Dataset Information

Protein stable isotope fingerprinting (P-SIF): Synechocystis sp. PCC6803 + Allochromatium vinosum DSM180 biomass mixture

ABSTRACT: Protein stable isotope fingerprinting (P-SIF) is a method to measure the carbon isotope ratios of whole proteins separated from complex mixtures, including cultures and environmental samples. The goal of P-SIF is to expose the links between identity and function in microbial ecosystems by (i) determining the values of δ13C for different taxonomic divisions, and (ii) using those values as clues to the metabolic pathways employed by the respective organisms. This project develops a two-dimensional HPLC protocol followed by tryptic digestion and isotope ratio mass spectrometry on fraction aliquots. Data from a mixture of Synechocystis sp. PCC6803 and A. vinosum DSM180 biomass show that P-SIF can predict the δ13C values of the original taxa. The measured values of δ13C along with the QTOF-MS/MS peptide signal intensity and protein assignments are used in an overdetermined set of linear equations to back-calculate the values of δAllo and δSyn for the individual species. These data suggest the approach will be useful to determine natural δ13C signatures in microbial ecosystems.

INSTRUMENT(S): 6520 Quadrupole Time-of-Flight LC/MS

ORGANISM(S): Allochromatium Vinosum (strain Atcc 17899 / Dsm 180 / Nbrc 103801 / Ncimb 10441 / D) (chromatium Vinosum) Synechocystis Sp. Pcc 6803

SUBMITTER: Susan Carter

LAB HEAD: Ann Pearson

PROVIDER: PXD003150 | Pride | 2015-11-10

REPOSITORIES: Pride

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Project description:The protozoan parasites Trypanosoma brucei spp. are responsible for important human and livestock diseases in sub Saharan Africa. In the mammalian blood, two developmental forms of the parasite exist: proliferative ?slender? forms and transmissible ?stumpy? forms that are quiescent, awaiting uptake in a tsetse fly bloodmeal. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission. The response is triggered by an elusive ?stumpy induction factor? (SIF) whose intracellular signaling pathway is also completely uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains cannot respond to SIF, but can generate forms with stumpy characteristics when exposed to cell permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNAi library screen to identify the signaling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to cell permeable cAMP or AMP analogues to select cells that remained proliferative and so were unresponsive to these signals. Genome-wide ion torrent-based RNA interference Target sequencing (RIT-seq) identified a cohort of genes implicated in all steps of the signaling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. The identified genes at each step have been validated in cells naturally capable of stumpy formation, confirming their role in SIF-induced density sensing and cellular quiescence. RNA was isolated from AnTat1.1 cells grown in mice with or without doxycycline. RBP7 (Tb927.10.12100) RNAi lines were grown in mice with or without doxycycline. Since the RBP7 RNAi is leaky (six independent cell lines were analysed and all were significantly leaky), transcripts that showed elevated or reduced abundance in both RNAi induced and uninduced populations were identified in comparison to the AnTat1.1 control. A similar approach was adopted for the RBP7 over-expressing lines.

Dataset Information

Protein stable isotope fingerprinting (P-SIF): Synechocystis sp. PCC6803 + Allochromatium vinosum DSM180 biomass mixture

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