Cell-cell interaction among Archaea: Ignicoccus and Nanoarchaeum

Focus of our investigations is the structural relationship of two hyperthermophilic Archaea Ignicoccus hospitalis and Nanoarchaeum equitans. Ignicoccus cells are strict chemolithoautotrophs; they gain their metabolic energy by the oxidation of molecular hydrogen with elemental sulfur. N.equitans, the smallest archaeal cell known today, can only grow in the presence of and direct contact with I. hospitalis cells. The analysis of its very small genome indicates the lack of important biosynthetic pathways for lipids, cofactors, aminoacids, nucleotides. Our main interest is the contact site between both cells: (A) What is the fine structure? We use different techniques for its visualization, like freeze-etching, (serial) ultrathin sections, and tomography (in collaboration with the MPI in Martinsried, Prof. Dr. W. Baumeister). (B) Which molecules are involved? N. equitans: an S-layer with 6fold symmetry; I. hospitalis: an outer membrane. (C) Which biomolecules or metabolites are transported by using which kind of 'transporters'?

Fig. "1": Ignicoccus hospitalis and Nanoarchaeum equitans, Platinum shadowed. Bar: 1 µm

So far, we were able to show that all cells of the genus Ignicoccus are cocci, with an unusual and unique ultrastructure (Rachel et al 2002; Näther und Rachel 2004; Paper et al. 2007). The tightly packed cytoplasm is delineated by a highly dynamic cytoplasmic membrane, forming round or elongated vesicles into the periplasm. The periplasm itself is variable in width, 20 to 400 nm in diameter, and its relative volume is – compared with other Archaea – unusually large, much larger than the cytoplasm. The outer boundary of the periplasm is an outer membrane, the first and still only one observed among the Archaea. It is composed of phytanyl ether lipids and pore-forming proteins. The major, dominating protein Imp1227 was recently characterized in detail (Burghardt et al. 2007). The mass of its monomer is only 6.2 kDa, forming stable oligomers (they do not dissociate even at 100°C in 2% SDS). These complexes with a diameter of 7 nm are tightly packed in the outer membrane and possess a central pore, 2 nm in diameter. Current investigations aim to purify it to homogeneity, to characterize its detailed structure by crystallography (in collaboration with the Biocentre in Basel, Prof. Dr. Tilman Schirmer), and to study its function, by analyzing the specificity of the pore, in artificial lipid membranes. In addition, we want to understand the function of this protein and of 'flagella'-like appendages (see project 2) for the docking of Nanoarchaeum equitans to Ignicoccus hospitalis.

Supported by a grant of the DFG (SPP 1112)

Coworker: Tillmann Burghardt, Carolin Meyer, Dr. Harald Huber, Reinhard Rachel

Literature:

• H. Huber, S. Burggraf, T. Mayer, I. Wyschkony, R. Rachel, and K.O. Stetter (2000). Ignicoccus gen. nov., a novel genus of hyperthermophilic, chemolithoautotrophic archaea, represented by two new species, Ignicoccus islandicus sp.nov. and Ignicoccus pacificus sp. nov. Int. J. Syst. Evol. Microbiol. 50: 2093-2100
• R. Rachel, I. Wyschkony, S. Riehl, and H. Huber (2002). The ultrastructure of Ignicoccus: Evidence for a novel outer membrane and for intracellular vesicle budding in an archaeon. Archaea 1: 9-18.
• H. Huber, M.J. Hohn, R. Rachel, T. Fuchs, V.C. Wimmer, and K.O. Stetter (2002). A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417: 63-67
• D.J. Näther and R. Rachel (2004). The outer membrane of the hyperthermophilic archaeon Ignicoccus: dynamics, ultrastructure and composition. Biochem. Soc. Transact. 32: 199-203
• T. Burghardt, D.J. Näther, B. Junglas, H. Huber and R. Rachel (2007). The dominating outer membrane protein of the hyperthermopilic Archaeum Ignicoccus hospitalis: a novel pore-forming complex. Mol. Microbiol. 63: 166-176
• W. Paper, U. Jahn, M.J. Hohn, M. Brandl, D.J. Näther, T. Burghardt, R. Rachel, K.O. Stetter and H. Huber (2007). Ignicoccus hospitalis sp. nov., the host of Nanoarchaeum equitans. Int J Syst Evol Microbiol 57: 803-808

