# Molecular insights into the dynamic relationship between respiration rate and sulfur isotope effect

## TL;DR

This study explores how microbial respiration rates and sulfur isotope effects are dynamically linked, revealing exceptions and their biochemical basis.

## Contribution

The paper provides a molecular and biochemical explanation for the dynamic relationship between respiration rates and sulfur isotope fractionation in sulfate-reducing bacteria.

## Key findings

- N2 fixation with malate increases metabolic sulfur flux and reversibility of sulfate reduction.
- N2 fixation with fructose increases respiration rate but reduces isotope fractionation.
- Elevated intracellular NADH may explain contrasting responses to N2 fixation.

## Abstract

Microbial sulfate reduction and its resulting sulfur isotope effects are crucial for understanding the past and present sulfur cycle. Microbial S-isotope effects have often been explained by their inverse correlation with the cell-specific sulfur reduction rate (csSRR), but exceptions exist. A notable example is when N2-fixing sulfate reducers fractionate sulfur isotopes more than those cultivated under fixed nitrogen, despite having a faster csSRR. To further understand the biochemical basis of the csSRR and S-isotope fractionation relationship, we monitored gene expression, ATP/AMP ratio, and triple S-isotope fractionation of a sulfate-reducing bacterium, DMSS-1 (Desulfovibrio sp.), under ammonium-repleted or depleted conditions with various electron donors. N2 fixation with malate occurred with an elevated metabolic sulfur flux as indicated by enhanced sulfate reduction gene expression, but also with an increased reversibility of sulfate activation and reduction, evidenced by a reduced ATP/AMP ratio. The two simultaneous molecular alterations potentially explain the higher fractionation values observed under N2 fixation. Meanwhile, N2 fixation with fructose, a more refractory but energy-rich electron donor, did not alter gene expression or ATP/AMP ratio, but led to increased csSRR and decreased isotope fractionation. Integrating our findings into the 32S, 33S, and 34S sulfur isotope model suggests that N2 fixation reduces leakage of sulfate across the cell membrane during fructose catabolism. Gene expression analysis of carbon catabolism indicates that elevated intracellular NADH may underlie this contrasting response to N2 fixation. While the csSRR and S-isotope fractionation relationship is demonstrably dynamic in this study, it reinforces the robustness of the typical inverse correlation in most natural environments, where csSRR high enough to overturn this relationship are rarely observed.

Sulfate-reducing microorganisms produce sulfide depleted in heavy sulfur isotopes during respiration, making the distribution of sulfur isotopes in natural environments an important clue for tracing their activity and physiology. An apparent inverse correlation between cell-specific respiration rate and sulfur isotope fractionation has been widely accepted as a primary control on naturally occurring sulfur isotope signatures. However, exceptions to this trend have been reported, warranting a better mechanistic understanding. Here, using the model sulfate-reducing bacterium DMSS-1, we manipulated carbon and nitrogen sources and monitored sulfur isotope fractionation, respiratory gene expression, and cellular energy status to provide a molecular and biochemical basis for the dynamic relationship between respiration rates and isotope effects. While this relationship is variable, our results suggest that reversing the inverse trend requires exceptionally fast respiration rates rarely achieved in natural environments. This highlights the robustness of the conventional inverse relationship in nature, despite intracellular complexity.