Chromogenicity of Streptomyces

A simplified technique to detect polyphenol oxidase and melanin formation by Streptomyces culture filtrates was developed. The procedure involves the direct assay of pigment formation by the culture filtrate with 3-(3,4-dihydroxyphenyl)-L-alanine (L-dopa) as a substrate. Among cultures of the International Streptomyces Project, 34 failed to produce a diffusible dark brown pigment on peptone-yeast extract-iron-agar and synthetic tyrosine-agar and gave a negative reaction to the melanin formation test. Sixteen cultures produced a diffusible dark brown pigment on both peptone-yeast extract-iron-agar and synthetic tyrosine-agar and gave positive reactions to the test with either L-tyrosine or L-dopa as substrate. Twenty-one cultures produced a diffusible dark brown pigment on peptone-yeast extract-iron-agar, but failed to do so on synthetic tyrosine-agar. Most of these cultures gave a positive reaction to the test when L-dopa was used as the substrate. The correlation between chromogenicity on complex organic media and melanin formation was more clearly established with L-dopa as substrate than with synthetic tyrosine-agar in the present test. The melanin formation test by the present technique, instead of chromogenicity on complex organic media, is recommended as a key feature for the classification of Streptomyces.     

Beta-lactam and aminoglycoside production from streptomycetes

Streptomyces cattleya, S. fradiae and S. griseus produced different amounts of growth when cultured sequentially through sporulation, vegetative and antibiotic production media. Only S. griseus grew well on all three types of medium. Streptomyces cattleya grew poorly on both sporulation and vegetative media. Growth was 1.6 and 8.0 mg/1/h respectively. For all three species, biomass yield in the final antibiotic production medium was dependent on amount of inoculum. Antibiotic yields were obtained only from production media. Under slow growth conditions L-cysteine and L-valine supplementation stimulated S. cattleya beta-lactam production, giving 1000 micrograms/ml beta-lactam equivalents compared with 45 micrograms/ml beta-lactam equivalents for no supplementation. For aminoglycosides the agar well diffusion bioassay was more sensitive towards the hydrochloride than the neutral salt. Paper chromatography confirmed the main antibiotic classes. RF values for replicate samples indicated aminoglycoside homogeneity and beta-lactam heterogeneity.     

Actinobacteria isolated from termite guts as a source of novel oxidative enzymes

A multi-faceted screening programme was designed to search for the oxidases, laccase, peroxidase and tyrosinase. Actinobacteria were selectively isolated from the paunch and colon region of the hindguts of the higher termite, Amitermes hastatus. The isolates were subjected to solid media assays (dye decolourization, melanin production and the utilization of indulin AT as sole carbon source) and liquid media assays. Eleven of the 39 strains had the ability to decolourize the dye RBBR, an indicator for the production of peroxidases in actinobacteria. Melanin production on ISP6 and ISP7 agar plates served as a good indicator for laccase and/or tyrosinase production and the ability of the strains to grow in the presence of indulin AT as a sole carbon source served as a good indicator of lignin peroxidase and/or general peroxidase production. Enzyme-producing strains were cultivated in liquid media and extracellular enzyme activities measured. Strains with the ability to produce oxidative enzymes under the conditions tested were identified to genus level by 16S rRNA gene analysis and compared to known oxidase producers. A strong relationship was observed between the environment sampled (termite guts where lignocellulose degradation occurs) and the dominant type of oxidative enzyme activity detected (laccases and peroxidases), which suggests the possibility of future targeted screening protocols linking the physical properties of the target enzymes with specific operational conditions required, such as lignocellulosic degradation in the preparation of biofuel feedstocks.     

