Proteomic and Isotopic Response of Desulfovibrio vulgaris to DsrC Perturbation

Abstract

Dissimilatory sulfate reduction is a microbial energy metabolism that can produce sulfur isotopic fractionations over a large range in magnitude. Calibrating sulfur isotopic fractionation in laboratory experiments allows for better interpretations of sulfur isotopes in modern sediments and ancient sedimentary rocks. The proteins involved in sulfate reduction are expressed in response to environmental conditions, and are collectively responsible for the net isotopic fractionation between sulfate and sulfide. We examined the role of DsrC, a key component of the sulfate reduction pathway, by comparing wildtype Desulfovibrio vulgaris DSM 644^T to strain IPFG07, a mutant deficient in DsrC production. Both strains were cultivated in parallel chemostat reactors at identical turnover times and cell specific sulfate reduction rates. Under these conditions, sulfur isotopic fractionations between sulfate and sulfide of 17.3 ± 0.5‰ or 12.6 ± 0.5‰ were recorded for the wildtype or mutant, respectively. The enzymatic machinery that produced these different fractionations was revealed by quantitative proteomics. Results are consistent with a cellular-level response that throttled the supply of electrons and sulfur supply through the sulfate reduction pathway more in the mutant relative to the wildtype, independent of rate. We conclude that the smaller fractionation observed in the mutant strain is a consequence of sulfate reduction that proceeded at a rate that consumed a greater proportion of the strains overall capacity for sulfate reduction. These observations have consequences for models of sulfate reducer metabolism and how it yields different isotopic fractionations, notably, the role of DsrC in central energy metabolism.

Keywords: chemostat; microbial energy metabolism; microbial sulfate reduction; proteomics; sulfur isotope fractionation.

FIGURE 1

The key enzyme-catalyzed reactions of MSR. Sulfate is imported to the cell through transporters, activated to APS by Sat, and reduced to sulfite by AprAB. Sulfite is reduced to H₂S by DsrAB at the siroheme active site (pink), which partially reduces sulfite to zero-valent S. Approximately zero-valent S is carried to the membrane by DsrC, where it undergoes final reduction to H₂S (Santos et al., 2015). There is potential for a branching flux of sulfur at DsrAB, which can produce ancillary H₂S by complete reduction of sulfite, or produce thionates by the partial reduction of sulfite and scavenging of that pool by intracellular sulfite (Leavitt et al., 2015). The amount of branching flux depends on the rate of electron supply, sulfur supply, and reduced-DsrC supply. Key steps predicted to affect fraction involve the reductive steps at AprAB, DsrAB, and DsrC.

FIGURE 2

Chemostat timepoints: for WT (A,C,E) and IPFG07 mutant (B,D,F). Panels A and B are calculated turnover times. Panels C and D are departures from steady-state. Panels E and F are sulfate and sulfide S isotopic compositions on the international scale (V-PDB). In all filled symbols are from the time points at which proteome samples were also collected.

FIGURE 3

Chemostat samples: (A) Steady-state doubling time; (B) Departure from steady-state (dilution rate and growth rate); Panel C shows cell specific sulfate reduction rates, each plotted versus sulfur isotope fractionation. Individual values for WT (blue squares) and IPFG07 (red circles) reactors from each of the five separate turnover time points with the mean in bold. In panels A and C, the dashed lines are the mean and shaded areas are the CI_95% for WT (blue) and IPFG07 (red). In panel B, the dark and light gray areas indicate ±5% and ±10% departures from steady-state, respectively.

FIGURE 4

Clusters of orthologous groups (COGs) for the 99 proteins identified in all five biological replicates from both IPFG07 and WT samples that were differentially expressed (see section “Materials and Methods” for statistical significance calculations).

FIGURE 5

Schematic representation of proteins directly involved in sulfate reduction pathway displayed according to their gene cluster arrangement. The color code of each protein follows the weighted abundance ratio data obtained for the group of 99 proteins that were differentially expressed between the two strains. Proteins not found in this dataset are denoted with dashed boxes.

FIGURE 6

Schematic representation of possible DsrC interacting proteins and their arrangement according to each gene cluster. These proteins belong to HdrB- and HdrD-like proteins, and are identified with CCG code. The color code of each protein follows the weighted abundance ratio data obtained for the group of 99 proteins that were differentially expressed between the two strains. Proteins not found in this set are denoted with dashed boxes. DsrMKJOP is also presented in Figure 5 for reference.