HYPERthermophiles and their BIOMINeralization mechanism – HYPERBIOMIN

A combination of multidisciplinary experimental approaches will allow to study the biology, mineralogy, biogeochemistry of Thermococcales. The combination of microbiology,
geochemistry, mineralogy, molecular biology and genetic technologies aimed to be pushed at a very high
level in each of the domains, ensures novelty and originality of the project.
Hyperthermophilic cultivation (by using bioreactors), biomineralization experiments, molecular biology and genetics of hyperthermophilic microorganisms are performed. Transcriptomic analyses are currently in progress to identify potential genes involved in biomineralization process. Biochemical techniques (such as chemical dosages, ATPmetry assays...) are also routinely used. For instance, the cellular activity will be monitored by ATPmetry assays that I have developed for hyperthermophiles in laboratory. Mineral phases produced and cell/mineral interactions will be studied by using a broad range of methods such as X-ray diffraction (XRD), transmission, scanning and Cryo-electron microscopy (TEM, SEM, Cryo-EM), energy dispersive X-Ray spectroscopy (EDXS) and electron energy loss spectroscopy in scanning transmission electron microscopy (STEM-EDX, STEM-EELS).

Since the start of the project, two articles have been published. The first paper, published in 01/22, is associated with axis 1 of the project. It demonstrates the impact of the metabolism employed by Thermococcales on the nature of the iron sulphide minerals produced. Thus, it is necessary that Thermococcales use sulfur and therefore metabolize S(0) to produce pyrites. Sulfur vesicles (initially used as intracellular sulfur detoxifying mechanism) act as a precursor for pyrite formation because they harbour reactive sulfur on the surface of cells. When cells grow in a medium with L-cystine (another sulfur compound, S-1 state), they do not produce pyrite. However, under both conditions, Thermococcales are able to produce greigites during the biomineralization process. It has been suggested that the production of greigite derives from the sulfurization of amorphous Fe (III) phosphates close to the surface of the cells.
Another important element is the cell revival observed in S(0) condition. I suggested that the precipitation of iron sulfides (from iron phosphates) could release phosphates (an important source for ATP) and other nutrients that can be used by part of the cell population (non-lysed and non-mineralized) thus allowing the cell revival.
The second published paper is associated with axis 2 of projext. This is a review explaining the role of vesicles in archaea (09/21). Sulfur vesicles (produced by Thermococcales in condition S(0)) play a key role in biomineralization process because allow the production of pyrite. In order to better understand the role of these vesicles in the production of minerals, it is now necessary to mineralize purified vesicles.
Finally, a third paper has recently been submitted. It shows that pyrite spherules produced within a few days in the presence of Thermococcales, consist of an assemblage of ultra-small nanocrystals of a few nanometers in size showing coherently diffracting domain sizes of few tens of nanometers. The production of these pyrite spherules involves a sulfur redox swing from S0 to S-2 and then to S-1, involving a comproportionation of -II and 0 oxidation states of sulfur. Moreover, organic compounds are detected in small but detectable quantities within the spherules. All these characteristics combined make those pyrite spherules possible candidates for biosignatures in extreme environments.

In order to better understand the role of vesicles in the formation and / or nucleation of minerals, it is necessary to mineralize purified vesicles.
In parallel, mineralization experiments with mutant strains deficient in vesicle production (some are already available in the laboratory) will be carried out to test whether these mutations affect the biomineralization steps and/or affect the formation of iron minerals.
Transcriptomic analyses are currently underway to identify potential genes involved in biomineralization process.

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.