Effect of regB on expression from the P1 and P2 promoters of the Pseudomonas aeruginosa regAB operon.

Exotoxin A production in Pseudomonas aeruginosa is dependent on two regulatory genes, regA and regB, which are located in tandem on the chromosome. Expression of regA and regB is controlled by two promoters (P1 and P2) situated upstream of the regAB locus. We have studied the effect of the regA and regB gene products on transcription from the regAB promoters. Transcriptional and translational fusions, under the control of the P. aeruginosa regA promoters, were used to analyze the regulation of these promoters in a variety of genetic backgrounds. When the regA P1 promoter was supplied in trans to strains lacking expression of regB (PAO1) or lacking transcription of the regAB operon (PA103-29), little activity from the P1 promoter was detected. In contrast, activity from the P2 promoter was not affected in either PAO1 or PA103-29. Sequence analysis of the regAB operon of PA103-29 detected two mutations. One of the mutations is predicted to result in a premature stop codon in the regA open reading frame. We complemented PA103-29 with a construction containing regA and an inactive regB or a construction containing both regA and regB to directly analyze the effect of regB on transcription of the regAB operon. When PA103-29 was complemented with regA but not regB, we could not detect any transcription from the P1 promoter. Complementation of PA103-29 with both regA and regB resulted in a high level of transcription from the P1 promoter and a corresponding early transcriptional activation of toxA. Our results indicated that induction of transcription from the P1 promoter requires the regB open reading frame and thus the regAB operon is autogenously regulated in P. aeruginosa.