A gene cluster similar to haem iron uptake loci of bacterial pathogens was identified in Pseudomonas aeruginosa. This phu locus (' Pseudomonas haem uptake ') consisted of the phuR receptor gene and the phuSTUVW operon encoding a typical ABC transporter. Expression of phuR and phuSTUVW from mapped transcriptional-start sites occurred under iron-restricted growth conditions and was directly controlled by the Fur protein. Binding of Fur was demonstrated by DNase footprinting of two adjacent ' Fur boxes ' that overlapped both the phuR and phuSTUVW promoters. Two tandem repeats of 154 bp were identified downstream of the phuSTUVW operon, each of which contained a strong Fur-dependent promoter driving expression of ironregulated RNAs antisense to phuSTUVW. Mutant strains with deletions in phuR and phuSTUV showed greatly reduced growth with either haem or haemoglobin as the only iron source : the defects were complemented by plasmids harbouring the phuR or the phuSTUV genes, respectively. Deletions of phuW or of the tandem repeats had only minor effects on haem utilization.The remaining haem and haemoglobin uptake still observed in the ∆phuR or ∆phuSTUV deletion mutants was due to a second haem-acquisition system, has, which was also under the direct control of Fur. This second haem-receptor gene, hasR, was identified upstream of and in an operon with hasA, encoding a haem-binding extracellular protein. A ∆hasR mutant also exhibited decreased utilization of haem and haemoglobin, and a ∆phuR ∆hasR double mutant was virtually unable to take up either compound. Both the PhuR and HasR proteins were detected in the outer-membrane fraction of P. aeruginosa grown in lowiron media. Taken together, the evidence suggests that the phu and has loci encode two distinct systems required for the acquisition of haem and haemoglobin in P. aeruginosa.

The expression of many virulence factors in Pseudomonas aeruginosa is dependent upon environmental conditions, including iron levels, oxygen, temperature, and osmolarity. The virulence of P. aeruginosa PAO1 is influenced by the iron-and oxygen-regulated gene encoding the alternative sigma factor PvdS, which is regulated through the ferric uptake regulator (Fur). We observed that overexpression of PvdS in strain PAO1 and a ⌬pvdS::Gm mutant resulted in increased pyoverdine production and proteolytic activity compared to when PvdS was not overexpressed. To identify additional PvdS-regulated genes, we compared extracellular protein profiles from PAO1 and the ⌬pvdS::Gm mutant grown under iron-deficient conditions. A protein present in culture supernatants from PAO1 but not in supernatants from ⌬pvdS::Gm was investigated. Amino acid sequence analysis and examination of the genomic database of PAO1 revealed that the N terminus of this 27-kDa protein is identical to that of protease IV of P. aeruginosa strain PA103-29 and is homologous to an endoprotease produced by Lysobacter enzymogenes. In this study, the gene encoding an endoprotease was cloned from PAO1 and designated prpL (PvdS-regulated endoprotease, lysyl class). All (n ‫؍‬ 41) but one of the strains of P. aeruginosa, including clinical and environmental isolates, examined carry prpL. Moreover, PrpL production among these strains was highly variable. Analysis of RNase protection assays identified the transcription initiation site of prpL and confirmed that its transcription is iron dependent. In the ⌬pvdS::Gm mutant, the level of prpL transcription was iron independent and decreased relative to the level in PAO1. Furthermore, transcription of prpL was independent of PtxR, a PvdS-regulated protein. Finally, PrpL cleaves casein, lactoferrin, transferrin, elastin, and decorin and contributes to PAO1's ability to persist in a rat chronic pulmonary infection model.

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...

Exotoxin A (ETA) is secreted by Pseudomonas aeruginosa under iron-limiting growth conditions. The ETA structural gene, toxA, is regulated at the transcriptional level by the gene products of the regAB operon. The expression of both toxA and regAB is repressed under iron-replete conditions, suggesting a role for the ferric uptake regulator (Fur) in regulation of ETA synthesis; however, the Fur protein does not interact directly with the toxA or the regAB promoters. Evidence is presented that the iron control of ETA synthesis is mediated by a Fur-regulated alternative sigma factor, PvdS, which had initially been identified as a positive activator for the production of the siderophore pyoverdin. In a delta pvdS deletion mutant, ETA was produced at low levels of less than 5% compared to wild type, but still in response to iron starvation, and introduction of a functional pvdS gene on a plasmid fully restored wild-type levels and normal iron regulation of ETA synthesis. Therefore, a functional pvdS locus is essential for ETA production. Neither toxA nor regAB mRNA was detectable in a delta pvdS mutant. Overexpression of pvdS from the tac promoter on a plasmid resulted in a high-level and iron-independent production of ETA in wild-type PAO1, in the delta pvdS strain, but not in a delta regA strain as a host. These findings suggest that PvdS is required for the activation of the regAB promoters. The transcription of regAB and toxA after induction of the P tac-pvdS gene was monitored in cells grown in high-iron medium. While both regAB and toxA were highly expressed during all growth phases under microaerobic conditions, toxA transcripts were detected only during the exponential but not the early stationary phase of growth under aerobic conditions. These results suggest that a second regulatory mechanism besides the Fur-PvdS system is involved in iron regulation of ETA production.

The activities of fumarase-and manganese-cofactored superoxide dismutase (SOD), encoded by the fumC and sodA genes in Pseudomonas aeruginosa, are elevated in mucoid, alginate-producing bacteria and in response to iron deprivation (D. J. Hassett, M. L. Howell, P. A. Sokol, M. L. Vasil, and G. E. Dean, J. Bacteriol. 179:1442-1451, 1997). In this study, a 393-bp open reading frame, fagA (Fur-associated gene), was identified immediately upstream of fumC, in an operon with orfX and sodA. Two iron boxes or Fur (ferric uptake regulatory protein) binding sites were discovered just upstream of fagA. Purified P. aeruginosa Fur caused a gel mobility shift of a PCR product containing these iron box regions. DNA footprinting analysis revealed a 37-bp region that included the Fur binding sites and was protected by Fur. Primer extension analysis and RNase protection assays revealed that the operon is composed of at least three major iron-regulated transcripts. Four mucoid fur mutants produced 1.7-to 2.6-fold-greater fumarase activity and 1.7-to 2.3-greater amounts of alginate than wild-type organisms. A strain devoid of the alternative sigma factor AlgT(U) produced elevated levels of one major transcript and fumarase C and manganase-cofactored SOD activity, suggesting that AlgT(U) may either play a role in regulating this transcript or function in some facet of iron metabolism. These data suggest that the P. aeruginosa fagA, fumC, orfX, and sodA genes reside together on a small operon that is regulated by Fur and is transcribed in response to iron limitation in mucoid, alginate-producing bacteria.