Biosynthesis of hexahydroxyperylenequinone melanin via oxidative aryl coupling by cytochrome P-450 in Streptomyces griseus

Dihydroxyphenylalanine (DOPA) melanins formed from tyrosine by tyrosinases are found in microorganisms, plants, and animals. Most species in the soil-dwelling, gram-positive bacterial genus Streptomyces produce DOPA melanins and melanogenesis is one of the characteristics used for taxonomy. Here we report a novel melanin biosynthetic pathway involving a type III polyketide synthase (PKS), RppA, and a cytochrome P-450 enzyme, P-450mel, in Streptomyces griseus. In vitro reconstitution of the P-450mel catalyst with spinach ferredoxin-NADP(+) reductase/ferredoxin revealed that it catalyzed oxidative biaryl coupling of 1,3,6,8-tetrahydroxynaphthalene (THN), which was formed from five molecules of malonyl-coenzyme A by the action of RppA to yield 1,4,6,7,9,12-hexahydroxyperylene-3,10-quinone (HPQ). HPQ readily autopolymerized to generate HPQ melanin. Disruption of either the chromosomal rppA or P-450mel gene resulted in abolishment of the HPQ melanin synthesis in S. griseus and a decrease in the resistance of spores to UV-light irradiation. These findings show that THN-derived melanins are not exclusive in eukaryotic fungal genera but an analogous pathway is conserved in prokaryotic streptomycete species as well. A vivid contrast in THN melanin biosynthesis between streptomycetes and fungi is that the THN synthesized by the action of a type III PKS is used directly for condensation in the former, while the THN synthesized by the action of type I PKSs is first reduced and the resultant 1,8-dihydroxynaphthalene is then condensed in the latter.     

Tyrosinase in the presumptive pigment cells of ascidian embryos: Tyrosine accessibility may initiate melanin synthesis

Tyrosinase activity appears in the presumptive pigment cells of ascidian embryos (Ciona intestinalis) several hours before the cells begin to synthesize melanin. These presumptive pigment cells develop into the otolith and ocellus pigment cells of the larval brain. Tyrosinase was identified by histochemical tests for tyrosine oxidase and dopa oxidase; both reactions were sensitive to tyrosinase inhibitors. Studies with puromycin suggested that tyrosinase was synthesized at the time it was first detected histochemically and that it was stable during the time interval before melanin synthesis. Supernumerary tyrosinase-containing cells were found adjacent to the presumptive pigment cells in three ascidian species examined (C. intestinalis, Styela partita, and Molgula manhattensis). Tyrosinase disappeared from the supernumerary pigment cells during larval development and these cells did not synthesize melanin.Tyrosinase in the presumptive and supernumerary pigment cells is apparently a functional enzyme which does not interact with substrate. External substrates ( -tyrosine and -dopa) did not react with enzyme in the living cells before the normal time of pigment synthesis, but gentle disruption of the cells (by freezing-and-thawing or osmotic shock) released active tyrosinase. Progessive enlargement of nonpigmented vesicles in the otolith cells of embryos exposed to phenylthiourea, an inhibitor of tyrosinase activity, suggested that tyrosinase vesicles actively accumulate tyrosine at the beginning of melanin synthesis. This tyrosine accumulation probably initiates melanin synthesis.     

Redefining the skin's pigmentary system with a novel tyrosinase assay

In mammalian skin, melanin is produced by melanocytes and transferred to epithelial cells, with the epithelial cells thought to receive pigment only and not generate it. Melanin formation requires the enzyme tyrosinase, which catalyzes multiple reactions in the melanin biosynthetic pathway. Here, we reassess cutaneous melanogenesis using tyramide-based tyrosinase assay (TTA), a simple test for tyrosinase activity in situ. In the TTA procedure, tyrosinase reacts with biotinyl tyramide, causing the substrate to deposit near the enzyme. These biotinylated deposits are then visualized with streptavidin conjugated to a fluorescent dye. In the skin and eye, TTA was highly specific for tyrosinase and served as a sensitive indicator of pigment cell distribution and status. In clinical skin samples, the assay detected pigment cell defects, such as melanocytic nevi and vitiligo, providing confirmation of medical diagnoses. In murine skin, TTA identified a new tyrosinase-positive cell type--the medullary cells of the hair--providing the first example of cutaneous epithelial cells with a melanogenic activity. Presumably, the epithelial tyrosinase originates in melanocytes and is acquired by medullary cells during pigment transfer. As tyrosinase by itself can generate pigment from tyrosine, it is likely that medullary cells produce melanin de novo. Thus, we propose that melanocytes convert medullary cells into pigment cells by transfer of the melanogenic apparatus, an unusual mechanism of differentiation that expands the skin's pigmentary system.     

