Comprehensive analytical method for the analysis of Coenzyme A activated Compounds in Actinomycetes.

CABRUJA, MATÍAS; GRAMAJO, HUGO AND GAGO, GABRIELA.

Congreso; IX Congreso de Microbiologia General; 2013

Acyl-CoAs are crucial compounds involved in essential metabolic pathways such as the Krebs cycle, lipid, carbohydrate, and amino acid metabolisms. Our group is actively studying lipid metabolism in Actinomycetes bacteria, especially the regulation of lipid homeostasis in Mycobacterium and Streptomyces. We found that acyl-CoAs are key signal molecules involved in the transcriptional regulation of lipid biosynthesis in these organisms. Therefore, the development of a method to determine the whole acyl CoA profile (short-, medium- and long- chain length) in these bacteria could be a useful tool for the analysis of variations in the acyl-CoA content in the different lipid related mutant constructed in our laboratory. Since acyl-CoAs represent a heterogeneous class of compounds, our aim was to develop a comprehensive, robust, and reproducible? analytical method for the determination of the acyl-CoA content in Actinomycetes. In this study we used an on-line HPLC?ESI-MS/MS method, based on the separation of the compounds by reversed-phase chromatography in a C18 column using triethylamine acetate and acetonitrile gradient followed by the detection by mass spectrometer consisting of an electro nanospray ionization and analysis of the time of flying. The high selectivity of mass spectrometry makes it a powerful tool for acyl-CoA determination from biological samples. The assay includes an extraction step, specifically adapted for the Mycobacterium lipid rich cell envelop. With this new analytical tool it will be possible to have new insights on lipid metabolism by following the fluctuations of the acyl CoA pools within the cells.