Reinhard Rachel

A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont

According to small subunit ribosomal RNA (ss rRNA) sequence comparisons all known Archaea belong to the phyla Crenarchaeota, Euryarchaeota, and—indicated only by environmental DNA sequences—to the ‘Korarchaeota’. Here we report the cultivation of a new nanosized hyperthermophilic archaeon from a submarine hot vent. This archaeon cannot be attached to one of these groups and therefore must represent an unknown phylum which we name ‘Nanoarchaeota’ and species, which we name ‘Nanoarchaeum equitans’. Cells of ‘N. equitans’ are spherical, and only about 400 nm in diameter. They grow attached to the surface of a specific archaeal host, a new member of the genus Ignicoccus. The distribution of the ‘Nanoarchaeota’ is so far unknown. Owing to their unusual ss rRNA sequence, members remained undetectable by commonly used ecological studies based on the polymerase chain reaction. ‘N. equitans’ harbours the smallest archaeal genome; it is only 0.5 megabases in size. This organism will provide insight into the evolution of thermophily, of tiny genomes and of interspecies communication.

Layer-by-Layer Assembled Gold Nanoparticles for siRNA Delivery

Although uptake into cells is highly complex and regulated, heterogeneous particle collectives are usually employed to deliver small interfering RNA (siRNA) to cells. Within these collectives, it is difficult to accurately identify the active species, and a decrease in efficacy is inherent to such preparations. Here, we demonstrate the manufacture of uniform nanoparticles with the deposition of siRNA on gold in a layer-by-layer approach, and we further report on the cellular delivery and siRNA activity as functions of surface properties.

Ferroglobus placidus gen. nov., sp. nov., a novel hyperthermophilic archaeum that oxidizes Fe2+ at neutral pH under anoxic conditions

Abstract A novel coccoid, anaerobic, Fe2+-oxidizing archaeum was isolated from a shallow submarine hydrothermal system at Vulcano, Italy. In addition to ferrous iron, H2 and sulfide served as electron donors. NO3– was used as electron acceptor. In the presence of H2, also S2O32– could serve as electron acceptor. The isolate was a neutrophilic hyperthermophile that grew between 65° C and 95° C. It represents a novel genus among the Archaeoglobales that we name Ferroglobus. The type species is Ferroglobus placidus (DSM 10642).

Electron Tomography of Ice-Embedded Prokaryotic Cells

Whole cells of archaea were embedded in vitreous ice by plunge freezing and investigated by automated energy-filtered electron tomography at 120 kV. The embedded cells were between 300 and 750 nm thick, and their structures were reconstructed to a resolution of 20-40 nm from tilt series comprising 50-140 images. The dose was kept within tolerable limits. A resolution of 20 nm allowed visualization of the individual stalks of the S-layer of Pyrobaculum aerophilum cells, which had undergone partial lysis, in three dimensions. The attainable resolution for low-dose electron tomography under different experimental conditions was theoretically investigated in terms of the specimen thickness. To obtain 2-nm resolution at 120 kV (300 kV), the specimen must not be thicker than 100 nm (150 nm). For a resolution of 10 nm, the maximum thickness is 450 nm (700 nm). An accelerating voltage of 300 kV is advantageous, mainly for specimens thicker than 100 nm. Experimental investigations so far have resulted in a resolution that is worse by a factor of 2-5 as compared to theory.

The ultrastructure of Ignicoccus: evidence for a novel outer membrane and for intracellular vesicle budding in an archaeon

A novel genus of hyperthermophilic, strictly chemolithotrophic archaea, Ignicoccus, has been described recently, with (so far) three isolates in pure culture. Cells were prepared for ultrastructural investigation by cultivation in cellulose capillaries and processing by high-pressure freezing, freeze-substitution and embedding in Epon. Cells prepared in accordance with this protocol consistently showed a novel cell envelope structure previously unknown among the Archaea: a cytoplasmic membrane; a periplasmic space with a variable width of 20 to 400 nm, containing membrane-bound vesicles; and an outer sheath, approximately 10 nm wide, resembling the outer membrane of gram-negative bacteria. This sheath contained three types of particles: numerous tightly, irregularly packed single particles, about 8 nm in diameter; pores with a diameter of 24 nm, surrounded by tiny particles, arranged in a ring with a diameter of 130 nm; and clusters of up to eight particles, each particle 12 nm in diameter. Freeze-etched cells exhibited a smooth surface, without a regular pattern, with frequent fracture planes through the outer sheath, indicating the presence of an outer membrane and the absence of an S-layer. The study illustrates the novel complex architecture of the cell envelope of Ignicoccus as well as the importance of elaborate preparation procedures for ultrastructural investigations.

Virology: Independent virus development outside a host

Viruses are thought to be functionally inactive once they are outside and independent of their host cell. Here we describe an exceptional property of a newly discovered virus that infects a hyperthermophilic archaeon growing in acidic hot springs: the lemon-shaped viral particle develops a very long tail at each of its pointed ends after being released from its host cell. The process occurs only at the temperature of the hosts habitat (75–90 °C) and it does not require the presence of the host cell, an exogenous energy source or any cofactors. This host-independent morphological development may be a strategy for viral survival in an environment that is unusually harsh and has limited host availability.

