Source of superoxide anion radical in aerobic mixtures consisting of NAD[P]H, 5-methylphenazinium methyl sulfate and nitroblue tetrazolium chloride

The source of superoxide anion radical (O2-.) in aerobic mixtures consisting of NAD[P]H, 5-methylphenazinium methyl sulfate (or its 1-methoxy derivative) and tetrazolium salt was investigated using superoxide dismutase (SOD), Mn(II), ferricytochrome-C, and epinephrine as probes. NAD[P]H + phenazine + O2 was found to reduce nitroblue tetrazolium, iodonitrotetrazolium, and thiazolyl blue in a manner sensitive to agents that dismutase O2-., viz., SOD and Mn(II). It also mediated the reduction of ferricytochrome-C, and augmented the autooxidation of epinephrine to the adrenochrome, without a tetrazolium salt present in the medium. The autooxidation of epinephrine, but not the reduction of ferricytochrome-C, was found to be sensitive to SOD. Nitroblue tetrazolium, either singly or in combination with SOD, did not stimulate the reduction of ferricytochrome-C. The oxidation of NADH, mediated by a catalytically low concentration of phenazine(+O2), was augmented two-fold by SOD. These observations are consistent with, and lend support to, a scheme of redox events (Scheme-3) wherein it is proposed that the source of O2-. in the NAD[P]H + phenazine + tetrazolium(+O2) system is the reduced phenazine, that the tetrazoinyl radical (a one-electron reduction product of tetrazolium) may not reduce O2 to O2-., that the redox reaction between semiquinone radicals of phenazine and O2 is reversible, and that the disproportionation of semiquinone radicals constitutes an important rate-limiting reaction in the expression of phenazine redox couple.     

phzO, a gene for biosynthesis of 2-hydroxylated phenazine compounds in Pseudomonas aureofaciens 30-84

Certain strains of root-colonizing fluorescent Pseudomonas spp. produce phenazines, a class of antifungal metabolites that can provide protection against various soilborne root pathogens. Despite the fact that the phenazine biosynthetic locus is highly conserved among fluorescent Pseudomonas spp., individual strains differ in the range of phenazine compounds they produce. This study focuses on the ability of Pseudomonas aureofaciens 30-84 to produce 2-hydroxyphenazine-1-carboxylic acid (2-OH-PCA) and 2-hydroxyphenazine from the common phenazine metabolite phenazine-1-carboxylic acid (PCA). P. aureofaciens 30-84 contains a novel gene located downstream from the core phenazine operon that encodes a 55-kDa aromatic monooxygenase responsible for the hydroxylation of PCA to produce 2-OH-PCA. Knowledge of the genes responsible for phenazine product specificity could ultimately reveal ways to manipulate organisms to produce multiple phenazines or novel phenazines not previously described.     

Effects of the microbial secondary metabolites pyrrolnitrin, phenazine and patulin on INS-1 rat pancreatic β-cells

The effects on pancreatic β-cell viability and function of three microbial secondary metabolites pyrrolnitrin, phenazine and patulin were investigated, using the rat clonal pancreatic β-cell line, INS-1. Cells were exposed to 10-fold serial dilutions (range 0-10 μg mL(-1)) of the purified compounds for 2, 24 and 72 h. After 2 h exposure, only patulin (10 μg mL(-1)) was cytotoxic. All compounds showed significant cytotoxicity after 24 h. None of the compounds altered insulin secretion with 2 and 20 mM glucose after 2 h. However, after 24 h treatment, phenazine and pyrrolnitrin (10 and 100 ng mL(-1)) potentiated insulin production and glucose-stimulated insulin secretion, whereas patulin had no effect. Exposure (24 h) to either phenazine (100 ng mL(-1)) or pyrrolnitrin (10 ng mL(-1)) caused similar increases in the Ca(2+) content of INS-1 cells. The outward membrane current was inhibited after 24 h exposure to either phenazine (100 ng mL(-1)) or pyrrolnitrin (10 or 100 ng mL(-1)). This study presents novel data suggesting that high concentrations of pyrrolnitrin and phenazine are cytotoxic to pancreatic β-cells and thus possibly diabetogenic, whereas at lower concentrations these agents are nontoxic and may be insulinotropic. The possible role of such agents in the development of cystic fibrosis-related diabetes is discussed.     

