Project description:We measured abundances of tRNAs by means of hydro-tRNA-seq (Gogakos et al., 2017), a method based on partial alkaline RNA hydrolysis that generates fragments suitable for sequencing, in the genome-reduced bacterium Mycoplasma pneumoniae.
Project description:Given the facilities for whole genome sequencing with next-generation sequencers, structural and functional gene annotation is now only based on automated prediction. However, errors in terms of gene structure are still frequently reported especially for the correct determination of initiation start codons. Here, we propose a strategy to enrich and detect protein N-termini by mass spectrometry in order to refine genome annotation. After selective protein N-termini derivatization using (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPPAc-OSu) as labeling reagent, protein digestion was performed with three proteases in parallel. TMPP-labeled N-terminal-most peptides were further resolved from internal peptides by the COmbined FRActional DIagonal Chromatography (COFRADIC) sorting methodology before analysis with tandem mass spectrometry. We refined the annotation of the genome of a model marine bacterium, Roseobacter denitrificans.
Project description:The Placozoa are an enigmatic group of simple marine metazoans where all taxa harbor intracellular bacteria. Despite four decades of research, the bacterial identities, their location and their roles remain elusive. Here we show that the placozoan Trichoplax H2 is associated with two intracellular bacteria. We detected the symbionts and reconstructed their physiology using metagenomic and metatranscriptomic evidence from the same single-animal specimens. One symbiont forms a new genus in the Midichloriaceae (Rickettsiales) and correlative fluorescent labelling and 3-D electron microscopic tomography showed that it inhabits the rough ER in the fiber-cells. It has mutualistic traits and occurs worldwide as we could detect it in 10% of all aquatic tag sequencing datasets. The second symbiont is an intracellular bacterium from the Margulisbacteria, a phylum-level clade previously only identified from DNA sequencing and not known to form intracellular associations. It resides in the digestive ventral epithelial cells, uses lipids digested by the host and has the physiological capacity to supplement the placozoan nutrition. Our single-individual approach revealed that this cultivable placozoan host forms a tripartite symbiosis and provides experimental access to microbial dark matter – a rickettsiales that inhabits a novel niche within eukaryote cells and an intracellular margulisbacterial symbiont.
Project description:We used culturing of fecal sample enrichments on solid medium containing gastric mucin as the main carbon source to isolate a novel bacterium that is largely restricted to using the N-acetylglucosamine and N-acetylgalactosamine sugars from mucin. This butyrate-producing bacterium accesses these sugars from both polymeric gastric mucin and chemically released oligosaccharides and has a genome with correspondingly restricted carbohydrate-active enzyme content. Sequencing data was curated to determine gene expression profiles when comparing N-acetylgalactosamine, N-acetylglucosamine, gastric mucin oligosaccharides and cellobiose.
Project description:The Rickettsiales Ehrlichia ruminantium (ER), the causal agent of the fatal tick-borne disease Heartwater, induces severe damage to the vascular endothelium in ruminants. Nevertheless, E. ruminantium-induced pathobiology remains largely unknown. Our work paves the way for understanding this phenomenon by using quantitative proteomic analyses (2D-DIGE-MS/MS, 1DE-nanoLC-MS/MS and biotin-nanoUPLC-MS/MS) of host bovine aorta endothelial cells (BAE) during the in vitro bacterium intracellular replication cycle. We detect 265 bacterial proteins (including virulence factors), at all time-points of the E. ruminantium replication cycle, highlighting a dynamic bacterium–host interaction. We show that E. ruminantium infection modulates the expression of 433 host proteins: 98 being over-expressed, 161 under-expressed, 140 detected only in infected BAE cells and 34 exclusively detected in non-infected cells. Cystoscape integrated data analysis shows that these proteins lead to major changes in host cell immune responses, host cell metabolism and vesicle trafficking, with a clear involvement of inflammation-related proteins in this process. Our findings led to the first model of E. ruminantium infection in host cells in vitro, and we highlight potential biomarkers of E. ruminantium infection in endothelial cells (such as ROCK1, TMEM16K, Albumin and PTPN1), which may be important to further combat Heartwater, namely by developing non-antibiotic-based strategies.