We use cryo electron tomography to obtain three-dimensional images of Caulobacter cells at successive stages of cell division. Our tomographic reconstructions show that the late stages of constriction and separation of the Caulobacter inner and outer membrane are separate events occurring one after the other and distinctly separated in time and space, consistent with FLIP assay results. A model for this process is advanced based on a series of 17 tomographic reconstructions. In Caulobacter, division proceeds by gradual constriction of the cylindrical part of the cell, and there is no septum as in B. subtilis or E. coli. The FtsZ ring forms at the division plane about 90 min into a 180 min cell cycle; about 30 min later, constriction of the cell is visible. Many proteins involved in cytokinesis are known in Escherichia coli and Bacillus subtilis and to a lesser extent in Caulobacter crescentus. In each of these species, the widely-conserved tubulin-like FtsZ protein initiates cell division by polymerizing into a ring at the future division site. Preliminary cryo electron tomography data shows for the first time the FtsZ ring in its intact native state. The details of the function of the FtsZ ring in late stages of bacterial cytokinesis are both less understood than earlier stages and more variable across different species. An important part of our work aims at the elucidation of these questions.We are also working with Deinococcus radiodurans, and the related Deinococcus grandis. These bacteria constitute a target of great relevance because of their unusual ability to repair DNA damage caused by ionizing radiation and other environmental stress. One goal of particular interest in studying these bacteria is to better understand the mechanical packaging of the DNA, which has been reported to be in a highly condensed, toroidal form. This in turn could help to further elucidate the mechanism of DNA repair in prokaryotes. This form of DNA packaging may be a general model for DNA protection under stress, and preliminary data shows the loose toroidal arrangement of Deinococcus grandis DNA. We are also studying the organization of the cell membrane separating individual cells in the dyads and tetrads, since it has been proposed that the exchange of material across these boundaries plays an important role in their unusual radiation resistance. Another part of our efforts targets the fully automation of data collection, tomographic reconstruction and feature extraction from finished tomograms. We present the first step in the development of a fully automatic membrane segmentation tool.

Bacteriophage are viruses that attack bacteria. To successfully invade their host bacterium, a bacteriophage must succeed in packaging, transporting, and delivering its genome; these processes necessitate the precise manipulation of DNA throughout the virus life cycle. The exquisite control that a bacteriophage has over its DNA is desirable in many different biological and technological settings. Therefore, establishing a fundamental understanding of the underlying physical principles in DNA manipulation by a bacteriophage lends insight into the methods used by Nature to control DNA conformation and aids in determining how we can mimic such methods.The bacteriophage phi29 assembles its protein capsid particle before the DNA is packaged. The phi29 genome is then forced into the empty capsid by an ATP-driven protein motor. As the capsid is filled with DNA, the packaging motor works against the increasing resistance associated with compacting the long polymer chain to near-crystalline density into the relatively small cavity. We study the evolving structure of the DNA within the phi29 capsid. Cryo EM reconstruction of the packaged bacteriophage exhibits a layered DNA structure, indicative of local order within the particle. Using Monte Carlo simulation, we predict the conformation of the DNA on the interior of the viral capsid and the forces required to achieve this structure. The resulting density plot from simulations show good agreement with the EM results, and the predicted forces are consistent with previous single-molecule measurements of the packaging forces. Conformations from the Monte Carlo simulations tend to exhibit local layer ordering with frequent defects associated with chain segments jumping across layers. We discuss the impact that such defects have on the overall order and the resistance to DNA packaging and ejection.

Metagenomic studies are revolutionizing our understanding of microbes in the biosphere. They have uncovered numerous proteins of unknown function in tens of essentially unstudied lineages that lack cultivated representatives. Notably, few of these microorganisms have been visualized, and even fewer have been described ultra-structurally in their essentially intact, physiologically relevant states. Here, we present cryogenic transmission electron microscope (cryo-TEM) 2D images and 3D tomographic datasets for archaeal species from natural acid mine drainage (AMD) microbial communities. Ultrastructural findings indicate the importance of microbial interconnectedness via a range of mechanisms, including direct cytoplasmic bridges and pervasive pili. The data also suggest a variety of biological structures associated with cell-cell interfaces that lack explanation. Some may play roles in inter-species interactions. Interdependences amongst the archaea may have confounded prior isolation efforts. Overall, the findings underline knowledge gaps related to archaeal cell components and highlight the likely importance of co-evolution in shaping microbial lineages.

Nafion is an ion-containing random copolymer used as a solid electrolyte in many electrochemical applications thanks to its remarkable ionic conductivity and mechanical stability. Understanding the mechanism of ion transport in Nafion, which depends strongly on hydration, therefore requires a complete picture of its morphology in dry and hydrated form. Here we report on a nanoscale study of dry versus hydrated as-cast 100 nm Nafion membranes using analytical transmission electron microscopy (TEM) and cryogenic TEM tomography, respectively. For the dry membrane, spherical clusters ∼3.5 nm in diameter corresponding to the hydrophilic sulfonic-acid-containing phase are identified. In contrast, cryo TEM tomography of the hydrated membrane reveals an interconnected channel-type network, with a domain spacing of ∼5 nm, and presents the first nanoscale 3D views of the internal structure of hydrated Nafion obtained by a direct-imaging approach.

