October 2017, № 9 (209), pages 91–95

doi: 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.

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About this article

Authors: Sidorov R.Yu., Tkachenko A.G.

Year: 2017

doi: 10.25198/1814-6457-209-91

Editor-in-chief

Sergey Aleksandrovich
MIROSHNIKOV

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