Stabilization of a Cu-binding site by a highly conserved tryptophan residue.

Copper serves as an essential cofactor for nearly all living organisms. There are still many gaps remaining in our knowledge of how Gram-positive bacteria import copper and maintain homeostasis. To obtain a better understanding of how these processes work, here we focus on the ycnKJI operon responsible for regulating copper levels in the Gram-positive bacterium Bacillus subtilis. This operon encodes three Cu-related proteins: a copper-dependent transcriptional repressor (YcnK), a putative copper importer (YcnJ), and a copper-binding protein of unknown function (YcnI). We previously found that YcnI's extracellular Domain of Unknown Function 1775 (DUF1775) houses a monohistidine brace motif that coordinates a single Cu(II) ion. The Cu(II) binding site includes a highly conserved tryptophan residue. Here, we investigate the role of that tryptophan and the ability of the protein to interact with other oxidation states of Cu. We find that YcnI exhibits strong preference for binding Cu in the oxidized Cu(II) state, and that the conserved tryptophan residue is not essential for the interaction. We determine the structure of a tryptophan variant to 1.95 Å resolution that indicates that the tryptophan is needed to stabilize the metal binding interaction, and find that this variant has weaker affinity for Cu(II) than the wild-type protein. Together, these data provide further insights into the DUF1775 domain family and reveal the role of the conserved tryptophan residue.

The YcnI protein from Bacillus subtilis contains a copper-binding domain.

Bacteria require a precise balance of copper ions to ensure that essential cuproproteins are fully metalated while also avoiding copper-induced toxicity. The Gram-positive bacterium Bacillus subtilis maintains appropriate copper homeostasis in part through the ycn operon. The ycn operon comprises genes encoding three proteins: the putative copper importer YcnJ, the copper-dependent transcriptional repressor YcnK, and the uncharacterized Domain of Unknown Function 1775 (DUF1775) containing YcnI. DUF1775 domains are found across bacterial phylogeny, and bioinformatics analyses indicate that they frequently neighbor domains implicated in copper homeostasis and transport. Here, we investigated whether YcnI can interact with copper and, using electron paramagnetic resonance and inductively coupled plasma-MS, found that this protein can bind a single Cu(II) ion. We determine the structure of both the apo and copper-bound forms of the protein by X-ray crystallography, uncovering a copper-binding site featuring a unique monohistidine brace ligand set that is highly conserved among DUF1775 domains. These data suggest a possible role for YcnI as a copper chaperone and that DUF1775 domains in other bacterial species may also function in copper homeostasis.

Light variability and mixotrophy: Responses of testate amoeba communities and shell δ13C values to a peatland shading experiment.

Paleoecological records suggest that growing season length and/or cloudiness may affect peatland carbon accumulation and testate amoeba-based environmental reconstructions, highlighting a need to understand how light intensity affects microbial communities. We shaded plots on two peatlands for two years to examine effects on testate amoeba communities, the relative abundance of mixotrophic and heterotrophic testate amoebae, transfer-function performance, and δ13C values of two species of mixotrophic testate amoebae. Surprisingly, relative abundance of mixotrophic species increased in shade, although compositional changes did not affect transfer-function performance. Shading did not affect δ13C values of Hyalosphenia papilio and Heleopera sphagni, which ranged from -23.5 to -19.6‰ and -23.2 to -19.2‰, respectively. These δ13C values were higher than those of potential food sources and lower than literature-derived values for Chlorella, the zoochlorellae inhabiting mixotrophic testate amoebae. δ13C values thus suggest that these mixotrophic species obtain some carbon from Chlorella, although coupled dietary and isotope studies are needed to quantify this contribution. More research is needed to assess impacts of light variability on peatland microbial communities; however, carbon sources are recorded by δ13C values of testate amoebae, indicating potential for studies of carbon cycling and how mixotrophy varies temporally and spatially.

The occurrence and dominant controls on arsenic in the Newark and Gettysburg Basins.

