Production by Bacillis subtilis of brown sporulation-associated pigments

The mode of production of the brown pigments of Bacillus subtilis 168 L-4, pigments frequently used as phenotypic markers for sporulation in this organism, has been studied. A defined liquid medium which promoted maximal pigment formation was developed. Five brown components, which could be resolved by thin-layer chromatography, were produced in the culture broth. Removal of cells from the medium at the end of logarithmic growth did not alter the type or amount of the pigments formed, indicating that the cells excreted pigment precursors into the medium during growth. Pigment formation from the precursors was found to occur by an oxygen-requiring, base-dependent, Mn2+-requiring, nonenzymatic pathway. Pigment production was also stimulated by the presence of tyrosine and histidine in the medium. The increases in extracellular pH often associated with spore formation in B. subtilis might be the cause of the concomitant appearance of brown pigments.     

The effect of different nitrogen sources on pigment production and sporulation of Monascus species in submerged, shaken culture.

Thirty-nine strains from the genus Monascus were cultivated aerobically to study the relation between nitrogen nutrition and sporulation and pigment production. The effects of yeast extract, nitrate, ammonium, and ammonium nitrate have been compared. During cultivation the pHs of the different media are not the same, resulting in the formation of different coloured pigments. When the source of nitrogen is yeast extract or nitrate the pH is around 6.5 and red pigments are formed, whereas with ammonium or ammonium nitrate the pH is around 2.5 and the pigments are orange. It is proposed that only the orange pigments, monascorubrin and rubropunctatin, are produced biosynthetically and that the other pigments are formed from these by chemical transformations depending on the cultural conditions. The presence of organic nitrogen is optimal for growth and unfavourable for pigment production. Reduced growth and best pigment formation occurs with the three other nitrogen sources. Nitrate stimulates conidiation and sexual reproduction, while ammonium is inhibitory. Pigment production is better when conidiation is reduced. A mechanism is proposed for the control of sporulation and pigment production.     

Pigment production from tryptophan by an Achromobacter species

Duerre, John A. (University of North Dakota, Grand Forks), and Patrick J. Buckley. Pigment production from tryptophan by an Achromobacter species. J. Bacteriol. 90:1686-1691. 1965.-A microorganism was isolated from the soil near the University of North Dakota. Biochemical and morphological characteristics indicated that this organism would best be classified as a member of the family Achromobacteraceae, genus Achromobacter, species unknown. The organism produced a red pigment when grown in a medium containing yeast extract and tryptophan. The pH optimum for pigment production was about 8.0 and the optimal temperature was 25 C. During a study of the nutritional requirements for growth and pigment production, it was found that the organism would grow and produce pigment in a medium containing tryptophan and nucleosides, but the rate of both growth and pigment formation in this medium was slower than that observed with tryptophan and yeast extract. The organism grew well in the presence of acid-hydrolyzed casein and nucleosides without producing pigment, indicating that the pigment is not necessary for growth. Resting-cell experiments definitely established tryptophan as the sole exogenous requirement for pigment production. The pigment was extracted from yeast extract-tryptophan medium with chloroform. Thin layer chromatographic analysis of the crude pigment extracted from this medium revealed the presence of two other pigments in addition to the major red pigment. One of these was a highly fluorescent orange pigment and the other a pink pigment. Only the red pigment was produced by resting cells in the presence of tryptophan alone. This pigment served as an electron acceptor when coupled with formic dehydrogenase, indicating its possible function as an oxidation-reduction pigment. The oxidized pigment had absorption peaks at 506 and 304 mmu. The peak at 506 mmu disappeared upon reduction with sodium sulfite. Shaking the reduced pigment in air proved to be an unsatisfactory method for returning the reduced pigment to the oxidized, colored state.     

Brown pigments produced by Yarrowia lipolytica result from extracellular accumulation of homogentisic acid

Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn(2+) accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn(2+). Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.     

