Isabelle
Daniel
a,
Philippe
Oger
b and
Roland
Winter
c
aLaboratoire de Sciences de la Terre, UMR 5570 CNRS – UCB Lyon1 – ENS Lyon, Bât. Géode, 2 rue Raphael Dubois, F-69622 Villeurbanne cedex, France. E-mail: isabelle.Daniel@univ-lyon1.fr
bLaboratoire de Sciences de la Terre, UMR 5570 CNRS – UCB Lyon1 – ENS Lyon, 46 allée d'Italie, F-69364 Lyon cedex 07, France. E-mail: philippe.oger@ens-lyon.fr
cUniversity of Dortmund, Department of Chemistry, Physical Chemistry I – Biophysical Chemistry, Otto-Hahn Str. 6, D-44227 Dortmund, Germany. E-mail: roland.winter@uni-dortmund.de
First published on 29th August 2006
Life on Earth can be traced back to as far as 3.8 billion years (Ga) ago. The catastrophic meteoritic bombardment ended between 4.2 and 3.9 Ga ago. Therefore, if life emerged, and we know it did, it must have emerged from nothingness in less than 400 million years. The most recent scenarios of Earth accretion predict some very unstable physico-chemical conditions at the surface of Earth, which, in such a short time period, would impede the emergence of life from a proto-biotic soup. A possible alternative would be that life originated in the depth of the proto-ocean of the Hadean Earth, under high hydrostatic pressure. The large body of water would filter harmful radiation and buffer physico-chemical variations, and therefore would provide a more stable radiation-free environment for pre-biotic chemistry. After a short introduction to Earth history, the current tutorial review presents biological and physico-chemical arguments in support of high-pressure origin for life on Earth.
Isabelle Daniel | Isabelle Daniel studied Earth Sciences at the Ecole Normale Supérieure of Lyon, where she obtained a PhD in 1995 in the field of mineralogy under extreme conditions of pressure and temperature. She spent one year at Arizona State University in the group of Prof. P. F. M. McMillan. In 1996 she was appointed assistant professor and in 2004 professor of geology at the University of Lyon 1. Her research interests include experimental measurements at high pressure and high temperature of the physical and chemical properties of materials relevant to the Earth’s interior, including minerals, fluids, and rocks. In collaboration with Philippe Oger, she has also developed an experimental program of in situ measurements on live micro-organisms at high pressure. |
Philippe Oger | Philippe Oger received his PhD in Plant Pathology in 1995 from the University of Paris, where he determined the alteration induced by the culture of genetically modified plants on the soil bacteria and their metabolism. From there he moved to the University of Illinois at Ubrana-Champaign to study the evolution of carbon metabolism in Agrobacterium tumefaciens. He joined the CNRS and the Laboratoire de Sciences de la Terre in Lyon in 2000. His current research focuses on the influence of high hydrostatic pressure on microbial metabolism in relation to geological processes. Several analytical techniques have been developed, or adapted, for the study in situ under controlled pressures and temperatures of the metabolism of model eukaryotes (yeast and Emiliana huxleyi) and several strains of model piezophilic and piezotolerant bacteria and archaea. |
Roland Winter | Roland Winter was born in Offenbach in 1954. He studied chemistry at the University of Karlsruhe and obtained his PhD degree in Physical Chemistry in 1982. He then joined Professsor Hensel's group at the University of Marburg as postdoctoral fellow where he worked on liquid matter under extreme conditions and where he received his habilitation and venia legendi in Physical Chemistry. After spending one year as a visiting scientist in Prof. Jonas' laboratory at the Department of Chemistry of the University of Illinois at Urbana-Champaign, where he started his work on high pressure molecular biophysics, he was appointed Professor at the University of Bochum. Since 1993 he has held a Chair of Physical Chemistry (Biophysical Chemistry) at the University of Dortmund. His research interests comprise the study of the structure, dynamics and phase behavior of model biomembranes and proteins. He also addresses pressure effects in molecular biophysics, such as pressure-induced phase transformations of lipid membranes and unfolding, denaturation and aggregation of proteins. |
Among the four aeons defined between the formation of the Earth and present day (Fig. 1), the geological timescale involved in the early evolution of the Earth until life emerged includes the hadean and archean periods. The Hadean is ordinarily defined as that time between the formation of the solar system and early accretion of the planets (4.6–4.5 Ga) and the onset of life. Consequently, the upper limit defined as the Archean has become older as the search for geological traces of life progresses, and it depends on what is taken to constitute evidence for life. It is currently located at around 4.0 ± 0.2 Ga, on the basis of the enrichment in 13C of inorganic carbonates. Alternatively, it could be defined as the end of the period of heavy bombardment in the solar system, i.e. 3.9 Ga, which can be deduced from the impact record of other planets like Mercury or Mars, or the Moon. Then, the Archean covers the early development of life until 2.5 Ga, when the Proterozoic begins.
