Isolation and identification of<Emphasis Type="Italic">Streptomyces</Emphasis> sp. and assay of its exocellular water-soluble blue pigments

A bacterial strain producing a great amount of blue pigment during submerse fermentation was isolated and identified. Based on morphological characteristics cell-wall chemotype and sequence of 16S rRNA gene, the strain should belong to the genusStreptomyces; it had 99.4% homology of 16S rRNA gene sequence with that ofStreptomyces indigocolor. The pigment production by the strain was affected by carbon and nitrogen sources. The main components of the pigment mixture (detected by HPLC and TLC) were tentatively classified as actinorhodin-related compounds. The pigment was relatively stable against light and higher temperature but was sensitive to low pH. The preliminary acute-toxicity determination showed that the pigment was nontoxic (LD₅₀>15 mg/g).     

Biocontrol potential of actinomycetes against Xanthomonas axonopodis pv. punicae,a causative agent for oily spot disease of pomegranate

India is a largest producer of pomegranate with high export value. The cultivation is affected with the oily spot disease caused by Xanthomonas axonopodis pv. punicae infection. The present study aims to control the disease with newer biocontrol methods. Thirty-six isolates of X. axonopodis were isolated from different varieties of infected pomegranates fruits from Maharashtra. Forty strains of actinomycete were also isolated from natural sources and screened for their antagonistic activity against X. axonopodis isolates. Eight strains of actinomycete were screened out for their high antagonistic activity and were optimized for maximizing antibiotic production. The extracted compound from A5 strain exhibited maximum inhibitory activity against all the pathogenic isolates with a MIC in the range of 0.625 to 1.25 mg mL^?1. It was identified as Streptomyces violaceusnige by 16SrRNA gene sequencing (Accession number KP208943). The extracted compound belonged to aminoglycosides with a molecular formula C₂₂H₂₈N₃O₆ determined by thin layer chromatography, high-performance liquid chromatography, gas chromatography-mass spectroscopy, nuclear magnetic resonance spectroscopy, fourier transform infrared spectroscopy and carbon hydrogen nitrogen ratio analysis. In vivo biocontrol studies with strain A5 and its extracted compound effectively prevented the growth 36 Xanthomonas isolates inoculated on pomegranate fruits, illustrating its biocontrol potential against the oily spot disease of pomegranate.     

Cytotoxic compounds from the marine actinobacterium Streptomyces corchorusii AUBN1/71

We isolated a bioactive streptomycete from marine sediment samples collected at Bay of Bengal, India, during our systematic study of marine actinobacteria. The taxonomic studies indicated that the isolate is related to Strepomyces corchorusii. However, it differed in certain aspects, and, hence, was designated as S. corchorusii AUBN₁/7. A solvent extraction followed by a chromatographic purification helped obtain from the isolate two cytotoxic compounds, which were identified as resistomycin, a quinone-related antibiotic, and tetracenomycin D, an anthraquinone antibiotic, on the basis of spectral data of pure compounds. They demonstrated in vitro a potent cytotoxic activity against cell lines HMO2 (gastric adenocarcinoma) and HePG2 (hepatic carcinoma) and also exhibited weak antibacterial activities against Gram-positive and Gram-negative bacteria. Published in Russian in Bioorganicheskaya Khimiya, 2006, Vol. 32, No. 3, pp. 328–334. The text was submitted by the authors in English.     

In Vitro Anticancer Activity of Staphyloxanthin Pigment Extracted from <Emphasis Type="Italic">Staphylococcus gallinarum</Emphasis> KX912244, a Gut Microbe of <Emphasis Type="Italic">Bombyx mori</Emphasis>

The present study reports the in vitro biological nature of the pigment produced by Staphylococcus gallinarum KX912244, isolated as the gut microflora bacterium of the insect Bombyx mori. The purified pigment was characterized as Staphyloxanthin based on bio-physical characterization techniques like Fourier transform infrared spectroscopy, high performance liquid chromatography, Proton nuclear magnetic resonance spectroscopy (¹H NMR), Liquid chromatography-Mass spectroscopy and Gas chromatography-Mass spectroscopy. The Staphyloxanthin pigment presented considerable biological properties including in vitro antimicrobial activity against pathogens Staphylococcus aureus, Escherichia coli and Candida albicans; in vitro antioxidant activity by % DPPH free radical scavenging activity showing IC₅₀ value of 54.22 µg/mL; DNA damage protection activity against reactive oxygen species and anticancer activity evaluated by cytotoxicity assay against 4 different cancer cell lines like the Dalton’s lymphoma ascites with IC₅₀ value 6.20?±?0.02 µg/mL, Ehrlich ascites carcinoma having IC₅₀ value 6.48?±?0.15 µg/mL, Adenocarcinomic human alveolar basal epithelial cells (A549 Lung carcinoma) bearing IC₅₀ value 7.23?±?0.11 µg/mL and Mus mucus skin melanoma (B16F10) showing IC₅₀ value 6.58?±?0.38 µg/mL and less cytotoxicity towards non-cancerous human fibroblast cell lines (NIH3T3) with IC₅₀ value of 52.24 µg/mL. The present study results suggest that Staphyloxanthin acts as a potential therapeutic agent especially due to its anticancer property.     

