Protein-SIP enables time-resolved analysis of the carbon flux in a sulfate-reducing, benzene-degrading microbial consortium

Abstract

Benzene is a major contaminant in various environments, but the mechanisms behind its biodegradation under strictly anoxic conditions are not yet entirely clear. Here we analyzed a benzene-degrading, sulfate-reducing enrichment culture originating from a benzene-contaminated aquifer by a metagenome-based functional metaproteomic approach, using protein-based stable isotope probing (protein-SIP). The time-resolved, quantitative analysis of carbon fluxes within the community supplied with either (13)C-labeled benzene or (13)C-labeled carbonate yielded different functional groups of organisms, with their peptides showing specific time dependencies of (13)C relative isotope abundance indicating different carbon utilization. Through a detailed analysis of the mass spectrometric (MS) data, it was possible to quantify the utilization of the initial carbon source and the metabolic intermediates. The functional groups were affiliated to Clostridiales, Deltaproteobacteria and Bacteroidetes/Chlorobi. The Clostridiales-related organisms were involved in benzene degradation, putatively by fermentation, and additionally used significant amounts of carbonate as a carbon source. The other groups of organisms were found to perform diverse functions, with Deltaproteobacteria degrading fermentation products and Bacteroidetes/Chlorobi being putative scavengers feeding on dead cells. A functional classification of identified proteins supported this allocation and gave further insights into the metabolic pathways and the interactions between the community members. This example shows how protein-SIP can be applied to obtain temporal and phylogenetic information about functional interdependencies within microbial communities.

Figure 1

Exemplary peptide mass spectra with ¹³C-incorporation patterns and development over time. (a–c) Examples of different ¹³C-incorporation patterns of the ¹³C-benzene cultivation at d₅₀ (black), d₆₃ (red), d₉₇ (green), d₁₈₀ (blue) and d₃₀₀ (purple): Peptide SYQIVEGTSNIQK with local incorporation pattern at 60.1% RIA (a), peptide TLTAGQITPYK with incorporation range from 0% to 60% RIA (best fit at 43.6% RIA) (b) and peptide IATAQAANR with incorporation range from 0% to 40% RIA (best fit at 24.8% RIA) (c). (d–f) Examples of different ¹³C-incorporation patterns of the ¹³C-carbonate cultivation at d₇₅ (black), d₁₅₁ (green) and d₃₀₀ (purple): Peptide APVVDDDGVR with local incorporation pattern at 40.6% RIA (d), peptide VFNIFGATGADmK (m=oxidized methionine) with incorporation range from 25% to 45% RIA (best fit at 39.6% RIA) (e) and peptide SVLDNEAIVSDPILAGSSK with incorporation range from 0% to 25% RIA (best fit at 12% RIA) (f). Averaged RIA (g, h) and doublings of peptide amount (i, j) of all peptides with incorporation patterns as shown in a and d (black diamond), b and e (white square) or c and f (black square) from all time points of the ¹³C carbonate and ¹³C benzene incubations, respectively.

Figure 2

Abundance of ¹³C-incorporation patterns in identified proteins. The detected groups of incorporation patterns for the ¹³C-benzene experiment (top row) and the ¹³C-carbonate experiment (left column) are shown together with the number of identified proteins in each category. These were used to form three major groups of proteins from putative organisms utilizing different carbon sources. ND=no ¹³C-incorporation detectable. Roman numeral=group of proteins. *Proteins were classified as group III as differentiation of the patterns from the ¹³C-carbonate cultivation was more reliable than from ¹³C-benzene cultivation.

Figure 3

Phylogenetic classification of identified proteins according to blast annotation of identified proteins. The broad bars show the phylogenetic groups present in the total amount of identified protein and in the three different groups of proteins that were formed based on incorporation patterns. The category ‘uncertain/others' contains proteins assigned to various other phylogenetic groups than listed and proteins that produced significant blast results, but allowed no clear phylogenetic classification. The category ‘unique' contains unique hypothetical proteins producing no significant blast results at all. The small bars show the phylogenetic distribution in selected phylogenetic marker proteins: chaperonines, subunits of F₀F₁ ATPases, ribosomal proteins and proteins involved in DSR. For group III, the number of phylogenetic marker proteins was too low and is not shown. The parenthetic numbers present the number of identified proteins in each group.

Figure 4

Functional classification of identified proteins. The numbers of proteins in different functional categories are shown for the protein groups with different ¹³C-incorporation patterns. Numbers within parentheses show the total number of identified proteins in a category.

Figure 5

Hypothetical overview of the community. Carbon sources and carbon flux through the identified groups of organisms as conceived by the interpretation of the stable isotope probing results.