Anne Summers | The University of Georgia (original) (raw)

Papers by Anne Summers

Research paper thumbnail of Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA

ABSTRACTRecent advances and lower costs in rapid high-throughput sequencing have engendered hope ... more ABSTRACTRecent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in clinical bacterial isolates. Despite its potential, several challenges should be addressed before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical laboratory. Here, with three distinct ESKAPE bacteria, we compared different approaches to identify best practices for detection of AMR genes, including: total genomic DNA and plasmid DNA extractions, solo assembly of Illumina short-reads and of ONT long-reads, two hybrid assembly pipelines, and three in silico AMR databases. We also determined the susceptibility of each strain to 21 antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also detectable in total genomic DNA indicating that, at least in these three enterobacterial genera, purification of plasmid DNA was not necessary to detect plasmid-borne AM...

Research paper thumbnail of Corrigendum to: Children with Amalgam Dental Restorations Have Significantly Elevated Blood and Urine Mercury Levels

Toxicological Sciences, 2022

Research paper thumbnail of Hg(II) Binding to Thymine Bases in DNA

Inorganic Chemistry, 2021

The compounds of mercury can be highly toxic and can interfere with a range of biological process... more The compounds of mercury can be highly toxic and can interfere with a range of biological processes, although many aspects of the mechanism of toxicity are still obscure or unknown. One especially intriguing property of Hg(II) is its ability to bind DNA directly, making interstrand cross-links between thymine nucleobases in AT-rich sequences. We have used a combination of small molecule X-ray diffraction, X-ray spectroscopies, and computational chemistry to study the interactions of Hg(II) with thymine. We find that the energetically preferred mode of thymine binding in DNA is to the N3 and predict only minor distortions of the DNA structure on binding one Hg(II) to two cross-adjacent thymine nucleotides. The preferred geometry is predicted to be twisted away from coplanar through a torsion angle of between 32 and 43°. Using 1-methylthymine as a model, the bis-thymine coordination of Hg(II) is found to give a highly characteristic X-ray spectroscopic signature that is quite distinct from other previously described biological modes of binding of Hg(II). This work enlarges and deepens our view of significant biological targets of Hg(II) and demonstrates tools that can provide a characteristic signature for the binding of Hg(II) to DNA in more complex matrices including intact cells and tissues, laying the foundation for future studies of mechanisms of mercury toxicity.

Research paper thumbnail of Integrating the Molecular Machines of Mercury Detoxification into Host Cell Biology

Metalloregulators of the MerR family activate transcription upon metal binding by underwinding th... more Metalloregulators of the MerR family activate transcription upon metal binding by underwinding the operator-promoter DNA to permit open complex formation by pre-bound RNA polymerase. Historically, MerR's allostery has been monitored only indirectly via nuclease sensitivity or by fluorescent nucleotide probes and was very specific for Hg(II), although purified MerR binds several thiophilic metals. To observe directly MerR's ligand-induced behavior we made 2-fluorotyrosine-substituted MerR and found similar, minor changes in 19 F chemical shifts of tyrosine residues in the free protein exposed to Hg(II), Cd(II) or Zn(II). However, DNA binding elicits large chemical shift changes in MerR's tyrosine residues and in DNAbound MerR Hg(II) provokes changes very distinct from those of Cd(II) or Zn(II). These chemical shift changes and other biophysical and phenotypic properties of wild-type MerR and relevant mutants reveal elements of an allosteric network that enables the coordination state of the metal binding site to direct metal-specific movements in the distant DNA binding site and the DNA-bound state also to affect the metal binding domain.

Research paper thumbnail of Organic and inorganic mercurials have distinct effects on cellular thiols, metal homeostasis, and Fe-binding proteins in Escherichia coli

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, Jan 26, 2015

The protean chemical properties of the toxic metal mercury (Hg) have made it attractive in divers... more The protean chemical properties of the toxic metal mercury (Hg) have made it attractive in diverse applications since antiquity. However, growing public concern has led to an international agreement to decrease its impact on health and the environment. During a recent proteomics study of acute Hg exposure in E. coli, we also examined the effects of inorganic and organic Hg compounds on thiol and metal homeostases. On brief exposure, lower concentrations of divalent inorganic mercury Hg(II) blocked bulk cellular thiols and protein-associated thiols more completely than higher concentrations of monovalent organomercurials, phenylmercuric acetate (PMA) and merthiolate (MT). Cells bound Hg(II) and PMA in excess of their available thiol ligands; X-ray absorption spectroscopy indicated nitrogens as likely additional ligands. The mercurials released protein-bound iron (Fe) more effectively than common organic oxidants and all disturbed the Na(+)/K(+) electrolyte balance, but none provoked ...

Research paper thumbnail of The Structure of the mer Operon

Plasmids in Bacteria, 1985

The DNA sequence has been determined for a 3.8-kb region which encodes the mercury-resistance (me... more The DNA sequence has been determined for a 3.8-kb region which encodes the mercury-resistance (mer) operon of the IncFII plasmid NR1. The sequence reveals 4 open reading frames which could encode proteins of 12,522, 9,429, 14,965, and 58,912 d corresponding to the 4 previously described Hg-inducible proteins detected in minicells carrying mer+ plasmids. The Hg(II) reductase protein sequence is about 90% homologous to that of Tn501, but the DNA sequence shows a homology of 60-70% to that of Tn501 except for short regions of very high homology. The entire mer region is 63.4% G-C overall. The region encoding the merR (positive regulatory) function has 3 possible open reading frames, 2 of which overlap in one direction and the third of which reads in the opposite direction. Attempts to visualize the polypeptide(s) encoded by the merR cistron were unsuccessful.

