Ignicoccus hospitalis and Nanoarchaeum equitans: ultrastructure, cell–cell interaction, and 3D reconstruction from serial sections of freeze-substituted cells and by electron cryotomography

Ultrastructure and intercellular interaction of Ignicoccus hospitalis and Nanoarchaeum equitans were investigated using two different electron microscopy approaches, by three-dimensional reconstructions from serial sections, and by electron cryotomography. Serial sections were assembled into 3D reconstructions, for visualizing the unusual complexity of I. hospitalis, its huge periplasmic space, the vesiculating cytoplasmic membrane, and the outer membrane. The cytoplasm contains fibres which are reminiscent to a cytoskeleton. Cell division in I. hospitalis is complex, and different to that in Euryarchaeota or Bacteria. An irregular invagination of the cytoplasmic membrane is followed by separation of the two cytoplasms. Simultaneous constriction of cytoplasmic plus outer membrane is not observed. Cells of N. equitans show a classical mode of cell division, by constriction in the mid-plane. Their cytoplasm exhibits two types of fibres, elongated and ring-shaped. Electron micrographs of contact sites between I. hospitalis and N. equitans exhibit two modes of interaction. One is indirect and mediated by thin fibres; in other cells the two cell surfaces are in direct contact. The two membranes of I. hospitalis cells are frequently seen in direct contact, possibly a prerequisite for transporting metabolites or substrates from the cytoplasm of one cell to the other. Rarely, a transport based on cargo vesicles is observed between I. hospitalis and N. equitans

Insight into the proteome of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis: the major cytosolic and membrane proteins

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic Crenarchaeon, is the host of Nanoarchaeum equitans. Together, they form an intimate association, the first among Archaea. Membranes are of fundamental importance for the interaction of I. hospitalis and N. equitans, as they harbour the proteins necessary for the transport of macromolecules like lipids, amino acids, and cofactors between these organisms. Here, we investigated the protein inventory of I. hospitalis cells, and were able to identify 20 proteins in total. Experimental evidence and predictions let us conclude that 11 are soluble cytosolic proteins, eight membrane or membrane-associated proteins, and a single one extracellular. The quantitatively dominating proteins in the cytoplasm (peroxiredoxin; thermosome) antagonize oxidative and temperature stress which I. hospitalis cells are exposed to at optimal growth conditions. Three abundant membrane protein complexes are found: the major protein of the outer membrane, which might protect the cell against the hostile environment, forms oligomeric complexes with pores of unknown selectivity; two other complexes of the cytoplasmic membrane, the hydrogenase and the ATP synthase, play a key role in energy production and conversion

Einblicke in die Interaktion zwischen Ignicoccus hospitalis und Nanoarchaeum equitans: Das Membranprotein Ihomp1 sowie Nachweis weiterer Proteine der Zellhüllen

Das Ziel der vorliegenden Arbeit war die strukturelle und funktionelle Charakterisierung des dominierenden Oberflächenproteins von I. hospitalis sowie die Identifikation weiterer, potenziell am Aufbau der Kontaktstelle zwischen I. hospitalis und N. equitans beteiligter Proteinspezies. Mit Ihomp1 (Ignicoccus hospitalis outer membrane protein 1) konnte das erste Protein einer bislang einzigartigen Membranstruktur in der Domäne der Archaeen charakterisiert werden. Es handelt sich entsprechend bioinformatischer Recherchen um ein kleines, funktionell uncharakterisiertes Protein (6,3 kDa), welches keine signifikanten strukturellen Homologien zu bekannten Proteinspezies aufweist. Wie im Rahmen der vorliegenden Arbeit gezeigt werden konnte, repräsentieren die durch Ihomp1 gebildeten homooligomeren 45 bzw. 50 kDa-Komplexe die funktionelle Einheit der dominierenden, Transmembranporen-formenden 7 nm-Partikel der Zelloberfläche von I. hospitalis und erfüllen möglicherweise eine Funktion in der Versorgung des Organismus mit Nährstoffen, wie dem terminalen Elektronenakzeptor Polysulfid. So konnte durch elektrophysiologische Einzelkanalstudien in künstlichen Systemen bereits eine signifikante Leitfähigkeit der Ihomp1-Komplexe nachgewiesen werden. Immunzytologische Studien an Gefrierätzpräparaten der drei bekannten Spezies der Gattung Ignicoccus belegten, dass es sich bei Ihomp1 um ein spezies-spezifisch exprimiertes Protein handelt. Vorläufige PCR-Analysen wiesen zudem auf ein ebenfalls auf I. hospitalis beschränktes Vorkommen des korrespondierenden Gens igni_1266 hin. Für die strukturelle und funktionelle Charakterisierung der nativen Ihomp1-Komplexe wurde ein mehrstufiges Aufreinigungsprotokoll etabliert. Mit Hilfe umfangreicher massenspektrometrischer sowie bioinformatischer und CD-spektroskopischer Analysen konnte so für die beiden stabilen 45 und 50 kDa Ihomp1-Komplexe eine einheitliche Stöchiometrie aus zehn Monomeren bestimmt werden, welche die Membran in alpha helikaler Form durchspannen. Eine röntgenkristallographische Untersuchung der Komplexe ist in Bearbeitung. Entsprechend vorläufiger gelchromatographischer Daten wird zudem eine Funktion des Proteins in der Initiation der Zell-Zell-Interaktion zwischen I. hospitalis und N. equitans als spezifisches Erkennungs- bzw. Bindemotiv für das S-Layer-Protein NEQ300 aus N. equitans diskutiert. Im Rahmen der proteinanalytischen Untersuchung der Membranfraktionen aus Rein- und Co-Kultur konnte die Expression von mehr als 280, in den Genomen von I. hospitalis und N. equitans annotierten Proteinspezies nachgewiesen werden. Die Interpretation der daraus gewonnenen Daten in Bezug auf eine mögliche Funktion dieser Proteine im Austausch zwischen I. hospitalis und N. equitans ist jedoch deutlich erschwert, da bioinformatischen Recherchen zufolge für einen Großteil der so identifizierten, mutmaßlichen Membranproteine der beiden Spezies gegenwärtig keine physiologische Funktion vorhergesagt werden kann