Fig. 1 Geological timescales, including the major early events that occurred on the young Earth. 1 Ga is one billion years, 1 Ma is one million years. Geological time is divided into four aeons, as presented in the left timescale. Hadean started 4.6–4.5 Ga ago, with the formation of the Earth, until the origin of life 4.0 ± 0.2 Ga ago. As included in the name, Archean covers the early stages of life to about 2.5 Ga. Proterozoic is from to 2.5 to about 0.56 Ga. Phanerozoic is not yet finished, and started with the appearance of skeletons or shells that could be fossilized in terranes. The onset of Phanerozoic is a time of evolutionary explosion. One should be aware that almost all marine invertebrate phyla appeared at that time. The right time scale details the major geological events that affected the young Earth until mid-Archean. (modified after13) |
The planets formed roughly in the mean time the Sun spent on the pre-main sequence, ca. 40 million years (Ma). Unfortunately, no outcrop has yet been found dating from the first 500 Ma of Earth's history. The oldest ‘real’ rocks are the 4.03 Ga old Acasta gneisses in the north of Canada, while the oldest terrestrial objects are highly refractory zircons, the oldest found to date being 4.40 Ga old.5 Therefore, deducing Earth's infancy relies entirely on isotopic geochemistry of short-lived nuclides, combined with theory and comparison with other solar system objects. Thanks to the improved performance of modern mass spectrometers, the timing of the evolution of early solar system, including the Earth, has been recently revised and it generally appears that it evolved faster than previously thought.3 The accretion of terrestrial planets may be envisaged as a four-stages process, although a variety of theories have been advanced (see Chambers,6 for a recent review, and references therein). First, the circumstellar dusts settle to the middle plane of the disk, within a few thousands of years. Second, planetisimals grow up to ca. 1 to 10 km in size, by collisions occurring at relatively low speed. Third, there is a “runaway” growth of 1,000 km-sized planetary embryos. This is thought to take place within a few hundred thousand years. It is actually possible that Mercury- or Mars-sized objects could originate in this fashion. All recent data for Martian meteorites indicate that accretion and core formation on Mars were extremely fast, maybe less than 1 million years. Consequently, Mars may represent a unique example of a large primitive planetary embryo, with a different accretion history from that of the Earth. In the fourth and late stage, the larger objects like Earth or Venus grew by prolonged collisions of planetary embryos, whose orbits become more elliptic and thereby cross each other as gravitational perturbations occur. Earth and Venus should have gained most of their mass in the first ten million years, but significant accretion continued for much longer. While accretion continued, the Earth's core was formed rapidly. The global differentiation of the silicate Earth was completed 4.53 Ga ago, within ca. 30 million years of Earth's formation.7 The most widely held theory for the formation of the Moon is that there was a giant impact between a Mars-sized planet and the Earth at around 40–50 Ma, when it was approximately 90% of its current mass, but already differentiated. Such a planetary collision would have been catastrophic. The energy released would have been sufficient to increase the temperature of the Earth by thousands of degrees.
Oxygen isotopes measured in zircons from Jack Hills (Australia) provide convincing evidence that rocks on Earth were being chemically altered at low temperature by liquid water before 4.2 Ga.10 Although controversial, this might suggest that Earth's oceans were already in place by 4.2 Ga, or maybe already by 4.4 Ga, depending on the author. Many Archean volcanic rocks appear to have erupted under water onto a continental substrate, as illustrated at Kambalda (Australia) by pillowed basalts, that contains old zircons and geochemical signature of assimilation of old continental crust. It seems that oceans flooded much of the continental crust during the late Archean, at the time these rocks formed. The actual oceanic level is controlled by several factors, among which, the volume of the oceans, and the buoyancy of the oceanic and continental crusts play a major role. The first explanation is that the volume of the ocean was certainly greater in the Hadean than at present. It may have been up to twice that of today's ocean. Indeed, this ocean probably contained most of the terrestrial water, since the Hadean mantle was hotter and thus dryer than its modern counterpart.
The hot Hadean mantle induced both a tectonic regime and topography for the Hadean Earth that is radically different from the modern one. The convection was vigorous, and Hadean hot spots were enormous. Oceanic crust was produced by a higher degree of partial melting, and it could be about 30 km thick, i.e. 4 times the modern one (7 km), and it could be overloaded by large amounts of magmatic products from hot spots. It reacted with water at the surface (independently of whether it is vapor or liquid) and formed serpentine and H2, which would promptly transform large amounts of CO2 and N2 into CH4 and NH3, respectively. The hydration of oceanic crust triggers that of continental crust. The degassing of CH4 and NH3 is a key point for the Hadean climate and the development of life. The large thickness of the oceanic crust would lead to an ocean floor, which could be shallower than at present time. This provides a second explanation to the conjecture that much of the continental surfaces were probably flooded during the late Archean. The consequence of a thick oceanic crust is also steep subducting slabs.2
Conversely, the continental lithosphere was thin, because both the crust and the lithospheric mantle were thinner than at modern time. The Hadean continental crust was enriched in short-lived radioactive elements, and consequently less viscous. The mantle being hot, the 1200 K isotherm that defines the bottom of the lithosphere was shallow. This also supports the assumption that most of the continental surfaces could be flooded at that time.
From the above discussion, it appears that the Hadean Earth could have been frozen over its surface most of the time, with ice sheets of thickness ranging from 0 up to 100 m, depending on the mean heat flow and its geographical distribution. Two cases can be envisaged: the ice blanket is thick, the sun does not get through, and the ocean is isolated from atmosphere; alternatively, the ice is thin enough that the sun penetrates, and that it can be broken by waves allowing exchanges. In both situations, the ice can be locally broken at hotspots, where intense volcanism might be present, creating warm ponds. Moreover, volcanoes always produce CO2, which will build up in the atmosphere if disconnected from oceans, ultimately leading to massive melting of the ice blanket.
