The formation of a blue pigment in the bacterial oxidation of isonicotinic acid

Summary A pseudomonad was isolated from soil which can utilize isonicotinic acid as its sole carbon source. Growth on this substrate leads to the accumulation of a compound with absorption maxima at 240 and 345 m which was isolated and identified as citrazinic acid. Subsequent to citrazinic acid formation a soluble blue pigment is produced. This pigment was crystallized and shown to be identical to one formed by the chemical oxidation of citrazinic acid. The pigment, although similar in properties, is distinct from those arising during the metabolism of 2-hydroxypyridine, nicotinic acid and nicotine by other bacteria. The possible structure of the pigment is discussed and a pathway of its formation from isonicotinic acid is suggested.     

Growth and pigment production by Arthrobacter pyridinolis n. sp.

A new bacterium capable of growing on 2-hydroxypyridine as sole source of carbon and nitrogen was isolated from soil. During its growth on solid medium, approximately 50% of this substrate was converted to a brilliant blue crystalline pigment which was deposited extracellularly in the colony mass. The pigment was identical to that produced by Arthrobacter crystallopoietes during its growth on 2-hydroxypyridine. The new isolate exhibited the typical cycle of morphogenesis characteristic of the genus Arthrobacter. The organism is different from all other reported species of Arthrobacter. It is proposed that the organism be named Arthorbacter pyridinolis n. sp.     

Growth and pigment production byArthrobacter pyridinolis n. sp.

A new bacterium capable of growing on 2-hydroxypyridine as sole source of carbon and nitrogen was isolated from soil. During its growth on solid medium, approximately 50% of this substrate was converted to a brilliant blue crystalline pigment which was deposited extracellularly in the colony mass. The pigment was identical to that produced byArthrobacter crystallopoietes during its growth on 2-hydroxypyridine. The new isolate exhibited the typical cycle of morphogenesis characteristic of the genusArthrobacter. The organism is different from all other reported species ofArthrobacter. It is proposed that the organism be namedArthrobacter pyridinolis n. sp.List of Abbreviations MSP mineral salts phosphate basal culture medium containing 2-hydroxypyridine, yeast extract and trace salts - 2-HP 2-hydroxypyridine - PFU plaque forming units - G+C guanine+cytosine - T _m midpoint of thermal denaturation     

Structure of green pigment formed by the reaction of caffeic acid esters (or chlorogenic acid) with a primary amino compound.

A marked greening observed in some foods such as sweet potato, burdock, and others during food processing was shown to be due to green pigment formation by the condensation reaction of two molecules of chlorogenic acid or caffeic acid ester with one molecule of a primary amino compound under aeration in alkaline solution. Reduction of the green pigment by ascorbic acid or NaBH4 gave a yellow product, which readily turn green and then blue in air. The reduced and acetylated product of the green pigment was identified to be a novel trihydroxy benzacridine derivative, and the yellowish ethanol solution of this product immediately turned green upon addition of butyl amine or diluted alkali. Therefore, the green pigment was assumed to be an oxidized quinone type product of trihydroxy benzacridine. This identification of the structure was supported by the correspondence of the measured absorption spectra with those calculated by the molecular orbital method. A possible charge transfer complex between products of different oxidation steps in green solution was proposed.     

A 2-Hydroxypyridine Catabolism Pathway in Rhodococcus rhodochrous Strain PY11

Rhodococcus rhodochrous PY11 (DSM 101666) is able to use 2-hydroxypyridine as a sole source of carbon and energy. By investigating a gene cluster (hpo) from this bacterium, we were able to reconstruct the catabolic pathway of 2-hydroxypyridine degradation. Here, we report that in Rhodococcus rhodochrous PY11, the initial hydroxylation of 2-hydroxypyridine is catalyzed by a four-component dioxygenase (HpoBCDF). A product of the dioxygenase reaction (3,6-dihydroxy-1,2,3,6-tetrahydropyridin-2-one) is further oxidized by HpoE to 2,3,6-trihydroxypyridine, which spontaneously forms a blue pigment. In addition, we show that the subsequent 2,3,6-trihydroxypyridine ring opening is catalyzed by the hypothetical cyclase HpoH. The final products of 2-hydroxypyridine degradation in Rhodococcus rhodochrous PY11 are ammonium ion and α-ketoglutarate.     

