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.     

Granadaene: Proposed Structure of the Group B Streptococcus Polyenic Pigment

The goal of this work was to determine the chemical nature of the red pigment produced by Streptococcus agalactiae, which has been thought to be a carotene. We extracted the pigment with 0.1 M KOH and purified it by column chromatography on Sephadex LH. Data from elemental analysis and mass and nuclear magnetic resonance spectra lead us to propose the structure to be that of a new ornithine rhamno-polyene with 12 conjugated double bonds, to which we have assigned the trivial name granadaene.     

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.     

A prodiginine pigment toxic to embryos and larvae of Crassostrea virginica

The red pigment produced by a marine Pseudomonas sp. which causes abnormal development and mortality in developing embryos of the American oyster, Crassostrea virginica, was analyzed. A comparative study of a nonpigmented and two pigmented mutants of the red parental strain indicated that virulence was associated and varied with pigmentation. The use of sonicated cells supported lysing of the pseudomonad cells as the most probable means of pigment release. Crude pigment extracted from the red parental strain and its yellow mutant was toxic to developing oyster embryos. Neither the “pigment” extracted from the white mutant nor dimethyl sulfoxide, used for dissolving the extract, was toxic. Three pigment fractions were demonstrated by thin-layer chromatography even after purification. Studies indicate that only the fraction corresponding to R₁ 0.41 was necessary for virulence. The virulent pigment fraction was identified as belonging to the prodiginine group.     

Isolation and Function of Allophycocyanin B of Porphyridium cruentum

Allophycocyanin B was purified to homogeneity from the eukaryotic red alga Porphyridium cruentum. This biliprotein is distinct from the allophycocyanin of P. cruentum with respect to subunit molecular weights, and spectroscopic and immunological properties. The purified allophycocyanin B has a long wavelength absorption maximum at 669 nm at room temperature and at 675 nm at −196 C while the fluorescence emission maximum is at 673 nm at room temperature and 679 nm at −196 C. The emission spectrum of allophycocyanin shifted only 1 nm, from 659 to 660 nm, on cooling to −196 C, and was the same with allophycocyanin crystals as it was with pure solutions of the pigment. Phycobilisomes from P. cruentum have a major fluorescence emission band at 680 nm at −196 C which emanates from the small amount of allophycocyanin B present in the phycobilisomes. Light energy absorbed by the bulk of the biliprotein pigments is transferred to allophycocyanin B with high efficiency.     

Starch Measurement in Plant Tissue Using Enzymatic Hydrolysis

This work explored completeness of starch hydrolysis in situ in relation to degree of gelatinization, starch content of tissue, evailable enzyme activity, and time allowed for hydrolysis. Maximum hydrolysis of starch in lyophilized red oak (Quercus rubra L.) root tissue with purified Diazyme (amyloglucosidase) or Clarase (Takadiastase) required high enzyme activity (2.4 U Diazyme or 48 U Clarase per mg starch). Results suggested that at least 70 U Clarase or 5 U Diazyme should be used per mg starch in routine analyses. Neither prolonging gelatinization (more than 15 min) nor hydrolysis (more than 24 to 48 lh) offset inadequate starch hydrolysis caused by insufficient enzyme activity. Starch was completely hydrolyzed in situ after 48 h without gelatinization by 5 U of Diazyme per mg starch. Tissue weight (5 to 100 mg) had no effect on starch hydrolysis by sufficient enzyme. Methanol: chloroform: water (12:5:3 by volume) freed tissues of solubles before starch hydrolysis. No interference with the glucose oxidase analysis of hydrolysates was encountered. In addition, the pigment free methanol–water fractions (soluble sugars, amino acids, organic acids) and chloroform fractions (lipids and pigments) were available or further analysis. Results obtained with red oak were verified with issue from other species such as jack pine (Pinus banksiana lamb.) and white spruce (Picea glauca (Moench) Voss). The resulting technique simply and reliably measured less than 5% starch in 5 mg lyophilized tissue, with a minimum of sample manipulation.     

