Cloning and sequence analysis of a gene (pchR) encoding an AraC family activator of pyochelin and ferripyochelin receptor synthesis in Pseudomonas aeruginosa

Abstract

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 and pyochelin was cloned. Nucleotide sequencing of the fragment revealed an open reading frame of 888 bp, designated pchR (pyochelin), capable 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 was produced off the 1.8-kb fragment, confirming that this open reading frame was indeed expressed. A region exhibiting homology to the consensus Fur-binding site of Escherichia coli was identified upstream of the pchR coding region overlapping a putative promoter. In addition, the C-terminal 80 amino acid residues of PchR showed approximately 50% homology (identity, 31%; conserved changes, 19%) to the carboxy terminus of AraC, a known transcriptional activator of gene expression in E. coli, Salmonella typhimurium, Citrobacter freundii, and Erwinia chrysanthemi. Within the C-terminal region of PchR, AraC, and a number of other members of the AraC family of transcriptional activators, there exists 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 transcriptional activator of pyochelin and ferripyochelin receptor synthesis in P. aeruginosa. In agreement with this, a PchR mutant obtained by in vitro mutagenesis and gene replacement was deficient in production of the ferripyochelin receptor and pyochelin.