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

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 effect.     

Campylobacter jejuni Contains Two Fur Homologs: Characterization of Iron-Responsive Regulation of Peroxide Stress Defense Genes by the PerR Repressor

Expression of the peroxide stress genes alkyl hydroperoxide reductase (ahpC) and catalase (katA) of the microaerophile Campylobacter jejuni is repressed by iron. Whereas iron repression in gram-negative bacteria is usually carried out by the Fur protein, previous work showed that this is not the case in C. jejuni, as these genes are still iron repressed in a C. jejuni fur mutant. An open reading frame encoding a Fur homolog (designated PerR for "peroxide stress regulator") was identified in the genome sequence of C. jejuni. The perR gene was disrupted by a kanamycin resistance cassette in C. jejuni wild-type and fur mutant strains. Subsequent characterization of the C. jejuni perR mutants showed derepressed expression of both AhpC and KatA at a much higher level than that obtained by iron limitation, suggesting that expression of these genes is controlled by other regulatory factors in addition to the iron level. Other iron-regulated proteins were not affected by the perR mutation. The fur perR double mutant showed derepressed expression of known iron-repressed genes. Further phenotypic analysis of the perR mutant, fur mutant, and the fur perR double mutant showed that the perR mutation made C. jejuni hyperresistant to peroxide stress caused by hydrogen peroxide and cumene hydroperoxide, a finding consistent with the high levels of KatA and AhpC expression, and showed that these enzymes were functional. Quantitative analysis of KatA expression showed that both the perR mutation and the fur mutation had profound effects on catalase activity, suggesting additional non-iron-dependent regulation of KatA and, by inference, AhpC. The PerR protein is a functional but nonhomologous substitution for the OxyR protein, which regulates peroxide stress genes in other gram-negative bacteria. Regulation of peroxide stress genes by a Fur homolog has recently been described for the gram-positive bacterium Bacillus subtilis. C. jejuni is the first gram-negative bacterium where non-OxyR regulation of peroxide stress genes has been described and characterized.     

Role of iron and superoxide for generation of hydroxyl radical, oxidative DNA lesions, and mutagenesis in Escherichia coli

We measured the generation of hydroxyl radical (OH(.)) and oxidative DNA lesions in aerobically grown Escherichia coli cells lacking in both superoxide dismutases (SodA SodB) and repressor of iron uptake (Fur) using electroparamagnetic resonance and gas chromatography-mass spectrometry with a selected-ion monitoring method. A specific signal corresponding to OH(.) generation and an increase in oxidative DNA lesions such as 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine were detected in the strain deficient in sodA sodB fur. We showed that iron metabolism deregulation in fur mutant produced a 2.5-fold iron overload. The sodA sodB fur strain was about 100-fold higher mutability than the wild-type strain. The mutation spectrum in the strain was found to induce GC --> TA and AT --> CG transversions predominantly. The hypermutability of the strain was suppressed by the tonB mutation which reduces iron transport. Thus, excess iron and excess superoxide were responsible for OH(.) generation, oxidative DNA lesion formation, and hypermutability in E. coli.     

Agrobacterium tumefaciens fur has important physiological roles in iron and manganese homeostasis, the oxidative stress response, and full virulence

In Agrobacterium tumefaciens, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A fur mutant was constructed to address the physiological role of the regulator. Atypically, the mutant did not show alterations in the levels of siderophore biosynthesis and the expression of iron transport genes. However, the fur mutant was more sensitive than the wild type to an iron chelator, 2,2'-dipyridyl, and was also more resistant to an iron-activated antibiotic, streptonigrin, suggesting that Fur has a role in regulating iron concentrations. A. tumefaciens sitA, the periplasmic binding protein of a putative ABC-type iron and manganese transport system (sitABCD), was strongly repressed by Mn(2+) and, to a lesser extent, by Fe(2+), and this regulation was Fur dependent. Moreover, the fur mutant was more sensitive to manganese than the wild type. This was consistent with the fact that the fur mutant showed constitutive up-expression of the manganese uptake sit operon. Fur(At) showed a regulatory role under iron-limiting conditions. Furthermore, Fur has a role in determining oxidative resistance levels. The fur mutant was hypersensitive to hydrogen peroxide and had reduced catalase activity. The virulence assay showed that the fur mutant had a reduced ability to cause tumors on tobacco leaves compared to wild-type NTL4.     