Elevated arsenic (As) concentrations in groundwater and rocks have been found in crystalline and sedimentary aquifers from New England to Pennsylvania, USA. The arsenic geochemistry and water-rock interactions of the Northern Appalachian Mountains and the Newark Basin have been researched at length, however, little is known about arsenic in the Gettysburg Basin. Both the Newark and Gettysburg Basins were formed during the breakup of Pangea, sediment deposition occurred during the Triassic and lithologies are of similar depositional environment. We compile and review the work done in the Newark Basin and collect new samples in the Gettysburg Basin for comparison. The Gettysburg Basin has 18%-39% of rock samples with arsenic concentrations greater than the crustal average of 2 mg/kg, while the Newark Basin has 73% to 95% of rock samples above the crustal average. The strongest controls on arsenic in rocks of the Gettysburg Basin are the relationship between arsenic and iron and silicon concentrations while the strongest controls in the Newark Basin are the relationship between arsenic and iron and organic carbon concentrations. The groundwater arsenic concentrations follow similarly with 8-39% of water samples from the Gettysburg Basin above 10 µg/L and 24-54% of water samples from the Newark Basin above 10 µg/L. The strongest controls on arsenic in water of the Gettysburg Basin are pH, alkalinity and silicon, while the strongest controls in the Newark Basin are pH and alkalinity.

Mercury emission from a temperate lake during autumn turnover.

Lakes in temperate regions stratify during summer and winter months, creating distinct layers of water differentiated by their physical and chemical characteristics. When lakes mix in autumn and spring, mercury cycling may be affected by the chemical changes that occur during mixing. Sampling was conducted in Lake Lacawac, Eastern Pennsylvania, USA, throughout the autumn of 2007 to characterize changes in emission of gaseous elemental mercury (Hg(0)) from the lake surface and dissolved mercury profiles in the water column during mixing. Water chemistry and weather parameters were also measured, including dissolved organic carbon (DOC), iron, and solar radiation which have been shown to interact with mercury species. Results indicate that emission of Hg(0) from the lake to the atmosphere during turnover was controlled both by solar radiation and by surface water mercury concentration. As autumn turnover progressed through the months of October and November, higher mercury concentration water from the hypolimnion mixed with epilimnetic water, increasing mercury concentration in epilimnetic waters. Dissolved absorbance was significantly correlated with mercury concentrations and with iron, but DOC concentrations were essentially constant throughout the study period and did not exhibit a relationship with either dissolved mercury concentrations or emission rates. Positive correlations between dissolved mercury and iron and manganese also suggest a role for these elements in mercury transport within the lake, but iron and manganese did not demonstrate a relationship with emission rates. This research indicates that consideration of seasonal processes in lakes is important when evaluating mercury cycling in aquatic systems.

Arsenic in groundwaters in the Northern Appalachian Mountain belt: a review of patterns and processes.

Naturally occurring arsenic in the bedrock of the Northern Appalachian Mountain belt was first recognized in the late 19th century. The knowledge of the behavior of arsenic in groundwater in this region has lagged behind nearly a century, with the popular press reporting on local studies in the early 1980s, and most peer-reviewed research articles on regional patterns conducted and written in the late 1990s and early 2000s. Research reports have shown that within this high arsenic region, between 6% and 22% of households using private drinking water wells contain arsenic in excess of 10 microg/L, the United States Environmental Protection Agency's maximum contaminant level. In nearly all reports, arsenic in drinking water was derived from naturally occurring geologic sources, typically arsenopyrite, substituted sulfides such as arsenian pyrite, and nanoscale minerals such as westerveldite. In most studies, arsenic concentrations in groundwater were controlled by pH dependent adsorption to mineral surfaces, most commonly iron oxide minerals. In some cases, reductive dissolution of iron minerals has been shown to increase arsenic concentrations in groundwater, more commonly associated with anthropogenic activities such as landfills. Evidence of nitrate reduction promoting the presence of arsenic(V) and iron(III) minerals in anoxic environments has been shown to occur in surface waters, and in this manuscript we show this process perhaps applies to groundwater. The geologic explanation for the high arsenic region in the Northern Appalachian Mountain belt is most likely the crustal recycling of arsenic as an incompatible element during tectonic activity. Accretion of multiple terranes, in particular Avalonia and the Central Maine Terrane of New England appear to be connected to the presence of high concentrations of arsenic. Continued tectonic activity and recycling of these older terranes may also be responsible for the high arsenic observed in the Triassic rift basins, e.g. the Newark Basin. There are only two well-known cases of anthropogenic contamination of the environment in the northern Appalachian Mountain belt, both of which are industrial sites with surface contamination at that infiltrated the local groundwater.

