A subcellular proteome atlas of the yeast Komagataella phaffii.

The compartmentalization of metabolic and regulatory pathways is a common pattern of living organisms. Eukaryotic cells are subdivided into several organelles enclosed by lipid membranes. Organelle proteomes define their functions. Yeasts, as simple eukaryotic single cell organisms, are valuable models for higher eukaryotes and frequently used for biotechnological applications. While the subcellular distribution of proteins is well studied in Saccharomyces cerevisiae, this is not the case for other yeasts like Komagataella phaffii (syn. Pichia pastoris). Different to most well-studied yeasts, K. phaffii can grow on methanol, which provides specific features for production of heterologous proteins and as a model for peroxisome biology. We isolated microsomes, very early Golgi, early Golgi, plasma membrane, vacuole, cytosol, peroxisomes and mitochondria of K. phaffii from glucose- and methanol-grown cultures, quantified their proteomes by liquid chromatography-electrospray ionization-mass spectrometry of either unlabeled or tandem mass tag-labeled samples. Classification of the proteins by their relative enrichment, allowed the separation of enriched proteins from potential contaminants in all cellular compartments except the peroxisomes. We discuss differences to S. cerevisiae, outline organelle specific findings and the major metabolic pathways and provide an interactive map of the subcellular localization of proteins in K. phaffii.

Enrichment and genome sequence of the group I.1a ammonia-oxidizing Archaeon "Ca. Nitrosotenuis uzonensis" representing a clade globally distributed in thermal habitats.

The discovery of ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota and the high abundance of archaeal ammonia monooxygenase subunit A encoding gene sequences in many environments have extended our perception of nitrifying microbial communities. Moreover, AOA are the only aerobic ammonia oxidizers known to be active in geothermal environments. Molecular data indicate that in many globally distributed terrestrial high-temperature habits a thaumarchaeotal lineage within the Nitrosopumilus cluster (also called "marine" group I.1a) thrives, but these microbes have neither been isolated from these systems nor functionally characterized in situ yet. In this study, we report on the enrichment and genomic characterization of a representative of this lineage from a thermal spring in Kamchatka. This thaumarchaeote, provisionally classified as "Candidatus Nitrosotenuis uzonensis", is a moderately thermophilic, non-halophilic, chemolithoautotrophic ammonia oxidizer. The nearly complete genome sequence (assembled into a single scaffold) of this AOA confirmed the presence of the typical thaumarchaeotal pathways for ammonia oxidation and carbon fixation, and indicated its ability to produce coenzyme F420 and to chemotactically react to its environment. Interestingly, like members of the genus Nitrosoarchaeum, "Candidatus N. uzonensis" also possesses a putative artubulin-encoding gene. Genome comparisons to related AOA with available genome sequences confirmed that the newly cultured AOA has an average nucleotide identity far below the species threshold and revealed a substantial degree of genomic plasticity with unique genomic regions in "Ca. N. uzonensis", which potentially include genetic determinants of ecological niche differentiation.

Higher nitrate-reducer diversity in macrophyte-colonized compared to unvegetated freshwater sediment.

Freshwater macrophytes stimulate rhizosphere-associated coupled nitrification-denitrification and are therefore likely to influence the community composition and abundance of rhizosphere-associated denitrifiers and nitrate reducers. Using the narG gene, which encodes the catalytic subunit of the membrane-bound nitrate reductase, as a molecular marker, the community composition and relative abundance of nitrate-reducing bacteria were compared in the rhizosphere of the freshwater macrophyte species Littorella uniflora and Myriophyllum alterniflorum to nitrate-reducing communities in unvegetated sediment. Microsensor analysis indicated a higher availability of oxygen in the rhizosphere compared to unvegetated sediment, with a stronger release of oxygen from the roots of L. uniflora compared to M. alterniflorum. Comparison of narG clone libraries between samples revealed a higher diversity of narG phylotypes in association with the macrophyte rhizospheres compared to unvegetated sediment. Quantitative PCR targeting narG- and 16S rRNA-encoding genes pointed to a selective enrichment of narG gene copies in the rhizosphere. The results suggested that the microenvironment of macrophyte rhizospheres, characterized by the release of oxygen and labile organic carbon from the root system, had a stimulating effect on the diversity and relative abundance of rhizosphere-associated nitrate reducers.