Cristina Siegerist

Diverse uncultivated ultra-small bacterial cells in groundwater

Bacteria from phyla lacking cultivated representatives are widespread in natural systems and some have very small genomes. Here we test the hypothesis that these cells are small and thus might be enriched by filtration for coupled genomic and ultrastructural characterization. Metagenomic analysis of groundwater that passed through a ~0.2-μm filter reveals a wide diversity of bacteria from the WWE3, OP11 and OD1 candidate phyla. Cryogenic transmission electron microscopy demonstrates that, despite morphological variation, cells consistently have small cell size (0.009±0.002 μm(3)). Ultrastructural features potentially related to cell and genome size minimization include tightly packed spirals inferred to be DNA, few densely packed ribosomes and a variety of pili-like structures that might enable inter-organism interactions that compensate for biosynthetic capacities inferred to be missing from genomic data. The results suggest that extremely small cell size is associated with these relatively common, yet little known organisms.

Three-dimensional analysis of the structure and ecology of a novel, ultra-small archaeon

Fully understanding the biology of acid mine drainage (AMD) is central to our ability to control and manipulate its environmental impact. Although genomics and biogeochemical methods are relatively well established in the field, their combination with high-resolution imaging of intact members of microbial biofilm communities has not yet reached its full potential. Here, we used three-dimensional (3D) cryogenic electron tomography to determine the size and ultrastructure of intact ARMAN cells, a novel ultra-small archaeon, and sought evidence for their interactions with other members of its community. Within acid mine drainage biofilms, apparently free-living ARMAN cells from a deeply branched archaeal lineage have volumes of 0.009–0.04 μm3 (mean ∼0.03±0.01 μm3), only ∼92 ribosomes, yet are frequent hosts for replicating viruses. Organization within the periplasm and partitioning of ribosomes to the inner surface of the cytoplasmic membrane may be factors in size minimization. Most cells contain enigmatic tubular structures of unknown function. The low ribosome copy number per unit volume, indicative of slow growth rates and targeting of cells by diverse viruses may account for the low abundance of ARMAN cells compared with other biofilm community members. Our results provide the first 3D analysis of structural features of these novel and enigmatic cells and their interactions with at least two types of viruses. Our findings also emphasize that new biological phenomena remain to be discovered among lower abundance organisms from novel uncultivated lineages.

Progress on H5Part: a portable high performance parallel data interface for electromagnetics simulations

Significant problems facing all experimental and computational sciences arise from growing data size and complexity. Common to all these problems is the need to perform efficient data I/O on diverse computer architectures. In our scientific application, the largest parallel particle simulations generate vast quantities of six-dimensional data. Such a simulation run produces data for an aggregate data size up to several TB per run. Motived by the need to address data I/O and access challenges, we have implemented H5Part, an open source data I/O API that simplifies the use of the Hierarchical Data Format v5 library (HDF5). HDF5 is an industry standard for high performance, cross- platform data storage and retrieval that runs on all contemporary architectures from large parallel supercomputers to laptops. H5Part, which is oriented to the needs of the particle physics and cosmology communities, provides support for parallel storage and retrieval of particles, structured and in the future unstructured meshes. In this paper, we describe recent work focusing on I/O support for particles and structured meshes and provide data showing performance on modern supercomputer architectures like the IBM POWER 5.

