Biosensors to Assess the Activity of Promoters and Chaperones in Bacillus subtilis Cells

Plasmids have been constructed to determine the activities of promoters in Bacillus subtilis cells. The plasmids contain constitutive (PfbaA, PymdA_mut3, PymdA) and inducible (PxylA, PxylA-cre) promoters with different activities and genes under their control that encode reporter proteins with different thermostabilities and structures. The following main parameters of the biosensors 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 amplitude of the response of the inducible PxylA promoter to a D (+)-xylose concentration of 1% was more than 100 times higher and that of the PxylA-cre promoter was about 80 times 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, which contain bacterial luciferases as reporter proteins, were used to assess the influence of DnaKJE and trigger-factor (TF) molecular chaperones on the synthesis level of active enzymes. It was shown that the absence of the DnaKJE and TF chaperones in B. subtilis cells led to a significant decrease in the synthesis of the native thermolabile Photobacterium leiognathi luciferase, while the absence of TF alone increased the activity of the thermostable Photorhabdus luminescens luciferase by about two times as compared to wild-type Bacillus subtilus 168. Erroneous translation under the streptomycin action and in the absence of the DnaKJE and TF chaperones significantly suppressed the synthesis of both luciferases.