Molecular bases of the adaptation to extreme environments
The M2E team has developed a unique savoir-faire in the study of adaptation to extreme environments, by combining the tools from different fields of science. This has led to two major discoveries about the proteome and the membrane, and has opened new perspectives.
Adaptation of the proteome
Only recently we published the first demonstration of the structural divergence between piezophilic and piezosensitive microorganisms, opening the way to deciphering the molecular basis of this adaptation. In contrast to accepted models, proteins of piezosensitive microorganisms are less sensitive to increasing HHP, while those of piezophiles are more flexible than that of the piezosensitive species, but undergo pressure-dependent rearrangements at a HHP threshold close to the pressure optimum of the organism. Thus, unexpectedly, the adaptation to HHP in piezophiles implies that the proteome of the cell is both more sensitive and more resistant to HHP. In the case of piezophiles, we observe that the hydration shale size is reduced and water appears less mobile, similarly to what is observed for osmoprotection by organic osmolytes. Our hypothesis is that structural modifications in piezophilic proteins affect mainly the water/protein interface, modifying their interactions with water and osmolyte molecules, which would explain the similarity between the HHP behavior of piezophile proteins and protein solution in presence of osmolytes. This would also explain why genome-wide genomics approaches failed to identity structural modifications in the proteome (AME team unpublished results). To circumvent the difficulties to identify structural modifications from genome sequence evolution, we propose to address this question starting from the determination of the physical alterations in the proteome in order to identify the putative imprint in the genomes of piezophiles, in a bottom-up approach. To achieve this goal, we propose to compare the physical (molecular structure, molecular dynamics) and biochemical characteristics of couples of proteins from near isogenic piezophile/piezosensitive model organisms: Thermococcus barophilus, a piezophile growing best at 400 bars, and T. kodakarensis, which cannot grow above 250 bars. Measures will be performed in the sub-ns to ms domain using state-of-the-art techniques under HHP (EINS, QENS, NMR, SANS, SAXS and other spectroscopies) to identify molecular motions at the atomic level. Comparisons within and between couples of proteins will identify the specific contribution of each atom/amino acid, on the high hydrostatic pressure (HHP), high temperature (HT) behavior of the studied proteins, which in turn will allow to draw a pattern of amino acid substitution to look for putative modifications in the genome. Such innovative approach was never tempted before and based on our preliminary results, we expect breakthrough progress in the understanding of HHP adaptation and protein folding by combining structural and dynamical aspects.
Adaptation of the membrane
Two major structural adaptations have been linked with the adaptation of the membrane to extreme environments: the synthesis of membrane-spanning, bipolar lipids and the binding of the glycerol moiety and the hydrocarbon chains by an ether bound. 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 and proven a thermally stable bilayer membrane architecture to explain the stability the hyperthermophilic archaeal membrane (Salvador Castell et al. submitted). 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 physico-chemical characteristics and stress behavior of that membrane still needs to be characterized in order to establish a the molecular model of the membrane. 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 have collaborators within our ANR performing 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.
This research 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. Our preliminary results clearly show that such a membrane architecture can exist in vitro. Obtaining the 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, demonstrate that lipid rafts, e.g. functionally distinct domains, exist in the membrane of Archaea. Thus, this project has numerous possible implications on cell physiology and functioning, and concrete biotechnological applications as well.