Anti-Legionella activity of staphylococcal hemolytic peptides

A collection of various Staphylococci was screened for their anti-Legionella activity. Nine of the tested strains were found to secrete anti-Legionella compounds. The culture supernatants of the strains, described in the literature to produce hemolytic peptides, were successfully submitted to a two step purification process. All the purified compounds, except one, corresponded to previously described hemolytic peptides and were not known for their anti-Legionella activity. By comparison of the minimal inhibitory concentrations, minimal permeabilization concentrations, decrease in the number of cultivable bacteria, hemolytic activity and selectivity, the purified peptides could be separated in two groups. First group, with warnericin RK as a leader, corresponds to the more hemolytic and bactericidal peptides. The peptides of the second group, represented by the PSMα from Staphylococcus epidermidis, appeared bacteriostatic and poorly hemolytic.     

Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.     

Pigment composition of the photosynthetic membrane and reaction center of the green bacterium Prosthecochloris aestuarii

We have performed a quantitative analysis of the pigment composition of different pigment-protein complexes present in the membrane of the green sulfur bacterium Prosthecochloris aestuarii, using the resolving power of reversed-phase high-performance liquid chromatography. The most purified photochemically active complexes contained only carotenoids (OH-chlorobactene and rhodopin), bacteriochlorophyll a and a chlorophyllous pigment with absorption maxima at 663 and 433 nm, like bacteriochlorophyll c. However, the lipophilicity of this pigment, labeled BChl 663, is quite high and indicates that it contains 5–6 additional methylene groups compared to the BChl c homologue known as most lipophilic. Comparison of the BChl 663 content of various pigment-protein complexes indicates that BChl 663 is present in an amount of 10–15 molecules per reaction center. BChl 663 absorbs at 670 nm in vivo, with a specific extinction coefficient of 85 (±10) mM⁻¹ · cm⁻¹. In view of the evidence that the primary electron acceptor in P. aestuarii is a pigment with absorption maximum at 670 nm (Nuijs, A.M., Vasmel, H., Joppe, H.L.P., Duysens, L.N.M. and Amesz, J. (1985) Biochim. Biophys. Acta 807, 24–34) a direct consequence of these experiments is the fact that only BChl 663 can be a likely candidate for the role of primary electron acceptor as no other pigments absorbing around 670 nm (e.g., bacteriopheophytin c) are present in a photochemically active pigment-protein complex derived from the membrane of this green bacterium.     

Granadaene: proposed structure of the group B Streptococcus polyenic pigment

The goal of this work was to determine the chemical nature of the red pigment produced by Streptococcus agalactiae, which has been thought to be a carotene. We extracted the pigment with 0.1 M KOH and purified it by column chromatography on Sephadex LH. Data from elemental analysis and mass and nuclear magnetic resonance spectra lead us to propose the structure to be that of a new ornithine rhamno-polyene with 12 conjugated double bonds, to which we have assigned the trivial name granadaene.     

Effect of carrier molecules on production and properties of extracellular hemolysin produced byStreptococcus agalactiae

Streptococcus agalactiae type la strain 090 produced a cell-associated hemolysin during exponential growth in medium lacking proteins. Growth of the organism in medium containing proteins or medium supplemented with Tween 40 resulted in the appearance of extracellular hemolytic activity that was filterable. Maximum extracellular hemolytic activity was obtained in the late exponential phase of growth corresponding to the maximum number of cells. Extracellular hemolysin released in medium containing proteins could be precipitated by ammonium sulfate. Cell-associated hemolysin could be extracted in the cold by purified lipoteichoic acid from the organism. Purification and characterization of the extracellular hemolysin by column chromatography showed that the hemolysin was associated with molecules eliciting its release. Hemolysin associated with lipoteichoic acid or Tween 40 had an apparent molecular weight of 1,800,000 or 60,000 daltons, respectively.     

