Transcriptional analysis of thePseudomonas aeruginosa toxAregulatory geneptxR

Abstract

The expression of the exotoxin A gene (toxA) in Pseudomonas aeruginosa is a complicated process that involves several regulators, including ptxR, which enhances toxA expression by 4- to 5-fold. Available evidence suggests that ptxR is expressed from two separate promoters, P1 and P2. Previous evidence indicated the presence, within the ptxR upstream region, of binding sites for several regulatory proteins, including PtxS, which negatively regulates ptxR expression. We utilized nested deletion and in vitro transcription analyses to examine the regulation of ptxR expression. The results from nested deletion analysis suggest that under aerobic conditions in iron-deficient medium, ptxR expression follows a biphasic curve that involves the P1 promoter only. Iron eliminated the second peak of ptxR expression but did not affect expression from the P2 promoter. Under microaerobic conditions, iron represses ptxR expression from subclones that carry P1 alone or P2 alone at both early and late stages of growth. Under anaerobic conditions, ptxR expression increases considerably. In addition, our results suggest that different segments of the ptxR upstream region play specific roles in ptxR expression; their deletion caused variations in the level as well as the pattern of ptxR expression. Our results also indicate that negative regulation of ptxR expression by PtxS does not occur through the PtxS binding site within the ptxR–ptxS intergenic region. In vitro transcription analysis using σ70-reconstituted P. aeruginosa RNA polymerase produced one transcript that closely resembles T1, indicating that P1 is recognized by σ70. RNA polymerase reconstituted with either RpoS or AlgU produced no transcripts. However, a transcript was produced by RpoH-reconstituted RNA polymerase.Key words: ptxR, regulation, Pseudomonas aeruginosa, PAO1.