Adaptations of life under high hydrostatic pressure in piezophilic microorganisms, the exemple of Thermococcus barophilus

Les environnements profonds marins ou continentaux représentent la majorité des biotopes sur Terre. Ils sont colonisés par des organismes, appelés piézophiles, adaptés aux fortes pressions hydrostatiques du milieu, conditions qui sont inhibitrices pour la croissance des organismes de surface. Dans le cadre de ce travail, j'ai cherché à élucider les spécificités de l’adaptation aux hautes pressions hydrostatiques. Pour cela, j'ai étudié un micro-organisme piézophile issu d'une source hydrothermale profonde, la souche MP de Thermococcus barophilus, dont l'optimum de croissance est de 400 fois la pression atmosphérique. J'ai caractérisé l'adaptation particulière de deux cibles cellulaires parmi les plus sensibles à la pression : les membranes et le protéome.Mes résultats montrent que la souche MP accumule des molécules de stress en condition de faible pression hydrostatique, c'est-à-dire que le protéome de cette souche est adapté aux conditions de hautes pressions. Il s'agit de la première démonstration d'une adaptation structurale chez un piézophile, et la démonstration que cette souche est une piézophile vraie. Par ailleurs, j'ai pu démontrer les mécanismes d'adaptation de la membrane en réponse à la pression et à la température. J'ai montré que cette réponse correspond à une adaptation homéovisqueuse de la composition membranaire, et que celle-ci est unique, car elle met en jeu trois mécanismes différents : une régulation du ratio di-/tetraéthers, une régulation du niveau d'insaturation des lipides, et la présence de lipides neutres dans la structure de la membrane. Ceci m'a amenée à proposer un nouveau modèle de membrane pour la souche modèle piézophile T. barophilus. La généralisation de ces observations et la confirmation de leur lien avec la piézophilie passe par l'étude d'autres organismes piézophiles.Deep marine and continental environments represent the major ecosystems on Earth. They are colonized by organisms named piezophiles, adapted to high pressures of the deep biosphere, conditions that inhibit the growth of surface organisms. My objectives were to elucidate the special features of adaptation to high hydrostatic pressures. My model of study was a piezophilic microorganism isolated from a deep-sea vent; Thermococcus barophilus strain MP, which grows optimally at a pressure of 400 times the atmospheric pressure. I characterized the specific adaptation of two cellular compartments amongst the most sensitive to pressure: membranes and proteome. My results show that strain MP accumulates stress molecules in conditions of low pressure, which mean T. barophilus proteome is adapted to high pressure conditions. This is the first demonstration of structural adaptation in a piezophile, and also shows that T. barophilus is a true piezophile. Besides, I proved membrane adaptation mechanisms in response to pressure and temperature. These mechanisms are based on homeoviscous adaptation of lipids composition. This adaptation is unique and involves three different mechanisms: the regulation of the di-/tetraether ratio, the modulation of lipid unsaturation, and the insertion of neutral lipids in the membrane structure. These results brought me to propose a new membrane model for the piezophilic strain T. barophilus. Before confirming these observations as a possible piezophilic trait of adaptation, this study needs to be extended to other piezophilic organisms

La vie sous pression des microorganismes piézophiles

La biosphère profonde regroupe un ensemble très disparate d’environnements localisés en profondeur dans les océans, sous le plancher océanique et dans le sous-sol. Dans la biosphère profonde, la pression hydrostatique augmente avec la profondeur pour atteindre des valeurs qui inhibent la croissance des organismes de surface. On y trouve des organismes, dits piézophiles, capables de vivre dans ces conditions extrêmes de pression. Les bactéries piézophiles connues sont principalement des γ-protéobactéries psychrophiles appartenant à cinq genres, Photobacterium, Shewanella, Colwellia, Psychromonas et Moritella, alors que les Archaea piézophiles sont toutes (hyper)thermophiles et appartiennent principalement aux Thermococcales. On ne connaît pas à ce jour de groupes microbiens limités à la biosphère profonde. La haute pression hydrostatique a un impact important sur les macromolécules biologiques et sur le cycle cellulaire, qui entraîne une baisse de fonctionnalité de nombreux composants cellulaires et éventuellement la mort cellulaire chez les organismes piézosensibles. Les différentes études physiologiques et génétiques menées à ce jour apportent une vision parcellaire des mécanismes adaptatifs possibles chez les piézophiles. Ainsi, il n’est pas clair si cette adaptation requiert la modification de seulement quelques gènes, de quelques voies métaboliques, une altération globale de nombreux gènes, et/ou une régulation compensatoire des gènes dont les produits sont les plus fortement affectés par la haute pression hydrostatique, ou une réponse compensatoire proche de la réponse au stress. L’adaptation piézophile est diffuse dans le génome, contrairement à l’adaptation thermophile ou halophile