Late-stage bombardment punctuated the Hadean with warm or maybe Inferno episodes. Tens of 10 to 100 km sized asteroids might statistically have hit the Earth ca. 3.9 Ga, after life had already emerged according to isotopic evidence. Impacts were still frequent as late as 3.2 Ga. Geological records, as shown by spherule beds, reveal four impact events between 3.5 and 3.2 Ga. The size of those spherule beds is large compared to the modern ones, and could they be produced by impacts big enough to boil off tens of meters of ocean water. Moreover, at least one impact of an asteroid larger than 500 km is predicted to have occurred between the formation of Earth and ∼3.8 Ga ago, leading to the complete evaporation of the oceans. Such cataclysmic events would have extirpated any life from the ocean. However, they may have favoured life in the deep regions of the Earth, at the ocean floor for instance, in environments protected from impacts. The occasional boiling of the oceans provides a useful explanation for the presence of hyperthermophilic (extreme heat-lovers) and piezophilic (pressure lover) organisms at the root of the tree of life (see below). This does not necessarily require that the first organism was a hyperthermophile or a piezophile, but it rather suggests that hyperthermophiles and piezophiles were privileged during one or several impacts. One should notice that if life emerged in hydrothermal vents on the Earth's ocean floor, life could be widespread in the solar system, wherever such hydrothermal systems are present with appropriate chemistry. Life might also have an extraterrestrial origin; it could have been brought on Earth by comets, meteorites or asteroids. Recent experiments indicate that some bacteria can survive shock pressures as high as 78 GPa.12
High pressure waters encompass 88% of the volume of the oceans, that have an average depth of 3800 m, and thus achieve an average hydrostatic pressure ca. 38 MPa. The maximum depth in the trenches can reach 11,000 m (110 MPa), but the volume of seawater below the “abyssal plain” (∼6000 m) is only 0.1% of the total. In the ocean, temperature decreases with depth until an almost constant 3 °C is reached below the thermocline (30–100 m). Thus, the high-pressure ocean is cold. In the continental system, on the contrary, the average geothermal gradient is ca. 25 °C km−1. Considering the actual temperature limit for life, e.g. 121 °C would thus place the “deep” limit for the putative continental biosphere ca. 5 km below ground on average, under maximal pressures of 150 MPa. At the time of emergence of life, the limits in pressure would be even smaller, due to the higher heat flux. Even though the maximal productivity of the high pressure continental or marine biosphere is orders of magnitude lower than that of the surface biotopes, due to their extremely large volume, these high pressure biotopes contribute significantly to the production and recycling of organic carbon on Earth.14
The deep ocean is characterized by the lack of sunlight, a stable average temperature of ca. 3 °C, low organic carbon or mineral and a constant oxygen concentration. Theoretical settling rates of phytoplankton, as well as the flocculent aggregates of particulate matter constituting “marine snow”, range from 1.0 to 0.1 m per day, or 5000 m in 1–50 years. It is generally estimated that about 99% of the organic matter produced photosynthetically in the surface waters is recycled in the upper 100–1000 m. Only about 1% of photosynthetically produced organic carbon reaches the deep-sea floor. Thus, the major nutritional characteristic of the deep sea as a habitat is the relatively low input of organic carbon. As a corollary, adaptations to oligotrophy (life with limited amount of nutrients) and psychrophily (optimal life at low-temperature) are common.
The deep-sea vents were discovered as the result of a systematic search for active volcanism at submarine spreading centers.15 In this zone, the contraction of freshly extruded lava upon cooling allows seawater to penetrate several kilometers downward into the newly formed crust. Reacting with basaltic rock at high pressures and temperatures exceeding 350 °C, the seawater is transformed into an acidic and highly reduced “hydrothermal fluid” enriched in metals, hydrogen sulfide, and molecular hydrogen. Going upwards, the hydrothermal fluid can contact sea water inside the porous basaltic rock and emerge as warm vents (3–50 °C). Alternatively, the fluid can emerge in the open ocean without prior mixing as hot vents (up to 400 °C). Upon mixing with the cold ocean seawater, the minerals present in the hot hydrothermal fluid precipitate to form chimney-like structures and a cloud-like smoke of mineral particles, hence the nickname “smokers”, around which the vent communities are spatially structured. From the time of their discovery, it was obvious that vent organisms were related to surface organisms. Similarly, from the beginning it was clear that vent organisms must obtain their energy from the hydrothermal fluids, since the organic carbon production of the vent organisms exceeded by orders of magnitude the amount of organic material that could possibly sediment from the surface.15
The diversity of bacterial and archaeal species isolated from the hydrothermal environment is large. In contrast to clones isolated from the open ocean, bacteria and archaea isolated from deep-sea hot vents are thermophiles. Most isolates are chimiolithotrophs, e.g. capable of gaining energy from the chemical transformation of dissolved minerals and to realize the fixation of dissolved carbonates into organic molecules. Many isolates are aerobes or facultative anaerobes, i.e. they use oxygen as a final electron acceptor. A growing number of obligate anaerobes have been characterized. The vent systems are also characterized by a great diversity of microscopic eukaryotes (ciliates, flagellates, nematodes, fungi, etc.), and especially predatory species that are supposed to feed on chimiosynthetic prokaryotes. Last, several vents are inhabited by large invertebrates, such as polykaetes, mussels, crabs or shrimps, which make these ecosystems crowded places, hence their nickname of deep-sea oases. Aside from their architectural resemblance to their closest non-vent cousins, the macro fauna of the vent ecosystems have a unique feeding strategy. This was characterized first in the vent clam, Clyptogena magnifica and in the vent worm Riftia pachyptila, a tube worm lacking a digestive tractus.16 Instead of feeding on smaller prey as would normally be the case, mussels and worms are fed by symbiotic, obligate anaerobic chimiolithotrophic bacteria that occupy a large portion of the animal's body. Bacteria use the energy from the hydrothermal vents fluids to fix dissolved carbon, much like their free-living counterpart. What the benefit to the bacterium would be apart from shelter is so far unclear. So, the vent ecosystems are organized very much like surface ecosystems, except that the vent ecosystem relies on prokaryotic chimiolithotrophs rather than on photosynthetic primary producers.
For several years, it was impossible to disconnect the deep chimiolithotrophic vent ecosystem from the photosynthetic surface ecosystem. Indeed, if oxygen was required as a final electron acceptor in absence of an appropriate anaerobic chimiolithotrophic prokaryotic compartment, then photosynthesis was required to produce the oxygen, and the vent ecosystem was indirectly dependent upon it. When the first obligate anaerobic archaea were isolated by Erauso et al. (1993),17 the demonstration was made that the vent ecosystem could function in the absence of oxygen, and therefore disconnected from sun light as an energy source.
First, all ecosystems on Earth depend directly or indirectly upon light as the source of energy for the fixation of atmospheric carbon through photosynthesis. Photosynthesis is a very complex energy harvesting process, which could not have appeared at the beginning of life on Earth. Harvesting chemical energy is a simpler mechanism, and it could have appeared earlier in the evolution of life on Earth. Thus, the vent chimiolithotrophy could represent a remnant energy harvesting mechanism, providing a window on the origin of life on Earth.
Second, the similarity between the top and bottom ocean biosphere clearly indicates that organisms from both environments originate from the same lineage. Whether the hot, anoxic deep-sea environments were colonized recently by surface Bacteria, Archaea and Eucarya, or whether the cold, irradiated surface environment was colonized gradually by microbes from the deep-sea remains an open question.
