Sidorov R.Yu., Tkachenko A.G.
INFLUENCE OF EXPRESSION OF REL GENE FROM MYCOBACTERIUM SMEGMATIS ON RPOS-LACZ-FUSION ACTIVITY IN ESCHERICHIA COLI ^{[№ 9 ' 2017]}
Guanosine 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.

Khaova E.A., Kashevarova N.M., Tkachenko A.G
REGULATION OF BACTERIAL PERSISTENCE BY POLYAMINES THROUGH THE STIMULATION OF EXPRESSION OF RPOS, YQJD AND RMF GENES ^{[№ 9 ' 2017]}
Persisters are rare dormant variants of regular cells and are highly tolerant to antibiotics. Persisters are able to resume growth. These observations suggest that persister cells may be the main culprit responsible for the recalcitrance of chronic infectious disease to antimicrobial therapy. General stress responses and polyamines are described to responsible for the formation of persister cells.
Showed that polyamines putrescine, spermidine and cadaverine upregulate the formation of Escherichia coli persister cells in a dynamic fashion through the stimulation of expression of rpoS, yqjD and rmf genes, relating to general stress responses. Results showed that rpoS gene is the most upregulated by spermidine. The expression of yqjD gene is slightly dependent on polyamines. We obtained that the expression of yqjD is dependent on rpoS. These results are in agreement with previously published data. So, although yqjD is not referred to as a “polyamine modulon”, polyamines still can upregulate its expression indirectly through positive effects on rpoS expression. The persister cells frequency of knockout strains decreased versus their parent during the time periods of stationary phase that were specific for each gene knockout mutant. The persister cell frequency of ΔrpoS strain exhibited maximal decrease versus parental strain at early stationary phase. Therefore, the persister formation at early stationary phase is primarily dependent on rpoS gene. The persister cell frequencies of rmf and yqjD mutants decreased nearer to the lowest values versus parent at late stationary phase. These results show that rmf and yqjD genes can significantly contribute to the persister formation at late stationary phase. These data are confirmed by results, obtained with strains harboring double and triple knockout mutations.
Therefore, rpoS gene is the most upregulated by spermidine and is involved in the persister cell formation predominating at early stationary phase. rmf gene, also relating to polyamine modulon, and yqjD gene, whose expression is slightly dependent on polyamines, are involved in the persister cell formation predominating at late stationary phase.

Kanygina O. N, Kravtzova O.S., Anisina I.N., Chetvertikova A.G., Salnikova E.V., Dostova T.M.,Tkachenko A.A.
DISPERSIVE ANALYSIS MONTMORILLONITE-BEARING OF CLAY OF ORENBURZHYE ^{[№ 12 ' 2011]}
The dispersive analysis montmorillonite-bearing clay of Orenburzhye, based on screen fractionation, sedimentation and optical microscopy is carried out. It is established that division of investigated clay on two fractions is expedient. From low-dispersed fractions by optimization of modes of synthesis probably reception of microporous ceramics of high density and durability.