Biodiversity Of Sorangium Cellulosum And Their Intraspecific Competition And Collaboration

Myxobacteria are Gram-negative bacteria that produce diverse and novel secondary metabolites that have anti-cancer, anti-bacterial, anti-fungal, and biologically immunoregulatory activities. As previously reported, approximately 100 basic structures and 600 structural variants have been discovered in myxobacteria, which account for about 3.5% of the known secondary metabolites produced by these microbes. Myxobacteria are unique among prokaryotes for their complicated multicellular behavior, especially the morphogenesis of fruiting bodies, and therefore are considered higher bacteria. Myxobacteria have become an important tool for studying competition and cooperation between bacterial strains because of their complicated social behavior. Work performed by Velicer and others has revealed the complicated interactions between Myxococcus xanthus lineages.Sorangium cellulosum is a species of cellulolytic myxobacteria with an excellent capacity of biological synthesis. The novel bioactive metabolic compounds isolated from Sorangium cellulosum strains account for up to 48.4% of the total discoveries from myxobacteria, some of which are promising as pharmaceuticals. For instance, epothilones, a type of 16-membered macrolide, have received attention because of their bioactivity on cancer cells by stabilizing microtubules, mimicking the mechanism of Taxol. In addition, epothilones also inhibit lower fungi. Only about 2% of Sorangium strains produce epothilones, but almost all strains display a broad and strong repression of fungi and cancer cells.More than 180 Sorangium cellulosum strains were isolated from 19 soil samples collected from different regions in China. These strains are diverse not only in the color and constriction of their fruiting bodies but also in the form of their swarms. They also have significant differences in their 16S rDNA and the synthesis of epothilones. Thus, S. cellulosum strains possess a huge degree of biological diversity.The specificity and biological diversity of various Sorangium cellulosum lineages present some interesting issues. How do different Sorangium strains from the same habitat interact? Is the secondary metabolism differentiation of lineages useful for the survival of the population? Does the phenomenon of complementation of metabolism character occur? What significance do these kinds of mechanisms of competition and cooperation have for the survival of Sorangium in the natural environment? We selected 6 Sorangium isolates from soil samples 0157 and 0003 (serial number in our lab) to study their competition and cooperation. There was remarkable growth inhibition between the isolates from the same and different habitats. Morphological effects were more diverse. At the level of secondary metabolism,83.3% of the pairwise mixed cultures changed the total yield of epothilones significantly; in 72%, the total yield of epothilones increased. The maximum increment was 41.5 times greater than that of the control. Bioactive assay results showed that the secondary metabolites synthesized by mixed cultures had a stronger inhibitory effect on Rhizopus. Further analysis was performed on two pairs. The unit yield represented a higher increment, and transcription of the epothilone biosynthetic gene cluster was analyzed. These results were exciting, especially considering the use of epothilone producers in industrial production, and they led us to further explore the survival of Sorangium in the natural environment.Based on the above work, we found that the production ratio of epothilones A and B comprised two groups:strains that only produced epothilone A (named epoB-strains) and strains that produced an approximately 2:1 ratio of A to B (named epoB+ strains). Sequencing and bioinformatic analysis of ATmodC2 revealed that four amino acid residues in the enzyme active center were altered in parallel to the shift in production ratio. Accordingly, site-directed mutagenesis was performed on the epoB+ ATModC2 gene at these four amino acids to change Val90, Ala91, Ala95, and Seri96 to Leu90, Val91, Ile95, and Phe196 (the corresponding residues in epoB- strains), respectively. These two genes were heterogeneously expressed in Escherichia coli, yielding inclusion body proteins. The ability of mutant ATModC2 to bind methylmalony-CoA was reduced nearly 80% in comparison with wide ATModC2·Epothiloneoside A, one of the new compounds identified, is the first glycosylated epothilone reported in the world. It is synthesized by glycosylation of epothilone A at the C3 site. It is a major component of glycosylated epothilones in Sorangium cellulosum strain So0157-2. The production and glycosylation ratios of epothiloneoside A in both solid and liquid culture conditions with various pH values and carbon sources were studied. The results revealed that glycosylation occurred whenever epothilones were produced, regardless of the changes in pH values, production time curves, or different carbon sources. We suggest that glycosylation is a stable process, paralleling the biosynthesis of epothilones in the So0157-2 strain.In addition to epothiloneoside A, many other novel epothilones were discovered. LF-8, LF-11 and epo-1 are the first 14-and 18-membered epothilones to be found in So0157-2. In this work, we analyzed the structures of these macrolides and conjectured regarding the existence of potential novel epothilones using MS analysis. These conclusions provide some guidance for the future discovery of more novel epothilones.