Applications of Mass Spectrometry in the Studies Concerning bldA Regulation of Natural Product Biosynthesis in Streptomyces sp.
The Streptomyces are a genus of soil-dwelling Gram-positive bacteria that produce a variety of secondary metabolites; they have even been dubbed as ‘antibiotic factories’ as a typical genome can contain as many as 30 clusters of genes responsible for biosynthesis of metabolites. Yet many of these compounds are not observed in laboratory cultures suggesting the products are locked by regulatory signals. One unique regulatory mechanism present in these organisms, the bldA gene, depends on the translation TTA codons within the genome. These codons are quite rare but are frequently found in other regulatory regions and in regions responsible for the biosynthesis of natural products. This thesis applies mass spectrometry to explore changes in the whole proteome of Streptomyces calvus in response to the bldA gene. S. calvus pTESa-bldA can undergo morphological differentiation and produces different secondary metabolites than the native strain. Hypothesized to be due to the action of a TTA codon containing pleotropic regulator AdpA, we observed that AdpA is expressed in the wild type at the protein level. Additionally, there was an increase in expression of other regulatory systems in reaction to bldA including the two-component system regulators controlling the phosphate management system and MtrAB system known to control cell division. Both systems have been previously demonstrated to have an impact on secondary metabolism and morphological differentiation. Furthermore, a combination of bioinformatics and high-resolution mass spectrometry were used to identify and guide purification of two natural products from Streptomyces curacoi in response to bldA. A non-ribosomal peptide, curacomycin, and a ribosomally synthesized and post-translationally modified peptide, curacozole, both were isolated and characterized from S. curacoi pTESa-bldA. Curacozole was produced with a high titre, 42 mg/L, that allowed for extensive antibacterial testing where it was found to be highly active against antibiotic resistant Gram-positive bacteria. Lastly, custom electrospray emitters were fabricated with significant signal enhancement to aid mass spectrometric investigations of these biological systems.
URI for this recordhttp://hdl.handle.net/1974/26512
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