Biodegradation of 2-methyl, 2-ethyl, and 2-hydroxypyridine by an Arthrobacter sp. isolated from subsurface sediment

A bacterium capable of degrading 2-methylpyridine was isolated by enrichment techniques from subsurface sediments collected from an aquifer located at an industrial site that had been contaminated with pyridine and pyridine derivatives. The isolate, identified as an Arthrobacter sp., was capable of utilizing 2-methylpyridine, 2-ethylpyridine, and 2-hydroxypyridine as primary C, N, and energy sources. The isolate was also able to utilize 2-, 3-, and 4-hydroxybenzoate, gentisic acid, protocatechuic acid and catechol, suggesting that it possesses a number of enzymatic pathways for the degradation of aromatic compounds. Degradation of 2-methylpyridine, 2-ethylpyridine, and 2-hydroxypyridine was accompanied by growth of the isolate and release of ammonium into the medium. Degradation of 2-methylpyridine was accompanied by overproduction of riboflavin. A soluble blue pigment was produced by the isolate during the degradation of 2-hydroxypyridine, and may be related to the diazadiphenoquinones reportedly produced by other Arthrobacter spp. when grown on 2-hydroxypyridine. When provided with 2-methylpyridine, 2-ethylpyridine, and 2-hydroxypyridine simultaneously, 2-hydroxypyridine was rapidly and preferentially degraded; however there was no apparent biodegradation of either 2-methylpyridine or 2-ethylpyridine until after a seven day lag. The data suggest that there are differences between the pathway for 2-hydroxypyridine degradation and the pathway(s) for 2-methylpyridine and 2-ethylpyridine.     

Bildung kristallinen Nicotinblaus beim Abbau von Nicotin durchArthrobacter oxydans

Zusammenfassung Es wird einArthrobacter oxydans beschrieben, derl-,DL- undd-Nicotin über 6-Hydroxynicotin abbaut und sogenanntes Nicotinblau bildet. Dieser Farbstoff kann unter Verwendung eines speziellen Mediums in Form brillantgrüner Kristalle isoliert werden, die sich auf Agarplatten in den Zentren der Kolonien abscheiden. Einige Eigenschaften des kristallinen Nicotinblaus werden angegeben.

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Formation of crystalline nicotine blue by a nicotine degradingArthrobacter oxydans

Summary A strain ofArthrobacter oxydans is described which degradesl-,DL-, andd-nicotine, forming nicotine blue. Under specified growth conditions this pigment is deposited as brillant green crystals in the center of colonies on agar. Some properties of the isolated crystalline nicotine blue are described.

     

The mechanism of the periodate–thiobarbituric acid reaction of sialic acids

1. The chromogen formation from N-acetylneuraminic acid in the periodate-thiobarbituric acid reaction was investigated. Measurement of periodate consumption showed an uptake of approx. 3moles/mole of substrate in neutral as well as in strongly acidic solution. Therefore the chromogen beta-formylpyruvic acid is not a direct product of the periodate oxidation; it is presumed to be formed from the true oxidation product, a hexos-5-uluronic acid, by aldol splitting during the reaction in hot acidic solution with thiobarbituric acid. 2. Methyl (methyl beta-l-threo-hexos-4-enepyranosid)uronate, an analogue of the pre-chromogen, has been shown to yield with thiobarbituric acid in acidic solution a pigment exhibiting an identical absorption spectrum and showing the same behaviour on paper chromatography as the pigment obtained from N-acetylneuraminic acid in the periodate-thiobarbituric acid assay. 3. The substitution at C-2 of methoxyneuraminic acid does not inhibit the periodate-thiobarbituric acid reaction. In neutral solution methoxyneuraminic acid is oxidized by periodate to a substance that reacts readily with thiobarbituric acid in acidic solution. When periodate oxidation is attempted in acidic solution, protonation of the amino group protects this group against oxidation, rendering methoxyneuraminic acid negative in the assay systems of Warren (1959a,b) and Aminoff (1959, 1961).     

