Differential Growth of and Nanoscale TiO2 Accumulation in Tetrahymena thermophila by Direct Feeding versus Trophic Transfer from Pseudomonas aeruginosa

Nanoscale titanium dioxide (TiO₂) is increasingly used in consumer goods and is entering waste streams, thereby exposing and potentially affecting environmental microbes. Protozoans could either take up TiO₂ directly from water and sediments or acquire TiO₂ during bactivory (ingestion of bacteria) of TiO₂-encrusted bacteria. Here, the route of exposure of the ciliated protozoan Tetrahymena thermophila to TiO₂ was varied and the growth of, and uptake and accumulation of TiO₂ by, T. thermophila were measured. While TiO₂ did not affect T. thermophila swimming or cellular morphology, direct TiO₂ exposure in rich growth medium resulted in a lower population yield. When TiO₂ exposure was by bactivory of Pseudomonas aeruginosa, the T. thermophila population yield and growth rate were lower than those that occurred during the bactivory of non-TiO₂-encrusted bacteria. Regardless of the feeding mode, T. thermophila cells internalized TiO₂ into their food vacuoles. Biomagnification of TiO₂ was not observed; this was attributed to the observation that TiO₂ appeared to be unable to cross the food vacuole membrane and enter the cytoplasm. Nevertheless, our findings imply that TiO₂ could be transferred into higher trophic levels within food webs and that the food web could be affected by the decreased growth rate and yield of organisms near the base of the web.     

The global nitrogen cycle in the twenty-first century

Global nitrogen fixation contributes 413 Tg of reactive nitrogen (N_r) to terrestrial and marine ecosystems annually of which anthropogenic activities are responsible for half, 210 Tg N. The majority of the transformations of anthropogenic N_r are on land (240 Tg N yr⁻¹) within soils and vegetation where reduced N_r contributes most of the input through the use of fertilizer nitrogen in agriculture. Leakages from the use of fertilizer N_r contribute to nitrate (NO₃⁻) in drainage waters from agricultural land and emissions of trace N_r compounds to the atmosphere. Emissions, mainly of ammonia (NH₃) from land together with combustion related emissions of nitrogen oxides (NO_x), contribute 100 Tg N yr⁻¹ to the atmosphere, which are transported between countries and processed within the atmosphere, generating secondary pollutants, including ozone and other photochemical oxidants and aerosols, especially ammonium nitrate (NH₄NO₃) and ammonium sulfate (NH₄)₂SO₄. Leaching and riverine transport of NO₃ contribute 40–70 Tg N yr⁻¹ to coastal waters and the open ocean, which together with the 30 Tg input to oceans from atmospheric deposition combine with marine biological nitrogen fixation (140 Tg N yr⁻¹) to double the ocean processing of N_r. Some of the marine N_r is buried in sediments, the remainder being denitrified back to the atmosphere as N₂ or N₂O. The marine processing is of a similar magnitude to that in terrestrial soils and vegetation, but has a larger fraction of natural origin. The lifetime of N_r in the atmosphere, with the exception of N₂O, is only a few weeks, while in terrestrial ecosystems, with the exception of peatlands (where it can be 10²–10³ years), the lifetime is a few decades. In the ocean, the lifetime of N_r is less well known but seems to be longer than in terrestrial ecosystems and may represent an important long-term source of N₂O that will respond very slowly to control measures on the sources of N_r from which it is produced.     

In vitro characterization of the inhibitor resistance of glutamate-1-semialdehyde aminotransferase from the cyanobacterium Synechococcus PCC630l GR6

Glutamate-l-semialdehyde (GSA) aminotransferase catalyses the final step in the C5 pathway converting glutamate to the tetrapyrrole precursor δ-aminolaevulinic acid. This enzyme is sensitive to gabaculine (2,3-dihydro-3-amino benzoic acid) and to 4-amino-5-fluoropentanoic acid (AFPA), which are irreversible, mechanism-based inhibitors of pyridoxal phosphatedependent enzymes. Spontaneous mutants of Synechococcus PCC6301 resistant to these inhibitors contain altered enzyme that displays corresponding resistance to high concentrations of the inhibitor. The enzyme from strain GR6, resistant to both inhibitors, contains a three-amino-acid deletion at positions 5–7 and a Met²⁴⁸ → Ile substitution. The enzyme from strain K40 resistant to AFPA but not to gabaculine, contains a Ser¹⁶³ → Thr substitution. GSA aminotransferases containing either the deletion or the substitution that are characteristic of the GR6 mutant were produced in Escherichia coli using the expression vector pMalc2. These engineered mutant enzymes were characterized in terms of their catalytic parameters and sensitivities to gabaculine and AFPA. Furthermore, maltose binding protein/aminotransferase fusion proteins were characterized spectrophotometrically to monitor the interaction of bound cofactor with diamino- and dioxocompounds related to the substrate and both inhibitors. Results were compared with those for similarly produced recombinant wild-type, K40 and GR6 GSA aminotransferases. The engineered products with either the N-terminal deletion or the Met²⁴⁸ → Ile substitution displayed catalytic efficiencies that were intermediate between the wild-type and GR6 or K40 enzymes. However, with respect to their absorption spectra, sensitivity to inhibitors and the reactivity of bound cofactor, they were essentially wild-type. These in vitro studies demonstrate that both changes in enzyme structure are necessary to obtain the distinctive properties of the GR6 aminotransferase, including resistance to high concentrations of gabaculine and AFPA.     

