Biosynthesis of phenazine pigments in mutant and wild-type cultures of Pseudomonas aeruginosa.

Pigmentation mutants of Pseudomonas aeruginosa, selected by observed visual differences in coloration from the wild-type strain, were examined for altered patterns of phenazine synthesis. Three classes of mutants that were incapable of pyocyanine production were identified. Pigmentation patterns that were found to characterize the various mutant classes implicated precursor-product relationships, and a biochemical scheme covering the terminal reactions of pyocyanine biosynthesis is proposed. Among compounds tested as inhibitors of pigmentation, two effectively inhibited pyocyanine production production while allowing cell growth. p-Aminobenzoate inhibited total pigmentation; i.e., no other phenazine accumulated. m-Aminobenzoate inhibited a presumptive methylation step in pyocyanine biosynthesis, abolishing the formation of pyocyanine and aeruginosin pigments but increasing the yields of phenazine 1-carboxylic acid and oxychlororaphin. D-[2,3,4,5(n)-14C]shikimate was most efficiently incorporated into phenazines in the middle to late exponential phase of growth. Label was incorporated predominantly into pyocyanine in the absence of inhibitors and into phenazine 1-carboxylic acid when the organism was grown in the presence of m-aminobenzoate.     

The physiologic role of pyocyanine synthesized by Pseudomonas aeruginosa

The physiological role of pyocyanine for Pseudomonas aeruginosa was studied. Its synthesis was shown to commence at the retardation growth phase. Pyocyanine was accumulated only in the growth medium. The addition of 2,6-dichlorophenolindophenol accepting the reducing equivalents from coenzyme Q and transferring them to cytochrome c inhibited the pigment accumulation. This was indicative of the connection between pyocyanine synthesis and the level of the reducing equivalents in the cells. Pyocyanine did not accept the reducing equivalents from coenzyme Q in the respiratory chain of P. aeruginosa. Only reduced pyridine nucleotides served as substrates for pyocyanine in the reaction of autooxidation. The kinetic parameters of this reaction and the affinity of NADH dehydrogenase for the substrate were measured. The kinetic data were analysed to show that, under the physiological conditions, pyocyanine could not apparently compete with the respiratory chain for the reducing equivalents and hence directly regulate the level of NAD(P)H in P. aeruginosa cells. In order to keep the oxidising activity at a level necessary for the cells, the latter decreased the content of the reducing equivalents either by synthesizing pyocyanine or owing to the activity of cyanide-resistant oxidase. These processes of releasing the reducing equivalents are in a reciprocal relationship.     

Studies on the mechanism of T cell inhibition by the Pseudomonas aeruginosa phenazine pigment pyocyanine

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.     

An electron spin resonance study of oxyradical generation in superoxide dismutase- and catalase-deficient mutants of Escherichia coli K-12

The response of superoxide dismutase- and catalase-deficient strains of Escherichia coli to redox active compounds was examined by electron spin resonance. Levels of radicals formed in response to pyocyanine in situ were extremely low and were found to be predominantly extracellular, even in a strain completely deficient in both superoxide dismutase and catalase. In cell-free extracts of superoxide dismutase-minus strains incubated with NADPH and pyocyanine, the primary accumulating radical was the superoxide anion (O2-), although low levels of the hydroxyl radical (.OH) were also detected. In contrast, extracts from strains lacking catalase were found to accumulate higher levels of hydroxyl radicals.     

Pyocyanine production by Pseudomonas aeruginosa.

Dextrose enhanced the growth of P. aeruginosa but suppressed the biosynthesis of pyocyanine. The preformed pigment could be released from dead cells. Pigmentation was not correlated directly with number of viable organisms in the culture. High concentration of maltose likewise inhibited pyocyanine production. Maltose contained in medium used for pyocyanine production by P. aeruginosa should be kept in low concentration or omitted.     

Mechanism of the antibiotic action pyocyanine.

Exposure of Escherichia coli growing in a rich medium to pyocyanine resulted in increased intracellular levels of superoxide dismutase and of catalase. When these adaptive enzyme syntheses were prevented by nutritional paucity, the toxic action of pyocyanine was augmented. The antibiotic action of pyocyanine was dependent upon oxygen and was diminished by superoxide dismutase and by catalase, added to the suspending medium. Pyocyanine slightly augmented the respiration of E. coli suspended in a rich medium, but greatly increased the cyanide-resistant respiration. Pyocyanine was able to cause the oxidation of reduced nicotinamide adenine dinucleotide, with O2- production, in the absence of enzymatic catalysis. It is concluded that pyocyanine diverts electron flow and thus increases the production of O2- and H2O2 and that the antibiotic action of this pigment is largely a reflection of the toxicity of these products of oxygen reduction.     

