Chromogenesis mirabilis in Streptomyces griseus

A number of chromogenic Streptomyces, producing diffusible melanoid pigment on complex organic media, fail to form melanin pigment on conventionally used synthetic tyrosine agar. By means of our new melanin formation test, almost all the chromogenic streptomyces can now be detected in chemically defined medium. In contrast to ordinary chromogenic streptomyces, two streptomyces species of the International Streptomyces Project, S. griseus ISP 5236 and S. ornatus ISP 5307, produce melanin pigment only on synthetic tyrosine agar, without showing chromogenicity on complex organic media. From the results obtained with S. griseus ISP 5236 and S. phaeochromogenes ISP 5073, it was revealed that melanin formation by Streptomyces, in general, is inhibited by L-cysteine present in organic nitrogen sources incorporated into natural media. Most chromogenic species of streptomyces produce a higher level of tyrosinase and rapidly utilize L-cysteine in the culture media which result in the manifestation of good chromogenicity on natural media. Peculiarity of chromogenicity of S. griseus and S. ornatus might be due to the lower ability to produce tyrosinase and to utilize L-cysteine in the culture medium.     

Melanin and novel melanin precursors from Aeromonas media

Many bacteria produce reddish brown to black pigments and some of these have been characterised. This report describes the isolation and characterisation of a diffusible brown melanin-like pigment from the bacterium Aeromonas media. Physico-chemical testing suggested that the pigment is a true melanin. New butanol-soluble yellow, red and brown pigments were isolated from the A. media strain under reducing conditions during melanogenesis and these pigments were shown to be unstable precursors of the polymeric brown melanin product.     

一株产蓝色素链霉菌18-A-5的鉴定与系统发育分析

从原始热带雨林土壤中,分离到一株产蓝色色素菌株18-A-5,对其进行了系统分类学研究。形态学特征观察表明,在高氏合成一号培养基上初产蓝绿色色素,日久为深蓝色,基内菌丝蓝色,气生菌丝灰白色,产灰色孢子,孢子丝直或柔曲,形成长孢子链,孢子圆柱形。其DNA的G＋C摩尔分数为62.4%,16S rDNA序列分析结果(GenBank登陆号为EU054353),18-A-5与生靛链霉菌Streptomyces indigoferus ATCC23924^T 、草绿色链霉菌Streptomyces herbaricolor ATCC23924^T具有极高的同源性,达100％,聚类分析表明,18-A-5与生靛链霉菌Streptomyces indigoferus、草绿色链霉菌Streptomyces herbaricolor两株菌聚类在一起,分支置信度为74%。结合生理生化特性、细胞壁化学组成分析、脂肪酸分析等将菌株18-A-5定名为草绿色链霉菌Streptomyces herbaricolor。并对该蓝绿色可溶性色素性质进行了耐酸碱性、热稳定性、抗菌谱等初步分析。     

Cloning of a DNA fragment involved in pigment production in Streptomyces avermitilis

Streptomyces avermitilis has the ability to synthesize a diffusible, brown, melanin-like pigment, a common property among many Streptomyces species. A region of the S. avermitilis chromosome involved in the production of this pigment was cloned in Escherichia coli. Production of the brown pigment was attained in E. coli, and is optimal when medium is supplemented with copper ions, tyrosine and IPTG. The cloned S. avermitilis pigment-producing DNA fragment is under the control of the lac promoter carried in the E. coli vector. The gene involved in pigment production could be used as a tool to analyse gene expression in S. avermitilis, and as an alternative cloning marker in Streptomyces-Escherichia coli vectors.     

Polymer production by Klebsiella pneumoniae 4-hydroxyphenylacetic acid hydroxylase genes cloned in Escherichia coli.

The expression of Klebsiella pneumoniae hpaA and hpaH genes, which code for 4-hydroxyphenylacetic acid hydroxylase in Escherichia coli K-12 derivative strains, is associated with the production of a dark brown pigment in the cultures. This pigment has been identified as a polymer which shows several of the characteristics reported for microbial melanins and results from the oxidative activity of 4-hydroxyphenylacetic acid hydroxylase on some dihydroxylated compounds to form o-quinones. A dibenzoquinone is formed from the oxidation of different mono- or dihydroxylated aromatic compounds by the enzyme prior to polymerization. We report a hydroxylase activity, other than tyrosinase, that is associated with the synthesis of a bacterial melanin.     