Third, the physico-chemical conditions in the deep biosphere are stable for geologically long periods, and ionizing radiation is low. In contrast, on the surface radiation is high and the physico-chemical environment variable, more so on the young Earth, when life appeared. Thus, the conditions of the deep biosphere could have been more appropriate for the emergence of life, and the deep biosphere might have witnessed its emergence.18
Fourth, the possibility that life can survive and proliferate in the absence of light, and that it might have emerged under high pressure, greatly expands the possibility that life could exist, or have emerged elsewhere in the Universe. For example, it becomes reasonable to assume that life could have emerged on other celestial bodies within our solar system, such as in the deep-oceans of Europa, or that life is still present within the Martian subsurface.19
The ubiquitous gene pool comprises 70 genes,20 among which 58 encode proteins involved in the translation of RNA into proteins, e.g. ribosomal proteins, amino acyl ARNt synthetases and protein modification enzymes. Therefore, the LUCA did possess a “modern” protein synthesis mechanism, capable of elaborating sophisticated proteins. The universal protein set also comprises the SRP54 protein of the SRP (signal recognition pathway) ribonucleoproteic complex, and Srα its cognate membrane receptor. These two proteins are involved in the translocation through the plasma membrane of proteins during protein synthesis. Thus, the LUCA already had a cytoplasmic membrane, a hypothesis also supported by the presence of two membrane associated ATP synthetases in the ubiquitous gene set. The ubiquitous gene set comprises only 3 proteins involved in the processing of DNA itself, a surprising result since DNA is the genetic storage material of all cells. Mushegian and Koonin have proposed that the LUCA had a RNA-based genome, and that DNA replication was invented twice, leading to the major dichotomy between the Archaea/Eucarya and Bacteria domains.20 In contrast, Forterre proposed that the LUCA had a DNA based genome, and that the separation of the Archaea/Eucarya and Bacteria domains occurred when the host DNA replication was displaced by the simpler, more effective DNA replication of a viral particle.21 The ubiquitous gene set does not contain any metabolic gene. This result is not a surprise, since the phylogenetic studies on metabolic genes clearly demonstrate their recurrent loss and acquisition during evolution.
Fig. 2 Piezophily in the tree of life. (a) The classical view of the tree of life. The topology of the tree is mainly based on rDNA comparison. (b) A revised topology of the universal tree of life, after correction of phylogenetic pitfalls, such as long branch attraction or the removal of uninformative site from the analysis. (c) and (d) Distribution of piezophilic and piezotolerant organisms in the two trees of life. Thick blacks lines highlight bacterial groups in which piezotolerant and piezophile organisms have been characterized. Thin black lines highlight groups for which only piezotolerant species are known. |
However, this consensus view was challenged by complete genome comparison that showed that many protein phylogenies contradict the universal tree of life, in that each domain was a mosaic of the two others in terms of gene contents, and that eukaryotes contained more bacterial genes than archaeal ones, and that archaea contained more bacterial than eukaryotic ones. The novel topologies of the universal tree of life (Fig. 2b) give less support to the high temperature LUCA, and they suggest a possible root between the Archaea and the Eucarya.25
Both complexification and simplification have occurred during evolution. For example, the evolution from the origin of life to the LUCA obviously must have been from simple (pre-biotic) to complex (DNA/RNA-based cellular organization), whereas the evolution from Gram negative bacteria to mitochondria and chloroplasts is one example of evolution from complex to simple. What happened in the case of the eukaryote/prokaryote transition?
Carl Woese has always argued that the LUCA must be primitive, a progenote, from which all life forms have evolved through stepwise complexification. The counter-intuitive hypothesis of a eukaryotic-like LUCA was proposed by Reanney who considers many RNA molecules typical of eukaryotes to be relics of the RNA world and ought thus to have been present in the LUCA.26 Two additional arguments can be put forward to support the “complex to simple” scenario in which a eukaryotic-like LUCA evolved by simplification to give birth to present-day prokaryotes. First, reductive evolution of central molecular mechanisms still occurs in bacteria. Second, most cellular functions require multiple enzymes physically interacting with one another. It is very difficult to imagine the displacement of a single component by several others simultaneously. In contrast, a replacement of several components by a single one is much easier, if the latter can perform the same task with similar or better efficiency. The occurrence of such events is shown by the well documented displacement of the original proteobacterial RNA polymerase (3 subunits) by a bacteriophage-like RNA polymerase (1 subunit) in the evolution of mitochondria and chloroplasts.
If life emerged in the deep sea, then one question remaining would be the colonization of the surface environment. The recent isolation of an obligate photosynthetic green-sulfur bacterium27 from a deep-sea hydrothermal vent might represent the missing link between the ancestral chimiolithotrophic energy harvesting metabolism which emerged in the depth of the ocean, and the photosynthetic light energy harvesting metabolism which eventually moved upward to colonize the ocean and thereafter the land surfaces.
Fig. 3 Definitions of the relations between growth rate of microorganisms and pressure. Piezophile microorganisms display a maximum growth at high pressure. They can either grow at atmospheric pressure or not, and are called strictly piezophile in the latter case. Piezotolerant microorganism grow best at atmospheric pressure, but can sustain high pressure, whereas mesophile or piezosensitive microorganisms totally stop growing at 40–50 MPa. (Redrawn after ref. 57.) |
A large number of piezophile microorganisms have been isolated and characterized. Pressure-regulated metabolism and gene expression have been explored in piezophiles as well as in mesophiles (lovers of temperate conditions), and in microorganisms as well as in higher eukaryotes.29 The physical and chemical effects of pressure on the major biomolecules found in cells are described in the next section of this review. However, the pressure-induced phenomena that occur in living organisms have not been systematically investigated due to their complexity.