Role of Iron and Sulfur in Pigment and Slime Formation by Pseudomonas aeruginosa

Media and an analytical scheme have been developed which allow both a qualitative and quantitative estimation of the formation of pyocyanine, related phenazines, pyorubrin, and a blue and a yellow-green fluorescent pigment by Pseudomonas aeruginosa. Use of the defined pyocyanine medium of Frank and DeMoss with sulfate or various organic sulfur sources allowed formation of pyocyanine, related phenazines, and pyorubrin. When sulfite was the sulfur source with or without iron, P. aeruginosa formed either a yellow-green or a blue fluorescent pigment. Formation of fluorescent pigments of P. aeruginosa is related to the ability of sulfite to act as a specific sulfur source. In an investigation of the role of both added iron and sulfur sources, complex patterns of pigment formation were observed. In addition to the fluorescent pigments, sulfite also supported the formation of slime by P. aeruginosa.     

Phenotype recognition of pyocyanine mutants in pseudomonas aeruginosa

Certain classes of pyocyanine mutants in Pseudomonas aeruginosa escape detection when screened in the presence of wild-type or other mutant cells. A technique is described for recognizing mutant phenotypes after cells are in individual agar wells. The procedure eliminates cross-feeding phenomena as well as the masking of mutant phenotypes by diffusing pyocyanine produced by nearby clones.     

Sensitivity of Haloarchaea to eubacterial pigments produced by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SB1

Pseudomonas aeruginosa SB1 was isolated from conventional well water. It produced an extracellular fluorescent bright green pigment which diffused into the growth medium. The purified pigment showed absorption maxima of 690, 370, 312 nm and was identified as pyocyanine, a known inhibitor of eubacterial and eukaryotic growth. The effect of this pigment when tested on orange-red pigmented halophilic archaeal isolates showed remarkable inhibitory activity and resulted in total growth inhibition at 4.67 mg/ml pyocyanine in 24 h. The present work is the first report on eubacterial Pseudomonas aeruginosa SB1 showing anti-Haloarchaeal activity.     

Influence of natural complex compounds with hipolipidemic activity on expression of bacteria virulence factors

Natural complex compounds with hipolipidemic activity, having considerable inhibitory effect on expression of bacteria virulence factors were isolated. Inhibitory properties of the compounds with respect to pyocyanine and protease formation, as well as their influence on the quorum sensing mechanism in Chromobacterium violacium were shown.     

Pattern of phenazine pigment production by a strain of Pseudomonas aeruginosa.

An atypical strain of Pseudomonas aeruginosa capable of synthesizing three phenazine pigments was isolated. Cultural conditions, under which the strain forms either chlororaphin, oxychlororaphin, or pyocyanine, are described. This broad spectrum of pigment production, as well as some other characteristics, sets this strain apart from previously described chlororaphin producers.     

Carbon monoxide:methylene blue oxidoreductase from Pseudomonas carboxydovorans.

The enzyme carbon monoxide:methylene blue oxidoreductase from CO autotrophically grown cells of Pseudomonas carboxydovorans strain OM5, was purified to homogeneity. The enzyme was obtained in 26% yield and was purified 36-fold. The enzyme was stable for at least 6 days, had a molecular weight of 230,000, gave a single protein and activity band on polyacrylamide gel electrophoresis, and was homogeneous by the criterion of sedimentation equilibrium. Sodium dodecyl sulfate gel electrophoresis revealed a single band of molecular weight 107,000. Carbon monoxide:methylene blue oxidoreductase did not catalyze reduction of pyridine or flavin nucleotides but catalyzed the oxidation of CO to CO2 in the presence of methylene blue, thionine, toluylene blue, dichlorophenolindophenol, or pyocyanine under strictly anaerobic conditions. The visible spectrum revealed maxima at 405 and 470 nm. The millimolar extinction coefficients were 43.9 (405 nm) and 395.5 (275 nm), respectively. Absorption at 470 nm decreased in the presence of dithionite, and the spectrum was not affected by the substrate CO. Maximum reaction rates were found at pH 7.0 and 63 degrees C; temperature dependence followed the Arrhenius equation, with an activation energy (delta H degree) of 36.8 kJ/mol (8.8 kcal/mol). The apparent Km was 53 microM for CO. The purified enzyme was incapable of oxidizing methane, methanol, or formaldehyde in the presence of methylene blue as electron acceptor.     

