Global Proteome Response to Deletion of Genes Related to Mercury Methylation and Dissimilatory Metal Reduction Reveals Changes in Respiratory Metabolism in Geobacter sulfurreducens PCA

Abstract

Geobacter sulfurreducens PCA can reduce, sorb, and methylate mercury (Hg); however, the underlying biochemical mechanisms of these processes and interdependent metabolic pathways remain unknown. In this study, shotgun proteomics was used to compare global proteome profiles between wild-type G. sulfurreducens PCA and two mutant strains: a ΔhgcAB mutant, which is deficient in two genes known to be essential for Hg methylation and a ΔomcBESTZ mutant, which is deficient in five outer membrane c-type cytochromes and thus impaired in its ability for dissimilatory metal ion reduction. We were able to delineate the global response of G. sulfurreducens PCA in both mutants and identify cellular networks and metabolic pathways that were affected by the loss of these genes. Deletion of hgcAB increased the relative abundances of proteins implicated in extracellular electron transfer, including most of the c-type cytochromes, PilA-C, and OmpB, and is consistent with a previously observed increase in Hg reduction in the ΔhgcAB mutant. Deletion of omcBESTZ was found to significantly increase relative abundances of various methyltransferases, suggesting that a loss of dissimilatory reduction capacity results in elevated activity among one-carbon (C1) metabolic pathways and thus increased methylation. We show that G. sulfurreducens PCA encodes only the folate branch of the acetyl-CoA pathway, and proteins associated with the folate branch were found at lower abundance in the ΔhgcAB mutant strain than the wild type. This observation supports the hypothesis that the function of HgcA and HgcB is linked to C1 metabolism through the folate branch of the acetyl-CoA pathway by providing methyl groups required for Hg methylation.

Quantitative Proteomic Analysis of Biological Processes and Responses of the BacteriumDesulfovibrio desulfuricansND132 upon Deletion of Its Mercury Methylation Genes

“…Because the hgcAB gene pair is required for Hg methylation by anaerobic microorganisms such as D. desulfuricans ND132, we first examined whether the HgcA and HgcB proteins could be identified and quantified as no protein‐level characterization of the two genes is currently available. Our previous global proteome analysis of G. sulfurreducens PCA did not identify any HgcA or HgcB proteins, leading us to speculate that these two proteins are expressed at very low levels, or their expression may be induced by the addition of Hg, or both. However, although their abundances were low, we were able to identify both HgcA and HgcB in the proteomes of WT D. desulfuricans ND132, as evidenced by their intensity ranking at the tail end among all the identified proteins (Figure A).…”

“…Of all the metal transporters that showed significant differential abundances, three RND family efflux proteins (UniProt IDs: F0JGV5, F0JGV3, and F0JGV2) are of particular interest since we previously identified a tripartite RND efflux pump (UniProt IDs Q74DI4, Q74DI5, and Q74DI6) that was less abundant in the Δ hgcAB mutant compared to the WT strain of G. sulfurreducens PCA . Interestingly, while F0JGV5, F0JGV3, and F0JGV2 show significant homology to the tripartite RND efflux pump that was identified in G. sulfurreducens PCA (F0JGV5 is 30% identical to Q74DI6, F0JGV3 is 35% identical to Q74DI5, and F0JGV2 is 46% identical to Q74DI4), their respective abundances increased in the Δ hgcAB mutant of D. desulfuricans ND132.…”

“…Protein Extraction and Proteolytic Digestion : Protein extraction and proteolytic digestion were carried out as described previously . Generally, cell pellets (about 1 × 10 11 cells) were suspended in 1 mL sodium dodecyl sulfate (SDS) lysis buffer (4% w/v SDS, 100 m m Tris‐HCl, pH 8.0) and vortexed.…”

“…Our recent proteomics studies with the FeRB Geobacter sulfurreducens PCA and its Δ hgcAB mutant strain indicated that the function of HgcA and HgcB may be linked to C1 metabolism. However, the absence of identifiable peptide fragments derived from HgcA or HgcB proteins in all samples of wild‐type (WT) and the Δ hgcAB deletion mutant of G. sulfurreducens PCA led us to speculate that these two proteins are expressed at very low levels and that their expression may be triggered by the addition of Hg, or both.…”

Reduced sulphur sources favour HgII reduction during anoxygenic photosynthesis by Heliobacteria

“…Much of the evidence published to date suggests that Hg methylation is a cometabolic process linked to one-carbon compound metabolism (Choi, Chase, & Bartha, 1994;Qian et al, 2016) in multiple clades of anaerobes (Gionfriddo et al, 2016;Parks et al, 2013;Podar et al, 2015). However, the role that common pools of nutrients (e.g., C, N or S) play in controlling different cometabolic Hg transformations is rarely discussed.…”