Conformational Transitions at an S‐Layer Growing Boundary Resolved by Cryo‐TEM

S-layers are two-dimensional protein or glycoprotein lattices that cover the surfaces of many bacteria and archaea. Because they constitute the first interface of interaction between microorganisms and their environment, hosts, and predators, they are of great biological interest. Moreover, owing to their nanoscale, periodic, porous structure and relative ease of manipulation, they have the potential to be useful for both nano-biotechnological and materials applications. However, details of the assembly process are not yet known for any Slayer and high resolution structural information is very limited. Herein, we report a two-dimensional (2D) structural analysis of the expanding boundary of an isolated Lysinibacillus sphaericus S-layer (SbpA) growing on a graphene support. The results reveal previously unknown steps in the conformational transformation that drives the well-documented non-classical pathway of S-layer assembly and show how the fully-folded oligomeric repeating unit is entropically locked into the ordered array. In addition, our results provide the first demonstration that the unique physical properties of graphene offer superior image quality for cryogenic transmission electron microscopy (cryo-TEM) of biological macromolecules. S-layers assemble from a single protein or glycoprotein sequence to form a 2D lattice that covers the entire cell surface of microorganisms, including the cell poles and division sites. They are non-covalently attached to peptidoglycans and related polymers of gram-positive cell walls, linked to the outer membrane of gram-negative bacterial cell walls, and integrated into the cytoplasmic membrane through trans-membrane domains in gram-negative archaea. The primary sequence of the single protein or glycoprotein species contains all the information needed for assembly. Although they are often called “crystalline” cell surface layers, they are better described as “quasi-crystalline” or “paracrystalline”. S-layers also self-assemble in vitro in the presence of Ca ions, either on support films or in bulk solution, into ordered arrays with long-range order, substantially larger than a single cell. Previous studies found that self-assembly of SbpA (1268 residues from Lysinibacillus sphaericus) on lipid bilayers follows a multi-step pathway. It starts with the aggregation of monomers that adsorb onto the lipids in an extended conformation to form amorphous or liquid-like clusters. These clusters subsequently crystallize into the characteristic lattice of homotetrameric units, which grows by addition of new tetramers to the lattice edge sites. The rate of tetramer addition increases linearly with protein concentration, implying that monomers are added one at a time. However, the pathway through which the individual monomeric units become integrated with the correct conformation into the homotetrameric units—arguably the single most important step in S-layer assembly—remains unknown. To gain insight into S-layer assembly at the level of the tetramer subunits in the intact solution state, we obtained cryo-TEM images of single sheets, plunge-frozen while growing on graphene. Because the active self-assembling Slayers are instantly frozen, all the conformational states present at the expanding boundary on the graphene flat support are captured. Image alignment and averaging provide a view of the steps leading to subunit recruitment and maturation in S-layer self-assembly. For this study, we chose the surface layer protein SbpA (1268 residues), from the gram-positive bacterium Lysinibacillus sphaericus, which naturally forms a 2D quasi-crystalline cell envelope. We carried out the reconstitution of SbpA on single graphene sheets supported by TEM grids designed for cryo-TEM. The great potential of graphene for use as a support for biological cryo-TEM samples has recently been discussed. While mechanically strong and elastic, the 0.246 nm lattice constant and one-atom thickness of a single graphene layer, approximately 0.34 nm, make them trans[*] L. R. Comolli Life Sciences Division, Lawrence Berkeley National Laboratory Berkeley, CA 94720 (USA) E-mail: lrcomolli@lbl.gov

Self-assembly of "S-bilayers", a step toward expanding the dimensionality of S-layer assemblies.

Protein-based assemblies with ordered nanometer-scale features in three dimensions are of interest as functional nanomaterials but are difficult to generate. Here we report that a truncated S-layer protein assembles into stable bilayers, which we characterized using cryogenic-electron microscopy, tomography, and X-ray spectroscopy. We find that emergence of this supermolecular architecture is the outcome of hierarchical processes; the proteins condense in solution to form 2-D crystals, which then stack parallel to one another to create isotropic bilayered assemblies. Within this bilayered structure, registry between lattices in two layers was disclosed, whereas the intrinsic symmetry in each layer was altered. Comparison of these data to images of wild-type SbpA layers on intact cells gave insight into the interactions responsible for bilayer formation. These results establish a platform for engineering S-layer assemblies with 3-D architecture.

Personal Display Wall

The LBNL Visualization Group has created a tiled display wall design that uses components that are readily available from a local hardware store and/or multiple online vendors, and requires minimal tools and skill to assemble. The result is a low-cost, easy to assemble tiled display device that is readily accessible to visualization researchers and domain scientists alike. The LBNL Personal Display (PD) Wall differentiates itself from other LCD-matrix displays because its design minimizes cost and complexity while retaining the functionality of its more expensive tiled display brethren. The PD-Wall occupies the same amount of desktop area as a large flatscreen LCD display panel. LBNL will be publishing and distributing simple plans so that any laboratory or user site can construct their own copies of this device.

VisPortal: Increasing Scientific Productivity by Simplifying Access to and Use of Remote Computational Resources

Our goal is to simplify and streamline the process of using remotely located visual data analysis software tools. This discussion presents an example of an easy-to-use interface that mediates access to and use of diverse and powerful visual data analysis resources. The interface is presented via a standard web browser, which is ubiquitous and a part of every researchers work environment. Through the web interface, a few mouse clicks are all that is needed to take advantage of powerful, remotely located software resources. The VisPortal project is the software that provides diverse services to remotely located users through their web browser. Using standard Globus-grid middleware and off-the-shelf web automation, the VisPortal hides the underlying complexity of resource selection and distributed application management. The portal automates complex workflows that would otherwise require a substantial amount of manual effort on the part of the researcher. With a few mouse clicks, a researcher can quickly perform complex tasks like creating MPEG movies, scheduling file transfers, launching components of a distributed application, and accessing specialized resources.