Study of the molecular and structural basis of adaptation to extreme conditions, from the molecule to the cell
Searching for the drivers of the adaptation to High Hydrostatic Pressure (HHP)
Why high hydrostatic pressure as an environmental driver ?
There is a large body of evidence which shows that life on Earth appeared 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 largely impede the emergence of life from a proto-biotic soup.
Some of the widely accepted scenarios suggest an origin for life in hydrothermal conditions in the depth of the Archaean ocean. The large body of water would have filtered the harmful radiation of the young sun preventing their deleterious effects on the nascent organic matter, as well as buffered physico-chemical variations, providing a stable radiation-free environment for pre-biotic chemistry.
As a direct consequence, the first molecules, and the first cells, appearing onEarth would need to be adapted to high temperature and HHP. Today, the so-called deep-biosphere still represents most of the biotopes on Earth and is colonized by very specific organisms, adapted to multiple extreme conditions such as extreme oligotrophy, pH, temperatures, or hydrostatic pressure. If the routes to the adaptation to temperature are starting to be quite well understood, adaptive strategies of piezophilic organisms (requiring HHP for growth) which inhabit the deep-biosphere remain elusive.
The effect of high hydrostatic pressure on biological systems
The reason HHP affects biological systems because pressure affects chemical equilibria and reaction rates. The behavior of all systems under high pressure is governed by Le Châtelier's principle, which predicts that the equilibrium is shifted towards the state of smaller volume. Hence, a process is favored under HHP if its volume gets smaller with increasing pressure, and reversely it will be unfavored. Hence, in a cell, HHP has a different impact on depending on the biomolecules. When considering the main biomolecules, DNA, RNA, lipids and proteins, HHP will tend to stabilize the nucleic acids and lipids and destabilize the proteins. If one considers the functional components of the cell, such as replication, transcription, translation or metabolism, HHP will impair them, because it negatively impacts proteins. Similalry, HHP strongly impacts the cell membranes because upon compression, the lipids adapt to volume restriction by changing their conformation and packing (they essentially go from oil to butter). As a consequence, with increasing pressure the membrane loses in fluidity, becomes impermeable, and the protein-lipid interactions, which are essential to the optimal function of the membrane are weakened.
Life under pressure, the essence of being piezophile
In the deep-biosphere,HHP often exceeds that inhibitory to surface organisms. Microorganisms inhabiting these biotopes are able to grow more efficiently under HHP than under atmospheric pressure. These have been called piezophiles (from the greek piezo = to press and philo = love). In short, often inhibitory pressures for surface organisms are those optimal for piezophilic growth. Piezophilic organisms have been isolated from all high pressure environments (the deep-ocean, hydrothermal vents, the sub-seafloor or the continental underground). They belong to a wide variety of bacterial and archaeal genera. The most extreme piezophiles cannot survive at atmospheric pressure, and are called obligate piezophiles. The ability of piezophiles to grow under HHP inhibitory to surface organisms, and more so, the inability of the obligate piezophiles to grow at atmospheric pressure are proof that piezophiles have adapted to HHP in the course of their evolution.
In the scope of axis 1, the M2E team is asking a very simple and fundamental question : What are the structural and genetic bases between a surface organism and a piezophile which inhibits the growth of the former and stimulates the growth of the latter. This research has been funded over the years by several national programs (ANR, CNRS MITI) and large instruments such as the ESRF (European Synchrotron Radiation Facility) or the Euroepan Neutron source of the ILL (Laue-Langevin Institute), which have supported our studies on the structural adaptation of the proteome in piezophiles or the on the structure of the membranes in pizophilic Archaea.
For more information on HHP adaptation in the piezophilic genome and proteome.
For more information on HHP adaptation in the membrane of piezophilic Archaea.
Archaeomembranes : How can a membrane bilayer be stable at temperature above the boiling point of water?Two major structural adaptations have been linked with the adaptation of the membrane to extreme pH and temperature environments: the synthesis of membrane-spanning, bipolar lipids and the binding of the glycerol moiety and the hydrocarbon chains by an ether bound. Bipolar lipids can form lipid monolayers, in which each polar headgroup points out on one side of the membrane. Monolayers are more rigid, less permeable and thermally more resistant than lipid bilayers. The presence of ether lipids also increases thermal stability of the lipids, allows a tighter packing, and consequently, a more impermeable membrane. Conversely, the lack of bipolar ether lipids is proposed to explain the limited temperature growth range of bacteria. However, several hyperthermophilic Archaea are known not to produce bipolar ether lipids, although growing optimally at up to 105°C, implicating that bilayers could also be stable above the boiling point of water. We have proposed a thermally bilayer membrane architecture to explain the stability the hyperthermophilic archaeal membrane. This novel membrane architecture predicts the presence of apolar lipids in the mid-plane of the bilayer, the presence of which would limit charge transfer between the two sides, decrease proton and water permeability, and increase membrane rigidity.The aim of the project is to demonstrate experimentally the validity of this novel membrane architecture and to explain the adaptation to hydrothermal stress in hyperthermophilic Archaea. To achieve this goal, we will compare the physico-chemical parameters of natural vs. reconstructed synthetic membranes, in presence or absence of apolar lipids, mimicking those of Archaea in order to identify the specific contribution of each lipid type, and each lipid moiety, on membrane stability. We will perform the total synthesis of di- and tetraether lipids. Working with synthetic lipids allows for the control of membrane composition and easier interpretation of molecular dynamics data, while working with natural lipids permits to test yet undetermined effects of polar headgroups/core lipids on membrane stability. Physical parameters will be determined from a combination of X-ray/neutron diffraction and diffusion, SAXS, Fourier-transform infrared (FTIR), fluorescence spectroscopies, liquid and solid state NMR and confocal fluorescence or electronic microscopies. The results will enable us to characterize the order parameters, size, shape and domain formation as well as permeability and viscosity of the lipid membranes. Due to the precise control of lipid compositions, variations in these parameter values can be readily attributed to specific lipid moieties. For the first time, it will allow the construction of a comprehensive model of the archaeal membrane and principles governing its stability, which will include a contribution for polar headgroups and apolar lipids.The ArchaeoMembranes project addresses a fundamental question of general and philosophical interest concerning life under extreme environmental conditions, and in fine, about the origin of life on Earth. In this project, we propose the groundwork for a novel membrane architecture, which demonstration would constitute a major breakthrough in membrane science, and on how we understand the cellular membrane. Our preliminary results clearly show that such a membrane architecture can exist in vitro. Obtaining definitive proof that it leads to the expected physical and physiological behavior will have a major scientific echo in the community since it will shed a new light on membrane adaptation to extreme conditions. If this novel ultrastructure can be proven, it implies that lipid rafts, e.g. functionally distinct domains, may coexist in the membranes of Archaea, which implications on cell physiology and functioning are numerous. Last, redefining the phase diagram of the membrane will also have concrete biotechnological applications. Project coordinationPhilippe Oger (Microbiologie, adaptation et pathogénie)The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents. PartnerLiPhy Laboratoire Interdisciplinaire de PhysiqueCBMN Chimie et Biologie des Membranes et des NanoobjetsICBMS Institut de Chimie et Biochimie Moléculaires et SupramoléculairesMAP Microbiologie, adaptation et pathogénieHelp of the ANR 478,796 euros Beginning and duration of the scientific project: December 2017 - 42 Months