The purpose of this study is to understand the distinct shape profile of the dividing Caulobacter crescentus (Cc) cell near the division midplane at various stages of cell division, visible in new high-resolution Cryo EM tomographic images. These images of Cc during cell division provide the needed resolution for understanding cell wall dynamics under the influence of the contractile ring at the division midplane (FtsZ ring). The bacterial cell wall is a peptidoglycan mesh that acts like a fabric that can support shear as well as distinct normal forces, as opposed to the lipid membranes which are usually assumed not to support shear or any differences in normal forces. New tomographic images clearly show the S-layer, inner, outer membranes and peptidoglycan mesh. The shape of the cell wall during division and the deflection at the division plane is a function of the material properties of the cell wall, the growth rate, and the force due to the FtsZ ring. Our hypothesis is that cell growth and cell contraction occur on similar timescales, and that the contractile force at the division midplane is minimal, serving to direct the growth of the peptidoglycan mesh very near the division plane so as to eventually pinch and isolate the two halves of the dividing cell. In this model the potential energy stored in the peptidoglycan mesh under the strain induced by the FtsZ ring is always minimal and the insertion of new cell wall material near division midplane at the point of inflection in the dividing cell is essential. Using this model we can predict the force due to the contractile ring given specific elastic parameters and growth rate.

A fundamental question in microbial ecology relates to community structure, and how this varies across environment types. It is widely believed that some environments, such as those at very low pH, host simple communities based on the low number of taxa, possibly due to the extreme environmental conditions. However, most analyses of species richness have relied on methods that provide relatively low ribosomal RNA (rRNA) sampling depth. Here we used community transcriptomics to analyze the microbial diversity of natural acid mine drainage biofilms from the Richmond Mine at Iron Mountain, California. Our analyses target deep pools of rRNA gene transcripts recovered from both natural and laboratory-grown biofilms across varying developmental stages. In all, 91.8% of the ∼ 254 million Illumina reads mapped to rRNA genes represented in the SILVA database. Up to 159 different taxa, including Bacteria, Archaea and Eukaryotes, were identified. Diversity measures, ordination and hierarchical clustering separate environmental from laboratory-grown biofilms. In part, this is due to the much larger number of rare members in the environmental biofilms. Although Leptospirillum bacteria generally dominate biofilms, we detect a wide variety of other Nitrospira organisms present at very low abundance. Bacteria from the Chloroflexi phylum were also detected. The results indicate that the primary characteristic that has enabled prior extensive cultivation-independent 'omic' analyses is not simplicity but rather the high dominance by a few taxa. We conclude that a much larger variety of organisms than previously thought have adapted to this extreme environment, although only few are selected for at any one time.

The shape of the bacteria cell wall during division and the deformation at the division plane are a function of the material properties of the cell wall, the growth rate, and the force due to the FtsZ ring. We have obtained 23 cryo-EM tomographic reconstructions through a time course following the cell division process in Caulobacter crescentus. We have also acquired over 300 images of the process by high resolution cryo-EM. The cell membranes have been segmented by an in-house developed utility, and represented on a canonical triangular mesh. These cell membrane surfaces provide us local curvatures; together with independently determined elastic parameters for the membrane, these data allow us to estimate the local forces exerted during the process. Our current models support the conclusion that cell growth and cell contraction occur on similar timescales, and that the contractile force at the division midplane is minimal, serving to direct the growth of the peptidoglycan mesh very near the division plane so as to eventually pinch and isolate the two halves of the dividing cell. The potential energy stored in the peptidoglycan mesh under the strain induced by the FtsZ ring is always minimal while insertion of new cell wall material near the division midplane at the point of inflection in the dividing cell is essential. During the late stages of the Caulobacter crescentus cell division process, the inner membrane (IM) separates from the cell wall and proceeds to compartmentalize the cytoplasmic spaces of mother and daughter cells. The outer membrane (OM) and S-layer lag behind forming a narrow tubular structure that keeps both cells connected until the IM process is completed. The shape of the IM surface evolves in a seemingly independent process. We compare the modeled membrane surfaces obtained from our data with simulated evolutions of close membrane surfaces, with realistic elastic parameters, to try to provide a first quantitative estimate of the work involved in this process. We also illustrate the combined use of cryo electron tomography and spectroscopic difference imaging in the study of sub-cellular structure and subcellular bodies in whole bacteria. We limit our goal and focus to bodies with a distinct elemental composition that is in sufficiently high concentration to provide the necessary signal-to-noise level at the relatively large sample thicknesses of the intact cell. This combination proves very powerful, as demonstrated by the characterization of a phosphorus-rich body in Caulobacter crescentus. We also confirm the presence of a body rich in carbon, demonstrate that these two types of bodies are readily recognized and distinguished from each other, and provide for the first time to our knowledge structural information about them in their intact state. In addition we also show the presence of a similar type of phosphorus-rich body in Deinococcus grandis, a member of a completely unrelated bacteria genus. Cryo electron microscopy and tomography allow the study of the biogenesis and morphology of these bodies at resolutions better than 10 nm, while spectroscopic difference imaging provides a direct identification of their chemical composition.

The aluminosilicate layers of the swelling clay mineral montmorillonite, and the saturated pores they delineate, control the mechanical properties and the transport of solutes in many natural and engineered environments. However, the structural basis of montmorillonite porosity remains poorly characterized due to the difficulty in visualizing hydrated samples in their native state. Here, we used cryogenic transmission electron microscopy (cryo-TEM) and cryo electron tomography (cryo-ET) to show that stacking defects within minimally altered, fully hydrated montmorillonite particles define multiscale porosity networks. Variations in layer lateral dimensions over tens to thousands of nanometers cause a range of topological and dynamic defects that generate pervasive curvature and introduce previously uncharacterized solute transport pathways. Observations of long-range rotational order between neighboring layers indicate that the layer-layer interactions that govern clay swelling involve three dimensional orienting forces that operate across nanoscale pores. These direct observations of the hierarchical structure of hydrated montmorillonite pore networks with nanoscale resolution reveal potentially general aspects of colloidal interactions in fluid-saturated clay minerals.