1. Investigations on the novel purely archaeal biocoenosis Ignicoccus hospitalis and Nanoarchaeum equitans.

From a submarine hydrothermal system north off Iceland (Kolbeinsey Ridge) a unique biocoenosis represented by two Archaea was discovered by us. It consists of the hyperthermophilic Archaea Ignicoccus hospitalis and Nanoarchaeum equitans, which form a so-called “intimate association” which at the moment cannot be assigned to one of the classic forms of a symbiosis, commensalisms or parasitism. Nevertheless, growth of N. equitans requires obligately the presence of its host I. hospitalis. Therefore, our data suggest a modulated parasitic lifestyle of N. equitans with its host I. hospitalis.
Our investigations demonstrate that both organisms exhibit a great variety of highly interesting features.

Fig 1

Electron-microscopic and fluorescence images of the co-culture of Nanoarchaeum equitans - Ignicoccus hospitalis
TEM a) Freeze etching of an I. hospitalis cell with four attached N. equitans cells.
TEM b) Ultrathin section of two Nanoarchaeum cells, attached to the outer membrane of an Ignicoccus cell.
TEM c) Ignicoccus cell with several Nanoarchaeum cells, platinum shadowed.
CLSM) Image from a confocal laser-scanning-microscope: co-culture of Nanoarchaeum equitans (small cocci, red) und Ignicoccus hospitalis (large cocci, green) after sequence specific (ss rRNA) fluorescence staining.

Scale bar for all figures: 1.0 µm

1.1. Nanoarchaeum equitans:
Nanoarchaeum equitans („the riding dwarf“), grows exclusively on the surface of Ignicoccus hospitalis („the friendly fire sphere“) (Abb. 1). It thrives in his biotope at temperatures up to 100 °C and could be cultivated for the first time in our lab. It is the smallest microorganism known to date, tiny spheres with a diameter of only 400 nm (= 0.0004 mm). By this they exhibit a cell volume of less than 1 % of an E. coli cell which is already in the range of large viruses, like the pox virus. The analysis of the genome of N. equitans revealed that its size is only about 490,000 base pairs and therefore the smallest archaeal genome and one of the smallest of all prokaryotic cells. Almost no genes for metabolic properties and biosynthesis could be detected which further demonstrates the dependence on its host I. hospitalis. The growth requirements of Nanoarchaeum equitans – temperatures up to 100 °C, no oxygen in the atmosphere, use of sulfur and volcanic gases - are in line with the environmental conditions of the ancient Earth, about 3.8 billon years ago. Therefore, N. equitans may probably represent a quite primitive form of live, possible even a kind of living fossil from the very beginning of live on Earth. Due to the unprecedented sequence of the ribosomal nucleic acid (16S rRNA) N. equitans resembles a separate kingdom within the Archaea, the Nanoarchaeota. Based on genome sequence data, they branch off very deep in the universal phylogenetic tree of live. As a further consequence, DNA of the Nanoarchaeota could not be detected in environmental samples by PCR techniques even with primers considered so far as “universal”. However, we were meanwhile able to detect gene sequences of further representatives of the Nanoarchaeota by the use of molecular techniques in other continental and marine hydrothermal systems.
Due to our results this novel kingdom of Archaea can be further investigated, now.
Growth studies and optimization experiments (also carried out in our 300 l fermenters) enable us to obtain at least some cell masses for biochemical and molecular investigations.

1.2. Ignicoccus hospitalis:
Like the two other members of the genus Ignicoccus (I. pacificus and I. islandicus) I. hospitalis is a hyperthermophilic (heat loving) microorganism, growing optimally at 90°C. All Ignicoccus species are strict anaerobes, i.e. they are unable to grow in the presence of oxygen. They produce all cell components from CO2. Therefore, they do not depend on organic substrates in the culture medium. They gain energy by reduction of elemental sulfur using molecular hydrogen as electron donor. Members of the genus Ignicoccus are the only Archaea which possess two membranes in their cell wall (Fig. 2). These membranes are highly distinct in their lipid- and protein composition. Ultrathin sections show a densely packed cytoplasm, surrounded by a typical archaeal membrane („inner membrane“, IM). A second membrane, „Outer Cellular Membrane (OMC)" encloses a huge InterMembrane Compartment (IMC) exhibiting a width up to 1 µm (= 2 to 3-times the volume of the cytoplasm). Despite the great similarity of the different Ignicoccus species, only I. hospitalis can serve as host for N. equitans.

Figure 2:
Ignicoccus hospitalis, ultrathin section

C = Cytoplasm
IM = inner membrane

IMC = Intermembrane compartment
OCM = outer cellular membrane
V = vesicles

Scale bar: 0,5 µm

1.3. Metabolic pathways in the biocenoesis of Ignicoccus hospitalis and Nanoarchaeum equitans:

Nanoarchaeum equitans has a highly reduced genome lacking nearly all genes for metabolic and biosynthetic pathways. It is therefore reasonable that all biosynthetic processes for the formation of cellular building blocks in this biocenosis are conducted by the host Ignicoccus hospitalis. Hence, a prerequisite for the understanding the metabolism of N. equitans is the knowledge on the metabolism of I. hospitalis.