Action Spectra for Step-Up Photophobic Response in Blepharisma

ABSTRACT The cells of Blepharisma which possess red pigment (blepharismin) show step-up photophobic response (temporal ciliary reversal induced by a sudden increase in light intensity). Bleaching of the cells by cold shock raised a threshold light intensity for the response, Oxidation of red pigment that produced blue pigment did not raise the threshold for the response. The action spectrum for the step-up photophobic response of the cells which possess normal red pigment had peaks at about 580, 540 and 490 nm, a value which coincided with peaks of an absorption spectrum of the red pigment. The absorption spectrum of oxidized pigment (blue pigment) shifted 20 nm toward infrared light. The action spectrum for the response of the cells which possess blue pigment also shifted 20 nm toward infrared light. Results suggest that red pigment might be involved in the step-up photophobic response. Key words. Blepharismin, ciliary reversal, photoreceptors, photoresponse.     

Der Lichteinfluss auf die Haarbildung am Hypokotyl vonSinapis alba L.

Summary In an earlier paper (Mohr 1957) it was described that the formation of anthocyanin and the inhibition of lengthening of the hypocotyl of dark grown seedlings (Sinapis alba) is governed by the action of two photomorphogenic action systems. The one system is the well known reversible red far-red reaction system (low energy reaction), the other one is a high energy reaction system which can be called — in reference to the action peaks in the far-red and in the blue — blue far-red reaction system. The chemical nature of the absorbing pigments is still unknown.In the present paper another photomorphogenic response of the young dark grown seedlings ofSinapis alba, the light dependent formation of unicellular hairs from epidermal cells of the hypocotyl, has been investigated. It has been shown that this response is also governed by these two reaction systems. These systems have been physiologically separated. Experiments have been presented which evaluate the importance of the assumption that the red absorbing pigment of the reversible red far-red pigment system is reformed from a precursor when the pigment present before irradiation is transformed into the far-red absorbing pigment by an irradiation with red.

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A visual pigment from chicken that resembles rhodopsin: amino acid sequence, gene structure, and functional expression.

The amino acid sequence of a rhodopsin-like visual pigment from chickens has been determined by isolating and sequencing its gene. The predicted sequence is between 70% and 80% identical to bovine, human, and chicken rhodopsins and between 40% and 50% identical to human blue, green, and red cone pigments, the chicken red cone pigment, and cavefish long-wave cone pigments. The encoded pigment, produced by transfection of cDNA into cultured cells, absorbs maximally at 495 nm as determined from photobleaching difference spectra and reacts at 20 degrees C with 50 mM hydroxylamine with a half-time of 16 min. These properties, together with a high pI predicted from the amino acid sequence, suggest that this cloned gene encodes the chicken green pigment previously identified by biochemical and spectroscopic studies. This sequence defines a new branch of the visual pigment gene family.     

The metabolism of naphthalene and its toxic effect on the eye

1. Naphthalene (1g./kg.) was fed daily by stomach tube to rabbits. 2. In more than half of the rabbits opacities in the lens and degeneration of the retina were visible in vivo. 3. Dissection of eye tissues revealed some or all of the following changes: a browning of the lens and eye humours, blue fluorescence of the eye humours and crystals in the retina and vitreous body. 4. The ascorbic acid concentration of the eye humours was decreased. 5. Some metabolites of naphthalene [1,2-dihydro-1,2-dihydroxynaphthalene, 2-hydroxy-1-naphthyl sulphate and (1,2-dihydro-2-hydroxy-1-naphthyl glucosid)uronic acid] are converted enzymically by the tissues of the eye into 1,2-dihydroxynaphthalene. 6. Changes in the eye are consistent with 1,2-dihydroxynaphthalene's being the primary toxic agent. The properties and reactions of this substance are described. 7. 1,2-Dihydroxynaphthalene is readily autoxidizable in neutral solution to form the yellow 1,2-naphthaquinone and hydrogen peroxide. This oxidation is reversed by ascorbate. 8. Ascorbate is oxidized catalytically by 1,2-naphthaquinone. This may account for the disappearance of ascorbate from the aqueous and vitreous humours of the eye after naphthalene feeding. It may also account for the appearance of crystals of calcium oxalate in the eye. 9. The brown colour of the lens of the naphthalene-fed rabbit is due to presence of naphthaquinone–protein compounds.     

