Comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea

Background Metal sulfide mineral dissolution during bioleaching and acid mine drainage (AMD) formation creates an environment that is inhospitable to most life. Despite dominance by a small number of bacteria, AMD microbial biofilm communities contain a notable variety of coexisting and closely related Euryarchaea, most of which have defied cultivation efforts. For this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring AMD archaea. Our analyses targeted members of the Thermoplasmatales and related archaea. These results greatly expand genomic information available for this archaeal order. Results We reconstructed near-complete genomes for uncultivated, relatively low abundance organisms A-, E-, and Gplasma, members of Thermoplasmatales order, and for a novel organism, Iplasma. Genomic analyses of these organisms, as well as Ferroplasma type I and II, reveal that all are facultative aerobic heterotrophs with the ability to use many of the same carbon substrates, including methanol. Most of the genomes share genes for toxic metal resistance and surface-layer production. Only Aplasma and Eplasma have a full suite of flagellar genes whereas all but the Ferroplasma spp. have genes for pili production. Cryogenic-electron microscopy (cryo-EM) and tomography (cryo-ET) strengthen these metagenomics-based ultrastructural predictions. Notably, only Aplasma, Gplasma and the Ferroplasma spp. have predicted iron oxidation genes and Eplasma and Iplasma lack most genes for cobalamin, valine, (iso)leucine and histidine synthesis. Conclusion The Thermoplasmatales AMD archaea share a large number of metabolic capabilities. All of the uncultivated organisms studied here (A-, E-, G-, and Iplasma) are metabolically very similar to characterized Ferroplasma spp., differentiating themselves mainly in their genetic capabilities for biosynthesis, motility, and possibly iron oxidation. These results indicate that subtle, but important genomic differences, coupled with unknown differences in gene expression, distinguish these organisms enough to allow for co-existence. Overall this study reveals shared features of organisms from the Thermoplasmatales lineage and provides new insights into the functioning of AMD communities.United States. Dept. of Energy. Genomics:GTL (Grant DE-FG02-05ER64134)National Science Foundation (U.S.). Graduate Research Fellowshi

Mariprofundus ferrooxydans PV-1 the First Genome of a Marine Fe(II) Oxidizing Zetaproteobacterium

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 6 (2011): e25386, doi:10.1371/journal.pone.0025386.Mariprofundus ferrooxydans PV-1 has provided the first genome of the recently discovered Zetaproteobacteria subdivision. Genome analysis reveals a complete TCA cycle, the ability to fix CO2, carbon-storage proteins and a sugar phosphotransferase system (PTS). The latter could facilitate the transport of carbohydrates across the cell membrane and possibly aid in stalk formation, a matrix composed of exopolymers and/or exopolysaccharides, which is used to store oxidized iron minerals outside the cell. Two-component signal transduction system genes, including histidine kinases, GGDEF domain genes, and response regulators containing CheY-like receivers, are abundant and widely distributed across the genome. Most of these are located in close proximity to genes required for cell division, phosphate uptake and transport, exopolymer and heavy metal secretion, flagellar biosynthesis and pilus assembly suggesting that these functions are highly regulated. Similar to many other motile, microaerophilic bacteria, genes encoding aerotaxis as well as antioxidant functionality (e.g., superoxide dismutases and peroxidases) are predicted to sense and respond to oxygen gradients, as would be required to maintain cellular redox balance in the specialized habitat where M. ferrooxydans resides. Comparative genomics with other Fe(II) oxidizing bacteria residing in freshwater and marine environments revealed similar content, synteny, and amino acid similarity of coding sequences potentially involved in Fe(II) oxidation, signal transduction and response regulation, oxygen sensation and detoxification, and heavy metal resistance. This study has provided novel insights into the molecular nature of Zetaproteobacteria.Funding has been provided by the NSF Microbial Observatories Program (KJE, DE), NSF’s Science and Technology Program, by the Gordon and Betty Moore Foundation (KJE), the College of Letters, Arts, and Sciences at the University of Southern California (KJE), and by the NASA Astrobiology Institute (KJE, DE). Advanced Light Source analyses at the Lawrence Berkeley National Lab are supported by the Office of Science, Basic Energy Sciences, Division of Materials Science of the United States Department of Energy (DE-AC02-05CH11231)

Cryo Electron Tomography Studies of Bacteria Cell Division and Chromosome Organization

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

Cryo Electron Tomography Studies of Bacteria Cell Division and Chromosome Organization

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

Inter-species interconnections in acid mine drainage microbial communities

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

DNA Structure within a Virus Particle

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