Untersuchungen zur Energiegewinnung des hyperthermophilen, schwefelreduzierenden Archaeons Ignicoccus hospitalis

Im Rahmen dieser Arbeit wurde versucht, die A1AO ATP-Synthase von I. hospitalis zu reinigen und die Untereinheitenzusammensetzung des Komplexes zu bestimmen. Obwohl der gekoppelte Komplex erfolgreich durch das Detergenz DDM (n-Dodecyl-β-D-Maltopyranosid) aus der Membran herausgelöst werden konnte, war eine Reinigung des Gesamtkomplexes bisher nicht möglich. Zahlreiche Versuche, das Enzym über säulenchromatographische Verfahren zu reinigen, führten lediglich zu einer Anreicherung der dissoziierten A1- und AO-Subkomplexe der ATP-Synthase. Eine Identifizierung der Untereinheiten war durch eine Kombination von 2D-Native/SDS-PAGE, Western-Blot-Analysen und MALDI-TOF MS/MS möglich. So konnte die in vivo Expression von acht annotierten Untereinheiten der ATP-Synthase (A, B, C, D, E, F, a(I) und c(K)) bestätigt und das Protein Igni1215 als Bestandteil der ATP-Synthase (Untereinheit H) identifiziert werden. Die beiden erhaltenen Subkomplexe bestanden aus A, B, E und F (A1) und aus C, D, H und a (AO). Die Untereinheit c war nur unregelmäßig als Bestandteil des AO-Subkomplexes nachzuweisen. Der A1-Subkomplex wurde erfolgreich über zwei Chromatographien gereinigt. Durch LILBID-MS wurde die molekulare Masse des A1-Subkomplexes auf 390 kDa und die Stöchiometrie zu A3B3EF bestimmt. Erste Kristallisationsversuche mit dem gereinigten A1-Komplex führten bereits zu einem Kristall, der eine Röntgenbeugung mit Reflexen von bis zu 2,8 Å erreichte. Allerdings war der Kristall zu klein, um einen vollständigen Datensatz für eine erfolgreiche Strukturaufklärung zu sammeln. Die ATP-Synthesefähigkeit der A1AO ATP-Synthase wurde durch sogenannte ‚pH-Jump‘ Experimente an frischen, intakten I. hospitalis-Zellen bewiesen. Hierbei konnte die ATP-Synthese durch den künstlich angelegten Protonengradienten signifikant stimuliert und durch die Behandlung mit den Inhibitoren DES, DCCD, TBT sowie dem Protonophor TCS inhibiert werden. Das Fehlen eines Na+-Bindemotivs in der c-Untereinheit deutet darauf hin, dass I. hospitalis Protonen als Kopplungsionen verwendet. Die subzelluläre Lage der A1AO ATP-Synthase wurde nach Immunmarkierung von ganzen I. hospitalis-Zellen und an Ultradünnschnitten durch fluoreszenz- und elektronenmikroskopische Untersuchungen geklärt. Überraschenderweise zeigten die Immunmarkierungsversuche, dass die ATP-Synthase nicht in der inneren, sondern in der äußeren Membran von I. hospitalis lokalisiert ist. In gleicher Weise wurde auch die H2:Schwefel-Oxidoreduktase in der äußeren Membran lokalisiert, was zu dem Schluss einer energetisierten äußeren Membran von I. hospitalis und einer ATP-Synthese im Intermembran-Kompartiment führte. Im Gegensatz dazu wurden DNA, RNA und Ribosomen im Cytoplasma lokalisiert. Folglich findet die Energiekonservierung in I. hospitalis räumlich getrennt von der Informationsprozessierung statt. Damit wurde für I. hospitalis eine funktionelle Kompartimentierung nachgewiesen, die derart für einen Prokaryonten noch nicht beschrieben wurde

Proteomic Characterization of Cellular and Molecular Processes that Enable the Nanoarchaeum equitans-Ignicoccus hospitalis Relationship