Mercury emission to the atmosphere from experimental manipulation of DOC and UVR in mesoscale field chambers in a freshwater lake.

Mesocosm experiments in an optically transparent lake allow the manipulation of both dissolved organic carbon (DOC) and incident ultraviolet radiation (UVR) in order to study mercury reduction and emission processes. In the absence of UVR and the presence of visible light, mercury emission is very low (approximately0.3 ng/m2/h). When UVR is permitted in the mesocosm chambers, mercury emission increases, with emission rates ranging from 0.3 to 2.5 ng/m2/h. At concentrations between 1.5 and 2.5 mg/L DOC, mercury emission does not appear to depend on either the concentration or the optical properties of the DOC. In particular, the addition of 1.0 mg/L DOC from a nearby wetland to a photobleached mesocosm did not increase the emission of mercury. The similarities between mercury emission from highly photobleached 1.5 mg/L DOC and from terrestrially enriched 2.5 mg/L DOC suggest that the moieties responsible for mercury reduction are far in excess of that needed for mercury reduction. Using the measured flux rate of mercury from the water surface, we calculated a dissolved gaseous mercury (DGM) concentration thatwould need to be present to drive the emissive flux. The buildup of DGM was used to approximate a kinetic rate constant for the net mercury reduction in this system of approximately 0.17 h(-1), which is consistent with existing published values.

Analgesic effects of the selective group II (mGlu2/3) metabotropic glutamate receptor agonists LY379268 and LY389795 in persistent and inflammatory pain models after acute and repeated dosing.

Group II (mGluR2/3) metabotropic glutamate receptors have been implicated in the mechanisms of persistent pain states. In the present study, the effects of the selective group II metabotropic glutamate receptor agonists LY379268 and LY389795 were evaluated in the formalin test, carrageenan-induced thermal hyperalgesia and mechanical allodynia, and capsaicin-induced mechanical allodynia in rats. The agonists LY379268 and LY389795 produced dose-dependent decreases in formalin-induced behaviors that were antagonized by the mGlu2/3 receptor antagonist LY341495. The group II antagonist LY341495 produced parallel shifts in the LY379268 dose-response curve, consistent with a competitive antagonism. LY379268 decreased formalin-induced behaviors after intracisternal but not intrathecal administration, suggesting primarily a supraspinal site of action. Both LY379268 and LY389795 produced a dose-related reversal of carrageenan-induced thermal hyperalgesia and capsaicin-induced mechanical allodynia, but had no effect on carrageenan-induced mechanical allodynia. Both agonists also increased response latencies in the hot plate test, but were without effect in the tail-flick test. However, both agonists produced motor impairment on the inverted screen at doses that were analgesic. Moreover, tolerance to the analgesic effects of LY379268 developed after 4 days of once-daily repeated administration in the formalin, carrageenan, capsaicin and hot plate tests. The present findings indicate that group II (mGluR2/3) metabotropic glutamate receptors may be involved in the mechanisms of hyperalgesia and allodynia, however tolerance rapidly develops to these effects.

Arsenic resistance in the archaeon "Ferroplasma acidarmanus": new insights into the structure and evolution of the ars genes.

Arsenic resistance in the acidophilic iron-oxidizing archaeon " Ferroplasma acidarmanus" was investigated. F. acidarmanus is native to arsenic-rich environments, and culturing experiments confirm a high level of resistance to both arsenite and arsenate. Analyses of the complete genome revealed protein-encoding regions related to known arsenic-resistance genes. Genes encoding for ArsR (arsenite-sensitive regulator) and ArsB (arsenite-efflux pump) homologues were found located on a single operon. A gene encoding for an ArsA relative (anion-translocating ATPase) located apart from the arsRB operon was also identified. Arsenate-resistance genes encoding for proteins homologous to the arsenate reductase ArsC and the phosphate-specific transporter Pst were not found, indicating that additional unknown arsenic-resistance genes exist for arsenate tolerance. Phylogenetic analyses of ArsA-related proteins suggest separate evolutionary lines for these proteins and offer new insights into the formation of the arsA gene. The ArsB-homologous protein of F. acidarmanus had a high degree of similarity to known ArsB proteins. An evolutionary analysis of ArsB homologues across a number of species indicated a clear relationship in close agreement with 16S rRNA evolutionary lines. These results support a hypothesis of arsenic resistance developing early in the evolution of life.