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Pseudomonas aeruginosa produces a blue pigment, pyocyanin. Pyocyanin is a redox-active phenazine compound that kills mammalian and bacterial cells through the generation of reactive oxygen intermediates. We examined the mechanisms by which P. aeruginosa resists pyocyanin. [14C]pyocyanin was taken up by both Escherichia coli and P. aeruginosa, though more slowly by the latter. Cyanide-insensitive respiration, used as an indicator of intracellular superoxide and/or hydrogen peroxide production, was 50-fold less in pyocyanin-treated P. aeruginosa than in E. coli. P. aeruginosa showed less cyanide-insensitive respiration than E. coli upon exposure to other redox-active compounds (paraquat, streptonigrin, and plumbagin). Electron paramagnetic resonance spectrometry and spin trapping showed that P. aeruginosa generated less pyocyanin radical and superoxide than E. coli. Cell extracts from E. coli contained an NADPH:pyocyanin oxidoreductase which increased the rate of reduction of pyocyanin by NADPH. Conversely, cell extracts from P. aeruginosa contained no NADPH:pyocyanin oxidoreductase activity and actually decreased the rate of pyocyanin-mediated NADPH oxidation. Antioxidant defenses could also reduce the sensitivity of P. aeruginosa to pyocyanin. Under culture conditions of limited phosphate, both pyocyanin production and catalase activity were enhanced. Superoxide dismutase activity was also increased under low-phosphate conditions. When cells were grown in a high-phosphate succinate medium, P. aeruginosa formed a previously described iron-superoxide dismutase as well as a manganese-cofactored superoxide dismutase. These results demonstrate that P. aeruginosa resists pyocyanin because of limited redox cycling of this compound and that under conditions favoring pyocyanin production, catalase and superoxide dismutase activities increase.
Production of pyocyanin enhances Pseudomonas aeruginosa virulence. Many of pyocyanin's in vitro and in vivo cytotoxic effects on human cells appear to result from its ability to redox cycle. Pyocyanin directly accepts electrons from NADH or NADPH with subsequent electron transfer to oxygen, generating reactive oxygen species. Reduced glutathione (GSH) is an important cellular antioxidant, and it contributes to the regulation of redox-sensitive signaling systems. Using the human bronchial epithelial (HBE) and the A549 human type II alveolar epithelial cell lines, we tested the hypothesis that pyocyanin can deplete airway epithelial cells of GSH. Incubation of both cell types with pyocyanin led to a concentration-dependent loss of cellular GSH (up to 50%) and an increase in oxidized GSH (GSSG) in the HBE, but not A549 cells, at 24 h. An increase in total GSH, mostly as GSSG, was detected in the culture media, suggesting export of GSH or GSSG from the pyocyanin-exposed cells. Loss of GSH could be due to pyocyanin-induced H(2)O(2) formation. However, overexpression of catalase only partially prevented the pyocyanin-mediated decline in cellular GSH. Cell-free electron paramagnetic resonance studies revealed that pyocyanin directly oxidizes GSH, forming pyocyanin free radical and O(2)(-). Pyocyanin oxidized other thiol-containing compounds, cysteine and N-acetyl-cysteine, but not methionine. Thus GSH may enhance pyocyanin-induced cytotoxicity by functioning as an alternative source of reducing equivalents for pyocyanin redox cycling. Pyocyanin-mediated alterations in cellular GSH may alter epithelial cell functions by modulating redox sensitive signaling events.
Pyocyanin, produced by Pseudomonas aeruginosa, has many deleterious effects on human cells that relate to its ability to generate reactive oxygen species (ROS), such as superoxide and hydrogen peroxide. Human cells possess several mechanisms to protect themselves from ROS, including manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (CuZnSOD), and catalase. Given the link between pyocyanin-mediated epithelial cell injury and oxidative stress, we assessed pyocyanin's effect on MnSOD, CuZnSOD, and catalase levels in the A549 human alveolar epithelial cell line and in normal human bronchial epithelial cells. In both cell types, CuZnSOD and MnSOD were unaltered, but over 24 h pyocyanin significantly decreased cellular catalase activity and protein content. Pyocyanin also decreased catalase mRNA. Overexpression of MnSOD in A549 cells prevented pyocyanin-mediated loss of catalase protein, but catalase activity still declined. Furthermore, pyocyanin decreased catalase activity, but not protein, in A549 cells overexpressing human catalase. These data suggest a direct effect of pyocyanin on catalase activity. Addition of pyocyanin to catalase in a cell-free system also decreased catalase activity. Mammalian catalase binds four NADPH molecules, helping maintain enzyme activity. Spin-trapping data suggest that pyocyanin directly oxidizes this NADPH, producing superoxide. We conclude that pyocyanin may decrease cellular catalase activity via both transcriptional regulation and direct inactivation of the enzyme. Decreased cellular catalase activity and failure to augment MnSOD could contribute to pyocyanin-dependent cytotoxicity.
