Engineering the rRNA decoding site of eukaryotic cytosolic ribosomes in bacteria

Structural and genetic studies on prokaryotic ribosomes have provided important insights into fundamental aspects of protein synthesis and translational control and its interaction with ribosomal drugs. Comparable mechanistic studies in eukaryotes are mainly hampered by the absence of both high-resolution crystal structures and efficient genetic models. To study the interaction of aminoglycoside antibiotics with selected eukaryotic ribosomes, we replaced the bacterial drug binding site in 16S rRNA with its eukaryotic counterpart, resulting in bacterial hybrid ribosomes with a fully functional eukaryotic rRNA decoding site. Cell-free translation assays demonstrated that hybrid ribosomes carrying the rRNA decoding site of higher eukaryotes show pronounced resistance to aminoglycoside antibiotics, equivalent to that of rabbit reticulocyte ribosomes, while the decoding sites of parasitic protozoa show distinctive drug susceptibility. Our findings suggest that phylogenetically variable components of the ribosome, other than the rRNA-binding site, do not affect aminoglycoside susceptibility of the protein-synthesis machinery. The activities of the hybrid ribosomes indicate that helix 44 of the rRNA decoding site behaves as an autonomous domain, which can be exchanged between ribosomes of different phylogenetic domains for study of function

Convergence of soil nitrogen isotopes across global climate gradients

Quantifying global patterns of terrestrial nitrogen (N) cycling is central to predicting future patterns of primary productivity, carbon sequestration, nutrient fluxes to aquatic systems, and climate forcing. With limited direct measures of soil N cycling at the global scale, syntheses of the (15)N:(14)N ratio of soil organic matter across climate gradients provide key insights into understanding global patterns of N cycling. In synthesizing data from over 6000 soil samples, we show strong global relationships among soil N isotopes, mean annual temperature (MAT), mean annual precipitation (MAP), and the concentrations of organic carbon and clay in soil. In both hot ecosystems and dry ecosystems, soil organic matter was more enriched in (15)N than in corresponding cold ecosystems or wet ecosystems. Below a MAT of 9.8°C, soil δ(15)N was invariant with MAT. At the global scale, soil organic C concentrations also declined with increasing MAT and decreasing MAP. After standardizing for variation among mineral soils in soil C and clay concentrations, soil δ(15)N showed no consistent trends across global climate and latitudinal gradients. Our analyses could place new constraints on interpretations of patterns of ecosystem N cycling and global budgets of gaseous N loss

Amphiphobic carbon nanotubes as macroemulsion surfactants

Single-walled carbon nanotubes (SWNTs) are insoluble in either water or oil. When mixtures of SWNTs, water, and toluene are sheared vigorously, a macroscopic emulsion of water droplets forms in toluene, with the SWNTs residing at the interface between the immiscible fluids, acting as a natural surfactant or interphase material. The average droplet dimension decreases as the SWNT/water mass ratio increases, implying a coarsening-and-pinning mechanism. The concept of emulsification via amphiphobicity will find applications in processing nanotubes, compatibilizing immiscible fluids, and creating new macroscopic emulsion materials with unique interfacial and structural properties

Sporocarp δ15N and use of inorganic and organic nitrogen in vitro differ among host-specific suilloid fungi associated with high elevation five-needle pines

Widespread decline of whitebark and limber pines in the northern Rocky Mountains (USA) has created an imperative to understand functional diversity in their ectomycorrhizal associates. Because suilloid fungi are likely important in successful reestablishment of pines the nitrogen-related functional traits of 28 high-elevation suilloid isolates were examined. Radial growth, mass accumulation and mycelial density were measured for isolates on six different nitrogen sources. The δ15N values of suilloid sporocarps used as sources for pure cultures were compared against growth parameters to investigate a possible link between these N-related functional traits. Isolates grew poorly on nitrate and BSA and grew well on glutamine, alanyl-glutamine and ammonium phosphate, with somewhat slower growth on alanine. Isolates and species varied considerably in their growth response to different nitrogen sources. Effective use of nitrate and BSA was uncommon and associated with isolates with high inherent growth rates. Sporocarp δ15N was negatively correlated with relative growth on alanine of the corresponding isolates. Our results suggest strong similarities in N source use patterns of suilloid fungi of whitebark pine origin and those of another high-elevation five-needle stone pine, the Swiss stone pine

Tracing metabolic pathways of lipid biosynthesis in ectomycorrhizal fungi from position‐specific 13C‐labelling in glucose

Six position‐specific 13C‐labelled isotopomers of glucose were supplied to the ectomycorrhizal fungi Suillus pungens and Tricholoma flavovirens. From the resulting distribution of 13C among fungal PLFAs, the overall order and contribution of each glucose atom to fatty acid 13C enrichment was: C6 (∼31%) \u3e C5 (∼25%) \u3e C1 (∼18%) \u3e C2 (∼18%) \u3e C3 (∼8%) \u3e C4 (∼1%). These data were used to parameterize a metabolic model of the relative fluxes from glucose degradation to lipid synthesis. Our data revealed that a higher amount of carbon is directed to glycolysis than to the oxidative pentose phosphate pathway (60% and 40% respectively) and that a significant part flows through these pathways more than once (73%) due to the reversibility of some glycolysis reactions. Surprisingly, 95% of carbon cycled through glyoxylate prior to incorporation into lipids, possibly to consume the excess of acetyl‐CoA produced during fatty acid turnover. Our approach provides a rigorous framework for analysing lipid biosynthesis in fungi. In addition, this approach could ultimately improve the interpretation of isotopic patterns at natural abundance in field studies