Cellular and Intracellular Insights Into Microbial Sulfate Reduction and Sulfur Disproportionation

Abstract

Microbially mediated processes are essential drivers of the sedimentary sulfur biogeochemical cycle. As such, they have governed the geologic sulfur isotopic record post the 'Great Oxidation Event'. The microbial sulfur isotopic fingerprint is a composite of the metabolism's identity, intracellular conditions, enzymatic parameters, and response to environmental forcing. These can be teased out with high-precision sulfur isotope measurements, provided we have a solid understanding of their respective mechanisms, and their relative contributions, carefully calibrated with well designed experiments. This thesis focused on furthering our current understanding of the biochemistry and evolutionary history of two major sulfur metabolisms, microbial sulfate reduction and microbial sulfur disproportionation. We adopted a fully integrated approach, and worked under the framework that microbial sulfur isotopic fingerprints reflect the metabolism's identity, bacterial physiology (intracellular conditions), and the nature of its specific response to extracellular factors, or local environment. This body of work represents substantial progress towards increasing the interpretability of these dissimilatory sulfur isotopic signatures. We aimed at refining our understanding of the biochemistry and intracellular dynamics of microbial sulfate reduction (Chapters 1 and 2). We made use of the availability of mutant strains of sulfate reducing bacteria with modified metabolic networks, which allows interrogating specific steps in isolation from the rest of the metabolic pathway. We used a deletion mutant strain with a metabolic network that exclusively comprises the sulfite reduction to sulfide step. We use its isotope effect as a calibration to enhance our understanding of the biochemistry of sulfite reduction by applying a metabolically informed isotope model and identified flavodoxin as the most likely electron carrier during dissimilatory sulfite reductase (Chapter 1). We also utilized MSR oxygen isotopic signature, which provides information on the intracellular flux of material that complements that provided by the sulfur isotopic signature. We built a metabolic isotope model rooted in thermodynamics to draw quantitative links between cell-specific sulfate reduction rates and active sedimentary cell abundances. This model is calibrated using data from a series of continuous culture experiments with two strains of sulfate reducing bacteria (fresh water bacterium Desulfovibrio vulgaris strain Hildenborough, and marine bacterium Desulfovibrio alaskensis strain G-20) grown on lactate across a range of metabolic rates and ambient sulfate concentrations. We identified key isotopic reactions and defined a new, calibrated framework for understanding oxygen isotope variability in sulfate. When used in combination with pore water sulfate/sulfide concentration data and diagenetic modeling, this allowed predicting the abundance of active cells of sulfate reducing bacteria, the result of which is consistent with direct biological measurements (Chapter 2). Finally, we focused on microbial sulfur disproportionation, its biochemistry and evolutionary history (Chapters 3 and 4). MSD possesses the full enzyme machinery for MSR, a vastly more energetically favorable pathway, yet sulfur disproportionating prokaryotes are incapable of growth with sulfate as electron acceptor. Understanding the mechanistic differences between MSR and MSD can provide unique insight into the biochemistry of MSD and refine our understanding of the evolutionary history of these metabolisms. We hypothesized that structural features in key enzymes are at the root of these differences, and we focused on adenosine phosphosulfate (APS) reductase. We found (1) a significantly shortened C-terminal domain in the beta subunit found in SDP, (2) altered surface electrostatic potential distribution around the channel entrance of the alpha subunit, and (3) the substituted amino acids within the substrate channel. We propose these have potential consequences for enzyme regulation via oligomerization, the nature of this enzyme's interaction with electron donors -specifically, QmoA-, and substrate recognition and binding. Phylogenetic relationships reveal a shared capacity for MSD among lineages possessing the C-terminal domain marker, specifically among the Desulfobulbaceae, and a potential to pinpoint evolutionary transitions between MSR and MSD (Chapter 3). We expand our search for further insights into the intracellular dynamics and biochemistry of sulfur disproportionation, with a focus on microbial sulfite and thiosulfate disproportionation. For this, we grew model organism Desulfocapsa sulfexigens as a thiosulfate disproportionator in the presence of either Fe(III) or Fe(II) as sulfide scavenger, and measured the yielded geochemical and isotopic signatures produced in these two conditions. We combine this data with additional data from the literature to calibrate a thermodynamically-rooted isotope model for sulfite and thiosulfate disproportionation, and provide unique insight into the intracellular dynamics during disproportionation. We find that during sulfite disproportionation, rubredoxin and rubrerythrin are the likely electron carriers during the reverse APS reduction reaction. During thiosulfate disproportionation, any electron carrier tested during reverse APS reduction (menaquinol, flavodoxin, specifically, F/FH and FH/FH2, rubredoxin and rubrerythrin) adequately reproduced observed isotope and geochemical data, for both Fe(III) and Fe(II) treatments. We interpret this difference in electron carrier requirement as reflecting the fact that during sulfite disproportionation, for reverse APS reduction to proceed, the electron carrier partner for that reaction must have a standard Gibbs free energy yield that can compete with sulfite reduction. This is not the case during thiosulfate disproportionation. These thermodynamic constraints could be the source of the stoichiometric relationship between products sulfate and sulfide during sulfite disproportionation. Together, the chapters of this thesis highlight the powerful insights provided when fully integrating the contribution of enzymatic, physiological, and environmental contributions to enhancing the interpretability of microbial sulfur isotopic signatures. Once these components and their contributions are fully calibrated, they will allow for a refined assessment of the contribution of microbial sulfur isotopic signatures to the long-term sulfur isotope record, and the physiological and environmental information enclosed within. Ultimately, this refined calibration of the microbial component of the sulfur cycle will allow placing new, improved constraints on paleo-environmental reconstructions over geological timescales. Future work will include testing link between geochemical signatures and the size of the microbial community that produces said signatures in a range of depositional environments, as well as refining our knowledge of the mechanism of microbial sulfur disproportionation and revisiting this metabolisms' evolutionary history and ecological significance. The dataset for microbial sulfur disproportionation will be expanded, both in quantity and across strains of sulfur disproportionators, in order to further refine the biochemistry of this pathway, and explore the implications for the metabolism's biochemical mechanism, and the metabolic plasticity shown by some sulfate reducers and disproportionators.