Research paper thumbnail of Gene expression controlled by the Hg(II) biosensor, merR

Journal of Inorganic Biochemistry, 1989

Research paper thumbnail of NMR Structural Studies Reveal a Novel Protein Fold for MerB, the Organomercurial Lyase Involved in the Bacterial Mercury Resistance System

Biochemistry, 2004

Mercury resistant bacteria have developed a system of two enzymes (MerA and MerB), which allows t... more Mercury resistant bacteria have developed a system of two enzymes (MerA and MerB), which allows them to efficiently detoxify both ionic and organomercurial compounds. The organomercurial lyase (MerB) catalyzes the protonolysis of the carbon-mercury bond resulting in the formation of ionic mercury and a reduced hydrocarbon. The ionic mercury [Hg(II)] is subsequently reduced to the less reactive elemental mercury [Hg(0)] by a specific mercuric reductase (MerA). To better understand MerB's unique enzymatic activity, we used nuclear magnetic resonance (NMR) spectroscopy to determine the structure of the free enzyme. MerB is characterized by a novel protein fold consisting of three noninteracting antiparallel-sheets surrounded by six R-helices. By comparing the NMR data of free MerB and the MerB/Hg/DTT complex, we identified a set of residues that likely define a Hg/DTT binding site. These residues cluster around two cysteines (C 96 and C 159) that are crucial to MerB's catalytic activity. A detailed analysis of the structure revealed the presence of an extensive hydrophobic groove adjacent to this Hg/DTT binding site. This extensive hydrophobic groove has the potential to interact with the hydrocarbon moiety of a wide variety of substrates and may explain the broad substrate specificity of MerB.

Research paper thumbnail of Thinking outside the (pill) box: Does toxic metal exposure thwart antibiotic stewardship best practices?

Plasmid, 2018

Multi-antibiotic resistant (MAR) bacteria cost billions in medical care and tens of thousands of ... more Multi-antibiotic resistant (MAR) bacteria cost billions in medical care and tens of thousands of lives annually but perennial calls to limit agricultural and other misuse of antibiotics and to fund antibiotic discovery have not slowed this MAR deluge. Since mobile genetic elements (MGEs) stitch single antibiotic resistance genes into clinically significant MAR arrays, it is high time to focus on how MGEs generate MAR and how disabling them could ameliorate the MAR problem. However, to consider only antibiotics as the drivers of MAR is to miss the significant impact of exposure to non-antibiotic toxic chemicals, specifically metals, on the persistence and spread of MAR. Toxic metals were among the earliest discovered targets of plasmid-encoded resistance genes. Recent genomic epidemiology clearly demonstrated the co-prevalence of metal resistances and antibiotic multiresistance, uniquely in humans and domestic animals. Metal resistances exploit the same, ancient "transportation infrastructure" of plasmids, transposons, and integrons that spread the antibiotic resistance genes and will continue to do so even if all antibiotic misuse were stopped today and new antibiotics were flowing from the pipeline monthly. In a key experiment with primates, continuous oral exposure to mercury (Hg) released from widely used dental amalgam fillings co-selected for MAR bacteria in the oral and fecal commensal microbiomes and, most importantly, when amalgams were replaced with non-metal fillings, MAR bacteria declined dramatically. Could that also be happening on the larger public health scale as use of amalgam restorations is curtailed or banned in many countries? This commentary covers salient past and recent findings of key metal-antibiotic resistance associations and proposes that the shift from phenotyping to genotyping in surveillance of resistance loci will allow a test of whether declining exposure to this leading source of Hg is accompanied by a decline in MAR compared to countries where amalgam is still used. If this hypothesis is correct, the limited success of antibiotic stewardship practices may be because MAR is also being driven by continuous, daily exposure to Hg, a non-antibiotic toxicant widely used in humans. 1.1. When was the Hg-MAR connection first noticed? The potential for metal co-selection of MAR was first observed in clinical isolates in the 1960's (Novick & Roth,

Research paper thumbnail of Phytoremediation of methylmercury pollution: merB expression in Arabidopsis thaliana confers resistance to organomercurials (environmentyheavy metalsyMinamata diseaseyorganic mercuryyremediation)

Methylmercury is an environmental toxicant that biomagnifies and causes severe neurological degen... more Methylmercury is an environmental toxicant that biomagnifies and causes severe neurological degenera- tion in animals. It is produced by bacteria in soils and sediments that have been contaminated with mercury. To explore the potential of plants to extract and detoxify this chemical, we engineered a model plant, Arabidopsis thaliana, to express a modified bacterial gene, merBpe, encoding organo- mercurial lyase

Research paper thumbnail of Molecular structure and dynamics in bacterial mercury resistance

Bacteria participate significantly in mercury transformation in natural and industrial environmen... more Bacteria participate significantly in mercury transformation in natural and industrial environments. Previous studies have shown that bacterial mercury resistance is mediated by the mer operon, typically located on transposons or plasmids. It encodes specific genes that facilitate uptake of mercury species, cleavage of organomercurials, and reduction of Hg(II) to Hg(0). Expression of mer operon genes is regulated by MerR, a

Research paper thumbnail of Structure and Conformational Dynamics of the Metalloregulator MerR upon Binding of Hg(II)

Journal of Molecular Biology, Mar 1, 2010

The bacterial metalloregulator MerR is the index case of an eponymous family of regulatory protei... more The bacterial metalloregulator MerR is the index case of an eponymous family of regulatory proteins, which controls the transcription of a set of genes (the mer operon) conferring mercury resistance in many bacteria. Homodimeric MerR represses transcription in the absence of mercury and activates transcription upon Hg(II) binding. Here, the average structures of the apo and Hg(II)-bound forms of MerR in aqueous solution are examined using small-angle X-ray scattering, indicating an extended conformation of the metal-bound protein and revealing the existence of a novel compact conformation in the absence of Hg(II). Molecular dynamics (MD) simulations are performed to characterize the conformational dynamics of the Hg (II)-bound form. In both small-angle X-ray scattering and MD, the average torsional angle between DNA-binding domains is ∼ 65°. Furthermore, in MD, interdomain motions on a timescale of ∼ 10 ns involving largeamplitude (∼20 Å) domain opening-and-closing, coupled to ∼ 40°variations of interdomain torsional angle, are revealed. This correlated domain motion may propagate allosteric changes from the metal-binding site to the DNA-binding site while maintaining DNA contacts required to initiate DNA underwinding.

