Several conditions of formation and isolation of violet pigment from culture fluid of Nocardia fructiferi var. ristomycin

A new water-soluble photochromogenic fluorescent violet pigment with the indicator properties was isolated from the culture fluid of Nocardia fructiferi var. ristomycini, strain 76. By its solubility and mobility in various solvent systems and by the absorption spectrum the new pigment is close but not identical to such a pigment as rubrocyanin. The presence of microelements, i.e. iron and magnesium in the medium was shown to be necessary for production of violet pigment.     

Effect of temperature on violacein production in a psychrotrophicChromobacterium from Lake Ontario sediment

A truly psychrotrophic strain ofChromobacterium, which was isolated from Lake Ontario sediment and characterized asChromobacterium lividum, was found to be capable of pigment production that was completely prevented at 0°C, although growth readily occurred. Normal pigment formation occurred at 15°C, 20°C, and 25°C. The prevention of synthesis of the pigment at 0°C, which was confirmed spectrophotometrically to be violacein, was not reversed by the presence of various carbon sources, although all except one acted as growth substrates. In addition, some of the carbon sources actually inhibited pigment production at 20°C, preventing violacein synthesis in the presence of pyruvate which was shown to allow pigmentation. Similar results were obtained under both liquid and solid media cultivation conditions.     

Kinetic analysis of red pigment and citrinin production by Monascus ruber as a function of organic acid accumulation

In submerged cultures performed in synthetic medium containing glucose and glutamate, the filamentous fungus Monascus ruber produced a red pigment and a mycotoxin, citrinin. In oxygen-limiting conditions, the production of these two metabolites was growth-associated, as was the production of primary metabolites. In oxygen-excess conditions, the profile of citrinin production was typical of a secondary metabolite, since it was produced mostly during the stationary phase. In contrast, the production of the pigment decreased rapidly throughout the culture, showing a profile characteristic of an inhibitory mechanism. The organic acids produced during the culture, L-malate and succinate, were shown to be slightly inhibitory against pigment production, while citrinin production was unaffected. However, this inhibition could not account for the observed profile of pigment production in batch cultures. Other dicarboxylic acids such as fumarate or tartrate showed a similar effect to that provoked by malate and succinate as regards pigment production. It was concluded that the decrease in red pigment production during the culture was due to the inhibitory effect of an unknown product whose accumulation was favored in aerobic conditions.     

Morphological change and enhanced pigment production of monascus when cocultured with saccharomyces cerevisiae or aspergillus oryzae

When a Monascus isolate, a producer of Monascus pigments, was cocultured with either Saccharomyces cerevisiae or Aspergillus oryzae in a solid sucrose medium, there were significant morphological changes in Monascus culture. Cocultures exhibited cell mass increases of 2 times and pigment yield increases of 30 to 40 times compared to monocultures of Monascus. However, enhanced cell growth, an increase in pigment production, and morphological change did not occur in coculture with Bacillus cereus. Saccharomyces cerevisiae was more effective at enhancing pigment production than Asp. oryzae. Enhanced cell growth and increased pigment production occurred only in conjunction with morphological changes. Culture filtrates of S. cerevisiae were also effective in inducing morphology change in Monascus, similar to culture broths of S. cerevisiae. The hydrolytic enzymes produced by S. cerevisiae, such as amylase, and chitinase, are thought to be the effectors. The commercial enzymes alpha-amylase and protease from Asp. oryzae both caused a morphological change in Monascus and were effective in enhancing pigment production. However, lysozyme, alpha-amylase and protease from Bacillus species, protease from Staphylococcus, and chitinase from Streptomyces were not effective. The hydrolytic enzymes which cause a morphological change of Monascus culture and enhancement of pigment production are thought to be capable of degrading Monascus cell walls. An approximate 10-fold increase in pigment production was observed in liquid cocultures with S. cerevisiae. Copyright 1998 John Wiley & Sons, Inc.     

A cloned regulatory gene of Streptomyces lividans can suppress the pigment deficiency phenotype of different developmental mutants.

We report here the cloning of a Streptomyces lividans gene that when introduced on a multicopy plasmid vector reversed the pigment deficiency phenotype of several distinct mutants blocked in development, pigment production, or both. Although this gene was shown by restriction enzyme analysis to be similar to a previously cloned afsB-complementing gene of Streptomyces coelicolor, we show that it does not correspond to the S. coelicolor chromosomal locus designated afsB. Thus, the cloned locus, which we propose to rename afsR, appears to complement the AfsB- phenotype by pleiotropic regulatory effects.     

