Intracellular metabolite levels shape sulfur isotope fractionation during microbial sulfate respiration

Abstract

We present a quantitative model for sulfur isotope fractionation accompanying bacterial and archaeal dissimilatory sulfate respiration. By incorporating independently available biochemical data, the model can reproduce a large number of recent experimental fractionation measurements with only three free parameters: (i) the sulfur isotope selectivity of sulfate uptake into the cytoplasm, (ii) the ratio of reduced to oxidized electron carriers supporting the respiration pathway, and (iii) the ratio of in vitro to in vivo levels of respiratory enzyme activity. Fractionation is influenced by all steps in the dissimilatory pathway, which means that environmental sulfate and sulfide levels control sulfur isotope fractionation through the proximate influence of intracellular metabolites. Although sulfur isotope fractionation is a phenotypic trait that appears to be strain specific, we show that it converges on near-thermodynamic behavior, even at micromolar sulfate levels, as long as intracellular sulfate reduction rates are low enough (<<1 fmol H2S⋅cell(-1)⋅d(-1)).

Keywords: dissimilatory sulfate reduction; flux–force relationship; sulfur isotope fractionation.

Fig. 1.

Two illustrations of the dissimilatory sulfate respiration network. (A) Sulfur-focused representation of S-isotope fractionation. Bidirectional arrows represent reversible S transformations. In this framework the “back flux” on any one step is a phenomenological constraint. (B) Metabolite-focused representation used here to quantify back flux. Arrows indicate net flux through the individual steps of the pathway, with the ratio of backward to forward flux controlled by the relative abundances of the reactants and products for each step as well as the kinetics of their associated enzymes. Sat is sulfate adenylyl transferase. Apr is APS reductase. dSiR is dissimilatory sulfite reductase. MK_red refers to the reduced form of menaquinone (menaquinol) and MK_ox refers to the oxidized form of menaquinone. ETC stands for “electron transfer complex.” The likely identities of these complexes in sulfate-reducing microbes are discussed in the text.

Fig. 2.

Predicted metabolite concentrations and isotopic fractionation in a model sulfate reducer. Shown are intracellular concentrations of sulfate (${\left[\mathrm{S}{\mathrm{O}}_4^{2-}\right]}_{\mathrm{in}}$, row A), APS ([APS], row B), PPi ([PPi], row C), and sulfite ($\left[\mathrm{S}{\mathrm{O}}_3^{2-}\right]$, row D) and the net isotopic fractionation between the substrate sulfate and product sulfide (${}^{34}\epsilon$, row E) as functions of extracellular sulfate (${\left[\mathrm{S}{\mathrm{O}}_4^{2-}\right]}_{\mathrm{out}}$, horizontal axis) and sulfide concentrations ([H₂S], vertical axis). All concentrations are shown on logarithmic scales. Intracellular metabolite levels are calculated from Eqs. S22–S25, whereas isotopic fractionation is calculated by application of Eqs. 2 and 5. Regions where calculated PPi concentrations (and associated fractionations) are physiologically unlikely are shown as gray shaded fields (SI Materials and Methods) (rows C and E).

Fig. 3.

Model calibration to experimental data. (A) Net isotopic fractionation $\left({}^{34}\epsilon \right)$ by A. fulgidus grown at 80 °C at constant csSRR (6, 7) as a function of extracellular sulfate concentrations $\left({\left[\mathrm{S}{\mathrm{O}}_4^{2-}\right]}_{\mathrm{out}}\right)$. Orange contours show the value of ${}^{34}\epsilon$ for different values of csSRR in fmol H₂S⋅cell⁻¹⋅d⁻¹. The black curve shows ${}^{34}\epsilon$ for the harmonic mean of the csSRR values that were reported for some of the experiments (black squares). The csSRR values were not reported for other experiments (white squares), resulting in the scatter around the black curve. (B) Predicted ${}^{34}\epsilon$ as a function of csSRR for ${\left[\mathrm{S}{\mathrm{O}}_4^{2-}\right]}_{\mathrm{out}}$ between 10 μM and 100 mM. Experiments for which ${\left[\mathrm{S}{\mathrm{O}}_4^{2-}\right]}_{\mathrm{out}}$, csSRR, and ${}^{34}\epsilon$ were all reported are also shown, color coded by ${\left[\mathrm{S}{\mathrm{O}}_4^{2-}\right]}_{\mathrm{out}}$. (C) Predicted fractionation exponent $\left({}^{33}\lambda \right)$ as a function of ${}^{34}\epsilon$ for the same ${\left[\mathrm{S}{\mathrm{O}}_4^{2-}\right]}_{\mathrm{out}}$ as in B. Experiments for which minor isotope data exist are also shown (14). (D) Predicted ${}^{34}\epsilon \mathrm{-}{\left[\mathrm{S}{\mathrm{O}}_4^{2-}\right]}_{\mathrm{out}}$ relationship for Desulfovibrio strain DMSS-1 grown at ∼20 °C, 21–14 mM ${\left[\mathrm{S}{\mathrm{O}}_4^{2-}\right]}_{\mathrm{out}}$, and 2–7 mM sulfide (–10). (E) Measured (white squares) and model (black curve) ${}^{34}\epsilon$ vs. csSRR for the experiments in D. These experiments were run in batch culture, so we assumed the average ${\left[\mathrm{S}{\mathrm{O}}_4^{2-}\right]}_{\mathrm{out}}$ and external sulfide concentrations for the interval over which ${}^{34}\epsilon$ vs. csSRRs were determined (SI Materials and Methods). (F) ${}^{33}\lambda$ vs. ${}^{34}\epsilon$ for the experiments in D. Error bars are 1σ reported in the experiments. (G–I) Same as D–F, but for D. vulgaris Hildenborough, grown at 25 °C and with precisely controlled ${\left[\mathrm{S}{\mathrm{O}}_4^{2-}\right]}_{\mathrm{out}}$ of 28 mM (11). Sulfide concentrations for the model curves were assumed to be 0.1 mM.

Fig. 4.

Sensitivity of ${}^{34}\epsilon$–csSRR (A) and ${}^{34}\epsilon -\left[\mathrm{S}{\mathrm{O}}_4^{2-}\right]$ (B) relationships to a halving (solid curves) and a doubling (broken curves) of the default [MK_red]/[MK_ox] (= 100) and [H₂S] (= 0.1 mM) values. Fractionation resulting from the default state is shown by the black curves. Shaded envelopes in A and B show the reversibility of the steps in the sulfate reduction pathway resulting from variation of [MK_red]/[MK_ox] and [H₂S] for a range of $\left[\mathrm{S}{\mathrm{O}}_4^{2-}\right]$ and csSRRs. Values of $f_{p,r}$ range from 0.45 to 0.99 for $\mathrm{S}{\mathrm{O}}_4^{2-}$ uptake, 0.98 to ∼1 for activation, ∼0 to 0.98 for APS reduction, and 0.99 to ∼1 for $\mathrm{S}{\mathrm{O}}_3^{2-}$ reduction.

Fig. 5.

Sensitivity of the ${}^{33}\lambda -{}^{34}\epsilon$ relationship to the ratio of reduced to oxidized menaquinone ([MK_red]/[MK_ox], orange) and the kinetic isotope fractionation during sulfate uptake (${}^{34}\epsilon _{\text{uptake}}^{\text{kin}}$, blue).