Panel 2 (Continued)

Abstract

Hyperthermophilic archaea with optimal growth temperatures above 80° C represent the upper temperature border of life on Earth, occurring in volcanic and deep subterranean hot environments. Most of them are anaerobes able to use inorganic energy and carbon sources. Individual cells from pure cultures of members of the genera Thermoproteus, Pyrobaculum, Thermofilum, Desulfurococcus, Staphylothermus, Thermodiscus, Pyrodictium, Thermococcus, and Pyrococcus exhibit an exceptional variation in size. The volume of cells in the same culture may vary by more than four orders of magnitude. The smallest cell sizes observed in hyperthermophilic archaea are rods 0.17 ^mm in diameter in Thermofilum, spheres 0.3 ^mm in diameter protruding from rod-shaped cells of Thermoproteus and Pyrobaculum, and disks, 0.2 to 0.3 ^mm in diameter and 0.08 to 0.1 ^mm wide in Thermodiscus and Pyrodictium. Pyrodictium forms web-like colonies in the centimeter range, in which the periplasmic space of the cells is connected to each other by a unique matrix of hollow tubules ("cannulae"). As a working hypothesis, the webs for the first time could enable an organism to use thermal gradients as an additional energy source. By their 16S rRNA-phylogeny, size-variable hyperthermophiles represent the shortest lineages closest to the root of the archaeal tree. Therefore, they may still be rather similar to their primitive ancestry at the early, much hotter Earth. The inability to keep their cell volumes constant may be seen as a primitive feature. However, by forming extremely small cells these organisms could be able to pass even pores of rocks in order to colonize deep subterranean environments.

Introduction

The first traces of life on Earth date back to the early Archaean age (Schopf, 1993; Mojzsis et al., 1996). Possibly, life had already existed about 3.9 billion years ago. At that time, there should have been an overall reducing atmosphere and a much stronger volcanism than today (Ernst, 1983). In addition, Earth's oceans were continuously heated by heavy impacts of meteorites. Therefore, within that scenario, early life had to be heat resistant to survive.

During the last decades, hyperthermophilic archaea had been isolated, which grow optimally (fastest) above 80° C, some even above 100° C (Stetter et al., 1981; Zillig et al., 1981; Stetter, 1982; Stetter and Zillig, 1985; Stetter, 1986; Stetter, 1996). Depending on the isolates, their minimum growth temperature is between 45 and 90° C, while their upper temperature border of growth is between 85 and 113° C (Table 1). Cultures of Pyrolobus and Pyrodictium, for the first time are even able to survive one hour autoclaving at 121° C, a kind of simulated "cosmic impact" scenario (Blöchl et al., 1997). Biotopes of hyperthermophiles are water-containing volcanic areas like terrestrial solfataric fields and hot springs, submari1ne hydrothermal systems, sea mounts, and abyssal hot vents ("Black Smokers"). The first evidence for the presence of communities of hyperthermophiles within geothermally heated subterranean rocks 3,500 meters below the surface of North Alaska was demonstrated recently (Stetter et al., 1993). Hyperthermophiles are well adapted to their biotopes, being able to grow at extremes of pH, redox potential, and salinity (see Table 1). Terrestrial hyperthermophiles usually require low salinity, while those of marine biotopes are adapted to the high salinity of sea water. Most hyperthermophiles are strict anaerobes. A great many exhibit a chemolithoautotrophic mode of nutrition: inorganic redox reactions serve as energy sources, and CO₂ is the only carbon source required to build up organic cell material (Table 2). Depending on the organisms, hyperthermophiles are able to use H2, ferrous iron, and reduced sulfur compounds as electron donors. On the other hand, oxidized sulfur compounds, nitrate, ferric iron, CO₂, and O₂ may serve as electron acceptors. Depending on the energy sources available, chemolithoautotrophic hyperthermophiles show great versatility: members of the same genera and even the same strains may be able to use different electron donors and acceptors (see Table 2). In addition, several hyperthermophilic archaea are facultative or obligate heterotrophs able to use organic compounds as energy and carbon sources (see Table 1). Within the 16S rRNA-based phylogenetic tree, hyperthermophiles establish all the short and deep lineages (Figure 1; Woese et al., 1990). Short phylogenetic branches indicate a rather slow evolution. Therefore, by 16S rRNA phylogeny, hyperthermophiles represent the most primitive organisms known so far. The conclusion of thermophily as a primordial feature is in agreement with our picture of the early Earth. In this paper, I present results about variation and lowest limits of cell size within hyperthermophilic archaea

Table 1 Growth Conditions of Some Hyperthermophilic Archaea

Table 2 Energy-yielding Reactions in Hyperthermophilic Archaea (Chemolithoautotrophes)

Figure 1. Hyperthermophiles (bulky lines) within the 16(18)S rRna-based phylogenetic tree.

Morphology and Limits and Variation in Cell Size in Hyperthermophilic Archaea

In line with their great phylogenetic diversity, hyperthermophilic archaea display a variety of different cell morphologies (Table 3). Cells may be regular to irregular cocci, sometimes lobed or wedge-shaped, irregular disks with ultraflat areas, regular rods, or rods with spheres protruding at their ends ("golf clubs"). As usual for prokaryotes, cells in (pure) cultures of the euryarchaeotal Methanothermus, Methanococcus, and Archaeoglobus contain normal-sized rod-shaped or coccoid cells with not much variation in cell volume (see Table 3). The same is true for the coccoid-shaped Sulfolobus and Acidianus within the Crenarchaeota. A very special case is Thermoplasma, a thermoacidophilic heterotrophic cell-wall-less pleomorphic member of the Crenarchaeota. Cells are very flexible and propagate by budding. Cultures of Thermoplasma contain highly irregular cells with great variation in shape and size. The smallest cells observed are very tiny cocci, about 0.2 m in diameter (see Table 3).