Cloning and regulation of Erwinia herbicola pigment genes.

The genes coding for yellow pigment production in Erwinia herbicola Eho10 (ATCC 39368) were cloned and localized to a 12.4-kilobase (kb) chromosomal fragment. A 2.3-kb AvaI deletion in the cloned fragment resulted in the production of a pink-yellow pigment, a possible precursor of the yellow pigment. Production of yellow pigment in both E. herbicola Eho10 and pigmented Escherichia coli clones was inhibited by glucose. When the pigment genes were transformed into a cya (adenylate cyclase) E. coli mutant, no expression was observed unless exogenous cyclic AMP was provided, which suggests that cyclic AMP is involved in the regulation of pigment gene expression. In E. coli minicells, the 12.4-kb fragment specified the synthesis of at least seven polypeptides. The 2.3-kb AvaI deletion resulted in the loss of a 37K polypeptide and the appearance of a polypeptide of 40 kilodaltons (40K polypeptide). The synthesis of the 37K polypeptide, which appears to be required for yellow pigment production, was not repressed by the presence of glucose in the culture medium, as was the synthesis of other polypeptides specified by the 12.4-kb fragment, suggesting that there are at least two types of gene regulation involved in yellow pigment synthesis. DNA hybridization studies indicated that different yellow pigment genes exist among different E. herbicola strains. None of six pigmented plant pathogenic bacteria examined, Agrobacterium tumefaciens C58, Cornyebacterium flaccumfaciens 1D2, Erwinia rubrifaciens 6D364, Pseudomonas syringae ATCC 19310, Xanthomonas campestris 25D11, and "Xanthomonas oryzae" 17D54, exhibited homology with the cloned pigment genes.     

Characterization of the Melanogenic System in Vibrio cholerae,ATCC 14035

The nature of the pigment formed by Vibrio cholerae and the characterization of its biosynthetic pathway is shown. This microorganism is able to synthesize melanin-like pigment when cultured in the presence of L-tyrosine. Other phenolic chemicals related to L-tyrosine do not lead to pigment production. The microorganism has no tyrosine hydroxylase activity, and the levels of dopa oxidase activity are very low, making the existence of a tyrosinase very unlikely. However, Vibrio cholerae contained transami-nases that transforms L-tyrosine into p-hydroxyphenylpyruvate. Moreover, Vibrio cholerae is able to go further in the catabolic pathway, releasing a great amount of homogentisic acid. This acid can spontaneously be oxidized to its p-quinone form, which subsequently polymerizes leading to pigment formation. It is concluded that the pigment formed by Vibrio cholerae is not synthesized by the Raper-Mason pathway, but by a L-tyrosine catabolism pathway leading to homogentisic acid. Some simple properties of that melanin are compared to model eu- and pheomelanin, but no clear distinction could be stated, indicating the similarity between all these pigments.     

Effect of jasmonates and exogenous polysaccharides on production of alkannin pigments in suspension cultures of Alkanna tinctoria

Summary The conditions for the efficient production of alkannin pigments by a suspension culture of Alkanna tinctoria were established. Pectin, polygalacturonic acid sodium salt and galactan increased the pigment production but not as much as agar did. A marked increase in the pigment content in cells and medium of suspension cultures after treatment with methyl jasmonate was observed. It was shown, applying a two-layer culture method, that mineral and olive oils intensified the pigment secretion from cells to the medium but did not enhance significantly their synthesis. Thin layer chromatography and high performance liquid chromatography methods showed that two main esters of alkannin are responsible for the characteristic colour of A. tinctoria suspension cultures.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole 3-acetic acid - NAA 1-naphthaleneacetic acid - MeJA methyl jasmonate - TLC thin layer chromatography - HPLC high performance liquid chromatography     

Melanin production encoded by a cryptic plasmid in a Rhizobium leguminosarum strain