Adaptation of the membrane in Archaea

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Exploring the deep marine biosphere : challenges, innovations, and opportunities

International audienceThe deep marine biosphere is one of the largest, and yet least explored, microbial habitats on the planet. Quantifying the extent, diversity, and activity of subsurface microbial communities is a crucial part of understanding their role in global biogeochemical cycles. Even though deep biosphere habitats can vary widely in chemistry, temperature, turnover rates, and energy sources, all subsurface microbes inherently experience high pressures. While not all subsurface microbes require elevated pressures, for many high pressures are essential to their cellular function and metabolism. Thus, when targeting this elusive portion of the biosphere, it is critical to maintain in situ pressure while sampling and cultivating subsurface microorganisms. In this perspective paper we highlight the sampling and cultivation technologies available to study these communities under in situ conditions. Maintaining elevated pressures throughout sampling, transfer, cultivation, and isolation is challenging, and more often than not samples are decompressed at some point during sample handling, potentially leading to biases in both community diversity and isolate physiology. The development of devices that maintain in situ pressures during sampling and allow for sample transfer without decompression have begun to address this challenge (like the PUSH – Pressurized Underwater Sample Handler). Such vessels can be used for both retrieval and enrichment of deep subsurface samples, as well as high-pressure growth and physiology experiments, thus expanding possibilities for deep biosphere exploration. Finally, we discuss the significant need to develop and share high-pressure facilities across the deep biosphere community, in order to expand the opportunities to discover novel piezophiles from the deep subsurface

Characterizing the Piezosphere: The Effects of Decompression on Microbial Growth Dynamics

The extent to which the full diversity of the subsurface microbiome can be captured via cultivation is likely hindered by the inevitable loss of cellular viability from decompression during sampling, enrichment, and isolation. Furthermore, the pressure tolerance of previously isolated strains that span surface and subsurface ecosystems can shed light into microbial activity and pressure adaptation in these transition zones. However, assessments of the effects of elevated pressure on the physiology of piezotolerant and piezosensitive species may be biased by high-pressure enrichment techniques. Here, we compared two high-pressure cultivation techniques-one that requires decompression of the whole cultures during sampling and one that employs the previously described isobaric PUSH devices-to explore the effects of repeated decompression during incubations performed to characterize isolates from deep environments. Two model sulfate-reducing prokaryotes were used to test the effects of decompression/ repressurization cycles on growth rates, cell yields, and pressure tolerance. The mesophilic bacterium Desulfovibrio salexigens was cultivated from 0.1 to 50 MPa, and the hyperthermophilic archaeon Archaeoglobus fulgidus was tested from 0.1 to 98 MPa. For both cultivation methods, D. salexigens showed exponential growth up to 20 MPa, but faster growth rates were observed for isobaric cultivation. Furthermore, at 30 MPa minor growth was observed in D. salexigens cultures only for isobaric conditions. Isobaric conditions also extended exponential growth of A. fulgidus to 60 MPa, compared to 50 MPa when cultures were decompressed during subsampling. For both strains, growth rates and cell yields decreased with increasing pressures, and the most pronounced effects of decompression were observed at the higher end of the pressure ranges. These results highlight that repeated decompression can have a significant negative impact on cell viability, suggesting that decompression tolerance may depend on habitat depth. Furthermore, sampling, enrichment, and cultivation in isobaric devices is critical not only to explore the portion of the deep biosphere that is sensitive to decompression, but also to better characterize the pressure limits and growth characteristics of piezotolerant and piezosensitive species that span surface and subsurface ecosystems

Rate and Extent of Growth of a Model Extremophile, Archaeoglobus fulgidus, Under High Hydrostatic Pressures

