Metschnikowia strains isolated from botrytized grapes antagonize fungal and bacterial growth by iron depletion

Noble-rotted grapes are colonized by complex microbial populations. I isolated pigment-producing Metschnikowia strains from noble-rotted grapes that had antagonistic activity against filamentous fungi, yeasts, and bacteria. A red-maroon pigment was formed from a diffusible colorless precursor released by the cells into the medium. The conversion of the precursor required iron and could occur both in the cells (red colonies) and in the medium (red halos around colonies). The intensity of pigmentation was correlated with the intensity of the antimicrobial activity. Mutants that did not form pigment also lacked antifungal activity. Within the pigmented halos, conidia of the sensitive fungi did not germinate, and their hyphae did not grow and frequently lysed at the tips. Supplementation of the medium with iron reduced the size of the halos and the inhibition zones, while it increased the pigment accumulation by the colonies. The iron-binding agent tropolone had a similar effect, so I hypothesize that pigmented Metschnikowia isolates inhibit the growth of the sensitive microorganisms by pigment formation, which depletes the free iron in the medium. As the pigment is a large nondiffusible complex produced in the presence of both low and high concentrations of ferric ions, the proposed mechanism is different from the mechanisms operating in microbes that release siderophores into the environment for iron acquisition.     

Pigment Production by Streptococcus agalactiae in Quasi-Defined Media

A quasi-defined medium that supports the growth of Streptococcus agalactiae as pigmented colonies has been developed. The medium contains starch, a peptic digest of albumin, amino acids, nucleosides, vitamins, and salts. The presence of free cysteine, which could be replaced with other sulphur-containing compounds and to a lesser degree by reducing agents, was required for pigment formation.     

Propionibacterium jensenii Produces the Polyene Pigment Granadaene and Has Hemolytic Properties Similar to Those of Streptococcus agalactiae

The red polyene pigment granadaene was purified and identified from Propionibacterium jensenii. Granadaene has previously been identified only in Streptococcus agalactiae, where the pigment correlates with the hemolytic activity of the bacterium. A connection between hemolytic activity and the production of the red pigment has also been observed in P. jensenii, as nonpigmented strains are nonhemolytic. The pigment and hemolytic activity from S. agalactiae can be extracted from the bacterium with a starch extraction solution, and this solution also extracts the pigment and hemolytic activity from P. jensenii. A partial purification of the hemolytic activity was achieved, but the requirement for starch to preserve its activity made the purification unsuccessful. Partially purified hemolytic fractions were pigmented, and the color intensity of the fractions coincided with the hemolytic titer. The pigment was produced in a soluble form when associated with starch, and the UV-visual spectrum of the extract gave absorption peaks of 463 nm, 492 nm, and 524 nm. The pigment could also be extracted from the cells by a low-salt buffer, but it was then aggregated. The purification of the pigment from P. jensenii was performed, and mass spectrometry and nuclear magnetic resonance analysis revealed that P. jensenii indeed produces granadaene as seen in S. agalactiae.     

A clonal approach to the problem of neural crest determination.

A fundamental question regarding neural crest development is the possible pluripotential nature of this embryonic tissue. As a first step in examining this problem, clonal techniques are used to produce homogeneous populations of crest cells. Primary cultures of these cells are obtained by explanting neural tubes from Japanese quail in vitro and allowing crest cells to migrate away. The explant is removed, the outgrowth is isolated, dissociated with trypsin, and the cells plated at clonal density. Colonies derived in this manner fall into the following categories: all cells of the colony pigmented; none of the cells pigmented; and some of the cells pigmented, the remainder unpigmented. Pigmented colonies generally arise from small, round cells whereas the non-pigmented colonies usually originate from large, flattened polymorphous cells. Differentiation of melanocytes does not preclude their continued proliferation. The pigment phenotype, in addition, is stable through at least 25 generations. That the mixed colonies, in fact, are clonally derived is shown by physically isolating single cells. The identity of the non-pigment cells was not established in the present work. A possible neural fate is suggested, however, since nerve-like cells develop after the petri plates become overgrown. Neural clones did not form even though nerve growth factor activity is present as a normal constituent of the culture medium and was added as a supplement in some instances. These techniques permit the preparation of large, homogeneous populations of neural crest cells and afford an opportunity to examine aspects of crest determination heretofore impossible to study.     