Degradation of melanin by Aspergillus fumigatus.

A strain of Aspergillus fumigatus from composted coffee and garden wastes utilized natural deproteinized insect, banana, hair, octopus, and synthetic tyrosine and dopa melanins as sole sources of carbon. With a sucrose supplement, degradation was essentially complete after 50 days in Czapek medium pH 6.5 at 30 degrees C. The catabolic rate differed for each substrate pigment, as did the molecular weight distribution of products accumulating in the medium. After incubation with L-[U-14C]melanin, over 50% was recovered in a dark fungal pigment, the remainder appearing as cell protein, chitin, lipid, CO2, and polar metabolites. When grown on melanin, the normally pale mycelia darkened with the production of a fungal allomelanin, with infrared spectrum and alkali fusion products differing from those of the substrate pigment. Isotope distribution in amino acids for A. fumigatus grown on labeled melanin supplemented with sucrose suggested separate pools for synthesis of cell proteins and melanoproteins. Deposition of allomelanin increased resistance of conidia, sterigma, and conidiophores to lytic carbohydrases as judged by scanning electron microscopy.     

Computer-assisted numerical analysis of colour-group data for dereplication of streptomycetes for bioprospecting and ecological purposes

Large numbers of alkaliphilic streptomycetes isolated from a beach and dune sand system were dereplicated manually based on aerial spore mass, colony reverse and diffusible pigment colours formed on oatmeal agar, and on their capacity to produce melanin pigments on peptone-yeast extract-iron agar. The resultant data were converted to their respective red, blue and green shade intensities. The Euclidean distances between each of the colours were calculated by considering red, green and blue shade intensity values as X, Y and Z coordinates in three dimensional space. The clusters of isolates delineated in the dendrogram generated using the distances were found to match those obtained by manual colour-grouping of the isolates. A reasonable linear correlation was found between the colour-group and corresponding rep-PCR data. The implications of the computer-assisted colour-grouping method for bioprospecting and ecological studies are discussed.     

Cryptococcus neoformans can utilize the bacterial melanin precursor homogentisic acid for fungal melanogenesis

Cryptococcus neoformans melanizes in the environment and in mammalian tissues, but the process of melanization in either venue is mysterious given that this microbe produces melanin only from exogenous substrates. Understanding the process of melanization is important because melanization is believed to protect against various stresses in the environment, including UV radiation, and pigment production is associated with virulence. Melanization in C. neoformans requires the availability of diphenolic precursors. In contrast, many bacteria synthesize melanin from homogentisic acid (HGA). We report that C. neoformans strains representing all four serotypes can produce a brown pigment from HGA. The brown pigment was acid resistant and had the electron paramagnetic resonance spectrum of a stable free radical, qualities that identified it as a melanin. Melanin "ghost"-like particles obtained from pigmented C. neoformans cells were hydrophobic, fluorescent under a variety of irradiation wavelengths, negatively charged, insoluble in organic solvents and alcohols, resistant to degradation by strong acids, and vulnerable to bleaching. HGA melanization was laccase dependent and repressed by high concentrations of glucose. The ability of C. neoformans to utilize a bacterial melanin precursor compound suggests a new substrate source for melanization in the environment.     

Assays for mammalian tyrosinase: a comparative study

This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed.     

A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli.

A 1.5-kb genomic fragment isolated from Streptomyces avermitilis that directs the synthesis of a brown pigment in Escherichia coli was characterized. Since pigment production in recombinant E. coli was enhanced by the addition of tyrosine to the medium, it had been inferred that the cloned DNA might be associated with melanin biosynthesis. Hybridization studies, however, showed that the pigment gene isolated from S. avermitilis was unrelated to the Streptomyces antibioticus melC2 determinant, which is the prototype of melanin genes in Streptomyces spp. Sequence analysis of the 1.5-kb DNA that caused pigment production revealed a single open reading frame encoding a protein of 41.6 kDa (380 amino acids) that resembled several prokaryotic and eukaryotic 4-hydroxyphenylpyruvate dioxygenases (HPDs). When this open reading frame was overexpressed in E. coli, a protein of about 41 kDa was detected. This E. coli clone produced homogentisic acid (HGA), which is the expected product of the oxidation of 4-hydroxyphenylpyruvate catalyzed by an HPD, and also a brown pigment with characteristics similar to the pigment observed in the urine of alkaptonuric patients. Alkaptonuria is a genetic disease in which inability to metabolize HGA leads to increasing concentrations of this acid in urine, followed by oxidation and polymerization of HGA to an ochronotic pigment. Similarly, the production of ochronotic-like pigment in the recombinant E. coli clone overexpressing the S. avermitilis gene encoding HPD is likely to be due to the spontaneous oxidation and polymerization of the HGA accumulated in the medium by this clone.     