The Pseudomonas aeruginosa secretory product pyocyanin damages lung epithelium, likely due to redox cycling of pyocyanin and resultant superoxide and H(2)O(2) generation. Subcellular site(s) of pyocyanin redox cycling and toxicity have not been well studied. Therefore, pyocyanin's effects on subcellular parameters in the A549 human type II alveolar epithelial cell line were examined. Confocal and electron microscopy studies suggested mitochondrial redox cycling of pyocyanin and extracellular H(2)O(2) release, respectively. Pyocyanin decreased mitochondrial and cytoplasmic aconitase activity, ATP levels, cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and mitochondrial membrane potential. These effects were transient at low pyocyanin concentrations and were linked to apparent cell-mediated metabolism of pyocyanin. Overexpression of MnSOD, but not CuZnSOD or catalase, protected cellular aconitase, but not ATP, from pyocyanin-mediated depletion. This suggests that loss of aconitase activity is not responsible for ATP depletion. How pyocyanin leads to ATP depletion, the mechanism of cellular metabolism of pyocyanin, and the impact of mitochondrial pyocyanin redox cycling on other cellular events are important areas for future study.
Pyocyanin (1-hydroxy-N-methylphenazine) is a cytotoxic pigment secreted by the bacterial species Pseudomonas aeruginosa, which frequently infects the lungs of immunosuppressed patients as well as those with cystic fibrosis. Pyocyanin toxicity results presumably from the ability of the compound to undergo reduction by NAD(P)H and subsequent generation of superoxide and H2O2 directly in the lungs. We report that in the presence of peroxidase mimics, microperoxidase 11, or hemin, pyocyanin undergoes oxidation by H2O2, as evidenced by loss of the pigment's characteristic absorption spectrum and by EPR detection of a free radical metabolite. The oxidation of pyocyanin is irreversible, suggesting an extensive modification of the pigment's phenazine chromophore. Oxidation of pyocyanin was observed also when exogenous H2O2 was replaced by a H2O2-generating system consisting of NADH and the pigment itself. That the oxidation involves the phenolate group of pyocyanin was verified by the observation that a related pigment, phenazine methosulfate, which is devoid of this group, does not undergo oxidation by microperoxidase 11/H2O2. In contrast to intact pyocyanin, oxidized pyocyanin was less efficient in NADH oxidation and stimulation of interleukin-8 release by human alveolar epithelial A549 cells in vitro, suggesting that oxidation of pyocyanin leads to its inactivation. This study demonstrates that pyocyanin may play a dual role in biological systems, first as an oxidant and ROS generator, and second as a substrate for peroxidases, contributing to H2O2 removal. This latter property may cause pyocyanin degradation and inactivation, which may be of considerable biomedical interest.
Pyocyanin added to suspensions of Pseudomonas aeruginosa P changes the structural organization of the cells depending on their physiological characteristics, in particular, the capability to liberate pyocyanin into the cultural broth. Exogenous pyocyanin does not interact with the cells of the parent strain producing the pigment. However, the structure of the isolated cell walls deformed after the fractionation becomes similar, upon contact with pyocyanin, to the structure of the freshly isolated cell walls. The fraction of the cytoplasmic membranes of the parent strain also slightly changes its spatial organization after addition of pyocyanin. The cells of the mutant which do not produce pyocyanin display the ability for structural interaction with exogenous pyocyanin. In contrast, the fraction of their cell walls does not react to addition of the pigment. The cytoplasmic membranes of the mutant interact with pyocyanin in the same manner as the whole cells. These changes caused by pyocyanin always involve certain parts of the molecules. As a result, the following features are observed in their spectra: the absorption at 1475 cm-1 becomes more intensive; the "knee" at the band Amide II is more pronounced in the range of 1500 cm-1; the absorption at 1520 cm-1 decreases as well as the intensity of the band Amide II as a whole. The interaction of pyocyanin with biological objects stems, apparently, from the presence of certain proteins in them since peroxidase was found to respond to pyocyanin in a manner similar to that described for the cells and their fractions.
