Биосенсоры для исследования активности промоторов и шаперонов в клетках Bacillus subtilis

Abstract

Plasmids to determine the activities of promoters in Bacillus subtilis cells have been constructed. Plasmids contain constitutive (PfbaA, PymdA_mut3, PymdA) and inducible (PxylA, PxylA-cre) promoters of different activities and genes under their control encoding reporter proteins with different thermostability and structure. The following main parameters were measured: maximal response, threshold concentration, minimal response time and the effect of catabolite repression for the inducible promoters. The activities of the constitutive promoters were compared. It was shown that the activity of the inducible PxylA promoter at a D(+)-xylose concentration of 1% was more than 100-fold higher and that of the PxylA-cre promoter was about 80-fold higher than the control level after 2 h of incubation. However, PxylA-cre is tightly closed and sensitive to glucose repression. The constructed lux sensors containing bacterial luciferases as reporter proteins were used to assess the influence of DnaKJE and trigger factor (TF) molecular chaperones on the level of synthesis of active enzymes. It was shown that the absence of both of the above chaperones in B. subtilis cells led to a significant decrease in the synthesis of the native thermolabile Photobacterium leiognathi luciferase, while the lack of TF increased the activity of the thermostable Photorhabdus luminescens luciferase by about 2 times as compared to wild type Bacillus subtilus 168. Erroneous translation under the streptomycin action and in the absence of both DnaKJE and TF chaperones significantly suppressed the synthesis of both luciferases. Bacillus subtilis, bacterial luciferase, biosensor, bioluminescence, promoter, chaperone, Trigger Factor, DnaKJ The work of I. Manukhov was supported by the Russian Science Foundation under the grant 20-16-00088. E. Gnuchikh's research on the design of lux-biosensors was partially funded by the Russian Foundation for Basic Research (project No. 20-34-70132).