EXOTOXIN-A PRODUCTION IN PSEUDOMONAS-AERUGINOSA REQUIRES THE IRON-REGULATED PVDS GENE ENCODING AN ALTERNATIVE SIGMA-FACTOR

Exotoxin A (ETA) is secreted by Pseudomonas aeruginosa under iron-limi ting growth conditions. The ETA structural gene, toxA, is regulated at the transcriptional level by the gene products of the regAB operon. T he expression of both toxA and regAB is repressed under iron-replete c onditions, suggesting a role for the ferric uptake regulator (Fur) in regulation of ETA synthesis; however, the Fur protein does not interac t directly with the toxA or the regAB promoters. Evidence is presented that the iron control of ETA synthesis is mediated by a Fur-regulated alternative sigma factor, PvdS, which had initially been identified a s a positive activator for the production of the siderophore pyoverdin . In a Delta pvdS deletion mutant, ETA was produced at low levels of l ess than 5% compared to wild type, but still in response to iron starv ation, and introduction of a functional pvdS gene on a plasmid fully r estored wild-type levels and normal iron regulation of ETA synthesis. Therefore, a functional pvdS locus is essential for ETA production. Ne ither toxA nor regAB mRNA was detectable in a Delta pvdS mutant. Overe xpression of pvdS from the tao promoter on a plasmid resulted in a hig h-level and iron-independent production of ETA in wild-type PAO1, in t he Delta pvdS strain, but not in a Delta regA strain as a host. These findings suggest that PvdS is required for the activation of the regAB promoters. The transcription of regAB and toxA after induction of the Ptac-pvdS gene was monitored in cells grown in high-iron medium. Whil e both regAB and toxA were highly expressed during all growth phases u nder microaerobic conditions, toxA transcripts were detected only duri ng the exponential but not the early stationary phase of growth under aerobic conditions. These results suggest that a second regulatory mec hanism besides the Fur-PvdS system is involved in iron regulation of E TA production.