Research paper thumbnail of Biomolecular Aspects of Mercury Transformations

Agu Fall Meeting Abstracts, Dec 1, 2008

Bacteria participate significantly in mercury transformation in natural and industrial environmen... more Bacteria participate significantly in mercury transformation in natural and industrial environments. Previous studies have shown that bacterial mercury resistance is mediated by the mer operon, typically located on transposons or plasmids. It encodes specific genes that facilitate uptake of mercury species, cleavage of organomercurials, and reduction of Hg(II) to Hg(0). Expression of mer operon genes is regulated by MerR, a metal-responsive regulator protein on the level of transcription. In vitro studies have shown that MerR forms a non-transcribing pre-initiation complex with RNA polymerase and the promoter DNA. Binding of Hg(II) induces conformational changes in MerR and other components of the complex resulting in the transcription of mer operon genes. As part of ongoing investigations on allosteric conformational changes induced by Hg(II) in dimeric MerR, and the implications on the binding of RNA polymerase to the promoter of the mer operon, we applied small angle scattering to study the regulatory mechanism of MerR in the presence and absence of Hg(II). Our results show that in the presence of Hg(II) the MerR dimer undergoes a significant reorientation from a compact state to a conformation revealing two distinct domains. Bacterial reduction of Hg(II) can also occur at concentrations too low to induce mer operon functions. Dissimilatory metal reducing bacteria, such as Shewanella and Geobacter are able to reduce Hg(II) in the presence of mineral oxides. This process has been linked to the activity of outer membrane multiheme cytochromes. We isolated and purified a decaheme outer membrane cytochrome OmcA from Shewanella oneidensis MR-1 and characterized its envelope shape in solution by small angle x-ray scattering. Structural features were identified and compared to homology models. These results show that OmcA is an elongated macromolecule consisting of separate modules, which may be connected by flexible linkers.

Research paper thumbnail of Biomolecular Mechanisms of Mercury Transfers and Transformations by Proteins of the Mer Operon

Agu Fall Meeting Abstracts, Dec 1, 2008

Aerobic bacteria exhibiting resistance to the toxic effects of Hg(II) and organomercurials [RHg(I... more Aerobic bacteria exhibiting resistance to the toxic effects of Hg(II) and organomercurials [RHg(I), e.g. MeHg(I)] and are widely found in both pristine and mercury contaminated environments. Resistance, afforded by a plasmid- or transposon-associated mer operon, involves an unusual pathway where Hg(II) and organomercurials [RHg(I)] undergo facilitated entry into the bacterial cytoplasm via an integral membrane transport protein (MerT) and are then "detoxified" by the concerted effort of two enzymes, organomercurial lyase (MerB), which catalyzes dealkylation (i.e., demethylation) of RHg(I) to Hg(II) and a hydrocarbon, and mercuric ion reductase (MerA), which catalyzes reduction of Hg(II) to Hg(0) as the ultimate detoxification for the organism. With a widespread distribution, these bacterial transformations play a significant role in the fate of mercury in the environment. Our focus is on elucidation of the molecular mechanisms for the transport and catalytic transformations of RHg(I) and Hg(II) by these proteins and the factors that influence the overall efficiency of the process. Current efforts are focused primarily on elucidating details of RHg(I) binding and dealkylation by MerB as well as the mechanism for transfer of the Hg(II) product to MerA. Key findings include the demonstration of a non-cysteine residue as essential for the catalytic activity and demonstration that direct transfer of Hg(II) to MerA proceeds more rapidly and more completely than transfer to small MW thiols such as cysteines or glutathione. Reuslts of these studies as well as an overview of our current understanding of the whole system will be presented.

Research paper thumbnail of Genetic Linkage and Horizontal Gene Transfer, the Roots of the Antibiotic Multi-Resistance Problem

Http Dx Doi Org 10 1080 10495390600957217, Feb 17, 2007

Bacteria carrying resistance genes for many antibiotics are moving beyond the clinic into the com... more Bacteria carrying resistance genes for many antibiotics are moving beyond the clinic into the community, infecting otherwise healthy people with untreatable and frequently fatal infections. This state of affairs makes it increasingly important that we understand the sources of this problem in terms of bacterial biology and ecology and also that we find some new targets for drugs that will help control this growing epidemic. This brief and eclectic review takes the perspective that we have too long thought about the problem in terms of treatment with or resistance to a single antibiotic at a time, assuming that dissemination of the resistance gene was affected by simple vertical inheritance. In reality antibiotic resistance genes are readily transferred horizontally, even to and from distantly related bacteria. The common agents of bacterial gene transfer are described and also one of the processes whereby nonantibiotic chemicals, specifically toxic metals, in the environment can select for and enrich bacteria with antibiotic multiresistance. Lastly, some speculation is offered on broadening our perspective on this problem to include drugs directed at compromising the ability of the mobile elements themselves to replicate, transfer, and recombine, that is, the three "infrastructure" processes central to the movement of genes among bacteria.

