HYPERthermophiles and their BIOMINeralization mechanism – HYPERBIOMIN

A combination of multidisciplinary experimental approaches was implemented to investigate the biology, mineralogy, and biogeochemistry of Thermococcales under biomineralization conditions. The integration of microbiology, geochemistry, mineralogy, and molecular biology ensured the novelty and originality of the project.

Cultivation of hyperthermophilic microorganisms and mineralization experiments were performed under a range of physicochemical conditions, including different temperatures (25 °C, 45 °C), iron sources (FeSO₄, FeCl₂) at variable concentrations, and in the presence or absence of cells. To monitor the successive stages of biomineralization, samples were collected at selected time points over a period of up to 30 days.

In parallel, abiotic pyrite synthesis experiments were conducted under the same physicochemical conditions (anaerobic atmosphere, 85 °C, presence of reactive sulfur), in the absence of intact cells. These experiments were performed both in the absence and in the presence of different organic materials (graphitic carbon, yeast extract, cell lysates), in order to generate abiotic references comparable to biological systems.

The physiological state and metabolic activity of non-mineralized cells were assessed using flow cytometry cell counts, ATP quantification by ATPmetry, confocal microscopy observations, and cryo-electron microscopy. The progression of fully mineralized cells, occurring as pyrite spherules, was quantified by transmission electron microscopy counts. The mineralized nature of the cells (iron- and sulfur-rich) was confirmed by EDXS spectroscopy.

Dissolved and precipitated iron were quantified using ferrozine-based colorimetric assays and MP-AES spectrometry.

In parallel, transcriptomic analyses were performed at different key time points of the biomineralization process. A dedicated workflow was specifically developed for transcriptomic analyses in Thermococcales and can be reused for future experiments by the entire research team.

The mineral phases produced and cell–mineral interactions were investigated using a wide range of techniques, including X-ray diffraction (XRD), transmission and scanning electron microscopy, cryo-electron microscopy (TEM, SEM, FIB-SEM, Cryo-EM), energy-dispersive X-ray spectroscopy (EDXS), and scanning transmission electron microscopy coupled with chemical analyses (STEM-EDX, STEM-EELS). Finally, selected samples were analyzed by X-ray absorption spectroscopy (XAS) at the SOLEIL synchrotron (Fe K-edge on the SAMBA beamline; C K-edge on the HERMES beamline) and at the Stanford Synchrotron Radiation Lightsource (SSRL; S K-edge on beamline 4-3).

The HYPERBIOMIN project has significantly advanced our understanding of microorganism–mineral interactions in deep-sea hydrothermal environments. All results obtained highlight the central role of hyperthermophilic archaea belonging to the order Thermococcales in the formation of iron sulfides and reveal strong links between biomineralization processes and adaptation of archaea to extreme environments rich in iron and sulfur. The HYPERBIOMIN project also enabled the identification and discussion of mineral and organic signatures associated with these processes, thereby contributing to a more refined definition of biosignature criteria in hydrothermal systems.

Thermococcales, hyperthermophilic sulfur-reducing archaea inhabiting hydrothermal systems, are able to rapidly induce the formation of iron sulfides, namely pyrite (FeS₂) and greigite (Fe₃S₄) (Fig 1). At 85 °C, under anaerobic conditions in iron- and sulfur-rich media, they produce pyrite spherules associated with cells and vesicles within a few hours. Pyrite nanocrystals nucleate at the surface of the cellular S-layer, forming smooth pyrite spherules. These biogenic pyrite spherules display specific morphological and mineralogical features and contain organic matter, which makes them of particular interest for biosignature research.

Pyrite formation depends on the presence of elemental sulfur (S⁰) in the medium. Under sulfur-rich conditions, Thermococcales accumulate polysulfides and produce sulfur-rich vesicles that play a key role in the mineralization process. Pyrite forms through a sulfur comproportionation reaction involving elemental sulfur in the medium and reduced sulfur (S²⁻) present in mackinawite (FeS), a metastable mineral phase initially produced by the interaction between Fe²⁺ and H₂S. In the absence of sulfur vesicles, no pyrite formation is observed, demonstrating the importance of the polysulfide pathway (Fig 3).

The kinetics of pyrite formation depend on both the nature and the concentration of iron. The production mechanism remains effective over a wide range of conditions, including lower temperatures (25–45 °C). However, intact cellular surfaces are essential: neither chemically fixed cells nor intracellular contents allow pyrite formation.

