive cell motility, but the structural basis of this function is unknown. Here, we report an atomic model of the archaella, based on the cryo electron microscopy (cryoEM) structure of the Methanospirillum hungatei archaellum at 3.4 Å resolution. Each archaellum contains ∼61,500 archaellin subunits organized into a curved helix with a diameter of 10 nm and average length of 10,000 nm. The tadpole-shaped archaellin monomer has two domains, a β-barrel domain and a long, mildly kinked α-helix tail. Our structure reveals multiple post-translational modifications to the archaella, including six O-linked glycans and an unusual N-linked modification. The extensive interactions among neighbouring archaellins explain how the long but thin archaellum maintains the structural integrity required for motility-driving rotation. These extensive inter-subunit interactions and the absence of a central pore in the archaellum distinguish it from both the bacterial flagellum and type IV pili

The structure of the endogenous ESX-3 secretion system.

The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the chromosome of the model organism Mycobacterium smegmatis. The structure reveals a stacked architecture that extends above and below the inner membrane of the bacterium. The ESX-3 protomer complex is assembled from a single copy of the EccB3, EccC3, and EccE3 and two copies of the EccD3 protein. In the structure, the protomers form a stable dimer that is consistent with assembly into a larger oligomer. The ESX-3 structure provides a framework for further study of these important bacterial transporters

High-Resolution Cryo-Electron Microscopy Structure Determination of <i>Haemophilus influenzae</i> Tellurite-Resistance Protein A via 200 kV Transmission Electron Microscopy

Membrane proteins constitute about 20% of the human proteome and play crucial roles in cellular functions. However, a complete understanding of their structure and function is limited by their hydrophobic nature, which poses significant challenges in purification and stabilization. Detergents, essential in the isolation process, risk destabilizing or altering the proteins’ native conformations, thus affecting stability and functionality. This study leverages single-particle cryo-electron microscopy to elucidate the structural nuances of membrane proteins, focusing on the SLAC1 bacterial homolog from Haemophilus influenzae (HiTehA) purified with diverse detergents, including n-dodecyl β-D-maltopyranoside (DDM), glycodiosgenin (GDN), β-D-octyl-glucoside (OG), and lauryl maltose neopentyl glycol (LMNG). This research not only contributes to the understanding of membrane protein structures but also addresses detergent effects on protein purification. By showcasing that the overall structural integrity of the channel is preserved, our study underscores the intricate interplay between proteins and detergents, offering insightful implications for drug design and membrane biology

Complete genome sequence of Methanospirillum hungatei type strain JF1.

Methanospirillum hungatei strain JF1 (DSM 864) is a methane-producing archaeon and is the type species of the genus Methanospirillum, which belongs to the family Methanospirillaceae within the order Methanomicrobiales. Its genome was selected for sequencing due to its ability to utilize hydrogen and carbon dioxide and/or formate as a sole source of energy. Ecologically, M. hungatei functions as the hydrogen- and/or formate-using partner with many species of syntrophic bacteria. Its morphology is distinct from other methanogens with the ability to form long chains of cells (up to 100 μm in length), which are enclosed within a sheath-like structure, and terminal cells with polar flagella. The genome of M. hungatei strain JF1 is the first completely sequenced genome of the family Methanospirillaceae, and it has a circular genome of 3,544,738&nbsp;bp containing 3,239 protein coding and 68 RNA genes. The large genome of M. hungatei JF1 suggests the presence of unrecognized biochemical/physiological properties that likely extend to the other Methanospirillaceae and include the ability to form the unusual sheath-like structure and to successfully interact with syntrophic bacteria