"Basic brown", a too little considered iris color]

The eye colour is mainly effected by the two components melanin and structural blue of the iris stroma which generally are present in various mixtures respectively combinations. Are these components lacking the dark pigment epithelium can produce a so-called "basic brown". The exact distinction between "brown" and "basic brown" is without doubt of considerable value for anthropological as well as for genetical examinations of eye colours.     

Cryptococcus neoformans Can Utilize the Bacterial Melanin Precursor Homogentisic Acid for Fungal Melanogenesis

Cryptococcus neoformans melanizes in the environment and in mammalian tissues, but the process of melanization in either venue is mysterious given that this microbe produces melanin only from exogenous substrates. Understanding the process of melanization is important because melanization is believed to protect against various stresses in the environment, including UV radiation, and pigment production is associated with virulence. Melanization in C. neoformans requires the availability of diphenolic precursors. In contrast, many bacteria synthesize melanin from homogentisic acid (HGA). We report that C. neoformans strains representing all four serotypes can produce a brown pigment from HGA. The brown pigment was acid resistant and had the electron paramagnetic resonance spectrum of a stable free radical, qualities that identified it as a melanin. Melanin “ghost”-like particles obtained from pigmented C. neoformans cells were hydrophobic, fluorescent under a variety of irradiation wavelengths, negatively charged, insoluble in organic solvents and alcohols, resistant to degradation by strong acids, and vulnerable to bleaching. HGA melanization was laccase dependent and repressed by high concentrations of glucose. The ability of C. neoformans to utilize a bacterial melanin precursor compound suggests a new substrate source for melanization in the environment.     

Melanosome capping of keratinocytes in pigmented reconstructed epidermis--effect of ultraviolet radiation and 3-isobutyl-1-methyl-xanthine on melanogenesis

Reconstructed pigmented epidermis was established by co-seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air-liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3-isobutyl-1-methyl-xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.     

Melanosome Capping of Keratinocytes in Pigmented Reconstructed Epidermis – Effect of Ultraviolet Radiation and 3‐Isobutyl‐1‐Methyl‐Xanthine on Melanogenesis

Reconstructed pigmented epidermis was established by co‐seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air–liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3‐isobutyl‐1‐methyl‐xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.     

The role of melanin in the antagonistic interaction between the apple scab pathogen Venturia inaequalis and Microsphaeropsis ochracea

The objective of this study was to examine the role of melanin in the interaction between the mycoparasite Microsphaeropsis ochracea and the apple scab pathogen Venturia inaequalis. Melanin was extracted from the cell wall of the pathogen and its chemical and physical properties determined on the basis of biochemical tests and visible and infrared spectra. The physical and chemical characteristics of V inaequalis melanin were similar to the those of synthetic dihydroxyphenylalanine (DOPA) melanin. Precursors of the four known melanin biosynthetic pathways were tested for their ability to restore the pigmentation of an albino strain of V inaequalis. Scytalone, an intermediate of the 1,8-dihydroxynaphthalene (DHN) pathway, was the only precursor to restore the dark-brown pigmentation. Tricyclazole and pyroquilon, two antipenetrant fungicides, specific inhibitors of DHN melanin synthesis in Pyricularia oryzae, were used to confirm the melanin pathway in V. inaequalis wild type. A reddish-brown pigment was obtained due to the accumulation of shunt products of the DHN melanin pathway instead of a dark-brown pigment, suggesting that the melanin extracted from V inaequalis was a DHN melanin. Furthermore, growth of an albino mutant of V. inaequalis on scytalone-amended medium resulted in the formation of dark granules similar to those seen in wild-type isolates. Transmission electron microscopic observations of M. ochracea grown in the presence of melanin showed that the granules accumulated gradually along fungal cell walls to form a uniform dark coating.     