Nanoarchaeum equitans, the only cultured representative of the Nanoarchaeota, is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. The molecular mechanisms that enable this relationship are unknown. Using whole-cell proteomics, differences in the relative abundance of >75% of predicted protein-coding genes from both Archaea were measured to identify the specific response of I. hospitalis to the presence of N. equitans on its surface. A purified N. equitans sample was also analyzed for evidence of interspecies protein transfer. The depth of cellular proteome coverage achieved here is amongst the highest reported for any organism. Based on changes in the proteome under the specific conditions of this study, I. hospitalis reacts to N. equitans by curtailing genetic information processing (replication, transcription) in lieu of intensifying its energetic, protein processing and cellular membrane functions. We found no evidence of significant Ignicoccus biosynthetic enzymes being transported to N. equitans. These results suggest that, under laboratory conditions, N. equitans diverts some of its host's metabolism and cell cycle control to compensate for its own metabolic shortcomings, thus appearing to be entirely dependent on small, transferable metabolites and energetic precursors from I. hospitalis

The Iho670 Fibers of Ignicoccus hospitalis: a New Type of Archaeal Cell Surface Appendage ▿

Ignicoccus hospitalis forms many cell surface appendages, the Iho670 fibers (width, 14 nm; length, up to 20 μm), which constitute up to 5% of cellular protein. They are composed mainly of protein Iho670, possessing no homology to archaeal flagellins or fimbrins. Their existence as structures different from archaeal flagella or fimbriae have gone unnoticed up to now because they are very brittle

Three Multiheme Cytochromes c from the Hyperthermophilic Archaeon Ignicoccus hospitalis: Purification, Properties and Localization.

Three different multiheme cytochromes c were purified from cell extracts of the hyperthermophilic archaeon Ignicoccus hospitalis. One tetraheme cytochrome, locus tag designation Igni_0530, was purified from membrane fractions together with the iron-sulfur protein Igni_0529. Two octaheme cytochromes, Igni_0955 and Igni_1359, were purified from soluble fractions but were also present in the membrane fraction. N-terminal sequencing showed that three of the four proteins had their signal peptides cleaved off, while results were ambigious with Igni_0955. In contrast, mass spectrometry of Igni_0955 and Igni_1359 resulted in single mass peaks including the signal sequences and eight hemes per subunit so that both forms might be present in the cell. Igni_0955 and Igni_1359 belong to the hydroxylamine dehydrogenase family (HAO; 29% mutual identity). HAO or reductase activities with inorganic sulfur compounds were not detected. Igni_0955 was reduced by enriched I. hospitalis hydrogenase at a specific activity of 243 nmol (min•mg hydrogenase)-1 while activity was non-existent with Igni_0530 and low with Igni_1359. Immuno-electron microscopy of ultrathin sections showed that Igni_0955 and Igni_1359 are located in both I. hospitalis membranes and also in the intermembrane compartment. We concluded that these cytochromes might function as electron shuttles between the hydrogenase in the outer cellular membrane and cellular reductases whereas Igni_0530 might be part of the sulfur-reducing machinery

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

Three multihaem cytochromes c from the hyperthermophilic archaeon Ignicoccus hospitalis: purification, properties and localization

Three different multihaem cytochromes c were purified from cell extracts of the hyperthermophilic archaeon Ignicoccus hospitalis. One tetrahaem cytochrome, locus tag designation Igni_0530, was purified from membrane fractions together with the iron–sulfur protein Igni_0529. Two octahaem cytochromes, Igni_0955 and Igni_1359, were purified from soluble fractions but were also present in the membrane fraction. N-terminal sequencing showed that three of the four proteins had their signal peptides cleaved off, while results were ambiguous for Igni_0955. In contrast, mass spectrometry of Igni_0955 and Igni_1359 resulted in single mass peaks including the signal sequences and eight haems per subunit and so both forms might be present in the cell. Igni_0955 and Igni_1359 belong to the hydroxylamine dehydrogenase (HAO) family (29% mutual identity). HAO or reductase activities with inorganic sulfur compounds were not detected. Igni_0955 was reduced by enriched I. hospitalis hydrogenase at a specific activity of 243 nmol min^-1 (mg hydrogenase)^-1 while activity was non-existent for Igni_0530 and low for Igni_1359. Immuno-electron microscopy of ultra-thin sections showed that Igni_0955 and Igni_1359 are located in both I. hospitalis membranes and also in the intermembrane compartment. We concluded that these cytochromes might function as electron shuttles between the hydrogenase in the outer cellular membrane and cellular reductases, whereas Igni_0530 might be part of the sulfur-reducing mechanism