A 5.9-kb DNA fragment was cloned from Pseudomonas aeruginosa PA103 by its ability to functionally complement a fur mutation in Escherichia coli. A fur null mutant E. coli strain that contains multiple copies of the 5.9-kb DNA fragment produces a 15-kDa protein which cross-reacts with a polyclonal anti-E. coli Fur serum. Sequencing of a subclone of the 5.9-kb DNA fragment identified an open reading frame predicted to encode a protein 53% identical to E. coli Fur and 49% identical to Vibrio cholerae Fur and Yersinia pestis Fur. While there is extensive homology among these Fur proteins, Fur from P. aeruginosa differs markedly at its carboxy terminus from all of the other Fur proteins. It has been proposed that this region is a metal-binding domain in E. coli Fur. A positive selection procedure involving the isolation of manganese-resistant mutants was used to isolate mutants of strain PA103 that produce altered Fur proteins. These manganese-resistant Fur mutants constitutively produce siderophores and exotoxin A when grown in concentrations of iron that normally repress their production. A multicopy plasmid carrying the P. aeruginosa fur gene restores manganese susceptibility and wild-type regulation of exotoxin A and siderophore production in these Fur mutants.

An iron-binding compound was isolated from ethyl acetate extracts of culture supernatant fluids of Pseudomonas aeruginosa and was purified by successive paper and thin-layer chromatographic procedures. The purified compound was characterized by UV, visible, infrared, and fluorescence spectroscopy. The compound possesses phenolic characteristics, with little or no similarity to dihydroxybenzoates and no indication of a hydroxamate group. P. aeruginosa synthesized the compound during active growth in culture media containing less than 5 x 10-6 M added FeCl3. When added to iron-poor cultures of P. aeruginosa, the compound promoted the growth of the bacterium and also reversed growth inhibition by the iron chelator ethylenediamine-di-(o-hydroxyphenylacetic acid).

Twelve mutants of Pseudomonas aeruginosa PAO defective in pyoverdin production were isolated (after chemical and transposon mutagenesis) that were nonfluorescent and unable to grow on medium containing 400 ,uM ethylenediaminedi(o-hydroxyphenylacetic acid). Four mutants were unable to produce hydroxamate, six were hydroxamate positive, one was temperature sensitive for pyoverdin production, and another was unable to synthesize pyoverdin on succinate minimal medium but was capable of synthesizing pyoverdin when grown on Casamino Acids medium (Difco Laboratories, Detroit, Mich.). The mutations were mapped on the PAO chromosome. All the mutations affecting pyoverdin production were located at 65 to 70 min, between catAl and mtu-9002.Pathogenic bacteria require iron for growth in a mammalian host (6). To obtain iron from the host, bacteria must effectively compete with the iron-sequestering proteins transferrin and lactoferrin (1). Many microbes possess iron acquisition systems mediated by siderophores, lowmolecular-weight products capable of binding and delivering iron to the cell via high-affinity transport systems (31).Pseudomonas aeruginosa is a major cause of nosocomial infections which result in high mortality. P. aeruginosa is known to produce two siderophores, pyochelin (9, 10, 25) and pyoverdin (8,42). Pyochelin is a phenolic siderophore that has two sulfur-containing heterocyclic rings (9, 10). Pyoverdin, previously termed bacterial fluorescein (A. Turfreijer, Ph.D. thesis, University of Amsterdam, Amsterdam, The Netherlands, 1941), has long been thought to be involved in iron metabolism because of its hydroxamate character (16) and the inhibition of its production by iron (14,17,38). Only recently has the siderophore activity of pyoverdin been demonstrated (8) and its molecular structure elucidated (42). This fluorescent siderophore produced by P. aeruginosa is a complex peptide containing two hydroxamate groups and a dihydroxyquinoline derivative (42) as its theorized chelating moieties. Yellow-green fluorescent peptides produced by other fluorescent Pseudomonas species and Azotobacter vinelandii (5,15,28,32,33,37,44) have similar spectral characteristics and extensive structural homology with pyoverdin.Recent investigations have suggested that iron acquisition by P. aeruginosa may play a role in its pathogenesis. The concentration of iron in culture medium has a significant effect on the production of the extracellular proteins, toxin A, alkaline protease, and elastase (3, 4). Pyochelin has been shown to increase the lethality of P. aeruginosa during infections in mice (7). A mutant unable to synthesize pyoverdin had an extremely depressed growth rate compared with that of wild-type strains when grown in human serum and transferrin (2).Advances in P. aeruginosa genetics have allowed the map positions of several reported virulence factors to be determined. Use of the chromosome-mobilizing plasmid R68.45 * Corresponding author.(19), R-prime plasmids (21), and transposon-facilitated recombination (24) has m...

Iron affects yields of toxin A, alkaline protease, elastase, pyochelin, and pyoverdin in Pseudomonas aeruginosa. Mutants of P. aeruginosa PAO1 resistant to the effect of iron on toxin (toxC) or elastase (elaC) yields were isolated. Two types of mutants were isolated: iron transport and iron regulatory mutants. The toxC regulatory mutants produced toxin A in medium containing iron; however, yields of elastase and alkaline protease remained sensitive to regulation by iron. The elaC regulatory mutants were resistant to the effect of iron on elastase yields, but toxin A and alkaline protease yields were decreased by iron, analogous to the parent strain. These data suggest that toxin A, elastase, and alkaline protease yields can be independently regulated by iron.

We isolated two mutants of Pseudomonas aeruginosa PAO with defective iron uptake. In contrast to the wild-type strain, the mutants produced extracellular protease actity in media containing high concentrations of salts or iron and hyperproduced elastase, staphylolytic enzyme, and exotoxin A in ordinary media (Xch mutants). The mutations were located in the 55' region of the chromosome, between the markers met-9011 and pyrD.