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Title: Identifying Key Proteins in Hg Methylation Pathways of Desulfovibrio by Global Proteomics, Final Technical Report

Abstract

Elemental mercury, Hg(0) is a contaminant at many DOE sites, especially at Oak Ridge National Laboratory (ORNL) where the spread of spilled Hg and its effects on microbial populations have been monitored for decades. To explore the microbial interactions with Hg, we have devised a global proteomic approach capable of directly detecting Hg-adducts of proteins. This technique developed in the facultative anaerobe, Escherichia coli, allows us to identify the proteins most vulnerable to acute exposure to organomercurials phenyl- and ethyl-mercury (as surrogates for the highly neurotoxic methyl-Hg) (Polacco, et al, 2011). We have found >300 such proteins in all metabolic functional groups and cellular compartments; most are highly conserved and can serve as markers for acute Hg exposure (Zink, et al. 2016, in preparation). We have also discovered that acute Hg exposure severely disrupts thiol, iron and redox homeostases, and electrolyte balance (LaVoie, et al., 2015) Thus, we proposed to bring these techniques to bear on the central problem of identifying the cellular proteins involved in bacterial uptake and methylation of mercury and its release from the cell.

Authors:
 [1];  [2];  [3];  [4]
  1. Univ. of Georgia, Athens, GA (United States). Dept. of Microbiology
  2. Univ. of California, San Francisco, CA (United States). Dept. of Pharmaceutical Chemistry
  3. Univ. of Missouri, Columbia, MO (United States). Dept. of Biochemistry
  4. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Publication Date:
Research Org.:
Univ. of Georgia, Athens, GA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1257709
Report Number(s):
10-21-RR182-404
DOE Contract Number:
SC0007173
Resource Type:
Technical Report
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; mercury; methylmercury; proteomics; transcriptomics

Citation Formats

Summers, Anne O., Miller, Susan M., Wall, Judy, and Lipton, Mary. Identifying Key Proteins in Hg Methylation Pathways of Desulfovibrio by Global Proteomics, Final Technical Report. United States: N. p., 2016. Web. doi:10.2172/1257709.
Summers, Anne O., Miller, Susan M., Wall, Judy, & Lipton, Mary. Identifying Key Proteins in Hg Methylation Pathways of Desulfovibrio by Global Proteomics, Final Technical Report. United States. doi:10.2172/1257709.
Summers, Anne O., Miller, Susan M., Wall, Judy, and Lipton, Mary. 2016. "Identifying Key Proteins in Hg Methylation Pathways of Desulfovibrio by Global Proteomics, Final Technical Report". United States. doi:10.2172/1257709. https://www.osti.gov/servlets/purl/1257709.
@article{osti_1257709,
title = {Identifying Key Proteins in Hg Methylation Pathways of Desulfovibrio by Global Proteomics, Final Technical Report},
author = {Summers, Anne O. and Miller, Susan M. and Wall, Judy and Lipton, Mary},
abstractNote = {Elemental mercury, Hg(0) is a contaminant at many DOE sites, especially at Oak Ridge National Laboratory (ORNL) where the spread of spilled Hg and its effects on microbial populations have been monitored for decades. To explore the microbial interactions with Hg, we have devised a global proteomic approach capable of directly detecting Hg-adducts of proteins. This technique developed in the facultative anaerobe, Escherichia coli, allows us to identify the proteins most vulnerable to acute exposure to organomercurials phenyl- and ethyl-mercury (as surrogates for the highly neurotoxic methyl-Hg) (Polacco, et al, 2011). We have found >300 such proteins in all metabolic functional groups and cellular compartments; most are highly conserved and can serve as markers for acute Hg exposure (Zink, et al. 2016, in preparation). We have also discovered that acute Hg exposure severely disrupts thiol, iron and redox homeostases, and electrolyte balance (LaVoie, et al., 2015) Thus, we proposed to bring these techniques to bear on the central problem of identifying the cellular proteins involved in bacterial uptake and methylation of mercury and its release from the cell.},
doi = {10.2172/1257709},
journal = {},
number = ,
volume = ,
place = {United States},
year = 2016,
month = 6
}

Technical Report:

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  • Hg is a wide-spread contaminant in the environment and is toxic in all of its various forms. Data suggest that RHg+ and Hg2+ are toxic in two ways. At low levels, Hg species appear to disrupt membrane-bound respiration causing a burst of reactive oxygen species (ROS) that further damage the cell. At higher Hg concentrations, RHg+ and Hg2+ may form adducts with cysteine- and selenocysteine-containing proteins in all cellular compartments resulting in their inactivation. Although these mechansims for toxicity are generally accepted, the most sensitive targets associated with these mechanisms are not well understood. In this collaborative project involving threemore » laboratories at three institutions, the overall goal was to develop of a mass spectrometry-based global proteomics methodology that could be used to identify Hg-adducted (and ideally, ROS-damaged) proteins in order to address these types of questions. The two objectives of this overall collaborative project were (1) to identify, quantify, and compare ROS- and Hg-damaged proteins in cells treated with various Hg species and concentrations to test this model for two mechanisms of Hg toxicity, and (2) to define the cellular roles of the ubiquitous bacterial mercury resistance (mer) locus with regards to how the proteins of this pathway interact to protect other cell proteins from Hg damage. The specific objectives and accomplishments of the Miller lab in this project included: (1) Development of algorithms for analysis of the Hg-proteomic mass spectrometry data to identify mercury adducted peptides and other trends in the data. (2) Investigation of the role of mer operon proteins in scavenging Hg(II) from other mer pathway proteins as a means of protecting cellular proteins from damage.« less
  • Final Technical Report: Isotopic Characterization of Biogeochemical Pools of Mercury and Determination of Reaction Pathways for Mercury Methylation.
  • Anaerobic aquatic sediments exposed to low-level inputs of inorganic mercury convert it to monomethylmercury, a highly hazardous environmental pollutant prone to biomagnification. Studies indicate that over 95% of mercury biomethylation is carried out by sulfate-reducing microorganisms. The importance of a corrinoid protein as a methyl transfer intermediate in the acetyl coenzyme A formation pathway of Clostridium thermoaceticum and other anaerobes suggests that enzymes of this pathway are responsible for generation and transfer of the methyl moiety to mercury by D. desulfuricans LS. The enzymes of this pathway include a methyltransferace and CO dehydrogenase/acetyl-CoA synthase in addition to formate dehydrogenase, themore » THF pathway enzymes and the corrinoid protein. This study reports on levels of these enzymes in D. desulfuicans and discusses their relationship to mercury methylation. 41 refs., 1 fig., 3 tabs.« less
  • The primary objectives of this proposal was to define the subset of proteins required for the ionizing radiation (IR) resistance of Deinococcus radiodurans R1, characterize the activities of those proteins, and apply what was learned to problems of interest to the Department of Energy.
  • We continue to utilize the oligonucleotide microarrays that were constructed through funding with this project to characterize growth responses of Desulfovibrio vulgaris relevant to metal-reducing conditions. To effectively immobilize heavy metals and radionuclides via sulfate-reduction, it is important to understand the cellular responses to adverse factors observed at contaminated subsurface environments (e.g., nutrients, pH, contaminants, growth requirements and products). One of the major goals of the project is to construct whole-genome microarrays for Desulfovibrio vulgaris. First, in order to experimentally establish the criteria for designing gene-specific oligonucleotide probes, an oligonucleotide array was constructed that contained perfect match (PM) and mismatchmore » (MM) probes (50mers and 70mers) based upon 4 genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were examined. Little hybridization was observed at a probe-target identity of <85% for both 50mer and 70mer probes. 33 to 48% of the PM signal intensities were detected at a probe-target identity of 94% for 50mer oligonucleotides, and 43 to 55% for 70mer probes at a probe-target identity of 96%. When the effects of sequence identity and continuous stretch were considered independently, a stretch probe (>15 bases) contributed an additional 9% of the PM signal intensity compared to a non-stretch probe (< 15 bases) at the same identity level. Cross-hybridization increased as the length of continuous stretch increased. A 35-base stretch for 50mer probes or a 50-base stretch for 70mer probes had approximately 55% of the PM signal. Mismatches should be as close to the middle position of an oligonucleotide probe as possible to minimize cross-hybridization. Little cross-hybridization was observed for probes with a minimal binding free energy greater than -30 kcal/mol for 50mer probes or -40 kcal/mol for 70mer probes. Based on the experimental results, a set of criteria were suggested for the design of gene-specific and group-specific oligonucleotide probes, and these criteria should provide valuable information for the development of new software and algorithms for microarray-based studies. Secondly, in order to empirically determine the effect of probe length on signal intensities, microarrays with oligonucleotides of different lengths were used to monitor gene expression at a whole genome level. To determine what length of oligonucleotide is a better alternative to PCR-generated probes, the performance of oligonucleotide probes was systematically compared to that of their PCR-generated counterparts for 96 genes from Shewanella oneidensis MR-1 in terms of overall signal intensity, numbers of detected genes, specificity, sensitivity and differential gene expression under experimental conditions. Hybridizations conducted at 42 C, 45 C, 50 C, and 60 C indicated that good sensitivities were obtained at 45 C for oligonucleotide probes in the presence of 50% formamide, under which conditions specific signals were detected by both PCR and oligonucleotide probes. Signal intensities increased as the length of oligonucleotide probes increased, and the 70mer oligonucleotide probes produced similar signal intensities and detected a similar number of ORFs compared to the PCR probes. cDNA, 70mer, 60mer and 50mer arrays had detection sensitivities at 5.0, 25, 100 and 100 ng of genomic DNA, or an approximately equivalent of 1.9 x 10{sup 6}, 9.2 x 10{sup 6}, 3.7 x 10{sup 7} and 3.7 x 10{sup 7} copies, respectively when the array was hybridized with genomic DNA. To evaluate differential gene expression under experimental conditions, S. oneidensis MR-1 cells were exposed to low or high pH conditions for 30 and 60 min, and the transcriptional profiling detected by oligonucleotide probes (50mer, 60mer, and 70mer) was closely correlated with that detected by the PCR probes. The results demonstrated that 70mer oligonucleotides can achieve the most comparable performance with PCR-generated probes. We have analyzed expression data as D. vulgaris transitioned during electron donor depletion. As the cells transitioned from exponential to stationary-phase a majority of the down-expressed genes were involved in translation and transcription, and this trend continued in the remaining time points. Interestingly, most phage-related genes were up-expressed at the onset of stationary-phase. This result suggested that nutrient depletion may impact community dynamics and DNA transfer mechanisms of sulfate-reducing bacteria via phage cycle. The putative feoAB system (in addition to other presumptive iron metabolism genes) was significantly up-expressed, and suggested the possible importance of Fe{sup 2+} acquisition under metal-reducing conditions. Namely, that iron availability should be considered when sulfate-reducing conditions are stimulated in the subsurface for heavy metal reduction.« less