To illustrate the effect of pressure on a microorganism, we can consider the brewing and baking yeast Saccharomyces cerevisiae, because it is the best characterized unicellular eukaryote in terms of genome characterization, and physiology. Yeast has proved to be a good eukaryotic model and evidence exists that mechanisms operating in yeast also occur in complex eukaryotes. Since 1996, the entire genome yeast has been sequenced. Gene features can be found in the Saccharomyces Genome Database (http://www.yeastgenome.org). The response of yeast to pressure has been recently reviewed30,31(Table 1). The prokaryotic counterpart to S. cerevisiae would be the mesophile model Escherichia coli, the behaviour of which at high pressure is reviewed by Welch et al.32 The characteristics of high-pressure adapted deep-sea prokaryotes belonging to Archea and Bacteria were reviewed by Bartlett.29
Pressure/MPa | Effects |
---|---|
0.1–50 MPa | Arrest of cell growth |
Metabolic changes | |
Inhibition of amino acid uptake | |
Stress-inducible expression | |
50 MPa– | Inhibition of ethanol fermentation |
Internal acidification | |
Stress-inducible expression | |
100 MPa– | Reduction in viability |
Membrane, and cell wall perturbations | |
Acquired piezotolerance | |
Stress-inducible expression | |
200 MPa– | Alteration of genome preservation |
Shrinkage and leakage of cells | |
Stress-inducible expression |
S. cerevisiae is piezotolerant. Yeast growth and cellular activity in virtually unaffected at pressures lower than 20–30 MPa depending on strain genotype. Higher pressures are however perceived as a stress in this micro-organism. Consequently, S. cerevisiae exhibits adaptation mechanisms through pressure-inducible genes and pressure-induced proteins. The major steps of yeast response to high pressure stress are summarized below.
Yeast viability decreases with increasing pressure and this effect is more pronounced above 100 MPa, until all wild type cells of yeast are killed at 220 MPa. However, a pressure of 50 MPa is neither sufficient to kill nor to alter the yeast cell morphology. Although not directly relevant to S. cerevisiae, this shows that pressures in the range of 200–500 MPa can be employed for sterilization of food. The piezotolerance of yeast cells depends on the duration of high pressure application. Although a short treatment at 50 MPa will not kill the cell, an incubation at 50 MPa for 24 h will result in 100% mortality. Piezotolerance also depends on the position in the cellular cycle. Yeast cells in stationary phase are more resistant to pressure than proliferating cells. A comparison of the yeast cell growth response to pressure stress with that to a classical heat-shock of 40 °C for 30 min, shows that pressurized cells at 50 MPa for 30 min have a slower response and also take longer to recover normal growth. This suggests that they still suffer metabolic changes after pressure is released.
The influence of pressure on gene expression has been recognised. In most cases gene expression is inhibited by pressure, but there are also specific proteins produced upon pressure-shock. Pressure has a specific effect on DNA, and shifts the double DNA helix towards a denser form. Also, protein association with DNA is less stable at high pressure, demonstrating that pressure may interfere with transcription, and alter genome expression. One issue was to determine whether the effect of pressure was mostly due to the relative instability of certain gene promoter binding or of mRNAs with pressure, or to a well defined stress response. Among the 6200 known or predicted genes of S. cerevisiae, 131 were induced more than 2-fold and 143 repressed greater than 2-fold by pressure, although the up-regulated genes are largely unknown. The genomic response of yeast to high pressure is a typical stress response. Genes involved in stress defence and carbohydrate metabolism are highly induced by pressure, while genes involved in cellular transcription, protein synthesis and cell cycle regulation are down-regulated. The stress response of yeast to pressure is similar to, but fundamentally different from, that of heat shock. Heat-shock induces a set of Heat Shock Proteins (HSPs), and activates the metabolism of trehalose (a nonreducing disaccharide), whose function is to prevent unfolding or to promote refolding of proteins, in order to keep the cell machinery working. The HSP genes induced by pressure in yeast are slightly different. For instance, high molecular weight HSPs or trehalose 6-phosphate synthetase genes are strongly induced by heat-shocks but are indifferent to pressure. In contrast, the small HSP26 gene is strongly induced by pressure. This gene codes for a small protein with a molecular chaperone activity, and like other members of the HSP family, it protects proteins from irreversible aggregation. The HSP26p complex probably dissociates under pressure, thus ensuring a chaperone activity of the protein, like at high temperature. After returning to ambient pressure, the complex probably reverts to the associated form and loses its chaperone activity. A number of yeast genes regulated by pressure also include a large set of Cold-Shock specific genes. This tends to show that yeast cells possess a mechanism to sense the stress of pressure, and to activate the appropriate gene expression machinery.
Protein synthesis is one of the most piezosensitive cellular functions, probably due to the disassembly of ribosome as a function of pressure. In contrast, RNA synthesis is maintained at higher pressures. Hence, even if the genes responsible for stress-induced proteins are up-induced, those proteins cannot be produced due to the inactivation of the protein synthesis apparatus during compression. S. cerevisiae has an improved piezo-resistance after being exposed to a mild stress, including heat-shock, cold-shock, ethanol-shock and hydrogen peroxide-shock. However, yeast cells subjected to a mild pressure do not acquire resistance to any subsequent severe pressure increase, unless they are incubated at room pressure between the two pressure treatments. This is likely related to the specific problems induced by pressure on the cells, that is reduction of membrane fluidity and impaired protein synthesis. When the stress is applied, the up-regulated genes are induced but cannot be translated. Once the cells return to ambient conditions, the biosynthesis can take place and protects the cells against further pressure increase for a relatively long period of time, compared to the duration observed for heat-shock.
Pressure changes the fluidity of the membrane in a similar way to low temperature, by enhancing the order of the phospholipid bilayers, and causing the fatty acid to pack more tightly. The fluidity of a membrane at 100 MPa and 2 °C (typical deep-sea conditions) is similar to that at atmospheric pressure and −18 °C. This is compensated by an increase in unsaturated fatty acids, which leads to highly disordered phospholipid bilayers that are less permeable to water molecules. Hence, this maintains the membrane in a functional liquid crystalline state despite the effect of pressure. The increased proportion of unsaturated fatty acids is common among deep-sea organisms. As stresses that both decrease membrane fluidity, the application of cold and pressure to yeast shows a slight induction of the ERG25 gene, whereas heat-shock down-regulates the ERG25 gene. ERG25 codes for the protein ERG25p, which is a sterol desaturase enzyme involved in ergosterol biosynthesis. Ergosterol is a sterol group with an unsaturated side chain, while cholesterol has a saturated one. Membranes enriched with ergosterol seem to be more resistant to ethanol and probably to temperature than cells enriched with cholesterol, whereas those containing cholesterol have proved to be more resistant to pressure than cholesterol-free ones.