Research paper thumbnail of Bacterial Oxidation of Mercury Metal Vapor, Hg(0)

Applied and Environmental Microbiology, Apr 1, 1998

We used metalloregulated luciferase reporter fusions and spectroscopic quantification of soluble ... more We used metalloregulated luciferase reporter fusions and spectroscopic quantification of soluble Hg(II) to determine that the hydroperoxidase-catalase, KatG, of Escherichia coli can oxidize monatomic elemental mercury vapor, Hg(0), to the water-soluble, ionic form, Hg(II). A strain with a mutation in katG and a strain overproducing KatG were used to demonstrate that the amount of Hg(II) formed is proportional to the catalase activity. Hg(0) oxidation was much decreased in stationary-phase cells of a strain lacking KatG, suggesting that the monofunctional hydroperoxidase KatE is less effective at this reaction. Unexpectedly, Hg(0) oxidation also occurred in a strain lacking both KatE and KatG, suggesting that activities other than hydroperoxidases may carry out this reaction. Two typical soil bacteria, Bacillus and Streptomyces, also oxidize Hg(0) to Hg(II). These observations establish for the first time that bacteria can contribute, as do mammals and plants, to the oxidative phase of the global Hg cycle.

Research paper thumbnail of Mercury resistance in a plasmid-bearing strain of Escherichia coli

Journal of Bacteriology, Dec 1, 1972

A strain of Escherichia coli carrying genes determining mercury resistance on a naturally occurri... more A strain of Escherichia coli carrying genes determining mercury resistance on a naturally occurring resistance transfer factor (RTF) converts 95% of 10-5 M Hg2+ (chloride) to metallic mercury at a rate of 4 to 5 nmoles of Hg2+ per min per 108 cells. The metallic mercury is rapidly eliminated from the culture medium as mercury vapor. The volatilizing activity has a temperature dependence and heat sensitivity characteristic of enzymatic catalysis and is inducible by mercuric chloride. Ag+ and Au3+ are markedly inhibitory of mercury volatilization.

Research paper thumbnail of Metal binding proteins, recombinant host cells and methods

Research paper thumbnail of A Rubrerythrin Operon and Nigerythrin Gene in Desulfovibrio vulgaris (Hildenborough)

Journal of Bacteriology, Jul 1, 1997

Rubrerythrin is a nonheme iron protein of unknown function isolated from Desulfovibrio vulgaris (... more Rubrerythrin is a nonheme iron protein of unknown function isolated from Desulfovibrio vulgaris (Hildenborough). We have sequenced a 3.3-kbp SalI fragment of D. vulgaris chromosomal DNA containing the rubrerythrin gene, rbr, identified additional open reading frames (ORFs) adjacent to rbr, and shown that these ORFs are part of a transcriptional unit containing rbr. One ORF, designated fur, lies just upstream of rbr and encodes a 128-amino-acid-residue protein which shows homology to Fur (ferric uptake regulatory) proteins from other purple bacteria. The other ORF, designated rdl, lies just downstream of rbr and encodes a 74-residue protein with significant sequence homology to rubredoxins but with a different number and spacing of cysteine residues. Overexpression of rdl in Escherichia coli yielded a protein, Rdl, which has spectroscopic properties and iron content consistent with one Fe 3؉ (SCys) 4 site per polypeptide but is clearly distinct from both rubrerythrin and a related protein, nigerythrin. Northern analysis indicated that fur, rbr, and rdl were each present on a transcript of 1.3 kb; i.e., these three genes are cotranscribed. Because D. vulgaris nigerythrin appears to be closely related to rubrerythrin, and its function is also unknown, we cloned and sequenced the gene encoding nigerythrin, ngr. The amino acid sequence of nigerythrin is 33% identical to that of rubrerythrin, and all residues which furnish iron ligands to both the FeS 4 and diiron-oxo sites in rubrerythrin are conserved in nigerythrin. Despite the close resemblance of these two proteins, ngr was found to be no closer than 7 kb to rbr on the D. vulgaris chromosome, and Northern analysis showed that, in contrast to rbr, ngr is not cotranscribed with other genes. Possible redox-linked functions for rubrerythrin and nigerythrin in iron homeostasis are proposed.

Research paper thumbnail of Cd(II)-Responsive and Constitutive Mutants Implicate a Novel Domain in MerR

Journal of Bacteriology, Jun 1, 1999

Expression of the Tn21 mercury resistance (mer) operon is controlled by a metal-sensing repressor... more Expression of the Tn21 mercury resistance (mer) operon is controlled by a metal-sensing repressor-activator, MerR. When present, MerR always binds to the same position on the DNA (the operator merO), repressing transcription of the structural genes merTPCAD in the absence of Hg(II) and inducing their transcription in the presence of Hg(II). Although it has two potential binding sites, the purified MerR homodimer binds only one Hg(II) ion, employing Cys82 from one monomer and Cys117 and Cys126 from the other. When MerR binds Hg(II), it changes allosterically and also distorts the merO DNA to facilitate transcriptional initiation by 70 RNA polymerase. Wild-type MerR is highly specific for Hg(II) and is 100-and 1,000-fold less responsive to the chemically related group 12 metals, Cd(II) and Zn(II), respectively. We sought merR mutants that respond to Cd(II) and obtained 11 Cd(II)-responsive and 5 constitutive mutants. The Cd(II)-responsive mutants, most of which had only single-residue replacements, were also repression deficient and still Hg(II) responsive but, like the wild type, were completely unresponsive to Zn(II). None of the Cd(II)-responsive mutations occurred in the DNA binding domain or replaced any of the key Cys residues. Five Cd(II)-responsive single mutations lie in the antiparallel coiled-coil domain between Cys82 and Cys117 which constitutes the dimer interface. These mutations identify 10 new positions whose alteration significantly affect MerR's metal responsiveness or its repressor function. They give rise to specific predictions for how MerR distinguishes group 12 metals, and they refine our model of the novel domain structure of MerR. Secondary-structure predictions suggest that certain elements of this model also apply to other MerR family regulators.