Biofilm-Specific Cross-Species Induction of Antimicrobial Compounds in Bacilli

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10^T and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B. licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10^T and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.     

Pigmentation and Acriflavine Resistance in Serratia marcescens

Stable, orange, acriflavine-resistant variants were selected by treatment of a wild-type, red, acriflavine-sensitive strain of Serratia marcescens with acriflavine. Visible, ultraviolet, infrared, and nuclear magnetic resonance spectra of purified pigment from the red strain were identical to those of the pigment from the orange strain, and the orange mutant was not due to a mutation affecting the structure of the pigment, prodigiosin. The color of the red strain was not affected by variations in pH between 5.0 and 8.0, whereas the color of the orange mutant changed from pink to orange over the same pH range. This variation was mimicked by the pH-induced variation in color of prodigiosin purified from either the red, wild-type or the orange, mutant strains. Density-gradient centrifugation of cell fragments after ultrasonic disintegration resulted in characteristic pigmented bands. Biochemical characterization of these pigmented bands showed that they contained pigment and a protein component, but no lipids, polysaccharides, sugars, glucosamine, or phosphates were detected. Further fractionation of these pigmented bands by zone electrophoresis on a sucrose density gradient indicated that some pigment in S. marcescens was specifically attached to protein components.     

Pseudomonas aeruginosa-Candida albicans Interactions: Localization and Fungal Toxicity of a Phenazine Derivative

Phenazines are redox-active small molecules that play significant roles in the interactions between pseudomonads and diverse eukaryotes, including fungi. When Pseudomonas aeruginosa and Candida albicans were cocultured on solid medium, a red pigmentation developed that was dependent on P. aeruginosa phenazine biosynthetic genes. Through a genetic screen in combination with biochemical experiments, it was found that a P. aeruginosa-produced precursor to pyocyanin, proposed to be 5-methyl-phenazinium-1-carboxylate (5MPCA), was necessary for the formation of the red pigmentation. The 5MPCA-derived pigment was found to accumulate exclusively within fungal cells, where it retained the ability to be reversibly oxidized and reduced, and its detection correlated with decreased fungal viability. Pyocyanin was not required for pigment formation or fungal killing. Spectral analyses showed that the partially purified pigment from within the fungus differed from aeruginosins A and B, two red phenazine derivatives formed late in P. aeruginosa cultures. The red pigment isolated from C. albicans that had been cocultured with P. aeruginosa was heterogeneous and difficult to release from fungal cells, suggesting its modification within the fungus. These findings suggest that intracellular targeting of some phenazines may contribute to their toxicity and that this strategy could be useful in developing new antifungals.     

Pigmentation and Antibacterial Activity of Fast Neutron- and X-Ray-induced Strains of Monascus purpureus Went

Seven new strains of Monascus purpureus Went were induced by neutron and x-ray irradiation. The quantity and quality of pigments produced by these strains differed. Strains N4S and N11S produced twice as much pigment as normal, while another strain, N14S, was albino. An unknown orange pigment was found in young colonies of the N11S strain. This orange pigment reacted with alcohols and malt extract medium to form red pigments. Strains N4S, N11S, X2P, and wild type inhibited the growth of certain bacteria, especially the Bacillus species. Strain N11S had more antibacterial activity than wild type. A major active compound was isolated with an ultraviolet absorption spectrum that was related to those of the red pigments found in this fungus. The active compound(s) was named monascidin.     