Fig. "2": 1. I. hospitalis and N. equitans, freeze-etched. Inset: Surface relief reconstruction of the N. equitans S-layer. 2. I. hospitalis and N. equitans, ultrathin section. Inset: electrophoretic separation of the cell envelope proteins of I. hospitalis

Cell appendages of species of the genus Ignicoccus

Ignicoccus cells were described to possess and express up to 10 extracellular appendages, which, in contrast to the initial description, are not seen to be used for motility. At least, in all observations done so far, there is no evidence for a specific motility of Ignicoccus cells. In contrast, Daniel Müller and Carolin Meyer (diploma theses) were able to show that Ignicoccus cells tightly adher to a large variety of surfaces, most likely using these appendages. For these structures of two species, I.hospitalis and I.pacificus, a purification protocol was established and the major protein identified; the main protein is similar, but clearly not identical. The structure of the filament is a three-stranded helix. At least in I. pacificus, two further appendages were identified, with a completely different architecture and unknown function.

Transmissions-Elektronenmikroskopische Aufnahme von I.hospitalis mit 'Anhängseln', nach Platin-Bedampfung. Balken: 1 µm

Supported by a grant of the DFG

Coworker: Carolin Meyer, Nadine Wasserburger, Prof. Dr. Reinhard Wirth, Reinhard Rachel

Directed dissolution of Pyrit using acidophilic Microorganisms: Cell-surface interactions

In this project, we employ fluorescence light microscopy and scanning and transmission electron microscopy, in order to study the etching of Pyrite by four different microorganisms, two Bacteria and two Archaea. The major factors investigated are: the influence of time of incubation, the surface of the pyrite crystal, on which the microorganisms prefer to adhere; and the type of microbial surface structures involved.

Supported by a grant of the DFG

Coworker: Andreas Klingl, Cordula Neuner, Harald Huber, Michael Thomm, Reinhard Rachel; in collaboration with Prof. Dr. G. Schmalz, University Hospital Regensburg, Dentistry, and Prof. Dr. W. Depmeier and Katja Etzel, University of Kiel, Institute for Geoscience.

Ultrastructure of Mouse Kidney

The major aim of this project is the investigation of kidney ultrastructure of mice in which selected genes have been "knocked-out". The preparation involves ultrathin sections of kidney samples prepared by standard embedding techniques following perfusion fixation; in addition, micro-biopsies are prepared by high-pressure freezing, freeze-substiution, resin-embedding and ultrathin sectioning. In particular, we want to study the ultrastructural changes at the glomerulum and at proximal and distal tubuli.

Supported by a grant of the DFG (SFB 699, Projekt Z2)

Principal investigators: Prof. Dr. R. Witzgall, Prof. Dr. E. Tamm, Prof. Dr. A. Kurtz, Prof. Dr. R. Warth

Niere, Podozylenmembran, Ultradünnschicht

Niere, distaler Tubulus, Ultradünnschnitt

The Faculty Centre for EM also has a preparation lab, including:

a high-pressure freezer (Leica EM-PACT 2) two freeze-substitution unit (Leica AFS 2; home-made unit, according to ETH Zürich, Dr. M.Müller and Dr. R.Hermann) a freeze-etching / metal shadowing unit, with three electron gun evaporation unit (two for Pt/C, 45 deg and 15 deg; one for pure Carbon 90 deg), incl. a sample rotation holder and two film thickness monitors (CFE 50; Cressington, Watford, England) embedding in resin (Epon; Lowicryl) two ultramicrotomes (Leica) Methods which are used include: freeze-etching heavy-metal shadowing negative staining (viruses, proteins) ultrathin sections immun-localization on sections or replicas digital image reconstruction, incl. crystallography and tomography

• Schwerpunktpraktikum Organism. Mikrobiologie im Sommersemester
• Elektronenmikroskopische Methoden in der Mikrobiologie (5st. Schwerpunktpraktikum als 14-tägiges Blockpraktikum; im März , nach Vereinbarung
• Literaturseminar "Membranproteine" (1st.)