Research paper thumbnail of Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA

ABSTRACTRecent advances and lower costs in rapid high-throughput sequencing have engendered hope ... more ABSTRACTRecent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in clinical bacterial isolates. Despite its potential, several challenges should be addressed before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical laboratory. Here, with three distinct ESKAPE bacteria, we compared different approaches to identify best practices for detection of AMR genes, including: total genomic DNA and plasmid DNA extractions, solo assembly of Illumina short-reads and of ONT long-reads, two hybrid assembly pipelines, and three in silico AMR databases. We also determined the susceptibility of each strain to 21 antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also detectable in total genomic DNA indicating that, at least in these three enterobacterial genera, purification of plasmid DNA was not necessary to detect plasmid-borne AM...

Research paper thumbnail of Corrigendum to: Children with Amalgam Dental Restorations Have Significantly Elevated Blood and Urine Mercury Levels

Toxicological Sciences, 2022

Research paper thumbnail of Hg(II) Binding to Thymine Bases in DNA

Inorganic Chemistry, 2021

The compounds of mercury can be highly toxic and can interfere with a range of biological process... more The compounds of mercury can be highly toxic and can interfere with a range of biological processes, although many aspects of the mechanism of toxicity are still obscure or unknown. One especially intriguing property of Hg(II) is its ability to bind DNA directly, making interstrand cross-links between thymine nucleobases in AT-rich sequences. We have used a combination of small molecule X-ray diffraction, X-ray spectroscopies, and computational chemistry to study the interactions of Hg(II) with thymine. We find that the energetically preferred mode of thymine binding in DNA is to the N3 and predict only minor distortions of the DNA structure on binding one Hg(II) to two cross-adjacent thymine nucleotides. The preferred geometry is predicted to be twisted away from coplanar through a torsion angle of between 32 and 43°. Using 1-methylthymine as a model, the bis-thymine coordination of Hg(II) is found to give a highly characteristic X-ray spectroscopic signature that is quite distinct from other previously described biological modes of binding of Hg(II). This work enlarges and deepens our view of significant biological targets of Hg(II) and demonstrates tools that can provide a characteristic signature for the binding of Hg(II) to DNA in more complex matrices including intact cells and tissues, laying the foundation for future studies of mechanisms of mercury toxicity.

Research paper thumbnail of Integrating the Molecular Machines of Mercury Detoxification into Host Cell Biology

Metalloregulators of the MerR family activate transcription upon metal binding by underwinding th... more Metalloregulators of the MerR family activate transcription upon metal binding by underwinding the operator-promoter DNA to permit open complex formation by pre-bound RNA polymerase. Historically, MerR's allostery has been monitored only indirectly via nuclease sensitivity or by fluorescent nucleotide probes and was very specific for Hg(II), although purified MerR binds several thiophilic metals. To observe directly MerR's ligand-induced behavior we made 2-fluorotyrosine-substituted MerR and found similar, minor changes in 19 F chemical shifts of tyrosine residues in the free protein exposed to Hg(II), Cd(II) or Zn(II). However, DNA binding elicits large chemical shift changes in MerR's tyrosine residues and in DNAbound MerR Hg(II) provokes changes very distinct from those of Cd(II) or Zn(II). These chemical shift changes and other biophysical and phenotypic properties of wild-type MerR and relevant mutants reveal elements of an allosteric network that enables the coordination state of the metal binding site to direct metal-specific movements in the distant DNA binding site and the DNA-bound state also to affect the metal binding domain.

Research paper thumbnail of Organic and inorganic mercurials have distinct effects on cellular thiols, metal homeostasis, and Fe-binding proteins in Escherichia coli

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, Jan 26, 2015

The protean chemical properties of the toxic metal mercury (Hg) have made it attractive in divers... more The protean chemical properties of the toxic metal mercury (Hg) have made it attractive in diverse applications since antiquity. However, growing public concern has led to an international agreement to decrease its impact on health and the environment. During a recent proteomics study of acute Hg exposure in E. coli, we also examined the effects of inorganic and organic Hg compounds on thiol and metal homeostases. On brief exposure, lower concentrations of divalent inorganic mercury Hg(II) blocked bulk cellular thiols and protein-associated thiols more completely than higher concentrations of monovalent organomercurials, phenylmercuric acetate (PMA) and merthiolate (MT). Cells bound Hg(II) and PMA in excess of their available thiol ligands; X-ray absorption spectroscopy indicated nitrogens as likely additional ligands. The mercurials released protein-bound iron (Fe) more effectively than common organic oxidants and all disturbed the Na(+)/K(+) electrolyte balance, but none provoked ...

Research paper thumbnail of The Structure of the mer Operon

Plasmids in Bacteria, 1985

The DNA sequence has been determined for a 3.8-kb region which encodes the mercury-resistance (me... more The DNA sequence has been determined for a 3.8-kb region which encodes the mercury-resistance (mer) operon of the IncFII plasmid NR1. The sequence reveals 4 open reading frames which could encode proteins of 12,522, 9,429, 14,965, and 58,912 d corresponding to the 4 previously described Hg-inducible proteins detected in minicells carrying mer+ plasmids. The Hg(II) reductase protein sequence is about 90% homologous to that of Tn501, but the DNA sequence shows a homology of 60-70% to that of Tn501 except for short regions of very high homology. The entire mer region is 63.4% G-C overall. The region encoding the merR (positive regulatory) function has 3 possible open reading frames, 2 of which overlap in one direction and the third of which reads in the opposite direction. Attempts to visualize the polypeptide(s) encoded by the merR cistron were unsuccessful.