β-Lactam and aminoglycoside production from streptomycetes

Streptomyces cattleya, S. fradiae and S. griseus produced different amounts of growth when cultured sequentially through sporulation, vegetative and antibiotic production media. Only S. griseus grew well on all three types of medium. Streptomyces cattleya grew poorly on both sporulation and vegetative media. Growth was 1·6 and 8·0 mg/1/h respectively. For all three species, biomass yield in the final antibiotic production medium was dependent on amount of inoculum. Antibiotic yields were obtained only from production media. Under slow growth conditions l -cysteine and l -valine supplementation stimulated S. cattleya β-lactam production, giving 1000 μg/ml β-lactam equivalents compared with 45 μg/ml β-lactam equivalents for no supplementation. For aminoglycosides the agar well diffusion bioassay was more sensitive towards the hydrochloride than the neutral salt. Paper chromatography confirmed the main antibiotic classes. R _F values for replicate samples indicated aminoglycoside homogeneity and β-lactam heterogeneity.     

Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.     

High molecular weight ribosomal ribonucleic acid from vegetative hyphae and spores of Streptomyces griseus.

High molecular weight ribosomal ribonucleic acids (rRNAs) were isolated from young vegetative cells and spores of a streptomycin non-producing Streptomyces griseus, and their electrophoretic mobility was compared to each other and to that of rRNAs of Escherichia coli K-12. The electrophoretic mobility of 23 and 16S rRNAs from vegetative cells and spores of S. griseus was identical, but the 23S rRNAs of streptomyces ribosomes migrated more slowly on polyacrylamide gel than those of E. coli ribosomes. Intact, electrophoretically homogenous rRNAs could be isolated from S. griseus (No. 45-H) only in the presence of diethyl 1 pyrocarbonate (DEP), and intact rRNAs could be obtained from spores only if DEP had been added before breaking the spores. Otherwise instead of two distinct bands, three were obtained on polyacrylamide gel.     

Isolation and characterization of tyrosinase produced by marine actinobacteria and its application in the removal of phenol from aqueous environment

The present study was focused on screening and characterization of tyrosinase enzyme produced by marine actinobacteria and its application in phenolic compounds removal from aqueous solution. A total of 20 strains were isolated from marine sediment sample and screened for tyrosinase production by using skimmed milk agar medium. Among 20 isolates, two isolates LK-4 and LK-20 showed zone of hydrolysis and these were taken for secondary screening by using tyrosiue agar medium. Based on the result of secondary screening LK-4 was selected for further analysis, such as tyrosinase assay, protein content and specific activity of the enzyme. The tyrosinase enzyme was produced in a SS medium and was partially purified by ammonium sulfate precipitation, dialysis and SDS PAGE. The isolate （LK-4） was identified as Streptomyces espinosus using 16S rRNA gene sequencing and named as ＂Streptomyces espinosus strain LK4 （KF806735）＂. The tyrosinase enzyme was immobilized in sodium alginate which was applied to remove phenolic compounds from water. The enzyme efficiently removed the phenolic compounds from aqueous solution within few hours which indicated that tyrosinasc enzyme produced by Streptomyces espinosus strain LK-4 can be potently used for the removal of phenol and phenolic compounds from wastewater in industries.     

Distribution of extracutaneous melanin pigment in Sparus auratus, Mugil cephalus, and Dicertranchus labrax (Pisces, Teleostei)

The morphological and biochemical characteristics of pigment accumulations found in the kidney, liver, spleen, and mesentery of three different species of teleost fishes have been studied. There are significant differences in number, distribution, and morphology of pigment accumulations in different organs of the three species. Biochemical studies have shown the existence of tyrosinase activity in the mesentery of Mugil cephalus and in the kidney and mesentery of Sparus auratus. No tyrosinase activity was found in any internal organs of Dicertranchus labrax. That activity was assayed using three methods: tyrosine hidroxylation, dopa oxidation, and melanin formation. The morphological and biochemical observations are in agreement. In those organs in which we have demonstrated melanin synthetic activity, the pigment cells are morphologically and like melanophores, while in the organs that show no melanin synthetic activity, the pigment cells resemble macrophages.     

