Diffusion of H2 S from anaerobic thiolated ligand biodegradation rapidly generates bioavailable mercury.

Methylmercury is a potent neurotoxin that biomagnifies through food webs and which production depends on anaerobic microbial uptake of inorganic mercury (Hg) species. One outstanding knowledge gap in understanding Hg methylation is the nature of bioavailable Hg species. It has become increasingly obvious that Hg bioavailability is spatially diverse and temporally dynamic but current models are mostly built on single thiolated ligand systems, omitting ligand exchanges and interactions, or the inclusion of dissolved gaseous phases. In this study, we used a whole-cell anaerobic biosensor to determine the role of a mixture of thiolated ligands on Hg bioavailability. Serendipitously, we discovered how the diffusion of trace amounts of exogenous biogenic H2 S, originating from anaerobic microbial ligand degradation, can alter Hg speciation - away from H2 S production site - to form bioavailable species. Regardless of its origins, H2 S stands as a mobile mediator of microbial Hg metabolism, connecting spatially separated microbial communities. At a larger scale, global planetary changes are expected to accelerate the production and mobilization of H2 S and Hg, possibly leading to increased production of the potent neurotoxin; this work provides mechanistic insights into the importance of co-managing biogeochemical cycle disruptions.

Golden Gate Assembly of Aerobic and Anaerobic Microbial Bioreporters.

Microbial bioreporters provide direct insight into cellular processes by producing a quantifiable signal dictated by reporter gene expression. The core of a bioreporter is a genetic circuit in which a reporter gene (or operon) is fused to promoter and regulatory sequences that govern its expression. In this study, we develop a system for constructing novel Escherichia coli bioreporters based on Golden Gate assembly, a synthetic biology approach for the rapid and seamless fusion of DNA fragments. Gene circuits are generated by fusing promoter and reporter sequences encoding yellow fluorescent protein, mCherry, bacterial luciferase, and an anaerobically active flavin-based fluorescent protein. We address a barrier to the implementation of Golden Gate assembly by designing a series of compatible destination vectors that can accommodate the assemblies. We validate the approach by measuring the activity of constitutive bioreporters and mercury and arsenic biosensors in quantitative exposure assays. We also demonstrate anaerobic quantification of mercury and arsenic in biosensors that produce flavin-based fluorescent protein, highlighting the expanding range of redox conditions that can be examined by microbial bioreporters. IMPORTANCE Microbial bioreporters are versatile genetic tools with wide-ranging applications, particularly in the field of environmental toxicology. For example, biosensors that produce a signal output in the presence of a specific analyte offer less costly alternatives to analytical methods for the detection of environmental toxins such as mercury and arsenic. Biosensors of specific toxins can also be used to test hypotheses regarding mechanisms of uptake, toxicity, and biotransformation. In this study, we develop an assembly platform that uses a synthetic biology technique to streamline construction of novel Escherichia coli bioreporters that produce fluorescent or luminescent signals either constitutively or in response to mercury and arsenic exposure. Beyond the synthesis of novel biosensors, our assembly platform can be adapted for numerous applications, including labeling bacteria for fluorescence microscopy, developing gene expression systems, and modifying bacterial genomes.

Aerobic and Anaerobic Bacterial Mercury Uptake is Driven by Algal Organic Matter Composition and Molecular Weight.

The biological mobilization of mercury (Hg) into microbes capable of Hg methylation is one of the limiting steps in the formation of the neurotoxin methylmercury (MeHg). Although algal dissolved organic matter (DOM) has been associated with increased MeHg production, the relationship between bacterial Hg uptake and algal DOM remains unexplored. In this study, we aimed to address how the quantity and quality of DOM, freshly harvested from several algae, affected the bacterial uptake of Hg with the use of a biosensor capable of functioning both aerobically and anaerobically. We combined biosensor measurements with high-resolution mass spectrometry and field-flow fractionation to elucidate how DOM composition and molecular weight influenced microbial Hg uptake. We showed that freshly harvested DOM from Chlorophyte and Euglena mutabilis strongly inhibited aerobic and anaerobic Hg uptake, whereas DOM harvested from Euglena gracilis did not exhibit this same pronounced effect. Once fractionated, we found that amino acids and polyamines, most abundant in Euglena gracilis DOM, were positively correlated to increase Hg uptake, suggesting that these molecules are potentially underappreciated ligands affecting Hg bioavailability. As water quality is affected by eutrophication, algal community assemblages will change, leading to variations in the nature of autochthonous DOM released in aquatic systems. Our results highlight that variations in the emergent properties of DOM originating from varying algal species can have a profound effect on bacterial Hg uptake and thus methylation.

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium.

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to bacteria control the amount that will accumulate in staple food crops. The bioavailability of these metals is dependent on their speciation in solution, but more particularly under anoxic conditions where Hg is methylated to toxic monomethylmercury (MeHg) and Cd persists in the rhizosphere. Whole-cell microbial biosensors give a quantifiable signal when a metal enters the cytosol and therefore are useful tools to assess metal bioavailability. Unfortunately, most biosensing efforts have so far been constrained to oxic environments due to the limited ability of existing reporter proteins to function in the absence of oxygen. In this study, we present our effort to develop and optimize a whole-cell biosensor assay capable of functioning anaerobically that can detect metals under anoxic condition in quasi-real time and within hours. We describe how the biosensor can help assess how chemical variables relevant to the environmental cycling of metals affect their bioavailability. The following protocol includes methods to (1) prepare Hg and Cd standards under anoxic conditions, (2) prepare the biosensor in the absence of oxygen, (3) design and execute an experiment to determine how a series of variable affects Hg or Cd bioavailability, and (4) to quantify and interpret biosensor data. We show the linear ranges of the biosensors and provide examples showing the method's ability to distinguish between metal bioavailability and toxicity by utilizing both metal-inducible and constitutive strains.

Ionic Strength Differentially Affects the Bioavailability of Neutral and Negatively Charged Inorganic Hg Complexes.

Mercury (Hg) bioavailability to bacteria in marine systems is the first step toward its bioamplification in food webs. These systems exhibit high salinity and ionic strength that will both alter Hg speciation and properties of the bacteria cell walls. The role of Hg speciation on Hg bioavailability in marine systems has not been teased apart from that of ionic strength on cell wall properties, however. We developed and optimized a whole-cell Hg bioreporter capable of functioning under aerobic and anaerobic conditions and exhibiting no physiological limitations of signal production to changes in ionic strength. We show that ionic strength controls the bioavailability of Hg species, regardless of their charge, possibly by altering properties of the bacterial cell wall. The unexpected anaerobic bioavailability of negatively charged halocomplexes may help explain Hg methylation in marine systems such as the oxygen-deficient zone in the oceanic water column, sea ice or polar snow.

Diagnosis and management of myocarditis in athletes.

Myocarditis is a pathologic entity that has serious potential consequences for competitive athletes. Myocarditis is an inflammation of the myocardium accompanied by myocellular necrosis. Cardiotropic viruses, in particular the Coxsackie B virus, have been implicated as the most common cause of acute myocarditis in the United States. A thorough history, physical examination, electrocardiogram, echocardiogram, elevated cardiac enzymes, and an elevated sedimentation rate help make the diagnosis. Athletes recovering from acute myocarditis should abstain from moderate to vigorous activity for 6 months, have normal cardiac function, and have no demonstrable arrhythmia before returning to sports.