Formation of dichlorofluorescein (DCF), the fluorescent oxidation product of 2',7'-dichlorodihydrofluorescein (DCFH2), in cells loaded with the latter compound is often used to detect ROS formation. We previously found that exposure of DCFH2-loaded A549 cells to the Pseudomonas aeruginosa secretory product pyocyanin results in DCF formation, consistent with ROS production. However, since pyocyanin directly accepts electrons from NAD(P)H, we hypothesized that pyocyanin might directly oxidize DCFH2 to DCF without an ROS intermediate. Incubation of DCFH2 with pyocyanin rapidly resulted in DCF formation, the rate of which was proportional to the [pyocyanin] and was not inhibited by SOD or catalase. Phenazine methosulfate, a pyocyanin analog, was more effective than pyocyanin in generating DCF. Mitoxantrone and ametantrone also produced DCF. However, menadione, paraquat, plumbagin, streptonigrin, doxorubicin, daunorubicin, and 5-iminodaunorubicin did not. Pyocyanin, phenazine methosulfate, mitoxantrone, and ametantrone also oxidized dihydrofluorescein and 5- (and 6-) -carboxy-2',7'-dichlorodihydrofluorescein, whereas dihydrorhodamine was oxidized only by pyocyanin or phenazine methosulfate. Under aerobic conditions, the interaction of DCFH2 with pyocyanin or phenazine methosulfate (but not mitoxantrone or ametantrone) produced superoxide, as detected by spin trapping. Direct oxidation of the fluorescent probes needs to be controlled for when employing these compounds to assess ROS formation by biological systems exposed to redox active compounds.
Pseudomonas aeruginosa is an n-alkane degrader that is frequently isolated from petroleum-contaminated sites and produces factors that enhance its competitiveness and survival in many environments. In this study, one such factor, pyocyanin, has been detected in an oil-degrading culture containing P. aeruginosa and is a redox-active compound capable of inhibiting microbial growth. To examine the effects of pyocyanin further, an oil-degrading culture was grown with and without 9.5 microM pyocyanin and microbial community structure and oil degradation were monitored for 50 days. Denaturing gradient gel electrophoresis (DGGE) analysis of cultures revealed a decrease in the microbial community diversity in the pyocyanin-amended cultures compared to that of the unamended cultures. Two members of the microbial community in pure culture exhibited intermediate and high sensitivities to pyocyanin corresponding to intermediate and low levels of activity for the antioxidant enzymes catalase and superoxide dismutase, respectively. Another member of the community that remained constant in the DGGE gels over the 50-day culture incubation period exhibited no sensitivity to pyocyanin, corresponding to a high level of catalase and superoxide dismutase when examined in pure culture. Pyocyanin also affected the overall degradation of the crude oil. At 50 days, the culture without pyocyanin had decreased polycyclic aromatic hydrocarbons compared to the pyocyanin-amended culture, with a specific reduction in the degradation of dibenzothiophenes, naphthalenes, and C(29) and C(30) hopanes. This study demonstrated that pyocyanin influenced the diversity of the microbial community and suggests the importance of understanding how interspecies interactions influence the degradation capability of a microbial community.
The effects of pyocyanine (phenazinium, 1-hydroxy-5-methyl-hydroxide, inner salt) on oxidative burst in human polymorphonuclear leukocytes were studied by several different approaches. In a cell- and enzyme-free system, pyocyanine oxidized NADPH. The reduced pyocyanine could be measured by its reaction with ferricytochrome c. It was shown by this assay that resting as well as phorbol myristate acetate- or zymosan-stimulated granulocytes reduced pyocyanine. The effect was independent of mitochondria, as cytoplasts were similarly active. Measurement of the hexose monophosphate shunt in intact granulocytes in the presence of pyocyanine indicated a concentration-dependent activation of the shunt without the generation of O2-, suggesting that pyocyanine oxidizes NADPH to NADP+ when it enters granulocytes. Intracellular NADPH in granulocytes was indeed lowered by almost 40% after incubation with pyocyanine. It is by this shuttling of reduction equivalents, leading to the partial depletion of NADPH, that pyocyanine affects the observed concentration-dependent partial inhibition of the phorbol myristate acetate- and zymosan-stimulated generation of O2-. A further consequence was that the intracellular killing of Staphylococcus aureus was also partially suppressed, particularly at higher loads of granulocytes with bacteria. Phagocytosis was not inhibited by pyocyanine concentrations as high as 500 microM. Pyocyanine did not affect the intracellular killing of Pseudomonas aeruginosa. The possible relevance of these findings to the course of mixed hospital infections in immunocompromised patients is discussed.
