Actinobacteria are Gram-positive bacteria that have a complex multicellular life cycle and are well known for their ability to produce a wide range of bioactive natural products (NPs). High... Show moreActinobacteria are Gram-positive bacteria that have a complex multicellular life cycle and are well known for their ability to produce a wide range of bioactive natural products (NPs). High throughput screening has failed to deliver the new antibiotics we so desperately need to combat multidrug-resistant pathogens. Therefore, new systematic approaches are needed to further explore the rich potential of Actinobacteria. The work described in this thesis entails systems biology approaches consisting of technologies such as proteomics, genomics, metabolomics and DNA binding studies. These were then applied to identify the biosynthetic gene clusters (BGCs) that are responsible for the production of novel antibiotics. Small molecules were thereby used as elicitors to activate the expression of cryptic BGCs in Streptomyces roseifaciens. Furthermore, S. coelicolor M1152 that was optimized for heterologous expression of antibiotics, was analysed for changes in protein expression, to understand which changes correlate to optimal antibiotic production. Finally, the role of the nucleoid associated protein SCO1839 in development and antibiotic production was studied. Chip-seq technology showed that it binds to thousands of DNA sequences on the S. coelicolor chromosome, which contain the motif GATC. I hope that this thesis contributes to utilizing multi-dimensional ‘omics approaches to answer major biological questions. Show less
A general overview of regulation of secondary metabolism in Pseudomonas species is given in Chapter 1. Several approaches were combined to identify novel genes involved in the regulation of PCN... Show moreA general overview of regulation of secondary metabolism in Pseudomonas species is given in Chapter 1. Several approaches were combined to identify novel genes involved in the regulation of PCN synthesis and to study their interactions with other regulators. Site-directed mutagenesis was used to test the hypothesis that rpoS is a regulatory gene of PCN synthesis (Chapter 2). To discover additional genes in the regulatory cascade, which already contains psrA and rpoS, a random DNA-fragment microarray of the PCL1391 genome was constructed and used for transcriptomics of the psrA and rpoS mutants (Chapter 3). A random mutagenesis approach resulted in the identification of pip, a novel gene that stimulates PCN production in PCL1391 (Chapter 4). Analyses on the role of Pip as a switch of PCN production depending on environmental conditions are described in Chapter 5. The results described in this thesis are summarized in Chapter 6, where in addition the regulatory network of PCN synthesis in P. chlororaphis PCL1391 is compared to regulatory networks of secondary metabolism in other Pseudomonas species. Show less