Mechanism of aromatic hydroxylation. Properties of a model for pyridine nucleotide-dependent flavoprotein hydroxylases

A model (NADH-phenazine methosulfate-O₂) formally similar to pyridine nucleotide-dependent flavoprotein hydroxylases catalyzed the hydroxylation of several aromatic compounds. The hydroxylation was maximal at acid pH and was inhibited by ovine Superoxide dismutase, suggesting that perhydroxyl radicals might be intermediates in this process. The stoichiometry of the reaction indicated that a univalent reduction of oxygen was occurring. The correlation between the concentration of semiquinone and hydroxylation, and the inhibition of hydroxylation by ethanol which inhibited semiquinone oxidation, suggested the involvement of phenazine methosulfate-semiquinone. Activation of hydroxylation by Fe³⁺ and Cu²⁺ supported the contention that univalently reduced species of oxygen was involved in hydroxylation. Catalase was without effect on the hydroxylation by the model, ruling out H₂O₂ as an intermediate. A reaction sequence, involving a two-electron reduction of phenazine methosulfate to reduced phenazine methosulfate followed by disproportionation with phenazine methosulfate to generate the semiquinone, was proposed. The semiquinone could donate an electron to O₂ to generate O₂ which could be subsequently protonated to form the perhydroxyl radical.     

Hexaammineruthenium as an electron donor to mitochondrial cytochrome oxidase: membrane potential generation in the absence of cytochrome c

Cytochrome c oxidase can generate membrane potential in the absence of cytochrome c (e.g., in cytochrome c-deficient mitochondria or in proteoliposomes) with hexaammineruthenium as an artificial electron donor. Of several other redox mediators tested, phenazine methosulfate was found to be an efficient artificial substrate for membrane energization by cytochrome oxidase, whereas TMPD, DAD, DCPIP or ferrocyanide are virtually ineffective. The ability of Ru(NH3)6(2+) and phenazine methosulfate to support the generation of delta psi by cytochrome c-oxidase correlates with their effectiveness as electron donors to cytochrome a in the cyanide-inhibited membrane-bound enzyme.     

Active transport of manganese in isolated membrane vesicles of Bacillus subtilis.

Membrane vesicles isolated from cells of bacillus subtilis W23 accumulate manganese in the presence of an energy source. The artificial electron donor system ascorbate and phenazine methosulfate or reduced nicotinamide adenine dinucleotide and phenazine methosulfate can supply the energy for the uptake. D-Lactate in the presence or absence of phenazine methosulfate would not support manganese accumulation. Anaerobiosis, cyanide, m-chlorophenyl carbonylcyanide hydrozone, valinomycin, gramicidin, and p-hydroxy-mercuribenzoate inhibit the uptake. The inhibition by p-hydroxymercuribenzoate is prevented by excess dithiothreitol. Potassium fluoride or sodium arsenate has no effect on the uptake. The manganese transport system in the B. subtilis vesicles exhibits Michaelis-Menten kinetics with a Km of 13 muM and a Vmax of 1.7 nmol/min per mg (dry weight) of membranes. The uptake of manganese is specific and is not inhibited by 0.1 mM CaCL2 or Mgcl2.     

Determination of serum and tissue levels of phenazines including clofazimine

A rapid and sensitive HPLC method is described for the analysis of synthetic phenazines, including clofazimine, from a variety of biological samples. Phenazines were extracted from serum, tissue and fat using a mixture of dichloromethane and sodium hydroxide. The drugs were then quantified on a reversed-phase C₁₈ column using a mobile phase consisting of 594 ml of water, 400 ml of tetrahydrofuran, 6 ml of concentrated acetic acid and 0.471 g of hexanesulfonic acid. In this mobile phase, each phenazine tested had its own retention time. This allowed one phenazine to be used as an internal standard for the analysis of other phenazines. The method was validated for clofazimine [3-(4-chloroanilino)-10-(4-chlorophenyl)-2,10-dihydro-2-(isopropylimino)phenazine] and B4090 [7-chloro-3-(4-chloranilino)-10-(4-chlorophenyl)-2,10-dihydro-2-(2,2,6,6-tetramethylpiperid-4-ylimino)phenazine] (VI) and shown to be accurate and precise across a broad concentration range from 0.01 to 50 μg/g (μg/ml). Extraction was 100% for each agent across this range. This system was used to measure clofazimine and VI levels following their administration to rats. The pharmacokinetic profile of VI was different to that of clofazimine, with high tissue concentrations but lower fat levels.     

