A.S. Nagel1, I.S. Kulbaev2, Zh.I. Andreeva-Kovalevskaya3, A.V. Siunov4, A.P. Karatovskaya5, N.V. Rudenko6, A.S. Solonin7

1–4, 7 FRC Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences, G.K. Scriabin Institute of Biophysical and Physiological Microbiology of the Russian Academy of Sciences (Pushchino, Russia)

5, 6 Branch of the Institute of Bioorganic Chemistry
named after Academicians M.M. Shemyakina and Yu.A. Ovchinnikov, RAS (Pushchino, Russia)

1 anagell@mail.ru, 2 islam.kulbaev@bk.ru, 3 hemolysin6@gmail.com, 4 av_siunov@rambler.ru, 5 annakaratovskaya@mail.ru, 6 nrudkova@mail.ru, 7 solonin@ibpm.pushchino.ru

One of the main problems of molecular biology is the study of DNA-protein recognition. Many negative transcriptional regulators are able to change the efficiency of recognition of the operator region depending on the bond with the induction factor. Detection and description of the regulatory protein complex containing the inducer allows us to determine the nature of the ligand. The necessity to determine the nature of small molecules involved in the transcriptional regulation of many biochemical processes occurring in a bacterial cell requires the accumulation of a complex of transcriptional regulators with an inducer. Currently, the common method for deciphering the nature of an inducer is X-ray structural analysis or gas chromatography followed by mass spectrometric analysis. Both of them require using high concentrations of a purified protein complex with an inducer. Identification of significant zones in the 3D structure of a protein containing small molecules will allow us to determine their nature. The study of the subtle mechanisms of regulation of the synthesis of proteins involved in the development of an infectious process will allow us to develop approaches to its suppression. Previously, the possibility of inducing expression of the Bacillus cereus hemolysin II gene (hlyII) depending on the composition of the culture medium was demonstrated. The presence of blood plasma in the culture medium provides an increase in the expression level of the hlyII gene by tens of times. The main negative regulator of the hlyII transcription is the HlyIIR protein, the 3D structure of which contains a site for binding potential inducer, the presence of which can lead to a change in the regulator conformation. It is assumed that conformational changes in the regulator lead to a decrease in the level of binding to the operator region. The discovered HlyIIR region is theoretically capable of binding to steroids of blood plasma. The system for expression of transcription regulators is proposed, in which the regulator gene is under the control of the promoter-operator region of the controlled gene. Using of this system for the expression of HlyIIR in a ligand-bound state under the control of the promoter of the Bacillus cereus hlyII gene controlled by it is demonstrated. Further development of the system allows developing a technology for isolation complex of the HlyIIR regulator associated with an inducer.

Nagel A.S., Kulbaev I.S., Andreeva-Kovalevskaya Zh.I., Siunov A.V., Karatovskaya A.P., Rudenko N.V., Solonin A.S. System for expression of transcriptional regulators in the ligand-bound state. Technologies of Living Systems. 2025. V. 22. № 4. Р. 76-85. DOI: https://doi.org/10.18127/j20700997-202504-09 (In Russian).