Pyocyanine was isolated by chloroform extraction of cultures of Pseudomonas aeruginosa, and purified by thin layer chromatography. The effects of pyocyanine on the various stages of T cell activation were studied with concanavalin A-stimulated CBA/J mouse splenocytes. At 12.5 microM concentration pyocyanine totally inhibited Con A-dependent proliferation and development of cytotoxic effector cells. Protein and RNA synthesis was only 50% inhibited at this concentration. Inhibitory doses of pyocyanine were nontoxic, in that cell viability was maintained, and the inhibitory effects were reversible after removal of the drug. Pyocyanine did not interfere with interleukin 2 synthesis, nor did it affect the lytic stage of cytotoxic effector T cells. However, T blasts generated by Con A in the presence of pyocyanine did not grow in response to IL2 even in the absence of pyocyanine, and IL2 receptors, detected by indirect immunofluorescence with the receptor-specific monoclonal antibody AMT-13, were diminished in pyocyanine-treated cells. Pyocyanine also inhibited IL2-dependent proliferation of T blasts with fully developed IL2 receptors. The substance thus interferes with several discrete stages of T cell activation.
Airway epithelial cells are the first targets of environmental stimuli and local cytokines. Pyocyanin-induced synergism with interleukin (IL)-1 or tumour necrosis factor (TNF) in triggering IL-8 release has been documented previously. In this study, IL-8 mRNA and protein expression were examined in cultured human bronchial epithelial cells (BEAS-2B) stimulated with pyocyanin alone, and in combination with IL-1beta or phorbol 12,13-dibutyrate (PDBu) in the absence and presence of a group of glucocorticoids. IL-8 mRNA was measured by RT-PCR, and IL-8 protein by ELISA (cell supernatants). Pyocyanin alone produced no increase in IL-8 mRNA and release. However, pyocyanin upregulated the stimulatory effect of IL-1beta or PDBu on the release of IL-8 in a dose-dependent manner. The stimulatory effect of pyocyanin on the IL-1beta- or PDBu-stimulated IL-8 release was reduced in the presence of dexamethasone, budesonide, and fluticasone. Budesonide and fluticasone were 10-fold more potent than dexamethasone. The protein kinase C (PKC) inhibitor, Go6976, also significantly reduced the stimulatory effect of pyocyanin on IL-1beta, and PDBu increased IL-8 release. In conclusion, this study shows that PKC signal pathway seems to be involved in the pyocyanin-mediated upregulation of the IL-1beta and PDBu-induced IL-8 release in BEAS-2B cells. These findings suggest that a vicious cycle perpetuating inflammation may exist in the biologic milieu of bronchiectatic patients infected with Pseudomonas aeruginosa due to the production of pyocyanin. The priming action of pyocyanin appears to be blocked by glucocorticoids, thus providing in vitro data in support of the clinical efficacy of inhaled glucocorticoids as anti-inflammatory drugs.
Pseudomonas aeruginosa 7NSK2 induces resistance in dicots through a synergistic interaction of the phenazine pyocyanin and the salicylic acid-derivative pyochelin. Root inoculation of the monocot model rice with 7NSK2 partially protected leaves against blast disease (Magnaporthe grisea) but failed to consistently reduce sheath blight (Rhizoctonia solani). Only mutations interfering with pyocyanin production led to a significant decrease in induced systemic resistance (ISR) to M. grisea, and in trans complementation for pyocyanin production restored the ability to elicit ISR. Intriguingly, pyocyanin-deficient mutants, unlike the wild type, triggered ISR against R. solani. Hence, bacterial pyocyanin plays a differential role in 7NSK2-mediated ISR in rice. Application of purified pyocyanin to hydroponically grown rice seedlings increased H202 levels locally on the root surface as well as a biphasic H202 generation pattern in distal leaves. Co-application of pyocyanin and the antioxidant sodium ascorbate alleviated the opposite effects of pyocyanin on rice blast and sheath blight development, suggesting that the differential effectiveness of pyocyanin with respect to 7NSK2-triggered ISR is mediated by transiently elevated H202 levels in planta. The cumulative results suggest that reactive oxygen species act as a double-edged sword in the interaction of rice with the hemibiotroph M. grisea and the necrotroph R. solani.
