Ferric uptake regulator mutants of Pseudomonas aeruginosa with distinct alterations in the iron‐dependent repression of exotoxin A and siderophores in aerobic and microaerobic environments

Abstract

Because the ferric uptake regulator (fur) appears to be an essential gene in Pseudomonas aeruginosa, resistance to manganese was used as an enrichment to isolate strains carrying point mutations in the fur gene in order to assess its role in the co-ordinate expression of siderophores and exotoxin A (ETA). This report describes a detailed molecular and phenotypic characterization of four mutants and one revertant, which carry point mutations in the fur gene. Two parental strains were used in this study. Three mutants were isolated from the widely used strain, PAO1. One of these, CS (cold sensitive), has a mutation in the 5' non-coding region of the fur gene while the two other mutants derived from this parent have mutations resulting in the following deduced changes in Fur: mutant A2, H86-->R; mutant A4, H86-->Y. The other mutant (C6) and its revertant (C6Rv) were derived from PAO6261, a mutant of PAO1 with a deletion in the anr gene (anaerobic regulation of arginine deiminase and nitrate reduction) that controls anaerobic respiration in P. aeruginosa. Fur from the C6 mutant has an A10-->G mutation while in the C6Rv spontaneous revertant the mutant Gly residue has been changed to Ser at this position. All mutants were examined for alterations in the iron-regulated expression of siderophores and ETA. The A2 and A4 mutants expressed higher levels of siderophores in iron-deficient media and in iron-replete media. The CS mutant constitutively expressed siderophores at 25 degrees C. At 42 degrees C siderophore biosynthesis was iron repressed as in the parental strain PAO1. The deletion of anr in PAO6261 had no apparent effect on the iron-mediated regulation of siderophore synthesis, but the C6 mutant derived from this strain produces siderophores constitutively. The iron-regulated production of siderophores by C6Rv was similar to the parental strain PAO6261 and PAO1. Because one of the parental strains used in this study is an Anr mutant, regulation of ETA production was assessed under aerobic and microaerobic conditions. Iron-dependent repression of ETA synthesis in both parental strains and A2 and A4 mutants was found to be 50-100-fold under aerobic and microaerobic conditions, as assayed by quantitative Western dot-blot assays. By contrast in the CS and C6 mutants, while iron-dependent repression os ETA synthesis was similar to both parental strains under aerobic conditions, ETA production in these mutants was constitutive in a microaerobic environment. RNase protection analysis of toxA and regAB transcription in PAO1, PAO6261 and the C6 mutant corroborated the results of quantitative dot-blot assays of ETA. The mutant Fur proteins were purified and examined for their ability to bind to the promoter of a gene (pvdS) that positively regulates the expression of siderophores and ETA. Fur from the A2 and A4 mutants and from the C6Rv revertant was able to bind to the target DNA, but with reduced affinity by comparison to wild-type Fur. Fur from the C6 mutant in DNase I footprint experiments failed to protect the promoter...

Isochorismate Synthase (PchA), the First and Rate-limiting Enzyme in Salicylate Biosynthesis of Pseudomonas aeruginosa

“…A similar regulatory mechanism has been observed for another siderophore of P. aeruginosa, pyoverdin (19). When cells have accumulated excess iron the Fur repressor is activated, which switches off the expression of the pyochelin and pyoverdin biosynthetic genes (15,16,20).…”

Iron and Pseudomonas aeruginosa biofilm formation

“…We tested a strain with a point mutation in this essential gene. This mutation results in low Fur activity (17). The influence of lactoferrin on the biofilm development of this mutant was not as severe as the influence of lactoferrin on the parent.…”

“…The locations of all of the mutations were confirmed by PCR with primers flanking the insertion sites. We also used PAO1⌬pvdS (20); PAO1⌬pvdA (14); PAO1⌬fpvA (11), a strain with a fur point mutation; PAO1furC6Tc (17,18), a PAO1⌬prrF1-F2 mutant (16); PAO1⌬pvdD ⌬pchEF (provided by Urs Ochsner, Replidyne Inc., Louisville, CO); and several strains with mutations generated as described below.…”

“…The major intracellular iron regulator in Proteobacteria including P. aeruginosa is Fur (6,15). The P. aeruginosa Fur appears to be an essential gene; however, Fur missense mutants have been isolated (17,18). Fur is an iron-dependent transcription repressor.…”

“…It activates gene expression indirectly through control of a pair of small regulatory RNAs (sRNAs), PrrF1 and PrrF2 (16). In P. aeruginosa, fur appears to be an essential gene, and the only fur mutants available either produce reduced levels of wild-type Fur or have missense mutations that exhibit high reversion rates (17,18).…”