The detection of monosomic colonies produced by mitotic chromosome non-disjunction in the yeast Saccharomyces cerevisiae

A diploid yeast strain, D6, is described which monitors mitotic non-disjunction by the phenotypic expression of a set of coupled and recessive markers flanking the centromere of chromosome VII. These markers are not expressed in the heterozygous condition prevailing in D6. The left arm of chromosome VII carries a tightly centromere linked marker, leu1 (leucine requirement), distal to leu1 in this order: trp5 (trytophan requirement), cyh2 (recessive resistance to cycloheximide) and met 13 (requirement for methionine). The right arm is marked with ade3 (simultaneous requirement for adenine and histidine). D6 is homozygous for ade2 and consequently, forms red rather than the normally white colonies. It shows no requirement for the above amino acids and it is sensitive to cycloheximide.Unmasking of all the markers on chromosome VII leads to colonies that are white because ade3 sets a block preceding the ade2 block (which causes the accumulation of a precursor of the red pigment), they require leucine, tryptophan and methionine, and grow on media with cycloheximide. Cells are plated on a cycloheximide medium where red and white colonies are formed. Colonies of spontaneous origin were tested. The majority of the white colonies expressed all the recessive markers whereas only few of the red colonies expressed all the markers on the left arm of chromosome VII.Basically expression of recessive markers on both sides of the centromere can be explained as a result of two coincident events of mitotic crossing over. However, the frequency of colonies expressing centromere linked leu1 was 14 times higher among the white types than the red ones. This suggested that the white, cycloheximide resistant, leucine requiring colonies arose by mitotic non-disjunction and not only by two coincident mitotic crossing over events.Presumptive spontaneous monosomic segregants were placed on sporulation medium. Only 8 out of 30 isolates sporulated, which showed that these eight segregants were diploid at the time of sporulation. They could have arisen by two coincident crossover events or through restoration of a normal disomic condition after non-disjunction had occurred. The genetic data thus leaves us with only its statistical argument in favour of non-disjunction. Further confirmation of monosomic nature of the white cycloheximide resistant colonies was provided by the estimates of their DNA contents. Compared to the stock wild type diploids the presumptive monosomics showed a reduction in DNA content.We have utilized D6 to investigate the possible induction of mitotic non-disjunction after treatment with gamma rays, heat shock at 52°C and ultraviolet irradiation. In all cases white, cycloheximide resistant colonies were produced at levels significantly higher than that found in untreated cultures. In order to detect the production of monosomic cells, treated cultures were grown for 48 h in non-selective medium after exposure to allow for “expression” of the monosomic condition.     

Correlation between Congo red binding and contact haemolysin production in Shigella species

Haemolytic strains of Shigella dysenteriae type 1, Shigella flexneri, Shigella boydii and Shigella sonnei cultured on Congo red agar produced pigmented colonies (Pcr+) whereas nonhaemolytic strains produced white colonies and did not bind Congo red (Pcr-). S. flexneri-1 haemolysin negative mutant (lacking plasmid) of haemolysin positive prototroph also did not bind Congo red and produced nonpigmented colonies. Among the twelve strains of Shigella included in this study, the characteristics of Congo red binding, plasmid profile and haemolytic activity appeared to be correlated. Congo red binding occurred comparatively more by haemolysin-producing strains. Congo red binding can be used as a quick and reliable method for virulence traits of pathogens, including haemolysin activity.     

Application of the complex of DNA with the congo red anionic diazo dye for detection of nuclease-producing colonies of marine bacteria

This study was aimed at the development of a method for detection of colonies of nuclease-secreting marine bacteria. The BAL nuclease-producing marine bacterium Pseudoalteromonas espejiana BAL-31 was used as the test object. A new method was developed involving the congo red (CR) anionic dye. The P. espejiana culture was plated on nutrient agar with CR and denatured DNA. In such media, CR was found to form complexes with DNA. After two days of incubation at 30°C, halos were found around the P. espejiana colonies. No halos appeared when DNA was not introduced, when BAL nuclease was inactivated, or when the plates were inoculated with Escherichia coli. It was concluded that the halos around the colonies indicated nuclease secretion. The halos were shown to result from the coagulation of CR released after digestion of the CR-DNA complex by the nuclease. This method for detection of nuclease-producing colonies can probably be used for all marine bacteria and possibly for halophilic bacteria as well.     

