ToxR (RegA)-mediated in vitro transcription of Pseudomonas aeruginosa toxA.

Exotoxin A (ETA) has been described as a major virulence factor produced by the opportunistic pathogen Pseudomonas aeruginosa. The transcription of the ETA structural gene (toxA) has been shown to be positively regulated by the product of the toxR gene (also called regA). However, the mechanism by which ToxR regulates toxA transcription is still under investigation. We have expressed toxR in Escherichia coli under the control of the T7 promoter and purified the wild-type ToxR protein. We have also produced ToxR as a fusion protein consisting of the first 12 amino acids of the T7 capsid protein attached to the N terminus of the intact ToxR protein. In the present study we have developed and used an in vitro transcription assay in order to investigate the mechanism of ToxR-mediated transcriptional regulation of toxA. Under the conditions of this in vitro assay toxA transcription requires the toxR product in addition to P. aeruginosa RNA polymerase (RNAP). Both the native and the T7::ToxR fusion proteins facilitate initiation of toxA transcription in vitro in the presence of Pseudomonas RNAP. Additional studies using (i) specific enzyme-linked immunosorbent assay; (ii) indirect immunoprecipitation; and (iii) gel-filtration chromatography, indicate that ToxR binds to the purified Pseudomonas RNAP and strengthens the possibility that ToxR may be an alternative sigma factor. Furthermore, the ToxR-mediated transcription of toxA is increased approx. threefold in the presence of crude cytoplasmic extracts from P. aeruginosa ToxR+ or ToxR-RegB- strains, indicating that additional factors play a role in the efficient and optimal transcription of toxA.