Comparative Proteomic Analysis of Desulfotomaculum reducens MI-1: Insights into the Metabolic Versatility of a Gram-Positive Sulfate- and Metal-Reducing Bacterium

Abstract

The proteomes of the metabolically versatile and poorly characterized Gram-positive bacterium Desulfotomaculum reducens MI-1 were compared across four cultivation conditions including sulfate reduction, soluble Fe(III) reduction, insoluble Fe(III) reduction, and pyruvate fermentation. Collectively across conditions, we observed at high confidence ~38% of genome-encoded proteins. Here, we focus on proteins that display significant differential abundance on conditions tested. To the best of our knowledge, this is the first full-proteome study focused on a Gram-positive organism cultivated either on sulfate or metal-reducing conditions. Several proteins with uncharacterized function encoded within heterodisulfide reductase (hdr)-containing loci were upregulated on either sulfate (Dred_0633-4, Dred_0689-90, and Dred_1325-30) or Fe(III)-citrate-reducing conditions (Dred_0432-3 and Dred_1778-84). Two of these hdr-containing loci display homology to recently described flavin-based electron bifurcation (FBEB) pathways (Dred_1325-30 and Dred_1778-84). Additionally, we propose that a cluster of proteins, which is homologous to a described FBEB lactate dehydrogenase (LDH) complex, is performing lactate oxidation in D. reducens (Dred_0367-9). Analysis of the putative sulfate reduction machinery in D. reducens revealed that most of these proteins are constitutively expressed across cultivation conditions tested. In addition, peptides from the single multiheme c-type cytochrome (MHC) in the genome were exclusively observed on the insoluble Fe(III) condition, suggesting that this MHC may play a role in reduction of insoluble metals.

Keywords: Desulfotomaculum reducens; Fe(III) reduction; comparative proteomic analysis; flavin-based electron bifurcation; gram-positive bacteria; heterodisulfide reductase; sulfate reduction.

Figure 1

A heat map of the proteomes generated for D. reducens. Replicates grouped together based on hierarchical clustering using the heat map command in R. The two Fe(III) reduction conditions form clusters that are more similar to each other than to any other condition. A similar relationship is observed for the pyruvate fermentation and sulfate reduction condition. Data input for each replicate (two biological duplicates and three technical triplicates per condition) consisted of the average ion intensity for all proteins observed, and data for all detected peptides was included. Identified proteins are displayed across the horizontal axis and ordered by hierarchical clustering with complete linkage. Relative average ion intensity is displayed from yellow (lowest) to red (highest) (R Development Core Team, 2012).

Figure 2

Identification of proteins encoded within heterodisulfide reductase (hdr)-containing loci. A number of proteins within hdr-containing loci were identified during proteomic analysis of D. reducens and many displayed differential abundance across cultivation conditions. Gene name abbreviations stand for iron sulfur proteins (FeS), methyl viologen hydrogenase, delta subunit (mvhD), annotated oxidoreductases (OR), electron transfer flavoproteins (etfA and etfB), sulfate adenyltransferase (sat), and APS reductase (aprA and aprB). Subunits of heterodisulfide reductases (hdr) are also shown. This figure is modified from an image published in the D. reducens genome paper and loci have been renumbered to match with locus tag position in the genome (Junier et al., 2010). ^*Refers to identification by only one unique peptide in at least half of the replicates.

Figure 3

Predicted pathways of lactate and pyruvate utilization in D. reducens based on proteomic analysis. (A) Dred_0367-9 is proposed to perform lactate oxidation in D. reducens. These proteins share similarity with a lactate-oxidizing complex recently described in Acetobacterium woodii to operate through flavin-based electron bifurcation (FBEB) (Awo_c08730, 20, and 10; Weghoff et al., 2015). Similarity between D. reducens proteins and those from A. woodii is displayed as percent sequence identity across percent query coverage (http://blast.ncbi.nlm.nih.gov). (B) Model of lactate and/or pyruvate utilization in D. reducens. In lactate-fed cultures, lactate is oxidized to pyruvate. Pyruvate is then converted to acetate (along with CO₂ or formate), and H₂ is produced during pyruvate fermentation. In lactate-fed conditions, H₂ is not formed, and instead reduced ferredoxin could be utilized in other pathways, including the lactate oxidation pathway. Pathways predicted to be involved in lactate-fed cultures are denoted with a blue L. Pathways predicted to be involved in pyruvate-fed fermentative cultures are denoted with a green F. Abundance comparisons for proteins displayed are in Supplementary Table 3.

Figure 4

Predicted pathway of sulfate reduction in D. reducens based on proteomic findings. The core sulfate reduction machinery (Sat, APS reductase, sulfite reductase) does not display significant differential abundance. Instead, certain clusters of proteins encoded within hdr-containing loci are increased in abundance during sulfate reduction and are predicted to be involved in the process. Abundance comparisons are shown for sulfate and Fe(III)-citrate reduction conditions. All protein abundance comparisons are displayed in Tables 3, 4.

Figure 5

Hdr-containing locus VI (Dred_1325-30) is proposed to transfer electrons to DsrC. (A) Dred_1325-30 is significantly increased in abundance during sulfate reduction relative to other cultivation conditions analyzed. This locus contains orthologs to proteins within the flavin-based electron bifurcation (FBEB) FlxABCD-HdrABC cluster described in Desulfovibrio vulgaris Hildenborough (DVU2399-2404) to be involved in electron transfer to DsrC (Ramos et al., 2015). Sequence similarity is displayed as percent sequence identity across percent query coverage (http://blast.ncbi.nlm.nih.gov). An open circle displays lack of an ortholog. (B) Hdr-containing locus I (Dred_0137-148) contains similarity to several proteins within locus VI and the FlxABCD-HdrABC cluster but is missing two of the Hdr-type proteins. Dred_0141 and Dred_0148 (identical proteins) are orthologs to the FlxA protein missing from the Dred_1325-30 cluster. It is possible that locus VI (Dred_1325-30) and locus I (Dred_0137-48), both of which were identified with high numbers of unique peptides in our proteomic data, work together to carry out FBEB in order to reduce DsrC.

Figure 6

The cluster of proteins most increased on Fe(III)-citrate relative to pyruvate fermentation has similarity to a described FBEB system. A significant number of peptides and overall increased abundance was observed for proteins within hdr-containing locus VII (Dred_1778-84) on the Fe(III)-citrate condition. Genes within this locus are orthologs to the FBEB complex BcdA–EtfBC from Clostridium kluyveri DSM 555 (Li et al., ; Buckel and Thauer, 2013). Similarity between this complex (CKL_0454-8) and proteins in D. reducens is displayed as percent sequence identity across percent query coverage (http://blast.ncbi.nlm.nih.gov). Furthermore, redundant genes for each of the proteins involved in the FBEB step are expressed solely on the Fe(III)-citrate condition and include the electron transfer flavoproteins Dred_0573 and Dred_0572 (45, 97% and 31, 80%), and the acyl-CoA dehydrogenase domain-containing protein Dred_0402 (53, 100%). Furthermore, the enoyl-CoA hydratase/isomerase Dred_0401 is a redundant protein (60, 100%) for the step leading to crotonoyl-CoA, also unique to the Fe(III)-citrate condition.