Project description:Planctomycetes of the genus Singulisphaera are common inhabitants of soils and peatlands. Although described members of this genus are characterized as possessing hydrolytic capabilities, the ability to degrade chitin has not yet been reported for these bacteria. In this study, a novel Singulisphaera representative, strain Ch08, was isolated from a chitinolytic enrichment culture obtained from a boreal fen in Northern European Russia. The 16S rRNA gene sequence of this isolate displayed 98.2% similarity to that of Singulisphaera acidiphila MOB10T. Substrate utilization tests confirmed that strain Ch08 is capable of growth on amorphous chitin. The complete genome of strain Ch08 determined in this study was 10.85 Mb in size and encoded two predicted chitinases, which were only distantly related to each other and affiliated with the glycoside hydrolase family GH18. One of these chitinases had a close homologue in the genome of S. acidiphila MOB10T. The experimental verification of S. acidiphila MOB10T growth on amorphous chitin was also positive. Transcriptome analysis performed with glucose- and chitin-growth cells of strain Ch08 showed upregulation of the predicted chitinase shared by strain Ch08 and S. acidiphila MOB10T. The gene encoding this protein was expressed in Escherichia coli, and the endochitinase activity of the recombinant enzyme was confirmed. The ability to utilize chitin, a major constituent of fungal cell walls and arthropod exoskeletons, appears to be one of the previously unrecognized ecological functions of Singulisphaera-like planctomycetes.
Project description:Ornithine lipids (OLs) are phosphorus-free membrane lipids widespread in bacteria but absent from archaea and eukaryotes. In addition to the unmodified OLs, a variety of OL derivatives hydroxylated in different structural positions has been reported. Recently, methylated derivatives of OLs were described in several planctomycetes isolated from a peat bog in Northern Russia, although the gene/enzyme responsible for the N-methylation of OL remained obscure. Here we identify and characterize the OL N-methyltransferase OlsG (Sinac_1600) from the planctomycete Singulisphaera acidiphila. When OlsG is co-expressed with the OL synthase OlsF in Escherichia coli, methylated OL derivatives are formed. An in vitro characterization shows that OlsG is responsible for the 3-fold methylation of the terminal δ-nitrogen of OL. Methylation is dependent on the presence of the detergent Triton X-100 and the methyldonor S-adenosylmethionine.
Project description:Background: Ependymomas encompass multiple, clinically relevant tumor types based on localization and molecular profiles. Although tumors of the methylation class “spinal ependymoma” (SP-EPN) represent the most common intramedullary neoplasms in children and adults, their developmental origin is ill-defined, molecular data are scarce, and the potential heterogeneity within SP-EPN remains unexplored. The only known recurrent genetic events in SP-EPN are loss of chromosome 22q and NF2 mutations, but neither types and frequency of these alterations nor their clinical meaning have been described in a large, epigenetically defined series. Methods: We mapped SP-EPN transcriptomes (n=76) to developmental atlases of the developing and adult spinal cord to uncover potential developmental origins of these tumors. In addition, transcriptomic, epigenetic (n=234), genetic (n=140), and clinical analyses (n=115) were integrated for a detailed overview on this entity. Results: Integration of transcriptomic ependymoma data with single-cell atlases of the spinal cord identified mature adult ependymal cells to display highest similarities to SP-EPN. Unsupervised hierarchical clustering of tumor data together with integrated analysis of methylation profiles identified two molecular SP-EPN subtypes. Subtype 1 predominantly contained NF2 wild type sequences with regular NF2 expression but revealed more extensive copy number alterations. Subtype 2 harbored previously known germline or sporadic NF2 mutations and was NF2-deficient in most cases, more often showed multilocular disease, and demonstrated a significantly reduced progression-free survival. Conclusion: Based on integrated molecular profiling of a large tumor series we identify two distinct SP-EPN subtypes with important implications for genetic counseling, patient surveillance, and drug development priorities.