This diploma thesis focuses on the phenotypic effect of 6S RNA in model organism Streptomyces coelicolor. 6S RNA is small non-protein-coding RNA. As it was shown in Escherichia coli, 6S RNA imitates structure of DNA promoter and traps sigma70-RNA polymerase, consequently blocking further transcription. Recently, 6S-like RNA was also discovered in Streptomyces (designated here as 6SSc for clarity), but its effect on their complex life cycle remains unknown. Here, we tested whether 6SSc co-regulates transition from the vegetative growth to the stationary phase. When 6SSc RNA was overexpressed in S. coelicolor, this transition was reflected by limited substrate mycelium growth hyper-sporulation, and an over-production of secondary metabolites, actinorhodin and undecylprodigiosin, whose production was measured spectrophotometrically. Hence, we hypothesize that this RNA regulator may also affect the production of other bioactive substances in Streptomyces, including clinically relevant antibiotics.
Anotace v angličtině
This diploma thesis focuses on the phenotypic effect of 6S RNA in model organism Streptomyces coelicolor. 6S RNA is small non-protein-coding RNA. As it was shown in Escherichia coli, 6S RNA imitates structure of DNA promoter and traps sigma70-RNA polymerase, consequently blocking further transcription. Recently, 6S-like RNA was also discovered in Streptomyces (designated here as 6SSc for clarity), but its effect on their complex life cycle remains unknown. Here, we tested whether 6SSc co-regulates transition from the vegetative growth to the stationary phase. When 6SSc RNA was overexpressed in S. coelicolor, this transition was reflected by limited substrate mycelium growth hyper-sporulation, and an over-production of secondary metabolites, actinorhodin and undecylprodigiosin, whose production was measured spectrophotometrically. Hence, we hypothesize that this RNA regulator may also affect the production of other bioactive substances in Streptomyces, including clinically relevant antibiotics.
This diploma thesis focuses on the phenotypic effect of 6S RNA in model organism Streptomyces coelicolor. 6S RNA is small non-protein-coding RNA. As it was shown in Escherichia coli, 6S RNA imitates structure of DNA promoter and traps sigma70-RNA polymerase, consequently blocking further transcription. Recently, 6S-like RNA was also discovered in Streptomyces (designated here as 6SSc for clarity), but its effect on their complex life cycle remains unknown. Here, we tested whether 6SSc co-regulates transition from the vegetative growth to the stationary phase. When 6SSc RNA was overexpressed in S. coelicolor, this transition was reflected by limited substrate mycelium growth hyper-sporulation, and an over-production of secondary metabolites, actinorhodin and undecylprodigiosin, whose production was measured spectrophotometrically. Hence, we hypothesize that this RNA regulator may also affect the production of other bioactive substances in Streptomyces, including clinically relevant antibiotics.
Anotace v angličtině
This diploma thesis focuses on the phenotypic effect of 6S RNA in model organism Streptomyces coelicolor. 6S RNA is small non-protein-coding RNA. As it was shown in Escherichia coli, 6S RNA imitates structure of DNA promoter and traps sigma70-RNA polymerase, consequently blocking further transcription. Recently, 6S-like RNA was also discovered in Streptomyces (designated here as 6SSc for clarity), but its effect on their complex life cycle remains unknown. Here, we tested whether 6SSc co-regulates transition from the vegetative growth to the stationary phase. When 6SSc RNA was overexpressed in S. coelicolor, this transition was reflected by limited substrate mycelium growth hyper-sporulation, and an over-production of secondary metabolites, actinorhodin and undecylprodigiosin, whose production was measured spectrophotometrically. Hence, we hypothesize that this RNA regulator may also affect the production of other bioactive substances in Streptomyces, including clinically relevant antibiotics.
The aim of this work is to verify whether in model organism Streptomyces coelicolor overproduction of 6S RNA shortens the exponential growth phase and leads to increased production of antibiotics. The experimental part of thesis is going to be done at Laboratory of Bacteriology, Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University trough following steps:
1. Cultivation of mutated strain (C6S) and control strain (C0) in different agar and liquid media
2. Preparation of spore suspension
3. Enumeration of viable spores in suspensions
4. Cultivation of S. coelicolor for isolation of secondary metabolites
5. Isolation of secondary metabolites (actinorhodin and undecylprodigiosin)
6. Spectrophotometric assay of S. coelicolor antibiotics
7. Phenotypic analysis
Zásady pro vypracování
The aim of this work is to verify whether in model organism Streptomyces coelicolor overproduction of 6S RNA shortens the exponential growth phase and leads to increased production of antibiotics. The experimental part of thesis is going to be done at Laboratory of Bacteriology, Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University trough following steps:
1. Cultivation of mutated strain (C6S) and control strain (C0) in different agar and liquid media
2. Preparation of spore suspension
3. Enumeration of viable spores in suspensions
4. Cultivation of S. coelicolor for isolation of secondary metabolites
5. Isolation of secondary metabolites (actinorhodin and undecylprodigiosin)
6. Spectrophotometric assay of S. coelicolor antibiotics
7. Phenotypic analysis
Seznam doporučené literatury
Kieser T., Bibb M. J., Buttner M. J., Chater K. F., Hopwood D. A. (2000): Practical Streptomyces genetics. Norwich: The John Innes Foundation
Mikulík K., Bobek J., Zídková J., Felsberg J. (2014): 6S RNA modulates growth and antibiotic production in Streptomyces coelicolor. Applied microbiology and biotechnology. 98, No. (16), pp. 7185-7197.
Wassarman K.M., Storz G. (2000): 6S RNA regulates E. coli RNA polymerase activity. Cell. 101, pp. 613-623.
Wassarman, K.M. (2007): 6S RNA: a small RNA regulator of transcription. Curr. Opin. Microbiol., 10, No. (2), pp. 164168.
Seznam doporučené literatury
Kieser T., Bibb M. J., Buttner M. J., Chater K. F., Hopwood D. A. (2000): Practical Streptomyces genetics. Norwich: The John Innes Foundation
Mikulík K., Bobek J., Zídková J., Felsberg J. (2014): 6S RNA modulates growth and antibiotic production in Streptomyces coelicolor. Applied microbiology and biotechnology. 98, No. (16), pp. 7185-7197.
Wassarman K.M., Storz G. (2000): 6S RNA regulates E. coli RNA polymerase activity. Cell. 101, pp. 613-623.
Wassarman, K.M. (2007): 6S RNA: a small RNA regulator of transcription. Curr. Opin. Microbiol., 10, No. (2), pp. 164168.