Pseudomonas aeruginosa colonizes and infects human tissues, although the mechanisms by which the organism evades the normal, predominantly neutrophilic, host defenses are unclear. Phenazine products of P. aeruginosa can induce death in Caenorhabditis elegans. We hypothesized that phenazines induce death of human neutrophils, and thus impair neutrophil-mediated bacterial killing. We investigated the effects of two phenazines, pyocyanin and 1-hydroxyphenazine, upon apoptosis of neutrophils in vitro. Pyocyanin induced a concentration- and time-dependent acceleration of neutrophil apoptosis, with 50 microM pyocyanin causing a 10-fold induction of apoptosis at 5 h (p < 0.001), a concentration that has been documented in sputum from patients colonized with P. aeruginosa. 1-hydroxyphenazine was without effect. In contrast to its rapid induction of neutrophil apoptosis, pyocyanin did not induce significant apoptosis of monocyte-derived macrophages or airway epithelial cells at time points up to 24 h. Comparison of wild-type and phenazine-deleted strains of P. aeruginosa showed a highly significant reduction in neutrophil killing by the phenazine-deleted strain. In clinical isolates of P. aeruginosa pyocyanin production was associated with a proapoptotic effect upon neutrophils in culture. Pyocyanin-induced neutrophil apoptosis was not delayed either by treatment with LPS, a powerfully antiapoptotic bacterial product, or in neutrophils from cystic fibrosis patients. Pyocyanin-induced apoptosis was associated with rapid and sustained generation of reactive oxygen intermediates and subsequent reduction of intracellular cAMP. Treatment of neutrophils with either antioxidants or synthetic cAMP analogues significantly abrogated pyocyanin-induced apoptosis. We conclude that pyocyanin-induced neutrophil apoptosis may be a clinically important mechanism of persistence of P. aeruginosa in human tissue.
The effect of cyclooxygenase-2 (COX-2) inhibitor, such (as meloxicam, and pyocyanin pigment of Pseudomonas aeruginosa) with and without praziquantel (PZQ) on worms, ova count, bone marrow and blood cells in 7 groups of Schistosoma mansoni infected mice was studied. The results revealed significant decrease of worm burden and ova count in all treated groups as compared to the infected untreated group, while those with combined treatment of PZQ and meloxicam or pyocyanin showed complete eradication of the worm with the highest reduction in the tissue egg load. EM showed extensive swelling and vesiculation of the tegument, completely implanted spines that overlie degenerated muscle layer were obvious in groups treated with either meloxicam or pyocyanin. Hematological study revealed significant increase (P<0.05) of total leucocytic count of PZQ treated group while that treated with either meloxicam or pyocyanin showed significant decrease (P<0.05), but in combination of PZQ with meloxicam or pyocyanin no significant difference as compared to the infected untreated group. The neutrophil was the main cell affected in groups treated with neither meloxicam nor pyocyanin alone with significant decrease (P<0.05), but with significant increase (P<0.05) in combination with PZQ as compared to the infected untreated group. Those treated with PZQ plus meloxicam showed significant increase as compared to that plus pyocyanin. Eosinophil count showed significant decrease (P<0.05) in all treated groups as compared to the infected untreated group. Inverse correlation between serum level of sFas and peripheral neutrophil count was detected. Ultrastructural study of the bone marrow explained the results as groups treated with meloxicam revealed dissociation between nuclear and cytoplasmic development in the neutophils with cytoplasm maintaining primitive appearance despite maturation of the nucleus, that is manifested by the persistent production of immature granules and the still orientation of Golgi cternae and the centriole around the nucleus. Groups treated with pyocyanin pigment revealed many abnormalities in neutophils as hypogranularity or early apoptotic morphology changes as intense perinuclear chromatin aggregation or nucleus fragmentation. In peripheral blood apoptotic morphology changes was detected in both groups treated with meloxicam or pyocyanin while most of cells of mice treated with PZQ were in an active state. Consequently, it is preferable to give meloxicam with PZQ for a short period of time (less side-effect) to eradicate S. mansoni worm completely but with continuous observation of the peripheral neutrophil count and function.