Brown pigmentation in Serratia marcescens cultures associated with tyrosine metabolism

Serratia marcescens produced a brown pigment when grown in minimal medium in the presence of tyrosine and high concentrations of copper(II) ion. The pigment was not related to the melanin pigments, but was similar to the pigment produced by autooxidation and polymerization of 3,4-dihydroxyphenylacetate, which is synthesized in S. marcescens from tyrosine through the 3,4-dihydroxyphenylacetate catabolic pathway. The enzymes of this pathway were induced under pigment production conditions; however, 3,4-dihydroxyphenylacetate 2,3-dioxygenase remained at low activity levels, permitting the accumulation and excretion of the substrate. Mutants unable to use tyrosine as a sole carbon and energy source were able to produce brown pigments only if the step blocked by the mutation was after the synthesis of 3,4-dihydroxyphenylacetate. The ability to produce brown pigments was common to all the S. marcescens strains tested.     

Brown Pigments Produced by Yarrowia lipolytica Result from Extracellular Accumulation of Homogentisic Acid

Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn²⁺ accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn²⁺. Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.     

Pigmentation and Sporulation Are Alternative Cell Fates in Bacillus pumilus SF214

Bacillus pumilus SF214 is a spore forming bacterium, isolated from a marine sample, able to produce a matrix and a orange-red, water soluble pigment. Pigmentation is strictly regulated and high pigment production was observed during the late stationary growth phase in a minimal medium and at growth temperatures lower than the optimum. Only a subpopulation of stationary phase cells produced the pigment, indicating that the stationary culture contains a heterogeneous cell population and that pigment synthesis is a bimodal phenomenon. The fraction of cells producing the pigment varied in the different growth conditions and occured only in cells not devoted to sporulation. Only some of the pigmented cells were also able to produce a matrix. Pigment and matrix production in SF214 appear then as two developmental fates both alternative to sporulation. Since the pigment had an essential role in the cell resistance to oxidative stress conditions, we propose that within the heterogeneous population different survival strategies can be followed by the different cells.     

Evaluation of Epicoccum nigrum for growth, morphology and production of natural colorants in liquid media and on a solid rice medium

Four nonpathogenic and nontoxigenic Epicoccum nigrum strains were evaluated for their growth, morphology and pigment producing ability in three complex and one defined liquid media. Epicoccum nigrum IBT 41028 produced pigments in all the four media tested with a maximum pigment of 3.68 AU at 410 nm in M1 medium (unoptimized) containing 5 g/l yeast autolysate. The color hue of the crude pigment extracts ranged from 74 to 102 exhibiting dark orange to green-yellow color. Pelleted morphology was shown to have a positive influence on the pigment production by E. nigrum strain IBT 41028 in the liquid media, and the use of Bis-tris buffer was found to diminish or reduce the pellet formation. Since Monascus is a well known pigment producer on rice. Pigment producing ability of E. nigrum IBT 41028 was tested on rice and compared to liquid media with Monascus ruber IBT 7904 as control. Though, both genera preferred rice but E. nigrum produced 4.6 folds higher pigment in the liquid unoptimized fermentation medium compared to M. ruber. Solid phase extraction and subsequently HPLC-DAD analysis of the crude pigment extracts showed qualitative as well as quantitative variation in the pigment composition under solid and liquid cultivations.     

Pigment Production on L-Tryptophan Medium by Cryptococcus gattii and Cryptococcus neoformans