Rhizobium leguminosarum strain VF39, isolated from nodules of field-grown faba beans in the Federal Republic of Germany, was shown to contain six plasmids ranging in molecular weight from 90 to 400 Md. Hybridisation to nif gene probes, plasmid curing, and mobilisation to other strains of Rhizobium and to Agrobacterium showed that the third largest plasmid, pRleVF39d (220 Md), carried genes for nodulation and nitrogen fixation. This plasmid was incompatible with pRL10JI, the Sym plasmid of R. leguminosarum strain JB300. Of the other plasmids, the two smallest (pRleVF39a and pRleVF39b, 90 and 160 Md respectively) were shown to be self-transmissible at a low frequency. Although melanin production is as yet unreported in strains of R. leguminosarum biovar viceae, strain VF39 produced a dark pigment, which, since it was not produced on minimal media and its production was greatly enhanced by the presence of tyrosine in the media, is probably melanin-like. Derivatives of VF39 cured of pRleVF39a no longer produced this pigment, but regained the ability to produce it when this plasmid was transferred into them. Strains of Agrobacterium tumefaciens, R. meliloti, and some strains of R. leguminosarum carrying pRleVF39a did not produce this pigment, indicating perhaps that some genes elsewhere on the VF39 genome are also involved in pigment production. Plasmid pRleVF39a appeared to be incompatible with the cryptic Rhizobium plasmids pRle336b and pRL8JI (both ca. 100 Md), but was compatible with the R. leguminosarum biovar phaseoli Sym plasmids pRP1JI, pRP2JI and pRph51a, all of which also code for melanin production. The absence of pRleVF39a in cured derivatives of VF39 had no effect on the symbiotic performance or competitive ability of this strain.     

The effect of pH and amino acids on conidiation and pigment production of Monascus major ATCC 16362 and Monascus rubiginosus ATCC 16367 in submerged shaken culture.

Monascus major ATCC 16362 and Monascus rubiginosus ATCC 16367 were cultivated aerobically on media containing nitrate or ammonium as nitrogen source to which the following modifications were made: (1) pH adjusted to 2.5 before sterilization; (2) addition of yeast extract; (3) addition of amino acids in identical proportions and concentrations to those found in yeast extract; (4) adjustment of pH to 2.5 after addition of amino acids. The addition of amino acids in the form of yeast extract increased mycelium formation and reduced conidiation and pigment production. The addition of an amino acid mixture did not increase mycelium formation to the same extent as yeast extract but increased the number of conidia, while pigment production was reduced, especially when nitrate was the nitrogen source. As the amino acids are taken up after conidial formation has started, it would appear that it is not the amino acids themselves which are directly responsible for the induction of conidiation. The addition of amino acids inhibits nitrate and ammonium uptake suggesting the need for an early intracellular nitrogen limitation to induce conidiation. Lowering the pH inhibits the formation of conidia and increases pigment production; also the effect of amino acid addition is totally annulled. The pH of the medium is all important in regulating the formation of conidia and pigment production. The possible effects of the pH on the uptake of certain medium components is discussed, as well as their possible control of certain metabolic pathways which ultimately determines the availability of intermediates for conidiation and pigment production.     

Yellow fluorescence of fusobacteria

Yellow fluorescence was shown to be a common property amongst seven species of fusobacteria. Pigment production varied depending on the basal medium used and the addition of cysteine hydrochloride to basal media was shown to stimulate yellow pigment production.     

Kinetic analysis of growth rate, ATP, and pigmentation suggests an energy-spilling function for the pigment prodigiosin of Serratia marcescens

Serratia marcescens is a gram-negative environmental bacterium and opportunistic pathogen. S. marcescens expresses prodigiosin, a bright red and cell-associated pigment which has no known biological function for producing cells. We present here a kinetic model relating cell, ATP, and prodigiosin concentration changes for S. marcescens during cultivation in batch culture. Cells were grown in a variety of complex broth media at temperatures which either promoted or essentially prevented pigmentation. High growth rates were accompanied by large decreases in cellular prodigiosin concentration; low growth rates were associated with rapid pigmentation. Prodigiosin was induced most strongly during limited growth as the population transitioned to stationary phase, suggesting a negative effect of this pigment on biomass production. Mathematically, the combined rate of formation of biomass and bioenergy (as ATP) was shown to be equivalent to the rate of prodigiosin production. Studies with cyanide inhibition of both oxidative phosphorylation and pigment production indicated that rates of biomass and net ATP synthesis were actually higher in the presence of cyanide, further suggesting a negative regulatory role for prodigiosin in cell and energy production under aerobic growth conditions. Considered in the context of the literature, these results suggest that prodigiosin reduces ATP production by a process termed energy spilling. This process may protect the cell by limiting production of reactive oxygen compounds. Other possible functions for prodigiosin as a mediator of cell death at population stationary phase are discussed.     

