CLONING AND NUCLEOTIDE-SEQUENCE ANALYSIS OF THE FERRIPYOVERDINE RECEPTOR GENE FPVA OF PSEUDOMONAS-AERUGINOSA

Pseudomonas aeruginosa K437 lacks the ferripyoverdine receptor and, as a result, grows poorly on an iron-deficient minimal medium supplement ed with ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and pyo verdine. By using a phagemid-based in vivo cloning system, attempts we re made to clone the receptor gene by complementing this growth defect . Several recombinant phagemids carrying P. aeruginosa chromosomal DNA which provided for good growth on EDDHA-pyoverdine-containing medium and which concomitantly restored production of the ferripyoverdine rec eptor in strain K437 were isolated. These phagemids contained a common 4.6-kb SphI fragment which similarly restored production of the recep tor in K437. Nucleotide sequencing of the SphI fragment revealed a sin gle large open reading frame, designated fpvA (ferripyoverdine uptake) , of 2439 bp. The predicted translation product of fpvA has a molecula r mass of 89,395 Da. N-terminal amino acid sequence analysis of the pu rified ferripyoverdine receptor confirmed fpvA as the receptor gene. M oreover, it indicated that the receptor is initially synthesized as a precursor with a signal sequence of 27 amino acids which is cleaved to yield the mature protein. The deduced FpvA polypeptide exhibited homo logy to regions shown to be conserved in TonB-dependent receptor prote ins. FpvA also shared strong homology (41.3% identity) with the PupA p rotein of Pseudomonas putida WCS358. This protein is the receptor for the iron-bound form of pseudobactin, a compound structurally very simi lar to pyoverdine.