Biomineralization constitutes a strategy for iron detoxification. The rapid precipitation of FeS initially protects the cells but induces a lethal physical stress. The transformation of a fraction of the population into pyrite enables the survival of another fraction, leading to the establishment of a heterogeneous population (Fig 5). Non-mineralized cells then overexpress genes involved in DNA repair, oxidative stress response, and detoxification mechanisms, thus promoting adaptation to iron- and sulfur-rich extreme environments (Fig 8).

The scientific and technical challenges highlighted by the HYPERBIOMIN project open new research directions for a better understanding of iron sulfide biomineralization processes in hydrothermal systems. In particular, multi-omics approaches (proteomics, metabolomics) will help to elucidate the molecular and metabolic mechanisms involved in Thermococcales. Furthermore, integrating the study of extracellular vesicles, whose role in biomineralization has been demonstrated but remains insufficiently characterized, as well as exploring the previously unknown role of viruses, represents a key step toward advancing beyond our current models.

C. Truong, S. Bernard, A. Gorlas, P. Le Pape, G. Morin, C. Baya, P. Merrot, P. Lefebvre and F. Guyot. Production of pyrite spherules by hyperthermophilic Thermococcales. (Submitted, Frontiers in Microbiology)

Gorlas A, Morey L, Mariotte T, Truong C, Bernard S, Guigner JM, Oberto J, Baudin F, Landrot G, Baya C, Le Pape P, Morin G, Forterre F and Guyot F (2022) Precipitation of greigite and pyrite by Thermococcales: adaptation to Fe- and S-rich environments? Environmental Microbiology 24: 626–642. doi: 10.1111/1462-2920.15915

Liu J, Soler N, Gorlas A, Krupovic V, Krupovic M, Forterre P (2021) Extracellular membrane vesicles and nanotubes in Archaea. Microlife 2: uqab007. doi: 10.1093/femsml/uqab007

Hydrothermal deep-sea vents are often iron- and sulfur-rich anaerobic systems. Whereas FeS2 pyrite is abiotically formed in the interior of chimneys at high temperatures (> 250°C), a major stock of FeS2 (pyrite and marcasite) is also produced in the cooler middle layers of the chimneys at lower temperatures (< 150°C) by a still unknown mechanism. In laboratory, Thermococcales, predominant inhabitants of the hot parts of hydrothermal sources, have been shown to rapidly produce abundant quantities of FeS2 within cells and vesicles, and of greigite (Fe3S4) on extracellular polymeric substances (EPS) suggesting that they may contribute to the geochemically important formation of “low temperature” FeS2 in their ecosystem. In order to provide a meaningful evaluation of the contribution of Thermococcales and more generally of the hyperthermophilic (T>80°C) biosphere to biogeochemical cycles and regulations, it is now time to progress in the mechanistic elucidation of those high temperature biomineralization phenomena. Thermococcus kodakarensis will be the organism of choice for HYPERBIOMIN project, since many interesting results have already been obtained for this strain and in our laboratory and genetic tools for this hyperthermophilic archaeon are well established. HYPERBIOMIN project addresses three main questions:
(1) What are the physiological conditions of the cells and physico-chemical parameters of the life medium which influence and control the rates of iron biominerals produced by Thermococcales?
(2) What are the biological entities and genes implied in the Thermococcales biomineralization mechanism?
(3) What are the adaptive strategies developed by hyperthermophiles to influence and cope with their highly mineralized high temperature environments?
To answer these questions, the project will follow three complementary approaches:
(1) The first approach consists in determining and analyzing quantitatively the iron-sulfide minerals produced under different physico-chemical conditions mimicking the fluctuating environment of hydrothermal chimneys. I have so far limited the biomineralization studies at the optimal growth parameters of Thermococcales strains. In collaboration with a PhD and a master student, we will determine the impacts of temperature and pH but also of the metabolic shifts of Thermococcales (H2S production vs H2 production) induced by environmental conditions on the composition, structure and properties of iron minerals formed during the biomineralization process.
(2) The second approach deals with the exploration of the molecular mechanism of biomineralization by Thermococcales. To date, no information is available about the relationships between production of biominerals and putative related-genes. We will detect and identify molecular partners involved in T. kodakarensis biomineralization mechanism focusing first on genes encoding ferritins and iron transporters because of their potential importance in iron biomineralization processes. I will then explore the genes involved in synthesis and expression of membranes vesicles which have been reported to contribute to the formation of pyrite by Thermococcales.
(3) In a third approach, I will investigate the significance of those biomineralization processes for the adaptation mechanisms of Thermococcales and Methanococcales in the hydrothermal ecosystem. Experiments involving both T. kodakarensis and Methanocaldococcus jannaschii in presence and absence of minerals will be carried out for deciphering adaptive responses to the harsh hydrothermal environment and the potential role of minerals in the adaptation of life in this simplified but yet complex and realistic ecosystem.