Chemical speciation and competitive cationic partitioning on a sandy aquifer material

Abstract

Coupled geochemical speciation/transport models are being developed to assess potential transport of metal contaminants in the subsurface environment. In a test of the geochemical speciation portion of the effort, MINTEQA2 model predictions are compared with laboratory data concerning the pH dependent partitioning behavior of eight cationic contaminants (Ba, Be, Cd, Cu, Ni, Pb, Tl and Zn) on a sandy aquifer material in an oxidized environment. MINTEQA2 contains provisions for describing potential attenuation due to both mineral phase precipitation processes and adsorption processes resulting from amorphous iron oxides in aquifer materials (MIT Diffuse Layer Model). In the comparison, several trends were discerned. (1) Adsorptive processes tend to better describe the pH-dependent partitioning behavior of transition metals (especially Pb, Zn and Ni). (2) Cd behavior is better described by precipitation as a cadmium carbonate phase. (3) Cu behavior is not reasonably described by the model. (4) Ba and Be comparisons are poor (although presumably their partitioning behavior results from adsorptive and/or pH sensitive solid solution processes). (5) unlike the other elements, the behavior of Tl is relatively insensitive to pH.     

Contrasting geographic patterns of genetic variation for molecular markers vs. phenotypic traits in the energy grass Miscanthus sinensis

Species and hybrids of Miscanthus are a promising energy crop, but their outcrossing mating systems and perennial life cycles are serious challenges for breeding programs. One approach to accelerating the domestication of Miscanthus is to harness the tremendous genetic variation that is present within this genus using phenotypic data from extensive field trials, high‐density genotyping and sequencing technologies, and rapidly developing statistical methods of relating phenotype to genotype. The success of this approach, however, hinges on detailed knowledge about the population genetic structure of the germplasm used in the breeding program. We therefore used data for 120 single‐nucleotide polymorphism and 52 simple sequence repeat markers to depict patterns of putatively neutral population structure among 244 Miscanthus genotypes grown in a field trial near Aberystwyth (UK) and delineate a population of 145 M . sinensis genotypes that will be used for association mapping and genomic selection. Comparative multivariate analyses of molecular marker and phenotypic data for 17 traits related to phenology, morphology/biomass, and cell wall composition revealed significant geographic patterns in this population. A longitudinal cline accounted for a substantial proportion of molecular marker variation (R² = 0.60, P = 3.4 × 10^?15). In contrast, genetic variation for phenotypic traits tended to follow latitudinal and altitudinal gradients, with several traits appearing to have been affected by divergent selection (i.e., Q_ST >> F_ST). These contrasting geographic trends are unusual relative to other plants and provide opportunities for powerful studies of phenotype–genotype associations and the evolutionary history of M. sinensis.     

Immunogenic and Invasive Properties of Brucella melitensis 16M Outer Membrane Protein Vaccine Candidates Identified via a Reverse Vaccinology Approach

Brucella is the etiologic agent of brucellosis, one of the most common and widely distributed zoonotic diseases. Its highly infectious nature, the insidious, systemic, chronic, debilitating aspects of the disease and the lack of an approved vaccine for human use in the United States are features that make Brucella a viable threat to public health. One of the main impediments to vaccine development is identification of suitable antigens. In order to identify antigens that could potentially be used in a vaccine formulation, we describe a multi-step antigen selection approach. We initially used an algorithm (Vaxign) to predict ORF encoding outer membrane proteins with antigenic determinants. Differential gene expression during acute infection and published evidence for a role in virulence were used as criteria for down-selection of the candidate antigens that resulted from in silico prediction. This approach resulted in the identification of nine Brucella melitensis outer membrane proteins, 5 of which were recombinantly expressed and used for validation. Omp22 and Hia had the highest in silico scores for adhesin probability and also conferred invasive capacity to E. coli overexpressing recombinant proteins. With the exception of FlgK in the goat, all proteins reacted to pooled sera from exposed goats, mice, and humans. BtuB, Hia and FlgK stimulated a mixed Th1–Th2 response in splenocytes from immunized mice while BtuB and Hia elicited NO release from splenocytes of S19 immunized mice. The results support the applicability of the current approach to the identification of antigens with immunogenic and invasive properties. Studies to assess immunogenicity and protective efficacy of individual proteins in the mouse are currently underway.     

Complex Responses of Intertidal Molluscan Embryos to a Warming and Acidifying Ocean in the Presence of UV Radiation

Climate change and ocean acidification will expose marine organisms to synchronous multiple stressors, with early life stages being potentially most vulnerable to changing environmental conditions. We simultaneously exposed encapsulated molluscan embryos to three abiotic stressors—acidified conditions, elevated temperate, and solar UV radiation in large outdoor water tables in a multifactorial design. Solar UV radiation was modified with plastic filters, while levels of the other factors reflected IPCC predictions for near-future change. We quantified mortality and the rate of embryonic development for a mid-shore littorinid, Bembicium nanum, and low-shore opisthobranch, Dolabrifera brazieri. Outcomes were consistent for these model species with embryos faring significantly better at 26°C than 22°C. Mortality sharply increased at the lowest temperature (22°C) and lowest pH (7.6) examined, producing a significant interaction. Under these conditions mortality approached 100% for each species, representing a 2- to 4-fold increase in mortality relative to warm (26°C) non-acidified conditions. Predictably, development was more rapid at the highest temperature but this again interacted with acidified conditions. Development was slowed under acidified conditions at the lowest temperature. The presence of UV radiation had minimal impact on the outcomes, only slowing development for the littorinid and not interacting with the other factors. Our findings suggest that a warming ocean, at least to a threshold, may compensate for the effects of decreasing pH for some species. It also appears that stressors will interact in complex and unpredictable ways in a changing climate.