Molecular and thermodynamic controls on cell envelope reactivity of a deep-sea hyperthermophilic methanogen: Solute-specific mechanisms governing archaeal envelope chemistry and nutrient acquisition

Abstract

Methanogenic archaea play an integral role the global carbon cycle, collectively generating well over half of annual methane production on Earth today. Methanogenesis represents one of the most influential and ancient metabolic strategies utilized by life on Earth. Biotic methane production is critically dependent on the bio-availability of nickel (Ni), which acts as an essential enzymatic cofactor in methanogenesis. Despite methanogens dependency on Ni, the processes governing its bioavailability remain limited. Nutrient bioavailability, including Ni, begins at the interface of the cellular envelope and the surrounding environment where non-metabolic, acid-base reactions control solute adsorption for subsequent transport into the cell to be utilized for metabolic processes. In archaea, this interface is dominated by a proteinaceous surface layer (S-layer) whose chemical reactivity and structural response to changing solution conditions remain poorly understood compared to bacterial systems. Here, I thoroughly explore this critical interface using the hyperthermophilic deep-sea methanogen Methanocaldococcus sp. FS406-22 as a model organism. This work employs a multi-technique approach integrating potentiometric titrations, isothermal titration calorimetry, attenuated total reflectance Fourier transform infrared spectroscopy, surface complexation modeling, and geochemical modeling to quantify proton-active surface functional groups at the cell envelope and evaluate the mechanisms governing the local chemical environment. Collectively, these data provide critical insights into how life mitigates outstanding environmental conditions. Ultimately, the scientific contributions herein better inform our understanding of the limitations on habitable conditions, both on Earth (spatially and temporally) and on extraterrestrial worlds Results demonstrate that the Methanocaldococcus sp. FS406-22 cell envelope contains multiple proton-reactive sites consistent with carboxyl, phosphate, and amine functional groups, and that their expression is strongly modulated by solution chemistry. Within a “simulated seawater" (SSW), the presence of divalent cations, particularly magnesium (Mg), reduces the contribution of amine sites to proton buffering at elevated pH and induces an enthalpy-entropy compensation indicative of solution driven surface reorganization. Spectroscopic analyses further reveal that the Mg containing SSW solution promote conformational changes in S-layer proteins, which coincide with altered spectral behavior in carbohydrate and phosphate associated regions attributed to glyco-protein components of the cell envelope. These observations indicate that protein conformation governs the hydration environment and accessibility of proton-active surface sites, linking molecular-scale structural changes to macroscopic surface reactivity. Building on these findings, bulk Ni adsorption experiments show that adsorption onto the cell envelope increases with increasing pH, corresponding to sequential deprotonation of carboxylic acid, phosphate, and amine surface sites in both sodium chloride (NaCl) and SSW solutions at matching ionic strengths. However, total Ni adsorption is maintained despite a reduction in amine-associated proton buffering in SSW, indicating that the amine sites that remain accessible and continue to participate in Ni coordination. Spectroscopic data further reveal protein-associated vibrational responses to Mg and Ni, suggesting that solution-driven protein reorganization (i.e. conformation) preserves Ni binding functionality at the cell envelope. Collectively, this work establishes archaeal cell envelopes as dynamic, chemically responsive interfaces that directly influence trace metal bioavailability. Through an integrated thermodynamic and spectroscopic approach aimed at probing archaeal surface reactivity, this dissertation advances understanding of Ni acquisition by methanogens and highlights the role of protein conformation in regulating nutrient availability. While this work explores only a single methanogen and its coordination with Ni, it provides foundational evidence to warrant the exploration of similar surface mediated mechanisms present in other archaea and trace metal systems. This dissertation has broad implications for constraining methanogenesis productivity, advancing models of trace metal cycling at the microbe-fluid interface, and interpreting biogeochemical signals preserved in the geologic record and planetary environments.