TOXR (REGA)-MEDIATED IN-VITRO TRANSCRIPTION OF PSEUDOMONAS-AERUGINOSATOXA

Exotoxin A (ETA) has been described as a major virulence factor produc ed by the opportunistic pathogen Pseudomonas aeruginosa. The transcrip tion 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 loxA transcription is still un der investigation. We have expressed toxR in Escherchia coil under the control of the T7 promoter and purified the wild-type ToxR protein. W e 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 us ed an in vitro transcription assay in order to investigate the mechani sm of ToxR-mediated transcriptional regulation of toxA. Under the cond itions of this in vitro assay tosA transcription requires the toxR pro duct in addition to P. aeruginosa RNA polymerase (RNAP). Both the nati ve and the T7::ToxR fusion proteins facilitate initiation of toxA tran scription in vitro in the presence of Pseunomonas RNAP. Additional stu dies using (i) specific enzyme-linked immunosorbent assay; (ii) indire ct immunoprecipitation; and (iii) gel-filtration chromatography, indic ate that ToxR binds to the purified Pseudomonas RNAP and strengthens t he possibility that ToxR may be an alternative sigma factor. Furthermo re, the ToxR-mediated transcription of toxA is increased approx. three fold 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.