TOXR (REGA) ACTIVATES ESCHERICHIA-COLI RNA-POLYMERASE TO INITIATE TRANSCRIPTION OF PSEUDOMONAS-AERUGINOSA TOXA

The Pseudomonas aeruginosa (Pa) structural gene (toxA), which encodes the exotoxin A protein has been shown to be regulated at the transcrip tional level by a protein designated ToxR (also known as RegA). We hav e previously reported that ToxR directly enhances toxA transcription i n vitro; however, in the absence of ToxR, Pa RNA polymerase (RNAP) tra nscribes toxA with low efficiency. In the present study, we have exami ned the ability of ToxR to initiate toxA transcription using the heter ologous Escherichia coli (Ec) RNAP and found that ToxR can function wi th Ec RNAP to efficiently transcribe toxA both in vitro and in vivo. A ntibodies produced against the sigma(70) subunit of Ec RNAP inhibit To xR-mediated enhancement of toxA transcription, suggesting that the RNA P holoenzyme (E sigma(70)) is required for transcriptional activation of toxA. We further demonstrate that ToxR is required for open-complex formation at the toxA promoter, By selectively deleting toxA upstream sequences, we have localized a 214-bp region containing both the toxA promoter and a putative ToxR-binding site sufficient for ToxR-mediate d transcription of toxA.