High hydrostatic pressure (HHP) batch cultivation of a model extremophile, Archaeoglobus fulgidus type strain VC-16, was performed to explore how elevated pressures might affect microbial growth and physiology in the deep marine biosphere. Though commonly identified in high-temperature and high-pressure marine environments (up to 2–5 km below sea level, 20–50 MPa pressures), A. fulgidus growth at elevated pressure has not been characterized previously. Here, exponential growth of A. fulgidus was observed up to 60 MPa when supported by the heterotrophic metabolism of lactate oxidation coupled to sulfate reduction, and up to 40 MPa for autotrophic CO2 fixation coupled to thiosulfate reduction via H2. Maximum growth rates for this heterotrophic metabolism were observed at 20 MPa, suggesting that A. fulgidus is a moderate piezophile under these conditions. However, only piezotolerance was observed for autotrophy, as growth rates remained nearly constant from 0.3 to 40 MPa. Experiments described below show that A. fulgidus continues both heterotrophic sulfate reduction and autotrophic thiosulfate reduction nearly unaffected by increasing pressure up to 30 MPa and 40 MPa, respectively. As these pressures encompass a variety of subsurface marine environments, A. fulgidus serves as a model extremophile for exploring the effects of elevated pressure on microbial metabolisms in the deep subsurface. Further, these results exemplify the need for high-pressure cultivation of deep-sea and subsurface microorganisms to better reflect in situ physiological conditions

High Temperature and High Hydrostatic Pressure Cultivation, Transfer, and Filtration Systems for Investigating Deep Marine Microorganisms

High temperatures (HT) and high hydrostatic pressures (HHP) are characteristic of deep-sea hydrothermal vents and other deep crustal settings. These environments host vast and diverse microbial populations, yet only a small fraction of those populations have been successfully cultured. This is due, in part, to the difficulty of sampling while maintaining these in situ conditions and also replicating those high-temperature and high-pressure conditions in the laboratory. In an effort to facilitate more HT-HHP cultivation, we present two HT-HHP batch culture incubation systems for cultivating deep-sea vent and subsurface (hyper)thermophilic microorganisms. One HT-HHP system can be used for batch cultivation up to 110 MPa and 121°C, and requires sample decompression during subsampling. The second HT-HHP system can be used to culture microorganisms up to 100 MPa and 160°C with variable-volume, pressure-retaining vessels that negate whole-sample decompression during subsampling. Here, we describe how to build cost effective heating systems for these two types of high-pressure vessels, as well as the protocols for HT-HHP microbial batch cultivation in both systems. Additionally, we demonstrate HHP transfer between the variable-volume vessels, which has utility in sampling and enrichment without decompression, laboratory isolation experiments, as well as HHP filtration

Le CO2 supercritique pour la régénération des masques FFP2

L' épidémie de Covid-19 (maladie à coronavirus), déclarée pandémie en mars 2020 par l’OMS, a mis en exergue la problématique mondiale de pénurie de protections faciales filtrantes, et tout particulièrement la disponibilité des masques respiratoires chirurgicaux et FFP2. Afin de faire face à la pénurie de masques et limiter la pollution liée à l’amoncellement de ces déchets usagés, plusieurs équipes de recherche, à l’échelle nationale, se sont organisées pour trouver dessolutions pérennes afin de décontaminer les protections faciales en vue de les réutiliser. Cette question se pose notamment pour la décontamination des FFP2 au service des soignants, les plus exposés au risque de contamination

Membrane homeoviscous adaptation in the piezo-hyperthermophilic archaeon Thermococcus barophilus

International audienceThe archaeon Thermococcus barophilus, one of the most extreme members of hyperthermophilic piezophiles known thus far, is able to grow at temperatures up to 103°C and pressures up to 80MPa. We analyzed the membrane lipids of T. barophilus by HPLC-MS as a function of pressure and temperature. In contrast to previous reports, we show that under optimal growth conditions (40 MPa, 85°C) the membrane spanning tetraether lipid GDGT-0 (sometimes called caldarcheol) is a major membrane lipid of T. barophilus together with archaeol. Increasing pressure and decreasing temperature lead to an increase of the proportion of archaeol. Reversely, a higher proportion of GDGT-0 is observed under low pressure and high temperature conditions. Noticeably, pressure and temperature fluctuations also impact the level of unsaturation of non-polar lipids with an irregular polyisoprenoid carbon skeleton (unsaturated lycopane derivatives), suggesting a structural role for these neutral lipids in the membrane of T. barophilus. Whether these apolar lipids insert in the membrane or not remains to be addressed. However, our results raise questions about the structure of the membrane in this archaeon and other Archaea harboring a mixture of di- and tetraether lipids

High hydrostatic pressure increases amino acid requirements in the piezo-hyperthermophilic archaeon Thermococcus barophilus

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