Identification of a Vibrio furnissii oligopeptide permease and characterization of its in vitro hemolytic activity

We describe purification and characterization of an oligopeptide permease protein (Hly-OppA) from Vibrio furnissii that has multifaceted functions in solute binding, in in vitro hemolysis, in antibiotic resistance, and as a virulence factor in bacterial pathogenesis. The solute-binding function was revealed by N-terminal and internal peptide sequences of the purified protein and was confirmed by discernible effects on oligopeptide binding, by accumulation of fluorescent substrates, and by fluorescent substrate-antibiotic competition assay experiments. The purified protein exhibited host-specific in vitro hemolytic activity against various mammalian erythrocytes and apparent cytotoxicity in CHO-K1 cells. Recombinant Hly-OppA protein and an anti-Hly-OppA monoclonal antibody exhibited and neutralized the in vitro hemolytic activity, respectively, which further confirmed the hemolytic activity of the gene product. In addition, a V. furnissii hly-oppA knockout mutant caused less mortality than the wild-type strain when it was inoculated into BALB/c mice, indicating the virulence function of this protein. Finally, the in vitro hemolytic activity was also confirmed with homologous ATP-binding cassette-type transporter proteins from other Vibrio species.     

Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone Bunodosoma caissarum: Purification and biological characterization

Two cationic proteins, C1 and C3, were purified to homogeneity from the hemolytic fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes employed gel filtration followed by ion exchange chromatography, being the purity and molecular mass confirmed by SDS-PAGE and mass spectrometry. Protein C1 represented the second major peak of the hemolytic fraction and was previously believed to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular mass of 15495 Da and was assayed for hemolysis, PLA₂ activity and acute toxicity in crabs and mice, showing no activity in these assays. It has an amino terminal with no similarity to all known hemolysins and, therefore, should not be considered a toxin, being its function completely unknown. The protein C3 (19757 Da), that also lacks PLA₂ activity, was recognized by antiserum against Eqt II and presented high hemolytic activity to human erythrocytes (ED₅₀ of 0.270 μg/ml), being named Caissarolysin I (Bcs I). Its activity was inhibited by pre-incubation with sphingomyelin (SM) and also when in presence of erythrocytes pre-treated with the SMase P2, a phospholipase D from the brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I. Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus Bunodosoma and belongs to the Actinoporin family of sea anemone hemolysins.     

Applications of red pigments from psychrophilic Rhodonellum psychrophilum GL8 in health,food and antimicrobial finishes on textiles

A psychrophilic bacterium (named GL8) producing red pigments was isolated from the high altitude Pangong Tso Lake located in Leh Ladakh, India. Based on 16S rDNA sequencing amplicon of 1370 bp, the psychrophilic bacterium was identified as Rhodonellum psychrophilum (R. psychrophilum GL8) and deposited in Gen Bank under accession no. MH031708.1.The red colored pigments from R. psychrophilum GL8 showed antimicrobial activity against E. coli, S. aureus, C. albicans, (MTCC 277, ATCC 90028) and S. cerevisiae (H1086) with minimum inhibitory concentrations from 31.25 μg/mL to 500 μg/mL. Red colored pigments also showed synergistic antifungal activity when combined with fluconazole and amphotericin B against C. albicans (MTCC 277 and ATCC 90028) and S. cerevisiae; vancomycin and erythromycin against E. coli and S. aureus. The red pigments showed antioxidant activity with an IC₅₀ of 13.159 μg/mL, LC–MS/MS analysis demonstrated that red pigment extracts contained a mixture of 2-methyl-3-butyl-prodigine, Prodigiosin, 2-methyl-3hexyl-prodigine, 3, 4-Didehydrorhodopsin, anhydrorhodovibrin, alloxanthin and Tetradecanoyl-hexadecanoyl compounds. Red pigment extracts were used to develop antimicrobial fabrics and did not show any cytotoxicity to U87MG human glioblastoma cell line, but showed a marginal growth inhibition to A172 human glioblastoma cell lines. In contrast, the red pigment extracts showed significant growth stimulation on L929 mouse fibroblast cell lines.     

