Genomic features and genetic evolution processes of Thermococcales

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Physico-chemical and thermodynamic changes due to high pressure

At pressures experienced by living organisms in natural habitats, i.e. between 0.1 MPa to less than 200 MPa, the energy conveyed by increased pressure is quite low compared to chemical bonds, so intermolecular distances and molecule conformations are affected, but covalent bond distances and bond angles are not influenced. Perturbations of covalent structures of low molecular mass biomolecules or of primary structure of macromolecules would require up to 2 000 MPa of pressure (Oger and Jebbar, 2010; Rivalain et al., 2010).
However, biochemical consequences can result from changes in reaction thermodynamics. Fundamentally, according to Le Chatelier’s principle, pressure can influence volume change (𝛥𝑉) in a reaction. Consequently, high pressure can inhibit a reaction which would be accompanied by a positive 𝛥𝑉, such as one forming gas, and can enhance a reaction which would be accompanied by a negative 𝛥𝑉. Thus, although temperature has a broader impact on such reactions, pressure holds different influences: while increased temperature induces acceleration of a biochemical reaction, pressure can either accelerate, inhibit, or have no effect on the reaction, according to the associated volume changes (Abe and Horikoshi, 2001).
The expression of Gibbs free energy (𝛥𝐺) implies a dependence on pressure (see detailed formulas in (Amend and Shock, 2001)). In the shallow layers of the ocean, i.e. at lower pressure ranges (typically 0.1 to 10 MPa, corresponding to hundreds of meters of water column), the effect of pressure on 𝛥𝐺 can be ignored. However, for piezophilic life, where optimal pressures for growth are rather in the dozens MPa, the impact of pressure on biochemical reactions should be acknowledged.

High pressure effects on piezosensitive and piezophilic organisms

Note: The bibliographical work implied in this 2nd section will be valorized and published within a book chapter on the microbiology of piezophiles (see BOOK CHAPTERS IN PREPARATION, P. 345). From those fundamental changes, many functional differences are induced over biological systems at high pressure. Piezophiles must cope with those extreme conditions, and those adaptations have led to several studies, while some of the exact mechanisms involved are yet to be determined.

Effects on proteins

The major effects of high pressure on proteins are compression and unfolding, which possibly lead to modulation or impairment of their functions (Bridgman, 1914; Ichiye, 2018). Multimeric proteins, such as ribosomes, can dissociate under high pressure, which may be a major reason for piezosensitivity (Gross and Jaenicke, 1994; Mozhaev et al., 1996). When pressure increases, proteins can either stabilize or weaken (Abe and Horikoshi, 2001; Boonyaratanakornkit et al., 2002; Chen and Makhatadze, 2017). For example, in Methanocaldococcus jannaschii, a piezophilic model, pressure stabilizes hydrogenases, but acts oppositely on homologs of the same order, in Methanotorris igneus, a strain isolated from shallow areas (Hei and Clark, 1994). Glutamate dehydrogenase from T. litoralis is stabilized by high pressure (Sun et al., 2001). DNA polymerases from Pyrococcus and Thermus species are stabilized at high pressure (Summit et al., 1998).

Effects on nucleic acids

In the range of known biotopes, DNA double-helixes are not very sensitive to pressure variations, probably as the hydrogen bonds, when disrupted, are associated to very small volume changes, positive up to 600 MPa as experimentally calculated with Clostridium perfringens DNA. High pressure, as encountered in known biotopes, thus stabilizes DNA structure (Girard et al., 2007; Hawley and Macleod, 1974; Macgregor, 1998; Wilton et al., 2008). The effects induced by pressure were also shown to depend on temperature and on melting temperature of DNA. With a Tm less than 50 °C, increased pressures destabilize DNA, but hold opposite effects when Tm are above 50 °C (Dubins et al., 2001). Spatial conformation could be impacted: high pressure, as encountered by piezophiles, experimentally shown to increase supercoiling of circular DNA, and to favor B conformation over Z conformation (although higher pressures might be required) (MacGregor and Chen, 1990; Tang et al., 1998) (FIGURE 10).

