Pyoverdine-mediated regulation of FpvA synthesis in Pseudomonas aeruginosa: involvement of a probable extracytoplasmic-function sigma factor, FpvI

Abstract

A search of the pvd pyoverdine biosynthesis locus of Pseudomonas aeruginosa identified an open reading frame, PA2387, whose product exhibited a sequence similar to those of a number of so-called extracytoplasmic- function sigma factors responsible for siderophore-dependent expression of iron-siderophore receptors in Escherichia coli and Pseudomonas putida. Deletion of this gene, dubbed fpvI, compromised pyoverdine-dependent FpvA ferric pyoverdine receptor production and fpvA gene expression, while the cloned gene stimulated fpvA expression. A Fur-binding site was identified immediately upstream of fpvI, consistent with the observed iron-regulated expression of fpvI and fpvA.

FIG. 1.

Immunoblots of iron-limited P. aeruginosa whole-cell extracts developed with anti-FpvA antibodies. (A) Lane 2, K1120 (FpvI⁺); lane 3, K2100 (FpvI⁻); lane 4, K2100 carrying pAR001 (FpvI⁺). An immunoblot of K1120 grown in iron-supplemented minimal medium is shown in lane 1. (B) Lane 1, K1120 (FpvI⁺); lanes 2 and 3, K1203 (FpvI⁺ PvdD⁻); lanes 4 and 5, K2102 (FpvI⁻ PvdD⁻); lane 6, K2102 carrying pAR001 (FpvI⁺ PvdD⁻). Samples in lanes 3, 5, and 6 were prepared from cells supplemented with pyoverdine (100 μg/ml) during growth.

FIG. 2.

fpvI expression in P. aeruginosa strain PAO1 K767, measured by RT-PCR of RNA (4 ng) isolated from cells cultivated under iron-sufficient (odd-numbered lanes) and iron-deficient (even-numbered lanes) conditions. The reactions were carried out for 26 (lanes 1 and 2), 27 (lanes 3 and 4), 28 (lanes 5 and 6), 29 (lanes 7 and 8), and 30 (lanes 9 and 10) cycles. fpvI and the rpsL (control) products are highlighted. Lane 11, 100-bp ladder with a 500-bp marker highlighted.

FIG. 3.

Nucleotide sequence of the 5′ upstream region of fpvI, highlighting a putative Fur box. The Fur box (underlined) and FpvI translational initiation codon (italicized) are highlighted. Matches to the consensus Fur box sequence are capitalized.