Research paper thumbnail of Gene expression controlled by the Hg(II) biosensor, merR

Journal of Inorganic Biochemistry, 1989

Research paper thumbnail of NMR Structural Studies Reveal a Novel Protein Fold for MerB, the Organomercurial Lyase Involved in the Bacterial Mercury Resistance System

Biochemistry, 2004

Mercury resistant bacteria have developed a system of two enzymes (MerA and MerB), which allows t... more Mercury resistant bacteria have developed a system of two enzymes (MerA and MerB), which allows them to efficiently detoxify both ionic and organomercurial compounds. The organomercurial lyase (MerB) catalyzes the protonolysis of the carbon-mercury bond resulting in the formation of ionic mercury and a reduced hydrocarbon. The ionic mercury [Hg(II)] is subsequently reduced to the less reactive elemental mercury [Hg(0)] by a specific mercuric reductase (MerA). To better understand MerB's unique enzymatic activity, we used nuclear magnetic resonance (NMR) spectroscopy to determine the structure of the free enzyme. MerB is characterized by a novel protein fold consisting of three noninteracting antiparallel-sheets surrounded by six R-helices. By comparing the NMR data of free MerB and the MerB/Hg/DTT complex, we identified a set of residues that likely define a Hg/DTT binding site. These residues cluster around two cysteines (C 96 and C 159) that are crucial to MerB's catalytic activity. A detailed analysis of the structure revealed the presence of an extensive hydrophobic groove adjacent to this Hg/DTT binding site. This extensive hydrophobic groove has the potential to interact with the hydrocarbon moiety of a wide variety of substrates and may explain the broad substrate specificity of MerB.

Research paper thumbnail of Thinking outside the (pill) box: Does toxic metal exposure thwart antibiotic stewardship best practices?

Plasmid, 2018

Multi-antibiotic resistant (MAR) bacteria cost billions in medical care and tens of thousands of ... more Multi-antibiotic resistant (MAR) bacteria cost billions in medical care and tens of thousands of lives annually but perennial calls to limit agricultural and other misuse of antibiotics and to fund antibiotic discovery have not slowed this MAR deluge. Since mobile genetic elements (MGEs) stitch single antibiotic resistance genes into clinically significant MAR arrays, it is high time to focus on how MGEs generate MAR and how disabling them could ameliorate the MAR problem. However, to consider only antibiotics as the drivers of MAR is to miss the significant impact of exposure to non-antibiotic toxic chemicals, specifically metals, on the persistence and spread of MAR. Toxic metals were among the earliest discovered targets of plasmid-encoded resistance genes. Recent genomic epidemiology clearly demonstrated the co-prevalence of metal resistances and antibiotic multiresistance, uniquely in humans and domestic animals. Metal resistances exploit the same, ancient "transportation infrastructure" of plasmids, transposons, and integrons that spread the antibiotic resistance genes and will continue to do so even if all antibiotic misuse were stopped today and new antibiotics were flowing from the pipeline monthly. In a key experiment with primates, continuous oral exposure to mercury (Hg) released from widely used dental amalgam fillings co-selected for MAR bacteria in the oral and fecal commensal microbiomes and, most importantly, when amalgams were replaced with non-metal fillings, MAR bacteria declined dramatically. Could that also be happening on the larger public health scale as use of amalgam restorations is curtailed or banned in many countries? This commentary covers salient past and recent findings of key metal-antibiotic resistance associations and proposes that the shift from phenotyping to genotyping in surveillance of resistance loci will allow a test of whether declining exposure to this leading source of Hg is accompanied by a decline in MAR compared to countries where amalgam is still used. If this hypothesis is correct, the limited success of antibiotic stewardship practices may be because MAR is also being driven by continuous, daily exposure to Hg, a non-antibiotic toxicant widely used in humans. 1.1. When was the Hg-MAR connection first noticed? The potential for metal co-selection of MAR was first observed in clinical isolates in the 1960's (Novick & Roth,

Research paper thumbnail of Phytoremediation of methylmercury pollution: merB expression in Arabidopsis thaliana confers resistance to organomercurials (environmentyheavy metalsyMinamata diseaseyorganic mercuryyremediation)

Methylmercury is an environmental toxicant that biomagnifies and causes severe neurological degen... more Methylmercury is an environmental toxicant that biomagnifies and causes severe neurological degenera- tion in animals. It is produced by bacteria in soils and sediments that have been contaminated with mercury. To explore the potential of plants to extract and detoxify this chemical, we engineered a model plant, Arabidopsis thaliana, to express a modified bacterial gene, merBpe, encoding organo- mercurial lyase

Research paper thumbnail of Molecular structure and dynamics in bacterial mercury resistance

Bacteria participate significantly in mercury transformation in natural and industrial environmen... more Bacteria participate significantly in mercury transformation in natural and industrial environments. Previous studies have shown that bacterial mercury resistance is mediated by the mer operon, typically located on transposons or plasmids. It encodes specific genes that facilitate uptake of mercury species, cleavage of organomercurials, and reduction of Hg(II) to Hg(0). Expression of mer operon genes is regulated by MerR, a

Research paper thumbnail of Structure and Conformational Dynamics of the Metalloregulator MerR upon Binding of Hg(II)

Journal of Molecular Biology, Mar 1, 2010

The bacterial metalloregulator MerR is the index case of an eponymous family of regulatory protei... more The bacterial metalloregulator MerR is the index case of an eponymous family of regulatory proteins, which controls the transcription of a set of genes (the mer operon) conferring mercury resistance in many bacteria. Homodimeric MerR represses transcription in the absence of mercury and activates transcription upon Hg(II) binding. Here, the average structures of the apo and Hg(II)-bound forms of MerR in aqueous solution are examined using small-angle X-ray scattering, indicating an extended conformation of the metal-bound protein and revealing the existence of a novel compact conformation in the absence of Hg(II). Molecular dynamics (MD) simulations are performed to characterize the conformational dynamics of the Hg (II)-bound form. In both small-angle X-ray scattering and MD, the average torsional angle between DNA-binding domains is ∼ 65°. Furthermore, in MD, interdomain motions on a timescale of ∼ 10 ns involving largeamplitude (∼20 Å) domain opening-and-closing, coupled to ∼ 40°variations of interdomain torsional angle, are revealed. This correlated domain motion may propagate allosteric changes from the metal-binding site to the DNA-binding site while maintaining DNA contacts required to initiate DNA underwinding.