Occurrence of the stringent response in Streptomyces sp. and its significance for the initiation of morphological and physiological differentiation

Streptomyces sp. MA406-A-1 produced formycin (a nucleoside antibiotic) in parallel with cell growth in a synthetic medium. When the synthetic medium was supplemented with 1% (w/v) Casamino acids, however, formycin was produced only after the end of exponential growth. The intracellular ppGpp pool increased gradually towards the end of exponential growth and was maximal at the beginning of formycin production. After shift down from Casamino acids medium to synthetic medium, the ppGpp pool increased immediately, while the GTP pool decreased; under such conditions, the ability to produce formycin increased eightfold. Relaxed (rel) mutants, the first isolated for a Streptomyces species, were found at high incidence (10%) among spontaneous thiopeptin-resistant isolates and had severely reduced abilities to accumulate ppGpp. These rel mutants also failed to produce formycin under the usual culture conditions and exhibited numerous pleiotropic effects such as an inability to produce melanin and an extended delay of aerial mycelium formation. Thus Streptomyces sp. exhibited a typical stringent response, and the response initiated (or was needed for) the induction of secondary metabolism. The response may have also participated in the initiation of aerial mycelium formation by decreasing the intracellular GTP pool.     

Characterization of melanin pigment produced by <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

Although most of the Ascomycetes present DHN-melanin, some reports suggest that A. nidulans does not produce this type of melanin. In this study, we analyzed the pigment extracted from highly melanized strains (MEL1 and MEL2) of Aspergillus nidulans to determine the type of melanin present in this fungus. Our results showed that the pigment produced by MEL1 and MEL2 mutants possesses physical and chemical properties and UV- and IR-spectra very similar to synthetic DOPA-melanin. The characterization of this pigment in terms of its degradation products indicated the presence of indolic units, which were also found in synthetic DOPA-melanin. The analyses of the elemental composition showed that the pigment extracted from these mutants has a high percentage of nitrogen and, therefore, it cannot be DHN-melanin, which presents only trace of nitrogen. This observation was confirmed in the test with tricyclazole because this inhibitor of DHN-melanin biosynthesis did not suppress pigment production in the MEL1 and MEL2 strains. On the other hand, in a medium containing tropolone, an inhibitor of DOPA-melanin biosynthesis, the dark pigmentation of the colonies was not observed indicating that this compound inhibited melanin production in these strains. Taken together, the results obtained in this study indicate that melanin produced by these mutants is DOPA type, representing the first report on characterization of this type of melanin in A. nidulans.     

Oxidation of NADH by melanin: effect of UV light and copper ions.

The oxidation reaction of NADH with synthetic melanin from L-dopa was investigated under various physicochemical conditions both by spectrophotometric measurements and ESR spectroscopy. The different amounts of NADH oxidized and the effects on the formation and decay of melanin free radicals were compared. Some hypotheses on a common physical mechanism involved in electron transfer reaction and in electron excitation through a liquid-solid interface are presented.     

The dark brown integumentary pigment of a barnacle (Balanus eburneus)

Summary A histochemical analysis involving tinctorial and solubility tests was pursued in conjunction with electron microscopy for the purpose of identifying the dark brown epidermal pigment of a barnacle (Balanus eburneus) as melanin and/or ommochrome. Histochemically, comparisons were made with other brown pigments located in the subcarapal epidermis of another crustacean, the fiddler crab (Uca pugilator), the dorsal skin of the red-backed salamander (Plethodon cinereus), the liver and testis of a slider turtle (Chrysemys sp.) and the substantia nigra of human brain. The solubility properties of the pigment of the two crustacean invertebrates were in general similar to one another and markedly different from the pigment of the three vertebrates. Insolubility in appropriate solvents classified the vertebrate pigment as melanin. The invertebrate pigment, however, which was soluble in the ommochrome solvents, concentrated sulfuric and formic acids and 2-chloroethanol, remained insoluble in the ommochrome solvents, dilute aqueous and methanolic hydrochloric acid and dilute sulfuric acid. On the basis of these solubilities, an unequivocal classification of the invertebrate pigment as either melanin or ommochrome did not appear possible. The tinctorial and electron microscopic properties of the barnacle pigment were also equally ambiguous in regard to its specific classification.