Pressure alters the structure of the cell wall and cytoskeleton. Those effects are counterbalanced by the up-regulation by pressure of the gene HSP12, which codes for HSP12p. HSP12p is a small hydrophilic protein located in the cell wall. It improves the flexibility of the cell wall, by disrupting interactions between adjacent polysaccharide layers that would else build a rigid structure. Once more, there is a common feature between pressure and low-temperature stress: they do not induce the majority of HSPs but small HSPs (like HSP12 and HSP26) related to membrane destabilization. In particular, the larger HSPs induced by heat-shock, and related to chaperone activity that prevents protein folding, are not induced by pressure.
Another effect of pressure on yeast cell is the acidification of the cytoplasm, and of the vacuole. At atmospheric pressure, cytoplasm and vacuole have a constant pH of 7.0 and 6.0, respectively. Increasing pressure to 50 MPa decreases the cytoplasm pH by 0.3 units, and the vacuole pH by 0.3 to 0.5 units. The increased acidity is due to the increased solubility of CO2 and greater dissociation of carbonic acid at high pressure. Intracellular acidification is also observed in the case of heat-shock, ethanol or osmotic stresses. The higher acidification of the vacuole has its origin in the induction of the gene HSP30, which codes for a down-regulator of the H+-ATPase activity in yeast plasma membrane. H+-ATPases pump out protons accumulated in the cytoplasm into the vacuole. As pressure increases, the yeast vacuole is assumed to serve as proton sequestrant to maintain favourable cytoplasmic pH.
Pressures used to investigate biochemical systems usually range from 0.1 MPa to about 1 GPa. Such pressures only change intermolecular distances and affect conformations, but do not change covalent bond distances or bond angles. The covalent structure of low molecular mass biomolecules (peptides, lipids, saccharides), as well as the primary structure of macromolecules (proteins, nucleic acids and polysaccharides), is not perturbed by pressures up to about 2 GPa. Pressure acts predominantly on the conformation and supramolecular structures of biomolecular systems. High pressure studies generally call for unique methods, which have been developed in recent years.33–40 Here, some basic concepts and results are discussed.
Fig. 4 T,P-phase diagram for the main (chain-melting) transition of different phospholipid bilayer systems. The fluid (liquid-crystalline) Lα-phase is observed in the low-pressure, high-temperature region of the phase diagram; the gel phase regions appear at low temperatures and high pressures, respectively. The acyl chains of the various phospholipids are denoted on the right hand side of the figure. |
Upon compression, the lipids adapt to volume restriction by changing their conformation and packing. A common slope of ∼220 °C GPa−1 has been observed for the gel–fluid phase boundary of the saturated phosphatidylcholines (Fig. 4). Using the Clapeyron relation, dTm/dP = TmΔVm/ΔHm, the positive slope can be explained by an endothermic enthalpy change, ΔHm, and a volume increase, ΔVm, for the gel–fluid transition that have been found in direct measurements of these thermodynamic properties. Similar transition slopes have been found for most phospholipid bilayer systems, such the mono-cis-unsaturated lipid POPC, the phosphatidylserine DMPS, and the phosphatidylethanolamine DPPE. Only the slopes of the di-cis-unsaturated lipids DOPC and DOPE are markedly smaller. The two cis-double bonds of DOPC and DOPE lead to very low transition temperatures and slopes, as they impose kinks in the linear conformations of the lipid acyl chains, thus creating significant free volume in the bilayer so that the ordering effect of high pressure is reduced.
It has been noted that applying high pressure can lead to the formation of additional gel phases that are not observed under ambient conditions, such as the interdigitated high pressure gel phase Lβi found for phospholipid bilayers with long acyl chain lengths. To illustrate this polymorph, the results of a detailed study of the P,T-phase diagram of DPPC in excess water are shown in Fig. 5a. At much higher pressures, further gel phases appear.34,35 The data demonstrate that biological organisms could modulate the physical state of their membranes in response to changes in the external environment by regulating fractions of the lipid components in the cell membrane that vary in chain length, chain unsaturation or headgroup structure via “homeoviscous adaption”. In fact, several studies have demonstrated that membranes are significantly more fluid in barophilic and/or psychrophilic species, which is principally a consequence of an increase in the unsaturated/saturated lipid ratio, as noted in the previous section.
Fig. 5 (a) T,P-phase diagram of DPPC bilayers in excess water (besides the Gel 1 (Pβ′), Gel 2 (Lβ′) and Gel 3 phase, an additional crystalline gel phase (Lc) can be induced in the low-temperature regime after prolonged cooling). (b) Phase diagram of DPPC-GD (5 mol%) in excess water as obtained from SAXS and FT-IR spectroscopy data. (c) Tentative P,T-phase diagram of the model raft mixture POPC/SM/Cholesterol (1 : 1 : 1) as obtained from spectroscopic and SAXS data. The lo + ld (+so) fluid/ordered domain coexistence region is hatched. |
However, Nature has further means to regulate the membrane fluidity. Biological membranes consist of lipid bilayers, which typically comprise a complex mixture of phospholipids and sterol, along with embedded or surface associated proteins. The sterol cholesterol is an important component of animal cell membranes that contain up to 50 mol%. Cholesterol thickens a liquid-crystalline bilayer and increases the packing density of lipid acyl chains in the plane of the bilayer in a way that has been termed a “condensing effect”. Measurements of the acyl chain orientational order of the lipid bilayer system demonstrated the ability of sterols to efficiently regulate their structure, motional freedom and hydrophobicity.34,35,44,45 Addition of increasing amounts of cholesterol leads to a drastic increase of the chain order parameter S in the lower pressure region. For concentrations above about 30–50 mol% cholesterol, the conformational order is almost independent of pressure and the fluid-to-gel phase transition can hardly be detected any more. Hence, sterols have the ability to regulate the structure, motional freedom and hydrophobicity of biomembranes, so that they can withstand drastic changes in environmental conditions, such as temperature and external pressure.
