Karyn L. Wilde

A Comparison of Copper Speciation Measurements with the Toxic Responses of Three Sensitive Freshwater Organisms

Environmental Context. A rapid Chelex resin method is shown to be a valuable speciation screening tool for use in a tiered risk assessment of copper toxicity in fresh waters. It is a more conservative measure than toxicity testing with sensitive biota, but a better indicator of toxicity than a dissolved copper measurement. Abstract. Twelve natural fresh waters with similar pH and hardness, but varying dissolved organic carbon (DOC) and copper concentrations, were assessed for (a) toxicity to an alga (Chlorella sp. 12), a cladoceran (Ceriodaph- nia cf. dubia) and a bacterium (Erwinnia sp.), and (b) copper speciation using a rapid Chelex extraction method, diffusive gradients in thin films (DGT) and anodic stripping voltammetry (ASV). In synthetic fresh water with no added DOC, at pH 7.0 and low hardness, the toxic responses (EC/IC50) of all three organisms to copper were similar. However, in the toxicity of copper added to natural water samples exhibited a negative linear relationship to DOC (r 2 = 0.82-0.83), with respective slopes for algae, cladocerans and bacteria decreasing in the ratio 7.4 : 3.5 : 1. The marked difference in responses in the presence of natural dissolved organic matter indicated that not all of the organisms conformed to the free ion activity model (FIAM). This was confirmed by copper ion selective electrode measurement of copper ion activity. Copper toxicity to algae in the presence of DOC was overestimated by free ion activity possibly due to surface binding of DOC. Copper toxicity to the bacteria was greater than predicted and was shown to be a result of bioavailability of some copper complexes formed with organic matter. Cladocerans appear to more closely follow FIAM predictions. These findings have important implications for attempts to extend predictive models of metal toxicity beyond fish to more sensitive freshwater species. The measured labile copper concentra- tions of copper-spiked natural waters varied from 0 to 70% of total copper concentrations. There was no clear relationship between the three measurement techniques. Good correlations were obtained between both algal and bacterial growth inhibitions measured on copper-spiked natural waters and the corresponding Chelex-labile copper concentrations. A single natural water sample was manipulated to different pH and hardness values, spiked with copper, and tested using the above three organisms with the Chelex method.Toxicity test results generally agreed with studies performed in synthetic fresh waters, showing that the relationships between toxicity, pH and hardness were organism-specific. Chelex-labile copper was always over-predictive of toxicity but significantly better (P ≤ 0.05) than dissolved copper concentrations, as it only detects the fraction of total copper that is reactive over biologically-relevant timescales. Colloidally-bound copper and copper associated with strong ligands are not detected. The Chelex method is therefore useful as a measure where speciation is accepted in water quality regulations.

Demonstration of the use of Scenedesmus and Carteria biomass to drive bacterial sulfate reduction by Desulfovibrio alcoholovorans isolated from an artificial wetland

A major factor limiting application of bacterial sulfate reduction to removal of sulfate and heavy metals in wetland systems is the requirement to supply carbon and energy to drive the process. Primary production by aquatic plants and algae is a cheap option for driving sustainable bacterial sulfate reduction and most operational systems have relied on plants. The use of harvested, non-growing algal biomass to support bacterial sulfate reduction was investigated. Two genera of green algae, strains N9 and A3, were isolated from treatment cells from the Artificial Wetland Filter at the Ranger uranium mine (Northern Territory, Australia) which successfully removes UO22+, Mn2+ and nitrate, but little sulfate, from mine waters. These algae were identified as Carteria sp. and Scenedesmus sp. and were used as the sole carbon and energy source to enrich a sulfate-reducing mixed bacterial culture from the constructed wetland. Bacterial sulfate reduction supported solely by degradation of algal biomass was demonstrated at laboratory scale using both algae. In excess of 300 mg/L, sulfate was reduced in 17 days following an initial period of approximately 8 days during which sulfate levels did not decrease. The amount and rate of reduction was shown to be dependent on the concentration of algal biomass added. Carteria algae at low concentration showed reduction earlier; however, yields at higher concentration were affected by unknown inhibition. Scenedesmus strain N9 produced a maximum specific yield of 94.3 g of sulfate reduced per gram biomass added compared with 43.5 for Carteria strain A3. Sequence analysis of the 16S rRNA gene of members of the bacterial consortium indicated that the sulfate-reducing bacteria (SRB) showed highest homology (98.5%) with Desulfovibrio alcoholovorans. A second bacterium, which showed homologies of 91–92% with organisms of the Clostridial assemblage, was also present in the culture and represents a new species, or possibly a new genus.