Research paper thumbnail of Biomolecular Aspects of Mercury Transformations

Agu Fall Meeting Abstracts, Dec 1, 2008

Bacteria participate significantly in mercury transformation in natural and industrial environmen... more Bacteria participate significantly in mercury transformation in natural and industrial environments. Previous studies have shown that bacterial mercury resistance is mediated by the mer operon, typically located on transposons or plasmids. It encodes specific genes that facilitate uptake of mercury species, cleavage of organomercurials, and reduction of Hg(II) to Hg(0). Expression of mer operon genes is regulated by MerR, a metal-responsive regulator protein on the level of transcription. In vitro studies have shown that MerR forms a non-transcribing pre-initiation complex with RNA polymerase and the promoter DNA. Binding of Hg(II) induces conformational changes in MerR and other components of the complex resulting in the transcription of mer operon genes. As part of ongoing investigations on allosteric conformational changes induced by Hg(II) in dimeric MerR, and the implications on the binding of RNA polymerase to the promoter of the mer operon, we applied small angle scattering to study the regulatory mechanism of MerR in the presence and absence of Hg(II). Our results show that in the presence of Hg(II) the MerR dimer undergoes a significant reorientation from a compact state to a conformation revealing two distinct domains. Bacterial reduction of Hg(II) can also occur at concentrations too low to induce mer operon functions. Dissimilatory metal reducing bacteria, such as Shewanella and Geobacter are able to reduce Hg(II) in the presence of mineral oxides. This process has been linked to the activity of outer membrane multiheme cytochromes. We isolated and purified a decaheme outer membrane cytochrome OmcA from Shewanella oneidensis MR-1 and characterized its envelope shape in solution by small angle x-ray scattering. Structural features were identified and compared to homology models. These results show that OmcA is an elongated macromolecule consisting of separate modules, which may be connected by flexible linkers.

Research paper thumbnail of Biomolecular Mechanisms of Mercury Transfers and Transformations by Proteins of the Mer Operon

Agu Fall Meeting Abstracts, Dec 1, 2008

Aerobic bacteria exhibiting resistance to the toxic effects of Hg(II) and organomercurials [RHg(I... more Aerobic bacteria exhibiting resistance to the toxic effects of Hg(II) and organomercurials [RHg(I), e.g. MeHg(I)] and are widely found in both pristine and mercury contaminated environments. Resistance, afforded by a plasmid- or transposon-associated mer operon, involves an unusual pathway where Hg(II) and organomercurials [RHg(I)] undergo facilitated entry into the bacterial cytoplasm via an integral membrane transport protein (MerT) and are then "detoxified" by the concerted effort of two enzymes, organomercurial lyase (MerB), which catalyzes dealkylation (i.e., demethylation) of RHg(I) to Hg(II) and a hydrocarbon, and mercuric ion reductase (MerA), which catalyzes reduction of Hg(II) to Hg(0) as the ultimate detoxification for the organism. With a widespread distribution, these bacterial transformations play a significant role in the fate of mercury in the environment. Our focus is on elucidation of the molecular mechanisms for the transport and catalytic transformations of RHg(I) and Hg(II) by these proteins and the factors that influence the overall efficiency of the process. Current efforts are focused primarily on elucidating details of RHg(I) binding and dealkylation by MerB as well as the mechanism for transfer of the Hg(II) product to MerA. Key findings include the demonstration of a non-cysteine residue as essential for the catalytic activity and demonstration that direct transfer of Hg(II) to MerA proceeds more rapidly and more completely than transfer to small MW thiols such as cysteines or glutathione. Reuslts of these studies as well as an overview of our current understanding of the whole system will be presented.

Research paper thumbnail of Genetic Linkage and Horizontal Gene Transfer, the Roots of the Antibiotic Multi-Resistance Problem

Http Dx Doi Org 10 1080 10495390600957217, Feb 17, 2007

Bacteria carrying resistance genes for many antibiotics are moving beyond the clinic into the com... more Bacteria carrying resistance genes for many antibiotics are moving beyond the clinic into the community, infecting otherwise healthy people with untreatable and frequently fatal infections. This state of affairs makes it increasingly important that we understand the sources of this problem in terms of bacterial biology and ecology and also that we find some new targets for drugs that will help control this growing epidemic. This brief and eclectic review takes the perspective that we have too long thought about the problem in terms of treatment with or resistance to a single antibiotic at a time, assuming that dissemination of the resistance gene was affected by simple vertical inheritance. In reality antibiotic resistance genes are readily transferred horizontally, even to and from distantly related bacteria. The common agents of bacterial gene transfer are described and also one of the processes whereby nonantibiotic chemicals, specifically toxic metals, in the environment can select for and enrich bacteria with antibiotic multiresistance. Lastly, some speculation is offered on broadening our perspective on this problem to include drugs directed at compromising the ability of the mobile elements themselves to replicate, transfer, and recombine, that is, the three "infrastructure" processes central to the movement of genes among bacteria.

