Isolation of Myxobacteria
Myxobacteria, as well as Chitinophaga, Actinobacteria, and Cytophaga, have long been known as reliable producers of bioactive secondary metabolites. The isolation of these bacteria from new environmental samples, collected in consideration of the Convention on Biological Diversity (CBD), and the subsequent screen for bioactive secondary metabolite production are in the focus of our research activities since more than 30 years. Our internal strain collection (see also working group “Microbial strain collection”) comprises, for example, more than 8000 myxobacterial isolates. Based on our permanent isolation activities the number of new isolates increases continuously. Within this procedure, the detection of new species, genera or even families, is not uncommon. The isolation of known, but in particular new groups is extraordinary important for us, because in general new species produce new secondary metabolite(s) – especially for myxobacteria this unwritten law could be confirmed in recent years. But also in well studied genera like Myxococcus new metabolites can be detected.
Two groups of Myxobacteria
Myxobacteria can be divided in two groups due to their nutritional-physiological properties: On the one hand there are the cellulose-decomposers like Sorangium or Byssovorax. At the other hand, and this is the majority, there are the non-cellulolytic but predatorily myxobacteria which are able to lyse other living bacteria by the secretion of special exo-enzymes. They utilize other bacteria as food source. Both groups show distinct swarming behaviour as well as fruiting body development.
For the isolation of myxobacteria we take advantage of their capability to degrade and metabolize cellulose or living microorganism. For cellulose-decomposers we use sterile filter paper on an agar plate, supplemented with a small amount of soil or other interesting substrates. For the bacteriolytic myxobacteria we offer living Escherichia coli on a water agar plate as food source. Swarms or even fruiting bodies of the myxobacteria can be detected after a few days of incubation at 30 °C. Here, on the E. coli plates the myxobacteria swarm over these food organisms, lyse them and produce their species specific fruiting bodies in case of nutrient limitation conditions. Fruiting bodies or material from the swarm edge are transferred to a new agar plate. This procedure is continued till the pure culture is obtained.
Interesting producer of new natural products or not?
The new isolated strains, as well as strains from the strain collection, are screened due to their microbial activity and also for other, non-active but new, metabolites. Therefore the bacteria are grown in different culture media in the presence of XAD-adsorber resin. The resin adsorbs the secondary metabolites secreted in the media. Solvent extraction of the XAD leads to crude extract which is tested in a standard procedure against different Gram-positive - and Gram-negative bacteria, yeasts, filamentous fungi and a eukaryotic cell line. The yield of the secondary metabolites is often quite low and needs to be optimized if the substance is classified as “interesting”. After optimization, the volume is up-scaled to 10, 70, 150 or even 300 liters. Then, the new substance is isolated and characterized by the chemists in the group.
Finally, the pure substance is tested against an enhanced panel of test organisms to get a broad spectrum of activity and the minimal inhibitory concentration (MIC). Additionally, new substances are forwarded to our numerous co-operation partners who have further test-systems with, for some cases, have to be conducted under high security levels (malaria, HIV...). If there is a good bioactivity, the next step is the elucidation of the mode of action. Information about the target can be used for selected modifications by derivatization. The colleagues from the medical chemistry then try to enhance the effect and selectivity.
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