Comparative Analysis Between Pseudomonas aeruginosa Genotypes and Severity of Symptoms in Patients with Unilateral or Bilateral Otitis Externa

Random amplified polymorphic DNA (RAPD) analysis was done on 32 isolates of Pseudomonas aeruginosa. These isolates were obtained from 22 patients who presented to the emergency room in a major medical center in Beirut, Lebanon, during a 5-month period with the diagnosis of either unilateral or bilateral otitis externa. Patients had yellowish to greenish discharge, moderate to severe external auditory canal swelling, moderate to severe pain, and periauricular cellulitis. None of these patients had intrinsic predisposing factors. An ear swab was obtained from both ears of patients, cultured on trypticase soy agar. P. aeruginosa was identified on the basis of pyocyanine production and API identification kits. RAPD analysis was done by using two primers (10 mer and 21 mer primers) and appropriate PCR conditions on extracted DNA. Our data have shown 23 RAPD patterns (A–W) distributed among the 32 P. aeruginosa isolates. RAPD patterns were reproducible. Twenty of 32 isolates were recovered from 10 patients with bilateral otitis externa. The remaining 12 of 32 isolates were recovered from 12 different patients with unilateral otitis externa. Eleven RAPD patterns (A,B,C,D,E,F,H,I,R,U,V) were associated with severe clinical symptoms, including severe pain, severe external auditory canal swelling, periauricular cellulitis, and a yellowish discharge. The remaining RAPD patterns were not associated with severe infections. This denotes a possible association between certain genotypes and severity of symptoms. Received: 6 July 2000 / Accepted: 5 September 2000     

Mechanism of photoinactivation in photosynthetic systems III. Site and mode of photoinactivation in photosystem I

The presence of ferredoxin, PMS, pyocyanine or FMN in the preincubationmixture completely protected chloroplast fragments from photoinactivationof the activity associated with photosystem I of photosynthesis.Neither ferredoxin, plastocyanin, ferredoxin-NADP reductasenor the cytochromes contained in chloroplast fragments werethe site of photoinactivation. The site and mode of photoinactivationin system I of photosynthesis are discussed. (Received September 3, 1969; )     

The effect of zinc and cobalt ions on the chromogenesis of Pseudomonas aeruginosa in the presence of methylphosphonic acid]

Adding zinc or cobalt sulphate into synthetic culture media of Pseudomonas aeruginosa was followed by partial or total inhibition of pyocyanine production, varying with the nature of the chemical source of phosphorus. An antagonism between Zn2+ and Co2+ ions was shown.     

Regulation of the chloroplast H+-ATPase by light. The involvement of Mg2+ ions

The involvement of Mg2+ ions in the light-dependent regulation of the chloroplast H+-ATPase was studied in both type C and osmotically shocked type A chloroplasts. The following results were obtained. ATPase activity measured under dark, partially uncoupling conditions, following light activation with dithiothreitol and pyocyanine, was markedly enhanced by the presence of Mg2+ in the activation stage. This Mg2+ effect required concentrations in the millimolar range, was rather slow (time range of minutes), reversible, rather unspecific and did not involve changes in the affinity to dithiothreitol. Dark deactivation of the ATPase in the absence of substrate was accelerated by Mg2+. The dark effect of Mg2+ also required millimolar concentrations, but was fast (time range of seconds), highly specific for Mg2+, and did not involve thiol oxidation. The major effect of the absence of Mg2+ from the light-activation stage or of its presence in the dark interval between activation and assay was the induction of an 'abnormal' sensitivity to uncouplers: after these treatments ATP hydrolysis was not stimulated but rather inhibited by NH4Cl or other uncouplers. The pretreatments in the light without Mg2+ or dark with Mg2+ did not affect the membrane proton permeability, nor the proton pumping coupled to ATPase activity. The results are discussed in terms of Mg2+-dependent regulation of the enzyme complex at the level of subunit interaction and its effect on the affinity to protons.     