Furrow-like invaginations of the yeast plasma membrane correspond to membrane compartment of Can1

Plasma membrane of the yeast Saccharomyces cerevisiae contains stable lateral domains. We have investigated the ultrastructure of one type of domain, the membrane compartment of Can1 (MCC). In two yeast strains (nce102Δ and pil1Δ) that are defective in segregation of MCC-specific proteins, we found the plasma membrane to be devoid of the characteristic furrow-like invaginations. These are highly conserved plasma membrane structures reported in early freeze-fracture studies. Comparison of the results obtained by three different approaches – electron microscopy of freeze-etched cells, confocal microscopy of intact cells and computer simulation – shows that the number of invaginations corresponds to the number of MCC patches in the membrane of wild-type cells. In addition, neither MCC patches nor the furrow-like invaginations colocalized with the cortical ER. In mutants exhibiting elongated MCC patches, there are elongated invaginations of the appropriate size and frequency. Using various approaches of immunoelectron microscopy, the MCC protein Sur7, as well as the eisosome marker Pil1, have been detected at these invaginations. Thus, we identify the MCC patch, which is a lateral membrane domain of specific composition and function, with a specific structure in the yeast plasma membrane – the furrow-like invagination.

Remarkable morphological diversity of viruses and virus-like particles in hot terrestrial environments.

Summary. Electron microscopic studies of the viruses in two hot springs (85 °C, pH 1.5–2.0, and 75–93 °C, pH 6.5) in Yellowstone National Park revealed particles with twelve different morphotypes. This diversity encompassed known viruses of hyperthermophilic archaea, filamentous Lipothrixviridae, rod-shaped Rudiviridae, and spindle-shaped Fuselloviridae, and novel morphotypes previously not observed in nature. Two virus types resembled head-and-tail bacteriophages from the families Siphoviridae and Podoviridae, and constituted the first observation of these viruses in a hydrothermal environment. Viral hosts in the acidic spring were members of the hyperthermophilic archaeal genus Acidianus.

Viral Diversity in Hot Springs of Pozzuoli, Italy, and Characterization of a Unique Archaeal Virus, Acidianus Bottle-Shaped Virus, from a New Family, the Ampullaviridae

ABSTRACT Virus-like particles with five different morphotypes were observed in an enriched environmental sample from a hot, acidic spring (87 to 93°C, pH 1.5) in Pozzuoli, Italy. The morphotypes included rigid rods, flexible filaments, and novel, exceptional forms. Particles of each type were isolated, and they were shown to represent viable virions of five novel viruses which infect members of the hyperthermophilic archaeal genus Acidianus. One of these, named the Acidianus bottle-shaped virus, ABV, exhibits a previously unreported morphotype. The bottle-shaped virion carries an envelope which encases a funnel-shaped core. The pointed end of the virion is likely to be involved in adsorption and channeling of viral DNA into host cells. The broad end exhibits 20 (± 2) thin filaments which appear to be inserted into a disk, or ring, and are interconnected at their bases. These filaments are apparently not involved in adsorption. ABV virions contain six proteins in the size range 15 to 80 kDa and a 23.9-kb linear, double-stranded DNA genome. Virus replication does not cause lysis of host cells. On the basis of its unique morphotype and structure, we propose to assign ABV to a new viral family, the Ampullaviridae.

Thermococcus chitonophagus sp. nov., a novel, chitin-degrading, hyperthermophilic archaeum from a deep-sea hydrothermal vent environment

Abstract From a hydrothermal vent site off the Mexican west coast (20°50′N, 109°06′W) at a depth of 2,600 m, a novel, hyperthermophilic, anaerobic archaeum was isolated. Cells were round to slightly irregular cocci, 1.2–2.5 μm in diameter and were motile by means of a tuft of flagella. The new isolate grew between 60 and 93°C (optimum: 85°C), from pH 3.5 to 9 (optimum: pH 6.7), and from 0.8 to 8% NaCl (optimum: 2%). The isolate was an obligate organotroph, using chitin, yeast extract, meat extract, and peptone for growth. Chitin was fermented to H2, CO2, NH3, acetate, and formate. H2S was formed in the presence of sulfur. The chitinoclastic enzyme system was oxygen-stable, cell-associated, and inducible by chitin. The cell wall was composed of a surface layer of hex- americ protein complexes arranged on a p6 lattice. The core lipids consisted of glycerol diphytanyl diethers and acyclic and cyclic glycerol diphytanyl tetraethers. The G+C content was 46.5 mol%. DNA/DNA hybridization and 16S rRNA sequencing indicated that the new isolate belongs to the genus Thermococcus, representing a new species, Thermococcus chitonophagus. The type strain is isolate GC74, DSM 10152.