In recent years strains previously grouped within Cryptococcus neoformans have been divided into two species C. neoformans and C. gattii, with Cryptococcus neoformans comprising serotypes A, D, and AD and C. gattii comprising serotypes B and C. Cryptococcus neoformans have also been subdivided into two varieties C. neoformans var. grubii, serotype A, and C. neoformans var. neoformans, serotype D. We analyzed the growth and pigment production characteristics of 139 strains of Cryptococcus spp. in L-tryptophan containing media. Nearly all strains of Cryptococcus, including each variety and serotype tested produced a pink water-soluble pigment (molecular weight of 535.2 Da) from L-tryptophan. Consequently, the partial separation of the species was based on whether the pink pigment was secreted into the medium (extracellular) or retained as an intracellular pigment. On L-tryptophan medium C. neoformans var. grubii and serotype AD produced a pink extracellular pigment. In contrast, for C. gattii, the pink pigment was localized intracellularly and masked by heavy production of brown pigments. Pigment production by C. neoformans var. neoformans was variable with some strains producing the pink extracellular pigment and others retained the pink pigment intracellularly. The pink intracellular pigment produced by strains of C. neoformans var. neoformans was masked by production of brown pigments. Cryptococcus laccase mutants failed to produce pigments from L-tryptophan. This is the first report that the enzyme laccase is involved in tryptophan metabolism. Prior to this report Cryptococcus laccase produced melanin or melanin like-pigments from heterocyclic compounds that contained ortho or para diphenols, diaminobenzenes and aminophenol compounds. The pigments produced from L-tryptophan were not melanin.     

Production of pigments byMonascus purpureus in solid culture

Summary The effect of physical and nutritional factors on the production of pigments byMonascus purpureus FRR 2190 was studied using cultures grown on both rice and a synthetic medium that was solidified with carrageenan and extruded into rice-like particles. Pigment yield was highly sensitive to physical parameters. Optimal pigment formation in rice cultures occurred at an initial pH of 6 and an initial moisture content of 56%. Lower moisture contents led to a large decrease in pigment concentration. Red and yellow pigment production on solidified gel media was increased up to three-fold compared to that of liquid cultures of the same medium composition, particularly when peptone was used as the sole nitrogen source. Solid state rice cultures gave the highest pigment yields.     

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.     

Biosynthesis of fumiquinazolines by the fungus Penicillium thymicola

Biosynthesis of fumiquinazolines F and G (FQs), PC-2, and pigments by the fungus P. thymicola VKM FW-869 is directly dependent on the content of carbon substrate (mannitol) in the medium. Pigment production prevailed at all of the tested mannitol concentrations. The necessary conditions for predominant FQ biosynthesis by the fungus P. thymicola are carbon source (mannitol) limitation and presence of NaCl in the cultivation medium. NaCl has a regulatory effect on the formation of secondary metabolites by enhancing FQ biosynthesis and reducing pigment formation. The maximum values of FQ biosynthesis and inhibition of pigment production are obtained at a mannitol concentration of 20 g/l and 2.5% NaCl in the medium.     

Biosynthesis of fumiquinazolines by the fungus Penicillium thymicola

Biosynthesis of fumiquinazolines F and G (FQs), PC-2, and pigments by the fungus P. thymicola VKM FW-869 is directly dependent on the content of carbon substrate (mannitol) in the medium. Pigment production prevailed at all of the tested mannitol concentrations. The necessary conditions for predominant FQ biosynthesis by the fungus P. thymicola are carbon source (mannitol) limitation and presence of NaCl in the cultivation medium. NaCl has a regulatory effect on the formation of secondary metabolites by enhancing FQ biosynthesis and reducing pigment formation. The maximum values of FQ biosynthesis and inhibition of pigment production are obtained at a mannitol concentration of 20 g/l and 2.5% NaCl in the medium.     

The fermentation of rice for anka pigment production

Optimal physical parameters of the solid state fermentation of rice to produce anka pigments and their influences on pigment production were studied. Anka pigment production, especially that of two orange anka pigments (rubropunctatin and monascorubrin), was highly sensitive to the moisture content of the rice substrate. Optimal initial moisture content of rice substrate was 24%. Pigment formation was retarded when extra water was added to the inoculated substrate during cultivation. High filling amount of rice substrate in a flask was unfavorable for pigment production. Loosening of the inoculated substrate once a day enhanced pigment production. With a high carbon dioxide level in the incubator, no orange pigments were detected. Freeze drying the fermented material produced a superior yield of anka pigments, while oven drying at 50°C for 24 h was a reasonable alternative. Journal of Industrial Microbiology & Biotechnology (2000) 25, 141–146. Received 27 December 1999/ Accepted in revised form 24 June 2000     