Solid-State NMR Spectroscopy of Functional Amyloid from a Fungal Hydrophobin: A Well-Ordered β-Sheet Core Amidst Structural Heterogeneity†

GrEASy fibrils: Hydrophobins are fungal proteins that assemble into an amphipathic fibrillar monolayer with amyloid properties and a hydrophobic face as water-resistant as Teflon. Solid-state NMR studies on EAS hydrophobin fibrils reveal direct evidence of a partial molecular rearrangement on assembly and an ordered β-sheet-rich core in the context of a whole protein in this functional amyloid.

In vivo deuteration strategies for neutron scattering analysis of bacterial polyhydroxyoctanoate

The cultivation of microorganisms on deuterated substrates has allowed us to control deuterium incorporation into biopolymer systems which is important for characterisation using neutron scattering techniques. Bacterial polyhydroxyoctanoate (PHO) is a polyester formed within inclusions inside bacterial cells and was deuterated in vivo under various conditions to characterise the formation of these inclusions by neutron scattering. Manipulation of deuterated media during microbial growth and PHO production phases resulted in polymer with partial or complete substitution of hydrogen by deuterium, as shown by gas chromatography. Sequential feeding of hydrogenated and deuterated forms of the same precursor was used to demonstrate that neutron scattering analysis could be used to differentiate between chemically similar phases in these polymer inclusions.

Structure and Characterisation of a Key Epitope in the Conserved C-Terminal Domain of the Malaria Vaccine Candidate MSP2

Merozoite surface protein 2 (MSP2) is an intrinsically disordered antigen that is abundant on the surface of the malaria parasite Plasmodium falciparum. The two allelic families of MSP2, 3D7 and FC27, differ in their central variable regions, which are flanked by highly conserved C-terminal and N-terminal regions. In a vaccine trial, full-length 3D7 MSP2 induced a strain-specific protective immune response despite the detectable presence of conserved region antibodies. This work focuses on the conserved C-terminal region of MSP2, which includes the only disulphide bond in the protein and encompasses key epitopes recognised by the mouse monoclonal antibodies 4D11 and 9H4. Although the 4D11 and 9H4 epitopes are overlapping, immunofluorescence assays have shown that the mouse monoclonal antibody 4D11 binds to MSP2 on the merozoite surface with a much stronger signal than 9H4. Understanding the structural basis for this antigenic difference between these antibodies will help direct the design of a broad-spectrum and MSP2-based malaria vaccine. 4D11 and 9H4 were reengineered into antibody fragments [variable region fragment (Fv) and single-chain Fv (scFv)] and were validated as suitable models for their full-sized IgG counterparts by surface plasmon resonance and isothermal titration calorimetry. An alanine scan of the 13-residue epitope 3D7-MSP2207-222 identified the minimal binding epitope of 4D11 and the key residues involved in binding. A 2.2-Å crystal structure of 4D11 Fv bound to the eight-residue epitope NKENCGAA provided valuable insight into the possible conformation of the C-terminal region of MSP2 on the parasite. This work underpins continued efforts to optimise recombinant MSP2 constructs for evaluation as potential vaccine candidates.

Transient antibody-antigen interactions mediate the strain-specific recognition of a conserved malaria epitope

Transient interactions in which binding partners retain substantial conformational disorder play an essential role in regulating biological networks, challenging the expectation that specificity demands structurally defined and unambiguous molecular interactions. The monoclonal antibody 6D8 recognises a completely conserved continuous nine-residue epitope within the intrinsically disordered malaria antigen, MSP2, yet it has different affinities for the two allelic forms of this antigen. NMR chemical shift perturbations, relaxation rates and paramagnetic relaxation enhancements reveal the presence of transient interactions involving polymorphic residues immediately C-terminal to the structurally defined epitope. A combination of these experimental data with molecular dynamics simulations shows clearly that the polymorphic C-terminal extension engages in multiple transient interactions distributed across much of the accessible antibody surface. These interactions are determined more by topographical features of the antibody surface than by sequence-specific interactions. Thus, specificity arises as a consequence of subtle differences in what are highly dynamic and essentially non-specific interactions.Krishnarjuna et al. show that multiple transient interactions mediate monoclonal antibody recognition of an epitope within a disordered malaria antigen, MSP2. These results explain the antibody’s differential affinities for two allelic forms of the antigen.