IRON-REGULATED TRANSCRIPTION OF THE PVDA GENE IN PSEUDOMONAS-AERUGINOSA - EFFECT OF FUR AND PVDS ON PROMOTER ACTIVITY

The pvdA gene, encoding the enzyme L-ornithine N-5-oxygenase, catalyze s a key step of the pyoverdin biosynthetic pathway in Pseudomonas aeru ginosa. Expression studies with a promoter probe vector made it possib le to identify three tightly iron-regulated promoter regions in the 5. 9-kb DNA fragment upstream of pvdA. The promoter governing pvdA expres sion was located within the 154-bp sequence upstream of the pvdA trans lation start site, RNA analysis showed that expression of PvdA is iron regulated at the transcriptional level. Primer extension and S1 mappi ng experiments revealed two 5' termini of the pvdA transcript, 68 bp ( T1) and 43 bp (T2) 5' of the PvdA initiation, The pvdA transcripts wer e monocystronic, with T1 accounting for 90% of the pvdA mRNA. Fur box- like sequences were apparently absent in the regions 5' of pvdA transc ription start sites. A sequence motif resembling the -10 hexamer of Al gU-dependent promoters and the iron starvation box of pyoverdin genes controlled by the sigma(E)-like factor PvdS were identified 5' of the TI. start site. The minimum DNA region required for iron-regulated pro moter activity was mapped from bp -41 to -154 relative to the ATG tran slation start site of pvdA, We used pvdA'::lacZ transcriptional fusion s and Northern (RNA) analyses to study the involvement of Pur and PvdS in the iron-regulated expression of pvdA. Two fur mutants of P. aerug inosa were much less responsive than wild-type PAO1 to the iron-depend ent regulation of pvdA expression. Transcription from the pvdA promote r did not occur in a heterologous host unless in the presence of the p vdS gene in trans and was abrogated in a pvdS mutant of P. aeruginosa. Interaction of the Fur repressor with a 150-bp fragment encompassing the pvdS promoter was demonstrated in vivo by the Pur titration assay and confirmed in vitro by gel retardation experiments with a partially purified Fur preparation. Conversely, the promoter region of pvdA did not interact with Fur. Our results support the hypothesis that the P. aeruginosa Fur repressor indirectly controls pvdA transcription throu gh the intermediary sigma factor PvdS; in the presence of sufficient i ron, Fur blocks the pvdS promoter, thus preventing PvdS expression and consequently transcription of pvdA and other pyoverdin biosynthesis g enes.