Regulation of toxA and regA by the Escherichia coli fur gene and identification of a Fur homologue in Pseudomonas aeruginosa PA103 and PA01

Abstract

A multicopy plasmid containing the Escherichia coli fur gene was introduced into Pseudomonas aeruginosa strain PA103C. This strain contains a toxA-lacZ fusion integrated into its chromosome at the toxA locus. Beta-galactosidase synthesis in this strain is regulated by iron, as is seen for exotoxin A production. Beta-galactosidase synthesis and exotoxin A production in PA103C containing multiple copies of E. coli fur was still repressed in low iron conditions. The transcription of regA, a positive regulator of toxA, was also found to be inhibited by multiple copies of the E. coli fur gene. In addition, the ability of PA103C containing multiple copies of E. coli fur to produce protease was greatly reduced relative to PA103C containing a vector control. A polyclonal rabbit serum containing antibodies that recognize E. coli Fur was used to screen whole-cell extracts from Vibrio cholerae, Shigella flexneri, Salmonella typhimurium and Pseudomonas aeruginosa. All strains tested expressed a protein that was specifically recognized by the anti-Fur serum. These results and those described above suggest that Fur structure and function are conserved in a variety of distinct bacterial genera and that at least some of these different genera use this regulatory protein to control genes encoding virulence factors.

FptA, the Fe(III)-pyochelin receptor of Pseudomonas aeruginosa: a phenolate siderophore receptor homologous to hydroxamate siderophore receptors

“…Siderophore receptors are believed to interact with the cytoplasmic membrane protein TonB to facilitate the movement of the outer membrane-bound ligand into the periplasiron box The strong regulation of fptA gene transcription by iron and the presence of a highly conserved Fur-binding site in the frtA promoter provide additional evidence of the importance of the Fur protein in iron regulation in P. aeruginosa (46,47). Expression of the FptA protein is iron repressible (31), and its probable regulation by Fur, as evidenced by the presence of the putative Fur-binding site, fits these observations well.…”

Dual regulation of catecholate siderophore biosynthesis in Azotobacter vinelandii by iron and oxidative stress The GenBank accession number for the sequence reported in this paper is AF238500.

“…Proteins were transferred to a nylon membrane, which was then blocked in 1i Blotto [10 mM Tris\HCl, pH 8n0, containing 0n15 M NaCl (TBS), 5 % skim milk, and 0n02 % sodium azide] for 30 min. A 1 : 500 dilution of polyclonal rabbit anti-Pseudomonas aeruginosa Fur antiserum (a gift from M. Vasil, University of Colorado) was added and incubated with shaking overnight (Prince et al, 1991). The blot was washed with TBS, and Fur was localized by incubating with secondary antibody (anti-rabbit IgG horseradish peroxidase conjugate, 1 : 1000 in TBS) for 3 h at room temperature, followed by development with TBS containing 0n5 mg ml − " chloronaphthol in methanol and 0n02 % H # O # .…”

Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities

“…Fur or Fur-like proteins have also been shown to regulate the expression of several virulence factors including a Shiga-like toxin in E. coli (10) and diphtheria toxin in Corynebacterium diphtheriae (49). Prince et al (48) have recently demonstrated that the E. coli fur gene product controls toxA and regAB expression in P. aeruginosa which was subsequently followed by the cloning, sequencing, and characterization of the fur gene from this organism (47). P. aeruginosa fur mutants were isolated from strain PA103 (an exotoxin A overproducer) which allowed constitutive biosynthesis of the powerful siderophores pyoverdin and pyochelin, even in the presence of 1 mM ferric chloride, which normally restricts siderophore biosynthesis.…”