Effects on lipids and membranes

Unlike nucleic acids, lipids are very sensitive to pressure, even in the living range of microorganisms. Such conditions raise the phase transition temperature (Tm) of the membrane (from liquid to gel) and encourage packing, which impairs lipid motion, and thus hinders membrane functionality. Lipids, being amphiphilic, can self-assemble; and an increased pressure leads to induced chain ordering, possibly leading to thicker membranes (FIGURE 11). This effect on membrane fluidity could be comparable to the application of low temperatures, and necessitates homeoviscous adaptation (Sinensky, 1974; Skanes et al., 2006; Winter and Jeworrek, 2009). It influences the curvature of a lipid membrane, as well as the structure of the bilayer itself, changing its micromechanic properties (Brooks, 2014; Salvador-Castell et al., 2020). Many proteins are associated to lipid membranes, and pressure can affect lipid-protein structures and interactions (Brooks, 2014; Lee, 2004).

Global physiological adaptations

All the local modifications described above, induced by high pressure environments, can cause global effects at the physiological level. For example, motility seems to be affected. The stress induced by high pressure in piezosensitive microorganisms (such as Escherichia coli or Photobacterium profondum 3TCK), as well as sub- and supra-optimal pressures for growth in the piezophile P. yayanosii, and low pressures for Photobacterium profundum SS9, seem to affect the rotation of flagella or archaellum. Note that the same pressure-responsive flagellum systems are found in other piezotolerant bacteria, such as Shewanella piezotolerans WP3. The resulting cell tumbling was interpreted as a way to escape from stressful conditions and seek nutrients (Eloe et al., 2008; Michoud and Jebbar, 2016; Nishiyama et al., 2013; Wang et al., 2008). Nonetheless, in T. barophilus, the need for amino acid increased at optimal pressure compared to Patm (Cario et al., 2015b). Cell cycles also are modulated. Filament formation has been observed in sub- or supra-optimal pressures for several piezophiles, and cell division is directly modified as some division proteins could be affected (Bartlett, 2002). Global metabolic responses to pressure were observed or hinted at in various microorganisms. Methanogenesis seem to be enhanced by pressure in Methanocaldococcus jannaschii (Miller et al., 1988a). Different respiratory chain proteins appear to be involved according to pressure of growth of Shewanella violacea (Chikuma et al., 2007). Evidences indicated that ATPase activity can be enhanced or impaired by pressure, while, in vivo, it is rather the state of the membrane that may be of importance in membrane ATPase activity (Kato et al., 2002; Souza et al., 2004).

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Table of contents :