Pyocyanine as a potent inhibitor for the growth ofRhodopseudomonas sphaeroides B5

Pyocyanine seveely inhibited the growth ofRhodopseudomonas sphaeroides B5 under photosynthetic conditions. Under these conditions, pyocynine at concentrations of 0.05 and 0.1 g/ml gave rise to about 61% and 99% inhibition, respectively, whereas pyocyanine at a concentration of 1 g/ml was required to inhibit the growth by about 55% under aerobic dark conditions. Antibiotic action of pyocyanine under photosynthetic conditions was reveersible and not bactericidal. Degradation of carotenoid was observe in pyocyanine-treated cultures.     

Kinetics of haemolytic activity in Proteus strains.

Proteus species produces toxins and constitutes a causative agent of some chronic and recurrent infections. For the study of haemolytic activity and the production and inhibition kinetics, a total of 140 local isolates were diagnosed and examined by the general biochemical methods, and their ability of haemolysis were tested by both direct and indirect methods utilizing the enrichment procedure for all strains. Two antibiotics, erythromycin and keflex (cephalexin), were tested for the study of haemolysis inhibition and its kinetisc. Rof further study, examples of Proteus species were selected; the new approach was based on mixing procedure between P. aeruginosa (also pyocyanine) and Proteus species for inhibition of haemolytic activity. Spectrophotometric analysis were used parallel to these studies to support quantitatively the observed results as all samples show an absorption centre at 542 +/- 1 nm. Results of such analysis of haemolytic activity and inhibition kinetics are presented.     

CORRELATION OF OXIDATION AND PHOSPHORYLATION IN HEMOLYZED BLOOD IN PRESENCE OF METHYLENE BLUE AND PYOCYANINE

1. The system:hemolyzed blood + glucose never exhibits glycolysis or, in the air, oxidation of glucose. When glucose is replaced by hexosephosphate ester, addition of methylene blue causes oxidation in air. 2. When cozymase is added also, the oxidation is increased, and a synthesis of hexosephosphate esters takes place. 3. When pyocyanine is used instead of methylene blue, the rate of oxidation is the same as with methylene blue, but a synthesis of phosphate esters takes place without addition of cozymase. 4. There is never a phosphate ester synthesis without oxidation going on, but oxidation does not necessarily go hand in hand with phosphate synthesis. 5. In order to couple the oxidation process with phosphate synthesis, two methods are available: either to start oxidation by methylene blue and to add coenzyme from yeast cells; or to start oxidation by pyocyanine, in which case coenzyme is unnecessary, though it improves the effect. 6. Iodoacetate always suppresses synthesis, but only under certain conditions decreases oxidation. Cyanide has no effect upon either process.     

Sulphydryl groups in photosynthetic energy conservation. I. Light-dependent inhibition of photophosphorylation by the sulphydryl reagent 2-2'dithio bis-(5-nitropyridine).

1. The sulphydryl reagent 2-2'dithio bis-(5-nitropyridine) (DTNP) inhibited photophosphorylation when the chloroplasts were preincubated with the reagent in the light. A maximum inhibition of about 50% was obtained in the presence of pyocyanine and MgCl 2 at 0.3 mumol DTNP per mg chlorophyll and was completed in about 40 s of preillumination. 2. Dithioerythritol, ADP plus Pi (or arsenate) and uncouplers prevented the inhibition when present during the preillumination while phloridzin, Dio-9 and discarine B were ineffective. Low concentrations of ADP or ATP afforded partial protection but other nucleotides had no effect. 3. DTNP inhibited the coupled electron transport rate to the basal level and had no effect on the uncoupled electron transport. The stimulation of proton uptake and inhibition of electron transport by ATP was prevented by DTNP. 4. The trypsin-activated but not the light- and dithioerythritol-triggered ATPase was inhibited by light preincubation of chloroplasts with DTNP. 5. Reversal of DTNP inhibition of photophosphorylation was obtained by a second preillumination in the presence of thiol groups. 6. More DTNP reacted with chloroplasts in the light than in the dark. Two mol of thione were formed in the light per mol of DTNP disappeared. 7. The results suggested that DTNP inhibition is related to the oxidation by DTNP of chloroplast vicinal dithiols probably exposed by a light-induced conformational change.