CLONING AND SEQUENCE-ANALYSIS OF A GENE (PCHR) ENCODING AN ARAC FAMILY ACTIVATOR OF PYOCHELIN AND FERRIPYOCHELIN RECEPTOR SYNTHESIS IN PSEUDOMONAS-AERUGINOSA

Pseudomonas aeruginosa K372 is deficient in the production of both the 75-kDa ferripyochelin receptor protein and pyochelin. A 1.8-kb EcoRI- SalI fragment which restored production of both the receptor protein a nd pyochelin was cloned. Nucleotide sequencing of the fragment reveale d an open reading frame of 888 bp, designated pchR (pyochelin), capabl e of encoding a 296-amino-acid protein of a 32,339-Da molecular mass. By using a phage T7-based expression system, a protein of ca. 32 kDa w as produced off the 1.8-kb fragment, confirming that this open reading frame was indeed expressed. A region exhibiting homology to the conse nsus Fur-binding site of Escherichia coli was identified upstream of t he pchR coding region overlapping a putative promoter. In addition, th e C-terminal 80 amino acid residues of PchR showed approximately 50% h omology (identity, 31%; conserved changes, 19%) to the carboxy terminu s of AraC, a known transcriptional activator of gene expression in E. coli, Salmonella typhimurium, Citrobacter freundii, and Erwinia chrysa nthemi. Within the C-terminal region of PchR, AraC, and a number of ot her members of the AraC family of transcriptional activators, there ex ists a highly conserved 17-residue domain where, in fact, two residues are strictly maintained and two others exhibit only conserved changes , suggesting a common functional significance to this region in all of these proteins. These data are consistent with a role for PchR as a t ranscriptional activator of pyochelin and ferripyochelin receptor synt hesis in P. aeruginosa. In agreement with this, a PchR mutant obtained by in vitro mutagenesis and gene replacement was deficient in product ion of the ferripyochelin receptor and pyochelin.