Red Pigment Produced by the Reaction of Dehydro-l-ascorbic Acid with α-Amino Acid

At the initial stage of the browning reaction of dehydro-l-ascorbic acid (DHA) with α-amino acid, a kind of red pigment was produced. The pigment was isolated as very hygroscopic red powder from non-aqueous reaction system, and its characterization was made. It was revealed that it had the same structure with that of the red pigment produced by the oxidation of l-scorbamic acid, an intermediate amino-reductone expected to be produced by Strecker degradation. Formation mechanism of the pigment which was considered to be an intermediate of browning reaction of DHA with α-amino acid was also discussed.     

Photooxidation of human serum albumin and its complex with bilirubin.

Irradiation with visible light of human serum albumin in aqueous solution at pH 8, in the presence of catalytic amounts of rose bengal or methylene blue, resulted in random oxidation of the histidine residues in the protein under consumption of one mole O2, and release of somewhat less than one proton, per histidine residue degraded. An increase of light absorption at 250 nm was proportional to the amount of oxygen consumed. Bilirubin bound to the oxidized protein showed an increased light absorption at its maximum, 460 nm, and a decreased binding affinity, indicating a conformational change of the protein on oxidation of histidine residues. This change also resulted in a slight perturbation of tyrosine light absorption, corresponding to a shift of the chromophore to more polar surroundings. Further, a sensitized oligomerization of albumin was observed, independent of oxidation of the histidine residues, and not consuming oxygen. Irradiation of a complex of human serum albumin with one molecule of bound bilirubin, in the absence of a sensitizing dye, resulted in a fast, non-oxygen consuming process whereby the light absorption maximum of the pigment was shifted 4 nm towards longer wavelength and part of the bilirubin was converted to a more polar pigment, bound less firmly to the protein. This was followed by a relatively slow oxidation of the pigment under uptake of one mole O2. Parallel photooxidation of the protein carrier could not be detected. It is considered possible that the fast, anaerobic process is operative in phototherapy of hyperbilirubinemia in the newborn. Serum albumin is probably not oxidized during this treatment.     

Biosynthesis and cytoplasmic accumulation of a chlorinated catechol pigment during 3-chlorobenzoate aerobic co-metabolism in Pseudomonas fluorescens

A strain of Pseudomonas fluorescens was capable of co-metabolizing 3-chlorobenzoic acid with the production of a chlorinated catechol black pigment. A peroxidase and another enzymatic activity referred to as a polyphenol oxidase were found to be involved in the oxidation of 4-chlorocatechol to 4-chloro-1,2-benzoquinone, i.e. in the production of highly reactive substrates for pigment formation. Therefore, P. fluorescens cells were seen to take an active part not only in 3-chlorobenzoate mineralization but also in overall pigment production. pH was found to be a key parameter in the regulation of the activity of P. fluorescens oxidoreductive enzymes. Ultrastructural investigations showed that electron dense granules of pigment were distributed throughout the cytoplasm of Pseudomonas fluorescens cells grown in presence of 3-chlorobenzoate, as confirmed also by Thiéry cytochemical investigations.In these cells, an extensive contraction of the cytoplasm as well as a significant damage to the cell wall after two days of incubation, suggested that pigment production caused a premature death of the cells accompanied by the leakage of the cell content. Pigment production seemed to occur mostly in the cytoplasmic context where the electron dense material accumulates until it is released in the medium after the cell lysis.Abbreviations 3-CBA 3-chlorobenzoic acid - BA benzoic acid - 4-CC 4-chlorocatechol - 3-CC 3-chlorocatechol - MBTH 3-methyl-2-benzothiazolinone hydrazone - l-DOPA l-3,4-dihydroxyphenyl-alanine - SPB sodium phosphate buffer     

Die Aufnahme von 14CO2 und die Verteilung des 14C auf freie Aminosäuren und auf Proteinaminosäuren im Hellrot und im Blaulicht. [Objekt: Farnvorkeime von Dryopteris filix-mas (L.) Schott]

