A thylakoid biogenesis BtpA protein is required for the initial step of tetrapyrrole biosynthesis in cyanobacteria.

Biogenesis of the photosynthetic apparatus requires complicated molecular machinery, individual components of which are either poorly characterized or unknown. The BtpA protein has been described as a factor required for the stability of photosystem I (PSI) in cyanobacteria; however, how the BtpA stabilized PSI remains unexplained. To clarify the role of BtpA, we constructed and characterized the btpA-null mutant (ΔbtpA) in the cyanobacterium Synechocystis sp. PCC 6803. The mutant contained only c. 1% of chlorophyll and nearly no thylakoid membranes. However, this strain, growing only in the presence of glucose, was genetically unstable and readily generated suppressor mutations that restore the photoautotrophy. Two suppressor mutations were mapped into the hemA gene encoding glutamyl-tRNA reductase (GluTR) - the first enzyme of tetrapyrrole biosynthesis. Indeed, the GluTR was not detectable in the ΔbtpA mutant and the suppressor mutations restored biosynthesis of tetrapyrroles and photoautotrophy by increased GluTR expression or by improved GluTR stability/processivity. We further demonstrated that GluTR associates with a large BtpA oligomer and that BtpA is required for the stability of GluTR. Our results show that the BtpA protein is involved in the biogenesis of photosystems at the level of regulation of tetrapyrrole biosynthesis.

GC-MS Metabolomic Profiling of Protic Metabolites Following Heptafluorobutyl Chloroformate Mediated Dispersive Liquid Microextraction Sample Preparation Protocol.

A simple analytical workflow is described for gas chromatographic-mass spectrometry (GC-MS)-based metabolomic profiling of protic metabolites, particularly amino-carboxylic species in biological matrices. The sample preparation is carried out directly in aqueous samples and uses simultaneous in situ heptafluorobutyl chloroformate (HFBCF) derivatization and dispersive liquid-liquid microextraction (DLLME), followed by GC-MS analysis in single-ion monitoring (SIM) mode. The protocol involves ten simple pipetting steps and provides quantitative analysis of 132 metabolites by using two internal standards. A comment on each analytical step and explaining notes are provided with particular attention to the GC-MS analysis of 112 physiological metabolites in human urine.