Table 3 Morphology and Size of Hyperthermophilic Archaea (Examples)

An unanticipated variation of cell sizes can be observed within pure cultures of members of the Thermococcales, Desulfurococcales, and Thermoproteales, which represent the deepest and shortest phylogenetic branches among the hyperthermophiles (Stetter, 1996): Cultures of Thermococcus and Pyrococcus usually show duplex-shaped irregular spheres, about 0.5 to 2 ^mm in diameter. However, during the early logarithmic growth phase, very tiny frog-egg-shaped cells about 0.2 ^mm in diameter arranged in clusters up to about 20 individuals may be observed. Sometimes, rather large cells show ribbon-like appendages that contain several very small cells in line (Stetter and Zillig, 1985). The function of these tiny cells is still unclear. However, after passing cultures of Pyrococcus through ultrafilters with 0.2 ^mm pore width, viable cultures could be obtained from the filtrates(Stetter, unpublished). Members of Thermoproteus and Thermofilum consist of stiff rectangular rods that show an extraordinary variation in length from about 1 to 100 m. As a rule, during the exponential growth phase, tiny spheres about 0.3 to 0.5^m m in diameter are protruding at one end under an angle of 135°. Similar-sized spheres can be seen in cultures also in free state and may represent an unusual way of cell propagation. Alternatively, cells of Thermoproteus and Thermofilum are able to multiply by regular cell division. Strains of Thermofilum exhibit much thinner rod-shaped cells than Thermoproteus. Sometimes, cells of Thermofilum are only 0.15 to 0.17^mm in diameter, and therefore can hardly be recognized under the phase contrast light microscope, while cells of Thermoproteus consist of rather slim rods, about 0.4 ^mm in diameter (see Table 3).

Cultures of the heterotrophic Staphylothermus and Desulfurococcus reveal spherical cells with enormous variation in diameter between 0.5 and 15 ^mm. Therefore, their cell volume varies by more than four orders of magnitude. At low-nutrient concentrations morphology of cells of Staphylothermus is shifted mainly to giant cells, about 10 to 15 m in diameter. The surface protein assembly of Staphylothermus (and possibly of the related Desulfurococcus) exhibits an unusual filamentous structure of extreme stability (Peters et al., 1995).

Cells of Thermodiscus consist of flat irregular disks, highly variable in diameter between 0.2 and 3 ^mm. They are about 0.1 to 0.2 ^mm wide. Sometimes pili-like structures of 0.01^mm in diameter and up to 15 ^mm in length connect the surfaces of two individuals. In the electron microscope, often extremely small disks, less than 0.2 ^mm in diameter, are seen (Stetter and Zillig, 1985). This observation could explain that the titer as determined by serial dilution is always at least 10 times higher than that determined by direct counting in the light microscope.

Cells of Pyrodictium consist of flat, irregular disks. They are 0.3 to 2.5 ^mm in diameter and may be up to 0.3 ^mm wide. As a rule, cells of Pyrodictium exhibit large ultraflat areas, only about 0.08 ^mm in width (Rieger et al., 1997). Remarkably, Pyrodictium never grows in suspension but in mold-like flakes, several centimeters in diameter (Pley et al., 1991). The flakes are made up by a unique matrix of hollow tubules ("cannulae") in which the cells are integrated (Rieger et al., 1995). Single cannulae are up to 40 ^mm long and 0.026 ^mm in diameter and consist of glycoprotein subunits in helical array. The cannulae penetrate into the periplasmic space of the cells and connect those to each other, building up a huge network and greatly extending the range of a single cell. The flakes of Pyrodictium may be seen even as very primitve multicellular prokaryotic organisms. Because the cannulae represent a great deal of the biomass of Pyrodictium cultures, they should be of great importance. Five different structural cannulae genes identified so far do not show significant homology to any genes in other organisms including hyperthermophiles (Mai, 1998).

The advantage of the huge Pyrodictium web is still not evident and only a working hypothesis can be presented. In considering the physical uniqueness of the natural hot vent biotope and the great extension of cell range by the cannulae, for the first time in the living world Pyrodictium could be able to use thermal energy. Pyrodictium grows within the porous walls of deep sea "Black Smoker" vents, several millimeters to centimeters wide. From inside, the walls are strongly heated by the 300 to 400° C vent fluids, while they are cooled from outside by the surrounding 3° C deep sea water. Therefore, these chimneys harbour very steep temperature gradients, in which the Pyrodictium webs are situated, having a cold and a hot end. Interestingly, although this organism is growing only up to 110° C, its cannulae are stable up to 140° C. Similar to a thermocouple, electrons could be shifted in between the cold and hot end of the Pyrodictium web. This could cause changes in the membrane potential and finally ATP formation within the cells. At present, we are designing experiments to try this working hypothesis.

The enormous variation in cell size and volume appears to be a rather primitive feature and is in line with the 16S rRNA phylogeny of the corresponding hyperthermophiles. The smallest cell sizes observed are in the 200 to 300 nanometer range and the ability of hyperthermophilic archaea to form those may be of great advantage to pass narrow pores of soils and rocks in order to colonize hot subterranean environments.

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