Summary Morphogenesis and metabolism of the early gametophytes (= sporelings) of the common male fern are controlled by light. The normal two-dimensional development of the gametophytes in white or blue light is correlated with an increase in protein content; inred light alone, on the other hand, the sporelings remain filamentous, and the protein content is markedly lower (cf. Mohr, 1965). The problem has been whether blue light increases the rate of protein synthesis or decreases the rate of protein degradation. This problem was solved in the present paper by the use of ¹⁴CO₂. Blue light promotes specifically the rate of protein synthesis as indicated by the increase of ¹⁴C incorporation into protein-bound amino acids under blue light as compared with red light.Using ¹⁴CO₂ we have analyzed the kinetics of free amino acid synthesis (Fig. 4) and protein synthesis (Fig. 5) under steady state conditions of photosynthetic CO₂ incorporation in blue or red light (Fig. 3). Under our conditions the rate of photosynthesis is about 1.5 times higher under blue light than under red light (Fig. 3, Table 1).The facts that the total pool sizes of the free amino acids are smaller in blue than in red light (v. Deimling and Mohr, 1967; Table 2) and that, on the other hand, the ¹⁴C-contents of the thoroughly labelled amino acid pools are virtually identical in blue and red (Table 3) indicate (a) that the pool sizes of these labelled amino acids may be equal in both light qualities and (b) that there is a compartmentation of free amino acid pools in the fern sporeling. This problem will be dealt with more in detail in a forthcoming paper on the behaviour of alanine in the fern sporeling (Payer, 1969).Protein synthesis is obviously much stronger under blue light than under red light. The detailed kinetics (Fig. 5b) indicate the involvement of two sorts of proteins: a relatively small part with high turnover which is rapidly labelled with a small but significant difference in red and blue, and a larger part with a slower turnover, the synthesis of which is strongly favored by blue light. — The first sort could be enzyme protein; the latter sort might be structural protein of the chloroplasts. These organelles increase dramaticly in size under the influence of blue light (Bergfeld, 1963). The amino acid composition of the protein, however, does not show any qualitative difference in gametophytes grown in blue or red light (v. Deimling and Mohr, 1967, Table 4).

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Die Aufnahme von ¹⁴CO₂ und die Verteilung des ¹⁴C auf freie Aminosäuren und auf Proteinaminosäuren im Hellrot und im Blaulicht. [Objekt: Farnvorkeime von Dryopteris filix-mas (L.) Schott]

     

Oxidation of cysteine to cystine by membrane fractions of Chlorella fusca

Isolated membrane fractions of Chlorella fusca 211-8b obtained by french-press treatment and sonication catalyzed the oxidation of l-cysteine to l-cystine. The pH-optimum of this reaction was determined to be around 8–8.5 and a stoichiometry of 4 SH-groups oxidized for one O₂ consumed was obtained. This thiol-oxidation system was specific for D-and l-cysteine; Dl-homocysteine and cysteamine were oxidized at about half the rate whereas all other thiols tested including glutathione, mercaptoethanol, mercaptopropionic acid and dithioerythritol were not oxidized by these membrane fractions. The apparent K_m for l-cysteine was determined as 3.3 mmol l^-1. Rates of 200 mol cysteine oxidized mg^-1 chlorophyll h^-1 were normally obtained. Extremely high rates of oxygen uptake were measured using l-cysteine methyl ester and l-cysteine ethyl ester. This thioloxidation system was not inhibited by mitochondrial electron-transport inhibitors such as rotenone or antimycin A, nor by the chloroplast electron-transport inhibitors 2,5-dibromothymochinone and 2,4-dinitrophenylether of iodonitrothymol. The cysteine oxidation catalyzed by C. fusca membranes was inhibited, however, by salicylhydroxamic acid, o-phenanthrolin, N,N-disalicyliden-1,3-diaminopropane 5,5-disulfonic acid, ethylenediaminetetraacetic acid, high KCN levels and by the buffers, N-[2-hydroxyl-1,1-bis(hydroxymethyl) ethyl] glycine and phosphate. This cysteine-oxidation system seems to function as a counterpart of thioredoxin-mediated light activation of enzymes, allowing reduced thiol groups to be oxidized again by O₂ (dark inactivation).Abbreviation DTNB 5,5-dithio-bis(-2-nitrobenzoic acid). Ellmann reagent     