Induction by Klebsiella aerogenes of a melanin-like pigment in Cryptococcus neoformans

While studying the interaction of Cryptococcus neoformans with Dictyostelium discoideum, we noticed that yeast colonies in agar with a feeder lawn of Klebsiella aerogenes were brown. This finding was intriguing because C. neoformans colonies are not pigmented unless they are provided with precursors for melanization. Strains of all C. neoformans serotypes produced brown pigment in response to K. aerogenes at 22, 30, and 37 degrees C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when C. neoformans was exposed to live Escherichia coli or heat-killed K. aerogenes. Analysis of K. aerogenes supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by C. neoformans. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for C. neoformans melanization in the environment.     

Beta-alanine and tanning polymorphisms

1. Beta-alanine is found in mycelial walls of mature tan, but not immature white, stage of Morchella esulenta, nor in any stage of a permanently white mutant. 2. Beta-alanine is also found in hydrolysed water-extracts of human hair, the concentration being higher in blond than in dark brown, and in pigmented than in unpigmented hair. 3. Beta-alanine, added to tyrosinase-oxidized tyrosine, dopa, or dopamine has only a slight yellowing influence on the final black pigment; but when the amino group of tyrosine is combined with leucine, added beta-alanine produces stable tan pigments. 4. With L-alanine substituted for beta-alanine in this reaction, green pigment results. 5. Gelatin filters stained with the tan pigment allow solar heating of underlaying water more quickly than do those stained with the black pigment. Unstained filters allow such heating even more quickly. 6. Beta-alanine enhances production of tan pigment when heated with the phospholipid, lecithin. Implications for pigmentary adaptation, and formation of lipofuscin-like age pigments are discussed.     

Ultrastructure and migration of screening pigments in the retina of Pieris rapae L. (Lepidoptera,Pieridae)

Summary The retinal morphology of the butterfly, Pieris rapae L., was investigated using light and electron microscopy with special emphasis on the morphology and distribution of its screening pigments. Pigment migration in pigment and retinula cells was analysed after light-dark adaptation and after different selective chromatic adaptations. The primary pigment cells with white to yellow-green pigments symmetrically surround the cone process and the distal half of the crystalline cone, whilst the six secondary pigment cells, around each ommatidium, contain dark brown pigment granules. The nine retinula cells in one ommatidium can be categorised into four types. Receptor cells 1–4, which have microvilli in the distal half of the ommatidium only, contain numerous dark brown pigment granules. On the basis of the pigment content and morphology of their pigment granules, two distal groups of cells, cells 1, 2 and cells 3, 4 can be distinguished. The four diagonally arranged cells (5–8), with rhabdomeric structures and pigments in the proximal half of the cells, contain small red pigment granules of irregular shape. The ninth cell, which has only a small number of microvilli, lacks pigment. Chromatic adaptation experiments in which the location of retinula cell pigment granules was used as a criterium reveal two UV-receptors (cells 1 and 2), two green receptors (cells 3 and 4) and four cells (5–8) containing the red screening pigment, with a yellow-green sensitivity.     

Effect of pH and nitrogen source on pigment production by Monascus purpureus

The effect of pH and nitrogen source on pigment production by Monascus purpureus 192F using glucose as the carbon and energy source, was studied in pH-controlled, batch fermentor cultures using HPLC analysis to determine individual pigment concentrations. A maximum of four pigments were detected in fungal extracts. These were the yellow pigments monascin and ankaflavin, the orange rubropunctatin and the red pigment monascorubramine. Monascorubramine was present as the major product in all instances. Fungal growth and ankaflavin synthesis were favoured at low pH (pH 4.0), whereas production of the other pigments was relatively independent of pH. The nature of the nitrogen source affected fungal growth and pigment production, independent of pH. Ammonium and peptone as nitrogen sources gave superior growth and pigment concentrations compared to nitrate. Ankaflavin was not detected in nitrate cultures. The highest red pigment production was obtained using a glucose-peptone medium at pH 6.5, due to the secretion of red pigments into the medium under these conditions. Correspondence to: M. R. Johns