We were able to show that I. hospitalis uses a so far unknown pathway for CO₂ fixation (Fig. 3a and 3b): As a first step, acetyl-CoA is reductively carboxylated to pyruvate, which is converted into phosphenolpyruvate (PEP). The second CO₂ fixation step is the carboxylation of PEP to oxaloacetate. Oxaloacetate is part of an incomplete reductive acid cycle lacking a 2-oxoglutarate: ferredoxin oxidoreductase. The regeneration of the primary acceptor molecule acetyl-CoA (Fig. 3b) starts with the reduction of oxaloacetate to succinyl-CoA. Succinyl-CoA is then reduced to 4-hydroxybutyrate, which is activated to the CoA thioester. 4-Hydroxybutyryl-CoA is dehydrated to crotonyl-CoA by the radical enzyme 4-hydroxybutyryl-CoA dehydratase. Finally, beta oxidation of crotonyl-CoA leads to two molecules of acetyl-CoA. Thus, the cyclic pathway forms an extra molecule of acetyl-CoA, with pyruvate synthase and PEP carboxylase as the carboxylating enzymes. The pathway the 6th known and is named dicarboxylate/4-hydroxybutyrate cycle.

The analyses of further pathways of the central carbon metabolism in I. hospitalis showed the presence of unconventional biosynthetic pathways, like the 2-aminoadipate pathway for biosynthesis of lysine, the citramalate pathway for biosynthesis of isoleucine and the ribulose monophosphate pathway for the biosynthesis of pentose phosphates.

These comprehensive analyses were performed in cooperation with Prof. G. Fuchs, Institute for Microbiology, University of Freiburg, and with PD Dr. W. Eisenreich, Institute for Biochemistry, Technical University of Munich.

fig.3a
fig 3b

Figure 4 (left):
A Electron micrograph of an Ignicoccus hospitalis cell. The black dots show the location of the ATP-synthase complexes in the outer cellular membrane (OCM) of I. hospitalis (obtained by specific antibody labelling). Ultrathin section, (C = cytoplasm; IM = inner membrane, OCM = outer cellular membrane: IMC = inter-membrane compartment).
scale bar: 1 µm

B: I. hospitalis cell: phase contrast image combined with specific DNA staining (by "DAPI").
Scale bar: 2 µm.

C: The same I. hospitalis cell as shown in „B“. Fluorescence image of a combination of DAPI staining (blue, cytoplasm) and labelling of the ATP-synthase-complexes with specific antibodies (green, outer cellular membrane). 
Scale bar: 2 µm.

Figure 5 (right):
Scheme of the cell envelope of I. hospitalis; included are the localisation of membrane protein complexes (hypothesis!)

2.) Isolation and characterization of novel hyperthermophilic Archaea from high temperature ecosystems:
Hyperthermophilic members of the Archaea (growing optimally at temperatures above 80 °C) have been isolated from numerous continental high temperature ecosystems, like Iceland, Italy, Yellowstone National Park or from submarine hydrothermal systems at the Mid Atlantic Ridge or the South Pacific Ridge. They represent deep branching lineages in the universal tree of life and are therefore highly interesting for the evolution of life. The main topics of our research activities are the enrichment of such microorganisms, the development of new cultivation techniques, the physiological, biochemical, and molecular characterization of the isolates, and the determination of their position in the universal phylogenetic tree of life. These organisms are also cultivated in large scale (up to 300 l) in our fermentation plant, producing cell masses for molecular investigations. This includes also optimization of the culture conditions and the culture media (e.g. by the use of ICP-analyses).

3.) Survival of thermophilic and hyperthermophilic Archaea in space:
Hyperthermophilic Archaea thrive in extreme biotopes, reminding to those of the ancient earth (absence of oxygen, high temperatures, presence of reducing gases like H2S, H2, NH3). Therefore, in recent times they became model organisms for investigations on the origin of life. In cooperation with the Deutsches Zentrum für Luft- und Raumfahrt (DLR, Cologne, Inst. of Aerospace Medicine) we investigate, if Archaea can survive under simulated space conditions. Questions to be answered are: can these organisms be spread by meteorites; can they grow on moons or planets which show appropriate growth conditions (e.g. like Mars). Hyperthermophilic Archaea seem to be highly suitable for these experiments, since they exhibit quite efficient repair mechanisms and other cellular adaptations to extreme environmental conditions, like a higher stability of cellular components. Therefore, it might be that they are able to compensate cell damages caused by impacts, high vacuum, extreme dryness or radiation. In preliminary experiments nine out of sixteen Archaea species exhibited high resistances to dryness and / or UV-radiation. Interestingly, great differences even between closely related species were obvious. In the future, data should be obtained for further organisms and the molecular bases for the resistances will be investigated.

(März 2011)

1.) Supported by DFG-Project HU703/2-1
2.) Supported by Spacelife-Project
3.) Collaborations with industries
4.) Former Projects: DFG-Project HU703/1-1 to HU703/1-3;  TH422/8-1

• Praktikum Mikrobiologie  (Praxismodul B.Sc.)
• Projektpraktikum Organismische Mikrobiologie I (Projektmodul, B.Sc.)
• Projektpraktikum Organismische Mikrobiologie II (Projektmodul, B.Sc.)
• Forschungspraktikum Mikrobiologie (Projektmodul III, B.Sc.)
• Mikrobiologisches Laborpraktikum für Masterstudenten (Schwerpunktmodul)
• Vorlesung und Praktikum: Einführung in die Biochemie, Mikrobiologie und Genetik für Realschule, Hauptschule, Grundschule