Research paper thumbnail of Bacterial Oxidation of Mercury Metal Vapor, Hg(0)

Applied and Environmental Microbiology, Apr 1, 1998

We used metalloregulated luciferase reporter fusions and spectroscopic quantification of soluble ... more We used metalloregulated luciferase reporter fusions and spectroscopic quantification of soluble Hg(II) to determine that the hydroperoxidase-catalase, KatG, of Escherichia coli can oxidize monatomic elemental mercury vapor, Hg(0), to the water-soluble, ionic form, Hg(II). A strain with a mutation in katG and a strain overproducing KatG were used to demonstrate that the amount of Hg(II) formed is proportional to the catalase activity. Hg(0) oxidation was much decreased in stationary-phase cells of a strain lacking KatG, suggesting that the monofunctional hydroperoxidase KatE is less effective at this reaction. Unexpectedly, Hg(0) oxidation also occurred in a strain lacking both KatE and KatG, suggesting that activities other than hydroperoxidases may carry out this reaction. Two typical soil bacteria, Bacillus and Streptomyces, also oxidize Hg(0) to Hg(II). These observations establish for the first time that bacteria can contribute, as do mammals and plants, to the oxidative phase of the global Hg cycle.

Research paper thumbnail of Mercury resistance in a plasmid-bearing strain of Escherichia coli

Journal of Bacteriology, Dec 1, 1972

A strain of Escherichia coli carrying genes determining mercury resistance on a naturally occurri... more A strain of Escherichia coli carrying genes determining mercury resistance on a naturally occurring resistance transfer factor (RTF) converts 95% of 10-5 M Hg2+ (chloride) to metallic mercury at a rate of 4 to 5 nmoles of Hg2+ per min per 108 cells. The metallic mercury is rapidly eliminated from the culture medium as mercury vapor. The volatilizing activity has a temperature dependence and heat sensitivity characteristic of enzymatic catalysis and is inducible by mercuric chloride. Ag+ and Au3+ are markedly inhibitory of mercury volatilization.

Research paper thumbnail of Metal binding proteins, recombinant host cells and methods

Research paper thumbnail of A Rubrerythrin Operon and Nigerythrin Gene in Desulfovibrio vulgaris (Hildenborough)

Journal of Bacteriology, Jul 1, 1997

Rubrerythrin is a nonheme iron protein of unknown function isolated from Desulfovibrio vulgaris (... more Rubrerythrin is a nonheme iron protein of unknown function isolated from Desulfovibrio vulgaris (Hildenborough). We have sequenced a 3.3-kbp SalI fragment of D. vulgaris chromosomal DNA containing the rubrerythrin gene, rbr, identified additional open reading frames (ORFs) adjacent to rbr, and shown that these ORFs are part of a transcriptional unit containing rbr. One ORF, designated fur, lies just upstream of rbr and encodes a 128-amino-acid-residue protein which shows homology to Fur (ferric uptake regulatory) proteins from other purple bacteria. The other ORF, designated rdl, lies just downstream of rbr and encodes a 74-residue protein with significant sequence homology to rubredoxins but with a different number and spacing of cysteine residues. Overexpression of rdl in Escherichia coli yielded a protein, Rdl, which has spectroscopic properties and iron content consistent with one Fe 3؉ (SCys) 4 site per polypeptide but is clearly distinct from both rubrerythrin and a related protein, nigerythrin. Northern analysis indicated that fur, rbr, and rdl were each present on a transcript of 1.3 kb; i.e., these three genes are cotranscribed. Because D. vulgaris nigerythrin appears to be closely related to rubrerythrin, and its function is also unknown, we cloned and sequenced the gene encoding nigerythrin, ngr. The amino acid sequence of nigerythrin is 33% identical to that of rubrerythrin, and all residues which furnish iron ligands to both the FeS 4 and diiron-oxo sites in rubrerythrin are conserved in nigerythrin. Despite the close resemblance of these two proteins, ngr was found to be no closer than 7 kb to rbr on the D. vulgaris chromosome, and Northern analysis showed that, in contrast to rbr, ngr is not cotranscribed with other genes. Possible redox-linked functions for rubrerythrin and nigerythrin in iron homeostasis are proposed.

Research paper thumbnail of Cd(II)-Responsive and Constitutive Mutants Implicate a Novel Domain in MerR

Journal of Bacteriology, Jun 1, 1999

Expression of the Tn21 mercury resistance (mer) operon is controlled by a metal-sensing repressor... more Expression of the Tn21 mercury resistance (mer) operon is controlled by a metal-sensing repressor-activator, MerR. When present, MerR always binds to the same position on the DNA (the operator merO), repressing transcription of the structural genes merTPCAD in the absence of Hg(II) and inducing their transcription in the presence of Hg(II). Although it has two potential binding sites, the purified MerR homodimer binds only one Hg(II) ion, employing Cys82 from one monomer and Cys117 and Cys126 from the other. When MerR binds Hg(II), it changes allosterically and also distorts the merO DNA to facilitate transcriptional initiation by 70 RNA polymerase. Wild-type MerR is highly specific for Hg(II) and is 100-and 1,000-fold less responsive to the chemically related group 12 metals, Cd(II) and Zn(II), respectively. We sought merR mutants that respond to Cd(II) and obtained 11 Cd(II)-responsive and 5 constitutive mutants. The Cd(II)-responsive mutants, most of which had only single-residue replacements, were also repression deficient and still Hg(II) responsive but, like the wild type, were completely unresponsive to Zn(II). None of the Cd(II)-responsive mutations occurred in the DNA binding domain or replaced any of the key Cys residues. Five Cd(II)-responsive single mutations lie in the antiparallel coiled-coil domain between Cys82 and Cys117 which constitutes the dimer interface. These mutations identify 10 new positions whose alteration significantly affect MerR's metal responsiveness or its repressor function. They give rise to specific predictions for how MerR distinguishes group 12 metals, and they refine our model of the novel domain structure of MerR. Secondary-structure predictions suggest that certain elements of this model also apply to other MerR family regulators.