Climate drivers alter nitrogen availability in surface peat and decouple N2 fixation from CH4 oxidation in the Sphagnum moss microbiome

Peat mosses (Sphagnum spp.) are keystone species in boreal peatlands, where they dominate net primary productivity and facilitate the accumulation of carbon in thick peat deposits. Sphagnum mosses harbor a diverse assemblage of microbial partners, including N₂-fixing (diazotrophic) and CH₄-oxidizing (methanotrophic) taxa that support ecosystem function by regulating transformations of carbon and nitrogen. Here, we investigate the response of the Sphagnum phytobiome (plant + constituent microbiome + environment) to a gradient of experimental warming (+0°C to +9°C) and elevated CO₂ (+500 ppm) in an ombrotrophic peatland in northern Minnesota (USA). By tracking changes in carbon (CH₄, CO₂) and nitrogen (NH₄-N) cycling from the belowground environment up to Sphagnum and its associated microbiome, we identified a series of cascading impacts to the Sphagnum phytobiome triggered by warming and elevated CO₂. Under ambient CO₂, warming increased plant-available NH₄-N in surface peat, excess N accumulated in Sphagnum tissue, and N₂ fixation activity decreased. Elevated CO₂ offset the effects of warming, disrupting the accumulation of N in peat and Sphagnum tissue. Methane concentrations in porewater increased with warming irrespective of CO₂ treatment, resulting in a ~10× rise in methanotrophic activity within Sphagnum from the +9°C enclosures. Warming's divergent impacts on diazotrophy and methanotrophy caused these processes to become decoupled at warmer temperatures, as evidenced by declining rates of methane-induced N₂ fixation and significant losses of keystone microbial taxa. In addition to changes in the Sphagnum microbiome, we observed ~94% mortality of Sphagnum between the +0°C and +9°C treatments, possibly due to the interactive effects of warming on N-availability and competition from vascular plant species. Collectively, these results highlight the vulnerability of the Sphagnum phytobiome to rising temperatures and atmospheric CO₂ concentrations, with significant implications for carbon and nitrogen cycling in boreal peatlands.     

Maturation of a eukaryotic cytochrome <Emphasis Type="Italic">c</Emphasis> in the cytoplasm of <Emphasis Type="Italic">Escherichia coli</Emphasis> without the assistance by a dedicated biogenesis apparatus

Maturation of c-type cytochromes involves the covalent and stereospecific enzymatic attachment of a heme b via thioether linkages to two conserved cysteines within apocytochromes. Horse cytochrome c is readily matured into its native holoform in the cytoplasm of E. coli when co-expressed with yeast cytochrome c heme lyase. Here we report the low yield formation of holocytochrome with covalently attached heme also in the absence of heme lyase. This is the first demonstration of in vivo maturation of a eukaryotic cytochrome c in a prokaryotic cytoplasm without the assistance by a dedicated enzymatic maturation system. The assembled cytochrome c can be oxidized by cytochrome c oxidase, indicating the formation of a functional protein. The absorption spectrum is typical of a low spin, six coordinated c-type heme. Nevertheless, minor spectral differences relative to the native cytochrome c, deviation of the midpoint reduction potential and slightly altered kinetic parameters of the interaction with cytochrome c oxidase emphasize the importance of cytochrome c heme lyase in folding cytochrome c into its native conformation.     

Reverse-phase high-performance liquid chromatography of proteins: The separation of hemoglobin chain variants

The potential of reverse-phase high-performance liquid chromatography for the separation of closely related proteins has been investigated. Using an octadecasilyl silica stationary phase and a propanol/pyridine formate solvent system normal α- and β-chains of human hemoglobin have been separated from several of their respective mutant chains which differ by single amino acid residues only. The results suggest that reverse-phase high-performance liquid chromatography is a powerful tool for the separation of medium-sized proteins with minimal structural difference.