Application of the complex of DNA with the congo red anionic diazo dye for detection of nuclease-producing colonies of marine bacteria

This study was aimed at the development of a method for detection of colonies of nuclease-secreting marine bacteria. The BAL nuclease-producing marine bacterium Pseudoalteromonas espejiana BAL-31 was used as the test object. A new method was developed involving the congo red (CR) anionic dye. The P. espejiana culture was plated on nutrient agar with CR and denatured DNA. In such media. CR was found to form complexes with DNA. After two days of incubation at 30 degrees C, halos were found around the P. espejiana colonies. No halos appeared when DNA was not introduced, when BAL nuclease was inactivated, or when the medium was inoculated with Escherichia coli. It was concluded that the halos around the colonies indicated nuclease excretion. The halos were shown to result from the coagulation of CR released after digestion of the CR-DNA complex by the nuclease. This method for detection of nuclease-producing colonies can probably be used for all marine bacteria and possibly for halophilic bacteria as well.     

A new polyphenoloxidase test for distinguishing between wood-rotting fungi

White-rot fungi causing the white decay of wood, secrete polyphenol oxidase type enzymes which catalyze the oxidation of diphenols to quinones. If the substrate also contains amino acids, they can be oxidized secondarily to a red pigment, the intensity of which depends on the activity of oxidation oxoenzymes. This finding was used for devising a quick test to demonstrate the oxidases secreted into the cultivation medium by wood-rotting fungi. As substrates we used hydroquinone and glycine. We tested a total of 89 species and 131 strains of wood-rotting fungi. Simultaneously, we compared the newly devised test with the most common test for the detection of polyphenoloxidases, with the guaiacol test. It follows from the results that the new test, in which the intensity of the red colour was estimated colorimetrically, permits to divide the wood-rotting fungi into two groups, the positively and the negatively reacting ones. This division does not fully coincide with that obtained by means of the guaiacol test.     

Possible demonstration of multipotential nature of embryonic neural retina by clonal cell culture.

The possible multipotential nature of the neural retina of early chick embryos was examined by the technique of clonal cell culture. Cultures were prepared from cells dissociated from freshly excised neural retinas of 3.5-day-old chick embryos or from cells harvested from primary highdensity cultures. The following four colony types were obtained: colonies differentiating into “lentoid bodies”; colonies with pigment cells; colonies with both “lentoid bodies” and pigment cells; and colonies comprised entirely of unidentifiable cells. Neuronal differentiation occurred frequently in the early stages of culture (up to about 10 days). In some of these neuronal colonies, “lentoid bodies” and, rarely, both “lentoid bodies” and pigment cells differentiated after a further culture period of up to 30 days. Secondary colonies established from primary colonies after 9–10 days demonstrated that these original colonies fell into four different categories: those giving rise to secondary colonies containing only “lentoid bodies,” those giving rise to pigmented colonies only, those developing both lentoid and pigmented colonies, and finally those which gave rise to secondary colonies of all three types, lentoid, pigmented, and mixed colonies. When primary pigmented colonies were recloned at about 30 days after inoculation, the differentiated pigment cells transdifferentiated into lens. Whether multispecific colonies were really of clonal origin or not is discussed. The possible presence of a multipotent progenitor cell able to give rise to multispecific clones in the neural retina of 3.5-day-old chick embryos is suggested. A sequence of differentiation starting from multipotent neural retinal cells to be terminated with lens through the differentiation of neuronal and pigment cells is hypothetically proposed.     

A New Solid Medium for Enumerating Cellulose-Utilizing Bacteria in Soil

A solid medium containing ashed, acid-washed cellulose and a dye, Congo red, has been developed for enumeration of cellulose-utilizing bacteria in soil. Bacteria able to use the cellulose in this medium produced distinct zones of clearing around their colonies. A vivid contrast between the uniform red color of the medium and these halos made this method of differentiation of these organisms superior to other methods.     

