engleski

Phosphoproteome analyses of oxytetracycline- producing Streptomyces rimosus strains

Streptomycetes are filamentous bacteria with a complex and tightly regulated developmental life cycle. These bacteria are best known for their capacity to produce a diverse range of pharmaceutically important antibiotics. Tetracyclines (TC), represent one of the most successful classes of antibiotics. On the global market TC consumption is increasing thus showing the need for the improvement of TC production strains. Among other TC producer strains, the Streptomyces rimosus is renowned for the oxytetracycline (OTC) production. Previous phosphoproteome studies of a model organism, Streptomyces coelicolor suggested that Ser/Thr/Tyr phosphorylation plays a role in regulating various aspects of S. coelicolor complex life style, including antibiotic production. Analysis of the S. rimosus genome predicted 33 eukaryotic-like Ser/Thr protein kinases and 25 eukaryotic type protein phosphatases. Our assumption was that the effect of the OTC overproduction in S. rimosus could be also a consequence of alteration in phosphorylation of the regulatory proteins or some other proteins involved in OTC biosynthesis. Thus, our strategy involved comparative phosphoproteome analysis of the parental strain vs. its derivative, a high OTC producer strain in order to possibly highlight differences of phosphorylation within regulatory networks responsible for elevated antibiotic production. First, evaluation of the culture conditions, mycelia differentiation and OTC production were performed for the parental S. rimosus strain and its derivatives. The strains displayed variation in sporulation, pigment and OTC production on solid media of different compositions. Growth kinetics and OTC production of selected S. rimosus strains were also examined in liquid medium. All strains exhibited growth arrest during early exponential phase of growth. This phase, also known as the “decision phase”, has been recognized as a key regulatory point which prepares mycelia for antibiotic production. However, it has never been described for S. rimosus. By fluorescence microscopy we found that various producer strains also exhibited variations in pellet formation or fragmentation. Furthermore, HPLC and mass spectrometry showed that the best S. rimosus producer accumulated ~ 35 times more OTC in the culture broth at the late stage of growth in comparison to the parental strain. Phosphorylation patterns examined by western blot during bacterial growth exhibited some interesting variations between six examined strains. Finally, proteome/phosphoproteome analyses were performed for two selected strains, S. rimosus parental strain and its derivative, a high OTC producer strain. Although this project is still ongoing, obtained results provide the first insight into the dynamics of protein synthesis and phosphorylation events of S. rimosus, which is industrially very important bacterium.

Phosphoproteome ; Serine ; Threonine and Tyrosine phosphorylation : Bacteria

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