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.

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.

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 microm(3) (mean approximately 0.03+/-0.01 microm(3)), only approximately 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.

Three-dimensional architecture of the bacteriophage phi29 packaged genome and elucidation of its packaging process.

The goal of the work reported here is to understand the precise molecular mechanism of the process of DNA packaging in dsDNA bacteriophages. Cryo-EM was used to directly visualize the architecture of the DNA inside the capsid and thus to measure fundamental physical parameters such as inter-strand distances, local curvatures, and the degree of order. We obtained cryo-EM images of bacteriophage that had packaged defined fragments of the genome as well as particles that had partially completed the packaging process. The resulting comparison of structures observed at intermediate and final stages shows that there is no unique, deterministic DNA packaging pathway. Monte Carlo simulations of the packaging process provide insights on the forces involved and the resultant structures.