Iron-induced pigment formation and ionizing radiation resistance in three strains of Micrococcus violagabriellae

Three strains of Micrococcus violagabriellae-Littlepig, Pig (Parental), and Superpig--were studied. The pulcherrimin pigment (which imparts a violet-red color to the colonies of the Parental and Superpig strains) is produced only when the cells are grown in the presence of excess iron and provides a unique system for the study of the radioprotection conferred by a pigment. Cell suspensions of each of the three strains (initially grown in the presence and in the absence of excess iron) were exposed to gamma rays and plated on media containing an excess of iron and on media without iron. The survival curves of the treated strains indicate a correlation between the color intensity of the pigment (pulcherrimin), and the ability of the organisms to survive moderate doses of ionizing radiation. Studies using radioactive iron (59Fe) showed that a correlation also exists between the intensity of the pigment and the amount of iron that the cells of each strain can remove from the medium. The effect of gamma irradiation on postirradiation cell growth is discussed for each strain irradiated during logarithmic growth and during a stationary state.     

A plate assay method for the detection of fungal polygalacturonase secretion

Abstract A method for the detection of polygalacturonase activity has been developed using ruthenium red staining of fungal colonies on polygalacturonate- agarose plates. Ruthenium red was shown to penetrate beneath the surface layers of the gel, in the regions surrounding a fungal colony where degradation of polygalacturonate had occurred. Without degradation of polygalacturonate ruthenium red did not penetrate the medium, was restricted to binding to the surface layers and was easily washed off. The medium containing undegraded polygalacturonate was a colourless clear background and areas of polygalacturonate degradation around the colonies were visualised as an intense purple-red halo. The method has been used to screen yeasts and filamentous fungi for polygalacturonase secretion.     

Water-Soluble Red Pigment Production by <Emphasis Type="Italic">Paecilomyces sinclairii</Emphasis> and Biological Characterization

Natural pigments have several advantages over synthetic colorants. In this study, the production of red pigment produced by Paecilomyces sinclairii in microbial fermentation was demonstrated and the pigment was purified and characterized. The red pigment was produced from submerged fungal fermentation and fractionated by medium pressure flash chromatography. After fractionation, the spectrophotometric characterization of the red pigment revealed an λ_max at 520 nm. Antimicrobial activity of the red pigment fraction was also studied against Escherichia coli O157 and Pseudomonas aeruginosa PAO1. The fraction (F2-F6) of the red pigment exhibited broad-spectrum antimicrobial activity in both bacteria. These results demonstrate the potential of this pigment in inhibiting bacterial growth and in food processing and other foodrelated applications.     

An ultraviolet light induced bacteriophage in Beneckea gazogenes.

An ultraviolet light induced prophage has been discovered in the red pigmented marine vibrio Beneckea gazogenes. Two spontaneously derived pigment mutants, one forming pink colonies and one lacking pigment and forming white colonies, were also irradiated. The presence of pigment was not related to phage induction; uv-induced cell lysis occurred in wildtype and mutant strains at the same dosages. Lysis was not prevented or retarded by exposure after irradiation to visible light indicating the phenomenon was not photoreactivable. Electron micrographs of the 'T-like' B. gazogenes phage are shown. A second beneckea was isolated form the anaerobic zone of cyanobacterial mats growing in the hypersaline environment of Laguna Mormona, Baja California. The Baja beneckea does not harbor a uv inducible prophage and is resistant to the B. gazogenes phage under all conditions tested.     

Selective medium for the isolation and enumeration of Klebsiella spp

A highly selective medium for the enumeration and isolation of Klebsiella pneumoniae and Klebsiella oxytoca was developed in which the typical colonies were convex and 1 to 2 mm in diameter. Their pigment was either a mucoid pink-red color or a more watery pale red with a dark red center. Relatively little colonial growth occurred for any other bacterial genera, and where such colonies did grow, they could be easily differentiated since the form was atypical. The medium already appears to have potential value as a means of assessing the efficiency of treating sewage and monitoring the microbiological quality of vegetables.