Metabolism fingerprinting of the antibiotic- producing bacterium Streptomyces coelicolor

Abstract

The aim of this master thesis project was to do a metabolism fingerprinting of streptomyces coelicolor strain A3(2)M145 growing in a fermentation vessel on a phosphate dependent medium. Three biological replicates were fermented and samples taken every three hours from 22 to 34 hours after inoculation and every four hours from 34 hours to 50.5 hours after inoculation.

Each sampling consisted of 3* 5ml samples from each biological replicate; these were washed and extracted for intracellular metabolic samples strictly following the already established protocols used by the Trondheim Partners on European SysMo-reseach project. The concentration of the sample was done using Freeze drying equipment before the samples were derivatized using Trimethylsylyl (TMS) derivatization. Gas Chromatography-Mass Spectrometry (GC-MS) instrument was used for sample analysis. GC_MS generated extract chromatograms were analysed using chemstation Qedit, AMDIS, and a revised version of Fiehn TMS metabolites library for identification and quantification of the compounds in sample extracts.

The results revealed the presence of TMS based metabolite pool in streptomyces coelicolor A3 (2) with varying concentration levels from one sampling point to another. Further, it was revealed that these metabolites form five different clusters some with high concentration early in sampling time interval and lower late. Some show almost constant concentration levels throughout the experiment and some others have low concentration levels early in sampling interval and high in the middle of the interval or late in the sampling time interval. The major metabolic switch takes place between 34 and35 hours after inoculation and coincides with phosphate depletion from the medium. The level of accumulated antibiotics was measured. These compounds accumulation coincided with the need of the cells to cope with depleted phosphate in the medium.