Recent studies have been carried out on more complex models for biomembrane systems, such as cholesterol-containing ternary mixtures that contain an unsaturated lipid like phosphatidylcholine and a saturated lipid like sphingomyelin. Such lipid systems are supposed to mimic distinct liquid-ordered (lo) lipid regions, called “rafts”, which coexist with liquid-disordered (ld), fluid-like domains. Rafts are also present in cell membranes and are thought to be important for cellular functions such as signal transduction and the sorting and transport of lipids and proteins. FT-IR spectroscopy in combination with calorimetry, fluorescence spectroscopy and synchrotron X-ray scattering has been used to characterize T- and P- dependent changes in the conformation, hydration, structure and phase behavior of the canonical lipid raft mixture POPC/SM/Cholesterol, and to establish a P,T-phase diagram of the system over an extended temperature and pressure range (Fig. 5c). The lo/ld phase coexistence region of the model raft mixture extends over a rather wide temperature range of about 40 °C. An overall fluid phase is reached at rather high temperatures (above ∼50 °C), only. At ambient temperature, a fully ordered lipid state is reached at 100–200 MPa. Interestingly, ceasing of membrane protein function in natural membrane environments has been observed for a variety of systems in this pressure range.34,35 This might be correlated with the membrane matrix reaching a physiologically unacceptable overall ordered state at these pressures.
Little is known about pressure effects on the motions of lipid bilayers at elevated pressures.34,35,37–39 Of particular interest is the effect of pressure on lateral diffusion, which is related to biological functions such as electron transport and some hormone-receptor interactions. Pressure effects on lateral diffusion of lipid molecules in relation to other membrane components have yet to be carefully studied, however. Pressure effects on the lateral self diffusion coefficient D of DPPC and POPC vesicles have been studied by Jonas.37 The lateral diffusion coefficient of DPPC in the liquid-crystalline phase decreases by about 30% from 1 to 30 MPa at 50 °C. A further 70% decrease in the D-value occurs at the pressure-induced Lα to gel phase transition. The effect of cholesterol incorporation into fluid lipid bilayers has a significant effect on the conformational order, but a less pronounced effect on the dynamic properties of the lipid membrane. Hence, lipid bilayers are able to regulate their structure and fluidity by an adjustment of their sterol composition as well as by a lateral redistribution of their various lipid components, and saturated (ordered) and unsaturated (fluid) domains. An increase in the sterol level in a membrane generally reduces the effect of variations in pressure.
Also with respect to the kinetics of the protein folding reaction, pressure studies are of particular use, as they allow us to evaluate the volume profile during the folding process and to characterize the nature of the barrier to folding or unfolding and the corresponding transition state. Moreover, pressure studies present an important advantage due to the generally observed positive activation volume for folding, the result of which is to slow down the folding reaction substantially, in turn allowing for relatively straightforward measurements of structural order parameters characteristic for folding intermediate states, that are difficult or even impossible to quantify on much faster timescales corresponding to ambient pressure conditions.
As an example, we show results of a study of the pressure-induced unfolding and refolding of staphylococcal nuclease (SNase), a small protein of 149 amino acids, consisting of about 26% α-helices and 25% β-sheets.46,47 The high pressure SAXS data at 25 °C revealed that over a pressure range from atmospheric to ∼300 MPa, the radius of gyration Rg of the protein doubles from roughly 17 Å for native SNase two-fold to nearly 35 Å (Fig. 6a). The scattering curves reveal a transition from a globular to an ellipsoidal structure. The FT-IR amide I′ absorption band reveals a pressure-induced denaturation process that is evidenced by an increase in disordered and turn structures and a drastic decrease in the content of β-sheets and α-helices. Contrary to the temperature-induced unfolded state, the pressure-induced denatured state retains some degree of β-like secondary structure and the protein molecules cannot be described as fully extended random polypeptide coil. Temperature-induced denaturation involves further unfolding of the protein molecule that is indicated by a larger Rg-value of 45 Å. There are many indications now that the conformation of a protein denatured by pressure is more compact than that of a protein denatured by temperature or chemical agents, and often resembles “molten globule”-type structures. This does not seem too surprising, as pressure is known to favour the formation of hydrogen bonds, which maintain the secondary-structure network, but is unfavourable for hydrophobic interactions, which are predominantly responsible for maintaining the tertiary structure of a protein. The idea is supported by theoretical results that suggest water penetration into the protein interior as a likely mechanism for pressure-denaturation of proteins due to a weakening of hydrophobic interactions, as opposed to the temperature-induced unfolding process. Assuming the pressure-induced unfolding transition of SNase to occur essentially as a two-state process, a standard Gibbs free energy change for unfolding of ΔG0 = 17 kJ mol−1 and a volume change for unfolding of ΔV = −80 ml mol−1 is obtained. Generally, proteins are 5–10 times less compressible than water. As a result, pressure-induced volume changes in proteins are quite small, typically <1%. For monomeric proteins such as SNase, the difference corresponds approximately to the volume of 4–5 water molecules (∼18 ml mol−1).