Pseudomonas aeruginosa and its products have been shown to inhibit mitogen-induced human lymphocyte blastogenesis as measured by [3H]TdR uptake. The phenazine pigment pyocyanine has been identified as one of the inhibitors present in cellfree culture supernatants. To determine the mechanism of the inhibitory action of pyocyanine, we studied its effect on the early stages of T cell activation. Pyocyanine inhibited lymphocyte stimulation induced by specific antigens, the lectin concanavalin A and the calcium ionophore, ionomycin, suggesting that its inhibitory effect is not dependent on interference with the T cell antigen receptor complex itself. Using quin-2, we showed that pyocyanine did not interfere with the mitogen-induced increase in cytosolic-free Ca2+. We also showed that pyocyanine did not interfere with the function of calmodulin stimulated Ca2+-Mg2+ ATPase activity, indicating that the mechanism of action of pyocyanine differs from that of the structurally related phenothiazine compounds. Analysis of IL 2 production and IL 2 receptor expression clearly showed that pyocyanine inhibits the production of this essential lymphokine as well as the expression of IL 2 receptors on the T cell membrane. This inhibition is dose dependent and not due to cellular toxicity. There was parallel inhibition of growth in cell volume as well as [3H]TdR uptake. Thus, our results demonstrate that pyocyanine inhibits T cell proliferation by decreasing the production of the critical lymphokine IL 2 and by decreasing the expression of the IL 2 receptor. Local suppression of lymphocyte stimulation by phenazine pigments such as pyocyanine may interfere with cellular immune responses that may be necessary for eradication of chronic infection with P. aeruginosa.
Pseudomonas aeruginosa is an important nosocomial pathogen that can cause acute and chronic infection, particularly of the respiratory system. Pyocyanin is a major P. aeruginosa virulence factor that displays redox activity and induces oxidative stress in cellular systems. The effect of pyocyanin on replicating human pulmonary epithelial (A549) cells was investigated. Cells were exposed to pyocyanin for 24 h and their subsequent growth and development were followed for 7 days. Pyocyanin (5-10 microM) arrested cell growth and resulted in the development of a morphological phenotype consistent with cellular senescence, that is, an enlarged and flattened appearance. The senescent nature of these cells was supported by positive staining for increased lysosomal content and senescence-associated beta-galactosidase activity. All cells treated with pyocyanin (10 microM) converted to the senescent phenotype, which remained stable for up to 7 days. Exposure to pyocyanin at 25 microM or greater resulted in cell death due to apoptosis. A549 cells exposed to pyocyanin generated hydrogen peroxide in a dose-dependent manner and the senescence-inducing effect of pyocyanin was inhibited by the antioxidant, glutathione, suggesting the involvement of reactive oxygen species. The induction of premature cellular senescence by redox-active bacterial toxins may be a hitherto unrecognized aspect of infection pathology and a limiting factor in the tissue repair response to infection.
Pseudomonas aeruginosa produces a blue pigment called pyocyanin. In the presence of oxidizable substrates, bacteria reduce this pigment to a colorless product, leukopyocyanin. Pyocyanin can also be nonenzymatically reduced by NADH. Leukopyocyanin formed by cell- or NADH-mediated reduction nonenzymatically reduces oxygen or Fe(III). Pyocyanin-dependent iron reduction by whole bacterial cells was measured by the formation of the ferrous-ferrozine complex. In addition, leukopyocyanin reduced chelated Fe(III) including ferric iron in complex with transferrin, the serum iron-binding protein. High-pressure liquid chromatography was used to display the reductive removal of iron from transferrin and the accumulation of iron in the ferrous-ferrozine complex. Pyocyanin stimulated the accumulation of 55Fe from [55Fe]transferrin when it was added to bacteria incubated under low-oxygen conditions. Although bacteria grown in the presence of 100 microM FeCl3 reduced pyocyanin just as rapidly as iron-limited bacteria, these cells did not accumulate iron in the presence or absence of pyocyanin. Therefore, P. aeruginosa participates indiscriminantly in the reduction of pyocyanin, but soluble or available iron generated by the pyocyanin is taken up specifically by iron-limited bacteria.