Studies on the Production of Pink Pigment in Pseudomonas extorquens NClB 9399 Growing in Continuous Culture

S ummary . The pink pigment of Pseudomonas extorquens was identified tentatively as an oxo-carotenoid similar to rhodoxanthin. The production of the pigment increased at the end of the logarithmic phase of growth of Ps. extorquens in batch cultures. It was produced during growth on a wide variety of carbon and nitrogen sources, with the exception of ethanol, but not in the presence of diphenylamine, an observation which was consistent with the identification of the pigment as a carotenoid. The organism was grown in continuous culture under conditions of oxygen and magnesium limitation which might be expected to restrict the oxidation of γ-carotenes to pink pigments. However, such limitations caused an increase in pigmentation over that of methanol-limited cultures. Non-pink mutants of Ps. extorquens , obtained after the use of the mutagen, ethyl methyl sulphonate, did not have growth-rates lower than those of the parent strain. The pigment would seem to have no central metabolic role in actively growing cells but carbon is diverted to pigment production when growth is restricted by other than carbon limitation.     

Pigment Production from Immobilized Monascus sp. Utilizing Polymeric Resin Adsorption

Pigment production by the fungus Monascus sp. was studied to determine why Monascus sp. provides more pigment in solid culture than in submerged culture. Adding a sterilized nonionic polymeric adsorbent resin directly to the growing submerged culture did not enhance the pigment production, thus indicating that pigment extraction is probably not a factor. Monascus cells immobilized in hydrogel were studied and exhibited decreased pigment production as a result of immobilization. This result is thought to be due to diffusional resistance of the pigment through the hydrogel beads. Addition of the adsorbent resin to the immobilized Monascus culture increased both the maximum pigment yield and the production rate above those of the free-cell fermentations. The provision of a support for the mycelium may explain enhanced pigment production by the solid-state culture. These results indicate that product diffusion from immobilized cell systems can be the limiting factor and that in situ extraction is one possible way to circumvent this problem.     

One-step enzymatic synthesis of blue pigments from geniposide for fabric dyeing

In this study, we describe a one-step chemoenzymatic reaction for the production of natural blue pigments, in which the geniposide from Gardenia extracts is transformed by glycosidases to genipin. Genipin is then allowed to react with amino acids, thereby generating a natural blue pigment. The β-glycosidases, most notably isolase (a variant of β-glucanase), recombinant β-glucosidase, Cellulase T, and amylases, were shown to hydrolyze geniposide to produce the desired pigments, whereas the α-glycosidases did not. Among the 20 tested amino acids, glycine and tyrosine were associated with the highest dye production yields. The optimal molar ratio of geniposide to glycine, two reactants relevant to pigment production, was unity. The natural blue pigments produced in this study were used to dye cotton, silk, and wool. The color yields of the pigments were determined to be significantly higher than those of other natural dyes. Furthermore, the color fastness properties of these dyes were fairly good, even in the absence of mordant.     

Study of methanol-induced phenotypic changes in a novel strain of Acinetobacter lwoffii

A Gram-negative bacterial strain designated LS2 isolated from Lahaul–Spiti valley of North India was shown to produce pink pigment while utilizing methanol as sole source of carbon and energy. Interestingly, pigment production was inducible in nature since the organism did not produce any pigment when grown on other carbon sources. Based on phenotypic and phylogenetic characterization the non-pigmented methylotroph was identified as a novel strain of Acinetobacter lwoffii MTCC 8288 (DQ144736). By means of spectral and mass analyses the pigment was characterized as bacterioruberin-like carotenoid molecule. Here, the carotenoid pigment may form an important part of the antioxidant defense mechanism against oxidative stress imparted by methanol. The methanol utilization pathway in strain LS2 was deciphered by showing the presence of functional methanol dehydrogenase and formaldehyde dehydrogenase genes. In addition, to investigate methanol induced physiological changes, comparative fatty acid profile was analysed and distinctive qualitative as well as quantitative differences in fatty acid content were observed. Therefore, we suggest that strain LS2 exhibiting such unique phenotypic property should be assigned a taxonomic position other than the pigmented and non-pigmented methylotrophs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Re-assessment of phenotypic identifications of Bacteroides putredinis to Alistipes species using molecular methods

Alistipes (previously Bacteroides) are strictly anaerobic gram-negative rods that resemble the Bacteroides fragilis group in that most species are bile-resistant and indole-positive; however, they are only weakly saccharolytic and most species produce light brown pigment only on laked rabbit blood agar. In this retrospective study, we re-identified 18 organisms previously identified phenotypically as "Bacteroides putredinis-like", but that did not produce pigment on routine media. The strains were identified with 16S rDNA sequencing and pigment production was evaluated on several different culture media. Only 12/18 strains had molecular identifications of Alistipes species, while the remaining strains phylogenetically resembled Butyricimonas and Odoribacter spp. Pigment production was not a reliable test for those Alistipes strains that are described as pigment producers.