The ultrastructure of the nauplius eye ofSapphirina (Crustacea: Copepoda)

Summary The ultrastructure of the specialized nauplius eye of three species of the copepod genusSapphirina was investigated. The gross morphology described earlier (Elofsson, 1966a) was confirmed. The ventral cup is covered by a red pigment and the lateral cups by a red and a black pigment. The ultrastructural configuration of the pigment granules was found to differ in the two kinds of pigment cells. The black pigment cell, moreover, contains a large number of transversely banded fibrils and is able to produce reflecting crystals. The pigment granules of the black pigment cell show a variation in electron density. An intimate connexion exists between the black pigment cell and large retinula cells in the lateral cups, indicating an exchange of material. The tapetal cells present in all three cups form crystal platelets contained in two sets of membranes. It is suggested that the ventral cup and part of the lateral cups function as thePecten-eye (Land, 1965). The rhabdomeres of the retinula cells are composed of microvilli measuring 400 Å. The orientation of these seems to exclude polarotactic behaviour. The ventral cup and the four small cells of the lateral cups contain some retinula cells with microvilli arranged parallel to the incoming light. The retinula cells further develop an intricate system of membrane-invaginations penetrating deep into the cell and associated with numerous mitochondria. Retinula cells of the ventral cup and part of the lateral cups contain clear portions filled with granular material only. Retinula and other cells contain attenuated mitochondria with parallel tubuli. The proximal lens in front of each lateral cup consists of one cell. A development from the conjunctival cells is suggested. The results are evaluated in terms of function and evolution.This work has been supported by a grant from the Swedish Natural Science Research Council (2760-2).     

金属离子对黑米花青苷色素吸收光谱的影响

以黑糯B糙米皮为实验材料 ,用 1 .5mol/L盐酸— 95 %乙醇 (V/V :1 5 / 85 )溶液提取黑米花青苷色素(BRAP) ,采用紫外可见分光光度法研究了 1 1种金属离子以及 (NH4 ) 1+ 离子对BRAP的作用。结果表明 ,未加离子条件下色素溶液可见光区λmax5 35nm ,紫外光区λmax2 80nm ,加入Al3 + 、Fe3 + 、Fe2 + 、Cu2 + 、Mn2 + 、Zn2 + 、Sn2 + 对其吸收光谱有显性影响。其中Al3 + 、Fe3 + 使 5 35nm特征吸收峰发生蓝移 ,Sn2 + 使其发生明显红移 ;Al3 + ,Fe2 + ,Mn2 + ,Zn2 +在 5 35nm附近有增加ABS值作用 ,Fe3 + 有减小ABS值作用 ;延长作用时间 ,Cu2 + 对BRAP吸收光谱的影响表现为λmax5 35nm发生蓝移 ,ABS值减小     

The intracellular blue pigment of Pseudomonas lemonnieri

Summary The large-scale production, isolation, and purification are described of the blue insoluble intracellular pigment of the bacterium Pseudomonas lemonieri. The pigment, C₂₆H₃₇N₅O₆, occurs in the cells as a salt (cation unknown) of 6-octanoylamino-3-hydroxy-2-aza-benzoquinone-(1,4)-4-[5-octanoylamino-2,6-dihydroxy-pyridyl-(3)-imide] (I). Nitric acid oxidation of pigment I yields IV, 6-octanoylamino-3-hydroxy-2-aza-benzoquinone-(1,4). Further hydrolysis of IV splits off n-octanoic acid, which is free of homologues. The structures given for the pigment and its degradation products have been proven by identification with authentic preparations.Although they have different chromophores, the pigment (I) of Pseudomonas lemonnieri and N,N-dioctanoyl-indigoidine (VI) nevertheless resemble one another in IR-absorbances, NMR-spectra, and chromatographic behavior, because of homogeneous functional groups and ring structures. I and VI are indeed chemically related, as can be seen from the facts that aminocitrazinic acid is a common starting material for the in vitro syntheses of both compounds, and that the diazaindophenol (I) can be converted to the diaza-diphenoquinone (VI) by hydrogenation and subsequent autoxidation.Dedicated with devotion and admiration to Professor C. B. Van Niel on his seventieth birthday.