On the mechanism of production of superoxide radical by reaction mixtures containing NADH, phenazine methosulfate, and nitroblue tetrazolium

In aerobic reaction mixtures containing NADH, phenazine methosulfate, and nitroblue tetrazolium, O2- production is mediated by the tetrazolium, not the phenazine. Thus, superoxide dismutase inhibited reduction of the tetrazolium, but when ferricytochrome c was substituted for the tetrazolium its reduction was not affected by this enzyme. Furthermore, NADH plus the phenazine did not accelerate the oxidation of epinephrine to adrenochrome unless the tetrazolium was present, and under those circumstances superoxide dismutase did inhibit adrenochrome formation. When the tetrazolium and ferricytochrome c were present simultaneously, addition of superoxide dismutase was seen to accelerate the reduction of the cytochrome. This is explainable by the reduction of O2- by the reduced phenazine, which thus competes with cytochrome c for the available O2-. When the O2- was eliminated by superoxide dismutase, more of the reduced phenazine was available for the direct reduction of cytochrome c.     

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction.     

A chemical method for the quantitative determination of 2-hydroxyestrone in human urine

A highly specific chemical procedure for the quantitative determination of 2-hydroxyestrone in the urine of pregnant women is described. The assay consists of the following steps: 1) Hot acid hydrolysis of 20 ml of urine, 2) purification of 2-hydroxyestrone by “reducing chromatography” on paper and silica gel column, 3) conversion of 2-hydroxyestrone to the phenazine compound, 4) purification of the phenazine derivative by alumina column chromatography, and 5) spectroscopic quantitation of the phenazine. For internal yield correction [4-¹⁴C]2-hydroxyestrone is added after urine hydrolysis. High specificity of the method is especially guaranteed by the specific transformation of 2-hydroxyestrone to a stable phenazine derivative and by rigorous chromatographic purification of the estrogen as well as of the phenazine. The method can be used for the determination of amounts of less than 1 μg of 2-hydroxyestrone/20 ml of urine. From the data obtained the coefficient of variation is calculated to be ±3.7%. The urinary excretion of 2-hydroxyestrone in late pregnancy was found to vary within a wide range of 30–800 μg of 2-hydroxyestrone/24 hours.It seems possible to extend this method to the determination of other 2-substituted estrogens present in urine.     

rpoS基因插入突变对铜绿假单胞菌两个吩嗪合成基因簇的调控

【目的】为了研究铜绿假单胞菌rpoS基因对吩嗪(Phenazine)合成基因簇phz1和phz2的调控方式与机制。【方法】采用抗庆大霉素基因(gentamycin resistance cassette,aacC1)插入失活的策略构建了rpoS基因突变株PA-SG;同时利用lacZ的翻译融合表达载体pME6015,构建了phz1′-′lacZ和phz2′-′lacZ翻译融合表达载体pMEZ1和pMEZ2。采用电转化法分别将pMEZ1、pMEZ2和pME6015导入铜绿假单胞菌突变株PA-SG和野生株PAO1,用Miller法检测融合β-半乳糖苷酶活性。【结果】在KMB或PPM培养基中,pMEZ1在突变株PA-SG中的表达均增强,为野生株的4-5倍;而pMEZ2在突变株PA-SG中的表达均降低,野生株是突变株的2-3倍。【结论】由此推测,铜绿假单胞菌rpoS基因对两个不同吩嗪合成基因簇的调控作用具有特异性,在一定程度上,rpoS负调控phz1,正调控phz2。     

Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici.

Pseudomonas fluorescens 2-79 (NRRL B-15132) and its rifampin-resistant derivative 2-79RN10 are suppressive to take-all, a major root disease of wheat caused by Gaeumannomyces graminis var. tritici. Strain 2-79 produces the antibiotic phenazine-1-carboxylate, which is active in vitro against G. graminis var. tritici and other fungal root pathogens. Mutants defective in phenazine synthesis (Phz-) were generated by Tn5 insertion and then compared with the parental strain to determine the importance of the antibiotic in take-all suppression on wheat roots. Six independent, prototrophic Phz- mutants were noninhibitory to G. graminis var. tritici in vitro and provided significantly less control of take-all than strain 2-79 on wheat seedlings. Antibiotic synthesis, fungal inhibition in vitro, and suppression of take-all on wheat were coordinately restored in two mutants complemented with cloned DNA from a 2-79 genomic library. These mutants contained Tn5 insertions in adjacent EcoRI fragments in the 2-79 genome, and the restriction maps of the region flanking the insertions and the complementary DNA were colinear. These results indicate that sequences required for phenazine production were present in the cloned DNA and support the importance of the phenazine antibiotic in disease suppression in the rhizosphere.     

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C.

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction.     

