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

Abstract

Pseudomonas aeruginosa is considered a strict aerobe that possesses several enzymes important in the disposal of toxic oxygen reduction products including iron-and manganese-cofactored superoxide dismutase and catalase. At present, the nature of the regulation of these enzymes in P. aeruginosa is not understood. To address these issues, we used two mutants called A4 and C6 which express altered Fur (named for ferric uptake regulation) proteins and constitutively produce the siderophores pyochelin and pyoverdin. Both mutants required a significant lag phase prior to log-phase aerobic growth, but this lag was not as apparent when the organisms were grown under microaerobic conditions. The addition of iron salts to mutant A4 and, to a greater extent, C6 cultures allowed for an increased growth rate under both conditions relative to that of bacteria without added iron. Increased manganese superoxide dismutase (Mn-SOD) and decreased catalase activities were also apparent in the mutants, although the second catalase, KatB, was detected in cell extracts of each fur mutant. Iron deprivation by the addition of the iron chelator 2,2-dipyridyl to wild-type bacteria produced an increase in Mn-SOD activity and a decrease in total catalase activity, similar to the fur mutant phenotype. Purified wild-type Fur bound more avidly than mutant Fur to a PCR product containing two palindromic 19-bp "iron box" regions controlling expression of an operon containing the sodA gene that encodes Mn-SOD. All mutants were defective in both ferripyochelin-and ferripyoverdin-mediated iron uptake. Two mutants of strain PAO1, defective in pyoverdin but not pyochelin biosynthesis, produced increased Mn-SOD activity. Sensitivity to both the redox-cycling agent paraquat and hydrogen peroxide was greater in each mutant than in the wild-type strain. In summary, the results indicate that mutations in the P. aeruginosa fur locus affect aerobic growth and SOD and catalase activities in P. aeruginosa. We postulate that reduced siderophore-mediated iron uptake, especially that by pyoverdin, may be one possible mechanism contributing to such effects.

The ferric uptake regulator of Pseudomonas aeruginosa has no essential cysteine residues and does not contain a structural zinc ion

“…Substitution of His-90 of the S. typhimurium Fur (equivalent to H89 of the E. coli protein) with arginine eliminated iron-mediated repression but had no effect on the role of Fur in the acid tolerance response (Hall & Foster, 1996). Although the fur gene of P. aeruginosa is thought to be essential (Hassett et al, 1996), some fur alleles with altered phenotypes have been characterized. Manganese resistance has been used as a screen for the isolation of fur alleles in P. aeruginosa, and amongst several characterized were two mutants with substitutions of arginine and tyrosine at His-86 (Barton et al, 1996).…”

“…The fur gene of P. aeruginosa is believed to be essential (Hassett et al, 1996), so it is difficult to evaluate the phenotypes associated with mutant alleles in a null background. The P. aeruginosa fur gene was originally cloned by complementation of an E. coli mutant (Prince et al, 1993), and P. aeruginosa Fur can bind to an E. coli Fur box in vitro (Ochsner et al, 1995).…”

Strain-specific proteome responses of Pseudomonas aeruginosa to biofilm-associated growth and to calcium

“…Biosynthesis and secretion of pyoverdine are regulated by means of the extracytoplasmic function (ECF) s factor PvdS, whose expression is regulated by iron concentration and the ferric uptake regulator (Fur) (Hassett et al, 1996;Ochsner et al, 1995;Vasil et al, 1998). Our results demonstrate that [Ca 2+ ] influences the production of iron-scavenging proteins and products, but the role of [Ca 2+ ] in this process is not clear.…”

TheSinorhizobium meliloti furGene Regulates, with Dependence on Mn(II), Transcription of thesitABCDOperon, Encoding a Metal-Type Transporter

“…In the classic model of Fur regulation established for Escherichia coli the Fur protein utilizes Fe(II) as a cofactor and binds to specific sequence elements in the promoter regions of these genes, termed Fur boxes, and thereby inhibits gene expression under iron-replete conditions (3, 13). Besides its role as a repressor of iron uptake mechanisms Fur has also been found to be involved in the regulation of alternative sigma factor and activator genes (45, 46), stress response genes (19,(28)(29)(30)(31), energy metabolism genes (39,59,64), and virulence-associated genes (9,11,22,23,40,69). Thus,…”