Necessity of OxyR for the hydrogen peroxide stress response and full virulence in Ralstonia solanacearum

The plant pathogen Ralstonia solanacearum, which causes bacterial wilt disease, is exposed to reactive oxygen species (ROS) during tomato infection and expresses diverse oxidative stress response (OSR) genes during midstage disease on tomato. The R. solanacearum genome predicts that the bacterium produces multiple and redundant ROS-scavenging enzymes but only one known oxidative stress response regulator, OxyR. An R. solanacearum oxyR mutant had no detectable catalase activity, did not grow in the presence of 250 μM hydrogen peroxide, and grew poorly in the oxidative environment of solid rich media. This phenotype was rescued by the addition of exogenous catalase, suggesting that oxyR is essential for the hydrogen peroxide stress response. Unexpectedly, the oxyR mutant strain grew better than the wild type in the presence of the superoxide generator paraquat. Gene expression studies indicated that katE, kaG, ahpC1, grxC, and oxyR itself were each differentially expressed in the oxyR mutant background and in response to hydrogen peroxide, suggesting that oxyR is necessary for hydrogen peroxide-inducible gene expression. Additional OSR genes were differentially regulated in response to hydrogen peroxide alone. The virulence of the oxyR mutant strain was significantly reduced in both tomato and tobacco host plants, demonstrating that R. solanacearum is exposed to inhibitory concentrations of ROS in planta and that OxyR-mediated responses to ROS during plant pathogenesis are important for R. solanacearum host adaptation and virulence.     

Fur in Magnetospirillum gryphiswaldense influences magnetosomes formation and directly regulates the genes involved in iron and oxygen metabolism

Magnetospirillum gryphiswaldense strain MSR-1 has the unique capability of taking up large amounts of iron and synthesizing magnetosomes (intracellular magnetic particles composed of Fe(3)O(4)). The unusual high iron content of MSR-1 makes it a useful model for studying biological mechanisms of iron uptake and homeostasis. The ferric uptake regulator (Fur) protein plays a key role in maintaining iron homeostasis in many bacteria. We identified and characterized a fur-homologous gene (MGR_1314) in MSR-1. MGR_1314 was able to complement a fur mutant of E. coli in iron-responsive manner in vivo. We constructed a fur mutant strain of MSR-1. In comparison to wild-type MSR-1, the mutant strain had lower magnetosome formation, and was more sensitive to hydrogen peroxide and streptonigrin, indicating higher intracellular free iron content. Quantitative real-time RT-PCR and chromatin immunoprecipitation analyses indicated that Fur protein directly regulates expression of several key genes involved in iron transport and oxygen metabolism, in addition it also functions in magnetosome formation in M. gryphiswaldense.     

Human copper-zinc superoxide dismutase complements superoxide dismutase-deficient Escherichia coli mutants

An Escherichia coli double mutant, sodAsodB, that is deficient in both bacterial superoxide dismutases (Mn superoxide dismutase and iron superoxide dismutase) is unable to grow on minimal medium in the presence of oxygen and exhibits increased sensitivity to paraquat and hydrogen peroxide. Expression of the evolutionarily unrelated eukaryotic CuZn superoxide dismutase in the sodAsodB E. coli mutant results in a wild-type phenotype with respect to aerobic growth on minimal medium and in resistance to paraquat and hydrogen peroxide. This supports the hypothesis that superoxide dismutation is the in vivo function of these proteins. Analysis of the growth of sodAsodB cells containing plasmids encoding partially active CuZn superoxide dismutases, produced by in vitro mutagenesis, shows a correlation between cell growth and enzyme activity. Thus, the sodAsodB strain provides a controlled selection for varying levels of superoxide dismutase activity.