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October 2017, № 9 (209), pages 91–95doi: 10.25198/1814-6457-209-91
Sidorov R.Yu., Tkachenko A.G. INFLUENCE OF EXPRESSION OF REL GENE FROM MYCOBACTERIUM SMEGMATIS ON RPOS-LACZ-FUSION ACTIVITY IN ESCHERICHIA COLIGuanosine tetraphosphate (ppGpp) is an important regulatory bacterial molecule involved in stringent response, regulation of persistence, stress adaptation, pathogenicity. The Rel protein from Mycobacterium smegmatis is responsible for synthesis and hydrolysis of ppGpp. The study is devoted to intracellular level detection of guanosine tetraphosphate via gene fusion of ppGpp-dependent rpoS gene and reporter gene lacZ in strain harbouring expression plasmid pMind::rel. The method of detection allows to quantify low concentrations of guanosine tetraphosphate, which is difficult to achieve when using analytical chemistry methods. At the moment gene fusion method is poorly developed for mycobacteria, so there is utility for finding approaches on E. coli. The mycobacterial expression plasmid pMind with a rel gene insert from Mycobacterium smegmatis have been constructed in following study. The resulting plasmid and precursory vector have been transformed into rpoS-lacZ-fusion strain. The cultures have been compared by measuring β-galactosidase activity. An increase in the expression of rpoS was observed in E. coli strain carrying the plasmid pMind with rel insert in comparison with the control strain containing plasmid without the insert. An addition of tetracycline, inducer of pMind expression, did not affect the expression of rpoS. Based on the data obtained, we can conclude that there is expression from tetA promoter in the pMind expression plasmid and keeping of mycobacterial Rel enzyme activity in Escherichia coli. The absence of tetracycline induction may result from the inability of the TetR repressor from pMind bind to regulatory sites in E. coli.Key words: guanosine tetraphosphate, persistence, expression plasmid pMind.
References:
1. Miller J. Experiments in molecular genetic /transl. from English by U.N. Zograf, T.S. Iljina, V.G. Nikiforov edited by S.I. Alichanjan — Moskow: Mir. — 1976. — 436 p.
2. Tkachenko A.G. Molecular mechanisms of stress responses of microorganisms/A.G. Tkachenko, — Ekaterinburg: Ural branch of the Russian Academy of Sciences. — 2012. — p. 107.
3. Tetracycline-inducible gene regulation in mycobacteria / M.C. Blokpoel [et al] // Nucleic Acids Res. — 2005. doi: 10.1093/nar/gni023
4. The relA Homolog of Mycobacterium smegmatis Affects Cell Appearance, Viability, and Gene Expression / J. Dahl [et al.] // J. of Bacteriol. — 2005. — Vol.187. — No7. — P. 2439-2447.
5. Transcripion profiling of the stringent response in Escherichia coli / T. Durfee [et al.] // J. Bacteriol. — 2007. — P.1084-1096.
6. Recent functional insights into the role of (p)ppGpp in bacterial physiology / V.Hauryliuk [et al] // Nature Reviews Microbiology. — 2015. — P.1-12. doi:10.1038/nrmicro3448
7. Maisonneuve, E. (p)ppGpp Controls Bacterial Persistence by Stochastic Induction of Toxin-Antitoxin Activity / E. Maisonneuve, M. Castro-Camargo, K.M. Gerdes // Cell. — 2013. — Vol. 154. — P.1140-1150.
8. Maniatis, T. Molecular Cloning: A Laboratory Manual / T. Maniatis, M.R. Green, J.Sambrook // Cold Spring Harbor Laboratory Press. — Изд. 4-е. — P.32-34, 84-87, 119-122.
9. Simons, R.W. Improved single and multicopy lac-based cloning vectors for protein and operon fusions / R.W. Simons, F. Houman, N. Kleckner // Gene. — 1987. — Vol. 53. — No 1. — P.85-96.
10. Weiss, L.A. Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp / L.A. Weiss, C.L. Stallings // J. of Bacteriol. — 2013. — Vol.195. — No. 24. — P.5629-5638.
11. BioCyc: Pathway/Genome Database Collection. Available at: https://biocyc.org.
About this article
Authors: Sidorov R.Yu., Tkachenko A.G.
Year: 2017
doi: 10.25198/1814-6457-209-91
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Editor-in-chief |
Sergey Aleksandrovich MIROSHNIKOV |
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