The interaction of Nanoarchaeum equitans with Ignicoccus hospitalis: proteins in the contact site between two cells

The two archaea Ignicoccus hospitalis and Nanoarchaeum equitans form a unique intimate association, the character of which is not yet fully understood. Electron microscopic investigations show that at least two modes of cell-cell interactions exist: (i) the two cells are interconnected via thin fibres; and (ii) the two cell surfaces are in direct contact with each other. In order to shed further light on the molecules involved, we isolated a protein complex, by using detergent-induced solubilization of cell envelopes, followed by a combination of chromatography steps. Analysis by MS and comparison with databases revealed that this fraction contained two dominant proteins, representing the respective major envelope proteins of the two archaea. In addition, a considerable set of membrane proteins is specifically associated with these proteins. They are now the focus of further biochemical and ultrastructural investigations

Analysis of the Ultrastructure of Archaea by Electron Microscopy

The ultrastructural characterization of archaeal cells is done with both types of electron microscopy, transmission electron microscopy, and scanning electron microscopy. Depending on the scientific question, different preparation methods have to be employed and need to be optimized, according to the special cultivation conditions of these—in many cases extreme—microorganisms. Recent results using various electron microscopy techniques show that archaeal cells have a variety of cell appendages, used for motility as well as for establishing cell–cell and cell–surface contacts. Cryo-preparation methods, in particular high-pressure freezing and freeze-substitution, are crucial for obtaining results: (1) showing the cells in ultrathin sections in a good structural preservation, often with unusual shapes and subcellular complexity, and (2) enabling us to perform immunolocalization studies. This is an important tool to make a link between biochemical and ultrastructural studies

Ignicoccus hospitalis sp. nov., the host of Nanoarchaeum equitans

A novel chemolithoautotrophic and hyperthermophilic member of the genus Ignicoccus was isolated from a submarine hydrothermal system at the Kolbeinsey Ridge, to the north of Iceland. The new isolate showed high similarity to the two species described to date, Ignicoccus islandicus and Ignicoccus pacificus, in its physiological properties as well as in its unique cell architecture. However, phylogenetic analysis and investigations on the protein composition of the outer membrane demonstrated that the new isolate was clearly distinct from I. islandicus and I. pacificus. Furthermore, it is the only organism known so far which is able to serve as a host for 'Nanoarchaeum equitans', the only cultivated member of the 'Nanoarchaeota'. Therefore, the new isolate represents a novel species of the genus Ignicoccus, which we name Ignicoccus hospitalis sp. nov. (type strain KIN4/I(T)=DSM 18386(T)=JCM 14125(T))

The dominating outer membrane protein of the hyperthermophilic Archaeum Ignicoccus hospitalis: a novel pore-forming complex

The membrane protein Imp1227 (Ignicoccus outer membrane protein; Imp1227) is the main protein constituent of the unique outer sheath of the hyperthermophilic, chemolithoautotrophic Archaeum Ignicoccus hospitalis. This outer sheath is the so far only known example for an asymmetric bilayer among the Archaea and is named 'outer membrane'. With its molecular mass of only 6.23 kDa, Imp1227 is found to be incorporated into the outer membrane in form of large, stable complexes. When separated by SDS-PAGE, they exhibit apparent masses of about 150, 50, 45 and 35 kDa. Dissociation into the monomeric form is achieved by treatment with SDS-containing solutions at temperatures at or above 113 degrees C. Electron micrographs of negatively stained samples confirm that isolated membranes are tightly packed with round complexes, about 7 nm in diameter, with a central, stain-filled 2 nm pore; a local two-dimensional crystalline arrangement in form of small patches can be detected by tomographic reconstruction. The comparison of the nucleotide and amino acid sequence of Imp1227 with public databases showed no reliable similarities with known proteins. Using secondary structure prediction and molecular modelling, an alpha-helical transmembrane domain is proposed; for the oligomer, a ring-shaped nonamer with a central 2 nm pore is a likely arrangement