The β-Hemolysin and Intracellular Survival of Streptococcus agalactiae in Human Macrophages

S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches.     

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.     

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.     

Isolation of Antibiotics Turbomycin A and B from a Metagenomic Library of Soil Microbial DNA

To access the genetic and biochemical potential of soil microorganisms by culture-independent methods, a 24,546-member library in Escherichia coli with DNA extracted directly from soil had previously been constructed (M. R. Rondon, P. R. August, A. D. Bettermann, S. F. Brady, T. H. Grossman, M. R. Liles, K. A. Loiacono, B. A. Lynch, I. A. MacNeil, M. S. Osburne, J. Clardy, J. Handelsman, and R. M. Goodman, Appl. Environ. Microbiol. 66:2541-2547, 2000). Three clones, P57G4, P89C8, and P214D2, produced colonies with a dark brown melanin-like color. We fractionated the culture supernatant of P57G4 to identify the pigmented compound or compounds. Methanol extracts of the acid precipitate from the culture supernatant contained a red and an orange pigment. Structural analysis revealed that these were triaryl cations, designated turbomycin A and turbomycin B, respectively; both exhibited broad-spectrum antibiotic activity against gram-negative and gram-positive organisms. Mutagenesis, subcloning, and sequence analysis of the 25-kb insert in P57G4 demonstrated that a single open reading frame was necessary and sufficient to confer production of the brown, orange, and red pigments on E. coli; the predicted product of this sequence shares extensive sequence similarity with members of the 4-hydroxyphenylpyruvate dioxygenase (4HPPD) family of enzymes. Another member of the same family of genes, lly, which is required for production of the hemolytic pigment in Legionella pneumophila, also conferred production of turbomycin A and B on E. coli. We further demonstrated that turbomycin A and turbomycin B are produced from the interaction of indole, normally secreted by E. coli, with homogentisic acid synthesized by the 4HPPD gene products. The results demonstrate successful heterologous expression of DNA extracted directly from soil as a means to access previously uncharacterized small organic compounds, serving as an example of a chimeric pathway for the generation of novel chemical structures.     

Purification and characterization of a dehalogenase from Pseudomonas stutzeri DEH130 isolated from the marine sponge Hymeniacidon perlevis

2-haloacid dehalogenases are enzymes that are capable of degrading 2-haloacid compounds. These enzymes are produced by bacteria, but so far they have only been purified and characterized from terrestrial bacteria. The present study describes the purification and characterization of 2-haloacid dehalogenase from the marine bacterium Pseudomonas stutzeri DEH130. P. Stutzeri DEH130 contained two kinds of 2-haloacid dehalogenase (designated as Dehalogenase I and Dehalogenase II) as detected in the crude cell extract after ammonium sulfate fractionation. Both enzymes appeared to exhibit stereo-specificity with respect to substrate. Dehalogenase I was a 109.9-kDa enzyme that preferentially utilized D-2-chloropropropionate and had optimum activity at pH 7.5. Dehalogenase II, which preferentially utilized L-2-chloropropionate, was further purified by ion-exchange chromatography and gel filtration. Purified Dehalogenase II appeared to be a dimeric enzyme with a subunit of 26.0-kDa. It had maximum activity at pH 10.0 and a temperature of 40 °C. Its activity was not inhibited by DTT and EDTA, but strongly inhibited by Cu²⁺, Zn²⁺, and Co²⁺. The K _m and V _max for L-2-chloropropionate were 0.3 mM and 23.8 μmol/min/mg, respectively. Its substrate specificity was limited to short chain mono-substituted 2-halocarboxylic acids, with no activity detected toward fluoropropionate and monoiodoacetate. This is the first report on the purification and characterization of 2-haloacid dehalogenase from a marine bacterium.