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

Because the ferric uptake regulator (fur) appears to be an essential g ene 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 cc-ordinate expression of sideroph ores and exotoxin A (ETA). This report describes a detailed molecular and phenotypic characterization of four mutants and one revertant, whi ch carry point mutations in the fur gene. Two parental strains were us ed in this study. Three mutants were isolated from the widely used str ain, 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 ch anges in Fur: mutant A2, H86 --> R; mutant A4, H86 --> Y. The other mu tant (C6) and its revertant (CGRv) were derived from PAO6261, a mutant of PAO1 with a deletion in the anr gene (anaerobic regulation of argi nine deiminase and nitrate reduction) that controls anaerobic respirat ion in P. aeruginosa. Fur from the C6 mutant has an A10 --> G mutation while in the C6Rv spontaneous revertant the mutant Gly residue has be en changed to Ser at this position. All mutants were examined for alte rations in the iron-regulated expression of siderophores and ETA. The A2 and A4 mutants expressed higher levels of siderophores in iron-defi cient media and in iron-replete media. The CS mutant constitutively ex pressed siderophores at 25 degrees C. At 42 degrees C siderophore bios ynthesis was iron repressed as in the parental strain PAO1. The deleti on of anr in PAO6261 had no apparent effect on the iron-mediated regul ation of siderophore synthesis, but the C6 mutant derived from this st rain produces siderophores constitutively. The iron-regulated producti on 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 a nd 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 quantit ative Western dot-blot assays. By contrast in the CS and C6 mutants, w hile iron-dependent repression of ETA synthesis was similar to both pa rental strains under aerobic conditions, ETA production in these mutan ts was constitutive in a microaerobic environment. RNase protection an alysis of toxA and regAB transcription in PAO1, PAO6261 and the C6 mut ant corroborated the results of quantitative dot-blot assays of ETA. T he mutant Fur proteins were purified and examined for their ability to bind to the promoter of a gene (pvdS) that positively regulates the e xpression 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 r educed affinity by comparison to wild-type Fur. Fur from the C6 mutant in DNase I footprint experiments failed to protect the promoter regio n of the pvdS gene, but it retained some weak binding activity in gel mobility shift assays. The data presented in this study not only furni sh some additional insights into the structure-function relationships of Fur, but also afford novel perspectives with regard to Fur and the iron-dependent regulation of virulence factors in P. aeruginosa under environmental conditions that have not previously been considered.