1. Browning D.F., Busby S.J.W. Local and global regulation of transcription initiation in bacteria. Nat Rev Microbiol. 2016. V. 14. № 10. P. 638–650.
2. Balleza E., López-Bojorquez L.N., Martínez-Antonio A. et al. Regulation by transcription factors in bacteria: beyond description. FEMS Microbiol Rev. 2009. V. 33. № 1. P. 133–151.
3. Lutz R., Bujard H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997. V. 25. № 6. P. 1203–1210.
4. Ulrich L.E., Koonin E.V., Zhulin I.B. One-component systems dominate signal transduction in prokaryotes. Trends Microbiol. 2005. V. 13. № 2. P. 52–56.
5. Hinrichs W., Kisker C., Düvel M. et al. Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance.
6. Science. 1994. V. 264. № 5157. P. 418–420.
7. Cuthbertson L., Nodwell J.R. The TetR family of regulators. Microbiol. Mol. Biol. Rev. 2013. V. 77. № 3. P. 440–475.
8. Orth P., Schnappinger D., Hillen W. et al. Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nat. Struct. Biol. 2000. V. 7. № 3. P. 215–219.
9. Aleksandrov A., Schuldt L., Hinrichs W., Simonson T. Tet repressor induction by tetracycline: a molecular dynamic, continuum electrostatics, and crystallographic study. J. Mol. Biol. 2008. V. 378. № 4. P. 898–912.
10. Baida G., Budarina Z.I., Kuzmin N.P., Solonin A.S. Complete nucleotide sequence and molecular characterization of hemolysin II gene from Bacillus cereus. FEMS Microbiol. Lett. 1999. V. 180. № 1. P. 7–14.
11. Budarina Z.I., Nikitin D.V., Zenkin N. et al. A new Bacillus cereus DNA-binding protein, HlyIIR, negatively regulates expression of B. cereus haemolysin II. Microbiology (Reading, Engl.). 2004. V. 150. № 11. P. 3691–3701.
12. Rodikova E.A., Kovalevskiy O.V., Mayorov S.G. et al. Two HlyIIR dimers bind to a long perfect inverted repeat in the operator of the hemolysin II gene from Bacillus cereus. FEBS Lett. 2007. V. 581. № 6. P. 1190–1196.
13. Ojanotko-Harri A., Nikkari T., Harri M.P., Paunio K.U. Metabolism of progesterone and testosterone by Bacillus cereus strain Socransky 67 and Streptococcus mutans strain Ingbritt. Oral Microbiol. Immunol. 1990. V. 5. № 4. P. 237–239.
14. Kovalevskiy O.V., Lebedev A.A., Surin A.K. et al. Crystal structure of Bacillus cereus HlyIIR, a transcriptional regulator of the gene for pore-forming toxin hemolysin II. J. Mol. Biol. 2007. V. 365. № 3. P. 825–834.
15. Andreeva-Kovalevskaya Zh., Siunov A., Budarina Zh. i dr. Zavisimost' effektivnosti transkripcii gena gemolizina II Bacillus cereus ot istochnika plazmy krovi. Vestnik biotekhnologii i fiziko-himicheskoj biologii im. Yu.A. Ovchinnikova. Informacionno-analiticheskij centr mediko-social'nyh problem. 2017. T. 13. № 1. S. 5–12. (in Russian).
16. Werten S., Waack P., Palm G.J. et al. Crystal structures of free and ligand-bound forms of the TetR/AcrR-like regulator SCO3201 from Streptomyces coelicolor suggest a novel allosteric mechanism. FEBS J. 2023. V. 290. № 2. P. 521–532.
17. Slamti L., Lereclus D. Molecular docking and molecular dynamic simulations of apoptosis proteins with potential anticancer compounds present in Clinacanthus nutans extract using gas chromatography-mass spectrometry. J. Biomol. Struct. Dyn. 2023. V. 41. № 13. P. 6104–6120.
18. Nagel' A.S., Andreeva-Kovalevskaya ZH.I., Siunov A.V., Solonin A.S. CHelnochnaya vektornaya plazmida s turbogfp v kachestve reporternogo gena dlya analiza promotornoj aktivnosti v kletkah E. coli i B. subtilis. Tekhnologii zhivyh sistem. 2020. T. 17. № 4. S. 42–50. (in Russian).
19. Rudenko N.V., Karatovskaya A.P., Zamyatina A.V. i dr. S-terminal'nyj domen gemoliticheskogo toksina ii bacillus cereus sposoben vzaimodejstvovat' s eritrocitami. Bioorganicheskaya himiya. 2020. T. 46. № 3. S. 280–285. DOI: 10.1134/S106816202003018819 (in Russian).
20. Sineva E., Shadrin A., Rodikova E.A. et al. Iron Regulates Expression of Bacillus cereus Hemolysin II via Global Regulator Fur. J. Bacteriol. 2012. V. 194. № 13. P. 3327–3335.
21. Rouches M.V., Xu Y., Cortes L.B.G., Lambert G. A plasmid system with tunable copy number. Nat. Commun. 2022. V. 13. № 1. P. 3908.
22. Park G., Yang J., Seo S.W. Dynamic control of the plasmid copy number maintained without antibiotics in Escherichia coli. J. Biol. Eng. 2024. V. 18. № 1. P. 71.