Fig. 6 (a) Radius of gyration Rg of SNase as a function of pressure at T = 25 °C. (b) Calculation of the three-dimensional free energy landscape of SNase (pH 5.5) using experimentally determined thermodynamic parameters. The Gibbs free energy of unfolding, ΔG, is plotted as a function of temperature and pressure. The slice of the three-dimensional free energy landscape for ΔG = 0 (dashed line) yields the P,T stability diagram of the protein. (c) P,T-stability diagram of SNase at pH 5.5 as obtained by SAXS, FT-IR spectroscopic and DSC measurements. |
The pressure midpoints at several temperatures obtained from the FT-IR spectroscopy and SAXS profiles are plotted as a P,T-phase diagram in Fig. 6b. Such a partially elliptic-like phase diagram is typical for monomeric proteins.33–36 Knowing experimentally obtained thermodynamic parameters, such as the changes in heat capacity, expansivity, compressibility, enthalpy and volume at the unfolding transition, allows the calculation of the three-dimensional free-energy landscape.34,35 The corresponding plot for the protein SNase (Fig. 6c) clearly shows that the protein is stable only (Gibbs free energy of unfolding ΔG > 0) within a limited P,T-phase-space. The good agreement between the experimental data points and the theoretical curve for ΔG = 0 justifies the two-state assumption for the unfolding transition of SNase. Certainly, the temperature and pressure dependence of the thermodynamic parameters involved must be considered to obtain quantitative agreement with the experimental data. Moreover, only if the denatured state has a rather well-defined average free energy, an effective two-state model may be a reasonable approximation. Generally, the unfolding process is best described by a funnel-like energy landscape picture.48 Certainly, the shape of the stability diagram depends on the individual protein structural composition and it may be more complicated, in particular for larger proteins. Also, additional regions in the phase diagram may appear, such as an extended region at high temperatures where aggregation occurs. We also note that the unfolded state ensemble in the P,T-plane can be of considerably different structure, and that long-lived metastable states may occur. Whereas monomeric proteins generally unfold at pressures above 200 MPa (at 400–800 MPa in most cases), oligomeric proteins dissociate at much lower pressures (mostly at ∼100–200 MPa).33–36
The perplexing effect of high pressure on protein aggregation consists in, on the one hand, inducing aggregation-prone intermediate states, and on the other hand the ability of high-pressure to prevent aggregation and to dissociate aggregates.51–54 The susceptibility of protein aggregates to pressure largely depends on the degree of the structural order of an aggregate. Fresh, amorphous aggregates are more sensitive to pressure and prone to refolding to the native state than mature amyloid fibrils. In the latter case, effectiveness of pressure-induced dissociation depends on the particular mode of polypeptide backbone and side chain packing that allows reducing remaining void volumes. The pressure-sensitivity of fresh aggregates and the virtual insensitivity of mature fibrils allows us not only to differentiate between various stages of the amyloid-formation, but also to obtain reliable thermodynamic data, such as Gibbs' free energy and volume changes, of the early stage of the protein transformation. This is only possible due to the reversibility of the process under high-pressure conditions. Such an approach has been successfully employed in studies on lysozyme, insulin, PrP and TTR amyloidogenesis.51–54 Our work on insulin fibrillation at high pressure conveyed an astonishing example of how studies employing HHP may shed new light on aggregation pathways and subfibrillar structure of amyloid. Though, as high pressure disfavors insulin aggregation, in fact it permits amyloidogenesis through an alternative, less effective pathway that brings about a negligible volume expansion, finally leading to insulin amyloid of a unique morphology. Concerning the free energy landscape of proteins, probably a more generalized protein landscape picture including an additional “aggregation funnel”—eventually consisting of different deep minima for different strains—must be envisaged. One of the most interesting prospects for application of high pressure in protein aggregation research was the idea of destroying prion infectivity through pressure treatment.55
How long did it take life to emerge from the limbs, invent the cell, the genetic information and the proteins, to finally reach the photosynthetic or methanogene outcome? Nobody knows, and the question is highly debated amongst the scientific community. However, as discussed in the introduction, catastrophic meteoritic events with the capability to vaporize the totality of the Earth' ocean waters occurred at least to −4.2 Ga, and most probably up to 3.9 Ga. Therefore, if life emerged on Earth, its path from nothingness to its almost full complexity must have taken between 100–400 million years. Several lines of evidence support the hypothesis that life could have originated under pressure.
One of the bottlenecks for the emergence of life is the synthesis of its first building blocks. On one hand, the thermal and physico-chemical conditions favorable to their “spontaneous” synthesis in the primordial soup are also those that favor their chemical instability. On the other hand, water in deep hydrothermal systems is under pressure (20–35 MPa) temperature (350–450 °C) conditions that correspond to the thermodynamic supercritical state. Under such conditions, experiments show that physicochemical properties like the dielectric constant ε, the viscosity η, the density ρ and the ionic hydration decrease in supercritical water (see review by Bassez56). Consequently, the solubility of ionic and polar compounds diminishes, while that of simple apolar molecules is enhanced. Hence, the apolar supercritical water in hydrothermal vents could concentrate prebiotic molecules, which would react more efficiently.56
High pressure allows the synthesis of a set of molecules that cannot be synthesized at ambient pressures, or at the expense of enzymatic activities. High pressure can shift the temperature requirement for a given chemical reaction towards lower temperature, lowering the ΔG0, allowing for prebiotic synthesis at lower temperatures. Furthermore, high pressure can stabilize several essential biological macro molecules such as DNA and RNA. Therefore, it could be possible for the proto-life to “invent” RNA, and make use of its catalytic properties, something that could not occur in the temperature conditions required for the spontaneous synthesis of RNA at surface conditions.
Perhaps, the most crucial indication about whether life originated from the deep waters of the proto-ocean is given to us by the study of the current life forms on Earth. When we compare the adaptations to different physico-chemical conditions that can be observed within the three domains of life, we are overwhelmed by the adaptation ability of life. However, only a few specific families of organisms for each environment can live in these extreme environments, whether hot, acidic, halophilic, etc. In fact, most organisms today can live in moderate physico-chemical conditions, from which only moderate variations can be tolerated. In contrast, most, if not all, organisms can live under a large range of hydrostatic pressures. In fact, surface organisms can withstand pressures as high as 20 MPa, e.g. the pressure equivalent to 2 km of water, without consequence on its life cycle or metabolism. Indeed, several will be able to live under much higher pressure before hydrostatic pressure is perceived as a stress. Tolerance to high hydrostatic pressure is the only physical or chemical parameter found in all organisms in which it was sought for, and may well indeed represent one of the most ancestral physical conditions under which life had to emerge.
GD | gramicidin D |
HHP | high hydrostatic pressure |
DMPC | 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (di-C14:0) |
DPPC | 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (di-C16:0) |
DPPE | 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (di-C16:0) |
DMPS | 1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (di-C14:0) |
DSPC | 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (di-C18:0) |
DOPC | 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (di-C18:1,cis) |
DOPE | 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (di-C18:1,cis) |
DEPC | 1,2-dielaidoyl-sn-glycero-3-phosphatidylcholine (di-C18:1,trans) |
POPC | 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (C16:0,C18:1,cis) |
SNase | staphylococcal nuclease |
DSC | differential scanning calorimetry |
SAXS | (WAXS) small(wide)-angle X-ray scattering |
SANS | small-angle neutron scattering |
TTR | transthyretin |
PrP | prion protein |
FT-IR | Fourier-transform infrared |
ΔV and ΔG0 | standard volume and free energy change |
ΔV≠ | activation volume of reaction |
This journal is © The Royal Society of Chemistry 2006 |