Biosynthesis and Functions of a Melanoid Pigment Produced by Species of the Sporothrix Complex in the Presence of l-Tyrosine

Sporothrix schenckii is the etiological agent of sporotrichosis, the main subcutaneous mycosis in Latin America. Melanin is an important virulence factor of S. schenckii, which produces dihydroxynaphthalene melanin (DHN-melanin) in conidia and yeast cells. Additionally, l-dihydroxyphenylalanine (l-DOPA) can be used to enhance melanin production on these structures as well as on hyphae. Some fungi are able to synthesize another type of melanoid pigment, called pyomelanin, as a result of tyrosine catabolism. Since there is no information about tyrosine catabolism in Sporothrix spp., we cultured 73 strains, including representatives of newly described Sporothrix species of medical interest, such as S. brasiliensis, S. schenckii, and S. globosa, in minimal medium with tyrosine. All strains but one were able to produce a melanoid pigment with a negative charge in this culture medium after 9 days of incubation. An S. schenckii DHN-melanin mutant strain also produced pigment in the presence of tyrosine. Further analysis showed that pigment production occurs in both the filamentous and yeast phases, and pigment accumulates in supernatants during stationary-phase growth. Notably, sulcotrione inhibits pigment production. Melanin ghosts of wild-type and DHN mutant strains obtained when the fungus was cultured with tyrosine were similar to melanin ghosts yielded in the absence of the precursor, indicating that this melanin does not polymerize on the fungal cell wall. However, pyomelanin-producing fungal cells were more resistant to nitrogen-derived oxidants and to UV light. In conclusion, at least three species of the Sporothrix complex are able to produce pyomelanin in the presence of tyrosine, and this pigment might be involved in virulence.     

Mutational mapping of the catalytic activities of human tyrosinase.

Tyrosinase (EC 1.14.18.1) is a copper-containing metalloglycoprotein that catalyzes several steps in the melanin pigment biosynthetic pathway; the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine (dopa) and the subsequent oxidation of dopa to dopaquinone. It has been proposed that tyrosinase is also able to oxidize 5,6-dihydroxyindole (DHI), a later product in the melanogenic pathway, to indole-5,6-quinone. Tyrosinase enzymatic activity is deficient in patients with classic type I oculocutaneous albinism (OCA), and more than 50 distinct mutations have now been identified in the tyrosinase genes of such patients. To determine the effects of the various tyrosinase gene mutations on the catalytic activities of the enzyme, we carried out site-directed mutagenesis of human tyrosinase cDNA, transiently expressed the mutant cDNAs in transfected HeLa cells, and assayed the resultant encoded proteins for tyrosine hydroxylase, dopa, and DHI oxidase activities, and resulting melanin production. The tyrosine hydroxylase activity of normal tyrosinase is thermostable, whereas its dopa oxidase and DHI oxidase activities are temperature-sensitive. Although all amino acid substitutions tested generally affected the dopa oxidase and DHI oxidase activities in parallel, several exerted distinctly different effects on the tyrosine hydroxylase activities. Together, these results confirm the DHI oxidase activity of mammalian tyrosinase and suggest that the dopa oxidase and DHI oxidase activities of tyrosinase share a common catalytic site, whereas the tyrosine hydroxylase catalytic site is at least partially distinct in the tyrosinase polypeptide.     

Radicals and melanomas

The synthesis of melanin involves the oxidation of phenolic substrates by the enzyme tyrosinase. In vertebrates tyrosinase is present only in specialized cells (melanocytes), where it catalyses the oxidation of tyrosine and certain diphenolic intermediate products to quinones which polymerize to give rise to melanin. This specialized metabolic pathway provides a possible approach to the specific chemotherapy of malignant tumours of pigment cells (malignant melanoma). Certain analogues of tyrosine are oxidized by tyrosinase generating reactive orthoquinones with cytotoxic potential. One such analogue, 4-hydroxyanisole, has been investigated as a possible specific melanocytotoxic precursor. The parent compound inhibits DNA synthesis but exhibits little general toxicity, while the tyrosinase oxidation products are highly toxic to cells. The mechanism of this toxicity may involve semiquinone radicals. Encouraging initial results have been obtained from clinical pilot studies using intra-arterial infusion of hydroxyanisole in patients with localized recurrences of malignant melanoma.     

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.