Novel synthetic isoquinolino[5,4-ab]phenazines: inhibition toward topoisomerase I, antitumor and DNA photo-cleaving activities

The novel DNA interactive isoquinolino[5,4-ab]phenazine derivatives were designed and synthesized. Their inhibitory abilities toward topoisomerase I, antitumor activities and DNA photo-cleaving abilities were examined. The substituents at peri sites of two phenazine N atoms played very important roles for all these biological activities. At a concentration of 100 microM, all these phenazine derivatives (but A2 and A6) exhibited an inhibitory activity toward topoisomerase I. A6 had efficient antitumor activities against both human lung cancer cell (A549) and murine leukemia cell (P388). A1, A5, and A6 exhibited antitumor activities selectively against P388. A2 was the most efficient DNA photocleaver, which had converted supercoiled DNA from form I to form II at <1 microM. Under anaerobic conditions, the electron transfer mechanism mainly contributed to DNA photo-induced cleavage, while under aerobic conditions, superoxide anion was also involved in this process.     

Population Structure and Diversity of Phenazine-1-Carboxylic Acid Producing Fluorescent Pseudomonas spp. from Dryland Cereal Fields of Central Washington State (USA)

Certain strains of the rhizosphere bacterium Pseudomonas fluorescens contain the phenazine biosynthesis operon (phzABCDEFG) and produce redox-active phenazine antibiotics that suppress a wide variety of soilborne plant pathogens. In 2007 and 2008, we isolated 412 phenazine-producing (Phz(+)) fluorescent Pseudomonas strains from roots of dryland wheat and barley grown in the low-precipitation region (<350 mm annual precipitation) of central Washington State. Based on results of BOX-PCR genomic fingerprinting analysis, these isolates, as well as the model biocontrol Phz(+) strain P. fluorescens 2-79, were assigned to 31 distinct genotypes separated into four clusters. All of the isolates exhibited high 16S rDNA sequence similarity to members of the P. fluorescens species complex including Pseudomonas orientalis, Pseudomonas gessardii, Pseudomonas libanensis, and Pseudomonas synxantha. Further recA-based sequence analyses revealed that the majority of new Phz(+) isolates (386 of 413) form a clade distinctly separated from P. fluorescens 2-79. Analysis of phzF alleles, however, revealed that the majority of those isolates (280 of 386) carried phenazine biosynthesis genes similar to those of P. fluorescens 2-79. phzF-based analyses also revealed that phenazine genes were under purifying selection and showed evidence of intracluster recombination. Phenotypic analyses using Biolog substrate utilization and observations of phenazine-1-carboxylic acid production showed considerable variability amongst members of all four clusters. Biodiversity indices indicated significant differences in diversity and evenness between the sampled sites. In summary, this study revealed a genotypically and phenotypically diverse group of phenazine producers with a population structure not seen before in indigenous rhizosphere-inhabiting Phz(+) Pseudomonas spp.     

Interaction of DNA with phenazine series compounds]

Complexes of DNA with phenazine compounds: neutral red, phenosaphranine; and 3-amino-7-dimethylamino-2-methyl-5-phenylphenazine were studied by viscosimetry and the dynamic birefringence technique. It is shown that, when complexing with DNA, each of the ligands induces specific changes in its macromolecular structure. Models of the binding of the compounds to DNA are proposed. It is shown that, depending on the location of bulk substituents of the phenazine chromophore, several variants of intercalation binding are realized, which differ by the orientation of the chromophore in the intercalation site.     

Role of glutamine synthetase in phenazine antibiotic production by Pantoea agglomerans Eh1087

Pantoea agglomerans strain Eh1087 produces the phenazine antibiotic D-alanylgriseoluteic acid. A glutamine auxotroph harboring an insertion in a putative glnA gene was obtained by transposon-mutagenesis of Eh1087 that produced less D-alanylgriseoluteic acid than the parental strain (strain Eh7.1). Cosmids encoding the Eh1087 glnA were isolated by their ability to complement the mutant for prototrophy. The role of the Eh1087 glnA locus was functionally confirmed by complementation of an Escherichia coli glnA mutant. Analysis of the nucleotide and deduced amino acid sequences of the Eh1087 glnA gene indicated a high degree of similarity to the glnA genes and glutamine synthetase enzymes of other Enterobacteriaceae. Isotopic labelling experiments with 15N-labelled ammonium sulfate demonstrated that wild-type Eh1087 incorporated 15N into griseoluteic acid more readily than the glnA mutant Eh7.1. We conclude that the 2 nitrogens in the phenazine nucleus originate from glutamine and the intracellular glutamine synthesized by Eh1087 is a source of the phenazine nucleus nitrogens even in glutamine-rich environments.