Bacteria have the ability to alter their morphology in order to adapt to changing environments. We have investigated the role of the cell envelope in the development and stress-adaptation... Show moreBacteria have the ability to alter their morphology in order to adapt to changing environments. We have investigated the role of the cell envelope in the development and stress-adaptation strategies of Actinobacteria. Here, we demonstrate how cryo-electron microscopy techniques can be used as efficient tools to study cell envelope of various bacteria. An in-depth study on the Streptomyces cell wall with cryo-electron tomography reveals the structural complexity of the Gram-positive cell wall in apically growing bacteria. Additionally, we report the formation of intracellular membrane accumulations in Streptomyces as a result of exposure to a stress-inducing agent. Furthermore, we studied the ability of the filamentous actinomycete Kitasatospora viridifaciens to extrude wall-deficient cells or S-cells upon exposure to hyperosmotic stress. We characterized the structural alterations associated with S-cell formation using cryo-electron microscopy and reveal that S-cell formation requires cytoskeletal protein FilP in K. viridifaciens. To summarize, this thesis provides new insights in the structural complexity and stress-induced alterations of the bacterial cell envelope. Show less
The explosive increase in infections by pathogens is a major problem in the clinic today. The theme of this thesis was to find novel antibiotics from actinomycetes. Next-generation... Show more The explosive increase in infections by pathogens is a major problem in the clinic today. The theme of this thesis was to find novel antibiotics from actinomycetes. Next-generation sequencing revealed that the biosynthetic potential of actinomycetes had been grossly underestimated. In this thesis, different antibiotics-eliciting strategies, including microbial cocultivation, streptomycin-resistant mutation, overexpression of pathway-specific activator, variation of culture conditions, were utilized to enforce fluctuations in the production of bioactive compounds in actinomycetes, after which, NMR-based metabolic profiling was used to facilitate uncovering those elicited molecules. This pipeline allowed the discovery of new antibiotics involving various chemical skeletons, such as 7-prenylisatin, methoxylated isocoumarins, endophenazines, and C-glycosylpyranonaphthoquinones. On the other hand, genome-mining methodology enabled the discovery of a group of endophenasides and leucanicidin in Kitasatospora sp. MBT66, whereby the rhamnosylation of both scaffold are executed by a same promiscuous glycosyltransferase. Last but not least, a novel antibiotic termed lugdunomycin with unprecedented chemical scaffold, as well as a number of new angucycline-type antibiotics, were characterized from Streptomyces sp. QL37. The biosynthetic pathway of lugdunomycin was deciphered by genetic knockout and OSMAC (One Strain MAny Compound) strategy. In summary, this thesis explores an interface of genomics and metabolomics to accelerate new antibiotics discovery. Show less
Actinomycetes produce 70% of all known antibiotics, most of which are produced by members of the genus Streptomyces. Furthermore, streptomycetes produce a plethora of other medically relevant... Show moreActinomycetes produce 70% of all known antibiotics, most of which are produced by members of the genus Streptomyces. Furthermore, streptomycetes produce a plethora of other medically relevant natural products as well as industrial enzymes. Streptomyces is a multicellular mycelial organism, and has a complex life cycle involving sporulation. Cell division in Streptomyces is very different from bacteria with planktonic growth, such as the model organisms E. coli and B. subtilis. Understanding the way cell division is controlled in Streptomyces is not only important for fundamental understanding of cell division in multicellular microorganisms, but may be applied to manipulate its morphology in liquid cultures, for more efficient industrial fermentation. Several novel cell division-related proteins were studied in this research, providing new functional insights into their function during growth and development and their localization in time and space in the Streptomyces mycelium. Cell division proteins do not function alone, but are members of a complex interaction network, which work in a disciplined team. The work described in this PhD thesis shows that streptomycetes have different control systems for cell division, which are complementary to each other to ensure cell division occurs at the right place and at the right time. Show less
This thesis may be regarded as a concept work, to see how feasible drug discovery approaches still are. For this, a strain collection was built up consisting of actinomycetes from soil in the... Show moreThis thesis may be regarded as a concept work, to see how feasible drug discovery approaches still are. For this, a strain collection was built up consisting of actinomycetes from soil in the Qinling and Himalaya mountains, which were subsequently tested for antibiotic production against multi-drug resistant clinical isolates. This resulted in close to 100 strains that showed strong antimicrobial activity, which were then analyzed in more detail. Two of the strains were subjected to extensive NMR-based metabolomics assisted by mass spectrometry, and several known and also novel antimicrobial compounds were elucidated. Finally, we also focused on the antibiotic activity of volatile organic compounds (VOCs) Show less