Acknowledgements – Remerciements
Context
List of figures
List of tables
Main abbreviations
Introduction – State of the art
I. The potential of hydrogen
1. The importance of energy in our modern societies
2. Hydrogen as the modern choice of future energy carrier
3. Hydrogen in natural systems and bio-production processes
II. The extreme microbial life of deep hydrothermal vents
1. Geochemical, physico-chemical and thermodynamical descriptions of hydrothermal vents
2. Microbial and biological diversity at hydrothermal vents
III. Life in hot areas
1. What happens when temperature increases?
1.1. Thermodynamical principles
1.2. Thermophilic metabolism
1.3. Cellular and molecular adaptations to high temperatures
2. (Hyper)thermophiles in biotechnology
IV. Life at high pressure
1. Physico-chemical and thermodynamical changes due to HP
2. High pressure effects on piezosensitive and piezophilic organisms
2.1. Effects on proteins
2.2. Effects on nucleic acids
2.3. Effects on lipids and membranes
2.4. Genomics and gene regulation
2.5. Global physiological adaptations
3. Applications of high pressure in biotechnological prospects
V. Thermococcales
1. Genetics, adaptation and evolution in Thermococcales and ensuing applications
1.1. Genomic features and genetic evolution processes of Thermococcales
1.2. Applications: genetic tools and adaptive engineering
2. Metabolism and physiology
2.1. General concept of the fermentation in Thermococcales
2.2. A wide panel of degradation possibilities
2.3. Substrate uptake
2.4. Carbohydrate catabolism
2.5. Peptide & amino acid catabolism
2.6. Other metabolic routes influencing catabolism
2.7. Energy conservation via hydrogenases in Thermococcales
2.8. Metabolic regulations occur mainly at the transcriptional level
3. Biotechnological applications of Thermococcales
VI. Studying Thermococcales as good chassis for HP H2 bio-production
1. Using high pressure to modulate the metabolism
2. Advantages of HP for H2 production
VII. Objectives Materials & Methods – General
1. Culture
1.1. Anaerobic culture
1.2. Media and substrates
1.2.1. Thermococcales Rich Medium (TRM)
1.2.2. Anoxic Artificial Sea Water (ASW) medium
1.2.3. Anoxic Low-Salt medium preparation
1.2.4. Colloidal sulfur
1.2.5. Colloidal chitin
1.2.6. Gas mixing
1.3. Cell density estimation
1.3.1. Optical counting
1.3.2. Flow cytometry
2. Metabolite measurements
2.1. Following the metabolite production over time
2.2. H2S assays
2.3. Gas chromatography
2.4. Ionic chromatography
3. Molecular biology
3.1. Total RNA extractions
3.2. Total DNA extractions
3.3. Verification of RNA and DNA quality and quantity
3.4. RT-qPCR
Matériels & Méthodes – Général (French version)
1. Culture
1.1. Culture anaérobie
1.2. Milieux et substrats
1.2.1. TRM (Thermococcales Rich Medium – milieu riche pour Thermococcales)
1.2.2. ASW (Anoxic Artificial Sea Water medium – milieu d’eau de mer anoxique artificielle)
1.2.3. Milieu anoxique à faible concentration en sels
1.2.4. Soufre colloïdal
1.2.5. Chitine colloïdale
1.2.6. Les mélanges de gaz
1.3. Estimation de la densité cellulaire
1.3.1. Comptages au microscope optique
1.3.2. Cytométrie en flux
2. Mesures de métabolites
2.1. Suivi de la production de métabolites au cours du temps
2.2. Tests H2S
2.3. Chromatographie en phase gazeuse
2.4. Chromatographie ionique
3. Biologie moléculaire
3.1. Extractions d’ARN total
3.2. Extraction d’ADN total
3.3. Vérification de la quantité et de la qualité de l’ARN et de l’ADN
3.4. RT-qPCR
Chapter I: Strain screening and fermentation overview
Introduction
Part I – Screening and choice candidate strain / substrate
Materials and methods
Results & Discussions
A) Selection of tested strains
B) Methodological adjustments
C) First phase: H2 tolerances
D) Second phase: substrate degradations
E) Choice of the candidate strain
Part II – Overview of the fermentation
Definition of the experimental plan
Materials and methods
Results
A) Choice of the substrate and H2 tolerances
B) Influence of substrate concentration
C) Influence of sulfur concentration
D) Influence of growth phase
E) Influence of product concentrations
F) Influence of temperature
G) Influence of pressure
H) Other strains
Fermentation overview: discussion
Chapter I: Screening and fermentation overview: Discussions and conclusions
Chapter II: Adaptation and optimization of high pressure tools and culture
methods
I – Introduction
II – Discontinuous high pressure culture
1. Presentation of the discontinuous incubators available at the LM2E
2. Methods for discontinuous HP culture
3. Development of a new device for gas-phase, gas-tight HP incubations
4. Discontinuous HP culture methods and tools: Conclusions
III – Continuous HP culture with the high pressure bioreactor (BHP)
1. Presentation of the initial state of the machine and protocol for HP culture
2. Results obtained on the BHP2
3. Further experiments and optimizations envisioned: concept of a new BHP3
IV – Chapter II: Conclusions and perspectives
Chapter III: Study of the hydrogeno/sulfidogenic metabolism at low and high
pressure in T. barophilus
Introduction
Materials and methods
Thermococcus barophilus strains
Culture conditions and physiological measurements
Results
T. barophilus strain Δ517 (wild type)
Δmbh
Δco-mbh
Δmbs
ΔshI
ΔshII
Discussions
Hydrogenogenic system
Sulfidogenic system
Maintenance of the redox balance
SurR regulation
Conclusions
Chapter IV: Adaptive laboratory evolution study of hydrogen tolerance in T.
barophilus MPT
Introduction
Materials and methods
Results
Adaptive laboratory evolution of T. barophilus towards higher H2 tolerance
Growth phenotype and adaptation
H2 tolerance
Sulfidogenic system
Accumulation of organic acids
Genomic modifications
Transcriptomic adaptation
Discussions & conclusions
General discussion – conclusion
I – Would our bioreactor solutions be used for applied H2 bio-production?
II – How to approach to the fundamental comprehension of T. barophilus metabolism?
III – Final conclusion
Valorization
Bibliography

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