Characterization and antifungal activity of the yellow pigment produced by a Bacillus sp. DBS4 isolated from the lichen Dirinaria agealita

This study emphasis the production of yellow pigment from endolichenic Bacillus sp. isolated from the lichen Dirinaria aegialita (Afzel. ex Ach.) B.J. Moore. Yellow pigment-producing twenty different strains were investigated. The hyperactive pigment-producing bacterial strain was identified as Bacillus gibsonii based on 99 % sequence similarity. Maximum bacterial pigment production appeared in Luria Bertani medium. Methanol extraction of the pigment and its partial purification using TLC was carried out. Furthermore, isolated pigments were characterized using UV-visible spectroscopy, FTIR spectroscopy, and GC-MS results related to the possibility of the carotenoid occurrence. The pigment also exhibited efficient antifungal activity against selected fungal pathogens of economic importance. Likewise, the pigment extract evaluated for the total antioxidant potential using Phosphomolybdenum and Ferric reducing antioxidant power assay and the results represented in Ascorbic Acid Equivalent (AAE)- 21.45 ± 1.212 mg/mL. The SC₅₀ of the pigment extract found to be 75.125 ± 0.18 µg/ml determined by the ABTS assay.     

Radio-protective and antioxidative activities of astaxanthin from newly isolated radio-resistant bacterium <Emphasis Type="Italic">Deinococcus</Emphasis> sp. strain WMA-LM9

A radio-resistant bacterium, designated as strain WMA-LM9, was isolated from desert soil. 16S rRNA gene sequencing indicated that the bacterium belongs to genus Deinococcus with maximum similarity to Deinococcus radiopugnans. Deinococcus sp. strain WMA-LM9 was found to be resistant to a ultraviolet (UV) dose of 5 × 10³ J/m², hydrogen peroxide (50 mM) and mitomycin C (10 μg/ml). A carotenoid pigment was extracted using chloroform/methanol/acetone (7:5:3) and purified by high-performance liquid chromatography on a C₁₈ analytical column. The compound was characterised as mono-esterified astaxanthin by ¹H, ¹³C nuclear magnetic resonance and mass spectrometry. It was tested for antioxidant activity, total flavonoids and phenolic content, radioprotective potential in correlation to the prevention of protein oxidation and DNA strand breaks in vitro. The carotenoid pigment showed a very potent antioxidant activity and significantly stronger scavenging ability against superoxides, with an IC₅₀ (concentration causing 50% inhibition of the desired activity) of 41.6 μg/ml. The total phenolic and flavonoid contents were 12.1 and 7.4 μg in terms of gallic acid and quercetin equivalents per milligram of dried mass, respectively. astaxanthin also showed a higher inhibitory action against oxidative damage to collagen, elastin and bovine serum albumin than did β-carotene. The carotenoid also inhibited breaks to DNA strands, as indicated by the results of the DNA damage prevention assay. We conclude that astaxanthin from Deinococcus sp. strain WMA-LM9 has protective effects against radiation-mediated cell damage, and it also protects cellular protein and DNA against oxidative stress and other anti-oxidant activities.     

Isolation and Characterization of Pediocin NV 5 Producing <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis> LAB 5 from Vacuum-Packed Fermented Meat Product

A potentially novel antimicrobial compound producing Pediococcus acidilactici LAB 5 was isolated from vacuum-packed fermented meat product. This compound was found active against some species of Enterococcus, Leuconostoc, Staphylococcus and Listeria, many of which are associated with food spoilage and food related health hazards. The strain was found to be a paired cocci which can utilize a broad range of carbohydrates and produce acid identical to the P. acidilactici and P. pentoseus. Since the antimicrobial agent was sensitive to proteolytic enzymes but quite resistant to heat, it was identified as a bacteriocin and was designated as Pediocin NV 5. The molecular weight of the bacteriocin was 10.3 kDa and the bacterium possessed a 5 kbp plasmid responsible for bacteriocin production and also for vancomycin resistance phenotype.     

2-[3-(2-Thioxopyrrolidin-3-ylidene)methyl]-tryptophan,a Novel Yellow Pigment in Salted Radish Roots

The structure of the yellow pigment found in salted radish roots was studied. It was found that 1-(2-thioxopyrrolidin-3-yl)-1,2,3,4-tetrahydro-β-carboline-3- carboxylic acid (TPCC) was unstable under neutral pH, and was easily converted into the yellow pigment. The yellow pigment was isolated and identified as 2-[3-(2-thioxopyrrolidin-3-ylidene)methyl]-tryptophan (TPMT) by IR, MS, ¹H-, and ¹³C-NMR spectroscopy. In addition, we proved that this compound was the main yellow pigment in salted radish roots. This compound induced no mutagenicity in Salmonella typhimurium TA98 and TA100, either with or without prior activation.     

Antifungal activity of novel indole derivative from endophytic bacteria Pantoea ananatis 4G-9 against Mycosphaerella musicola

An endophytic bacterium isolated from banana G-9 (AAA genotype) leaves exhibited strong antagonistic activity against Mycosphaerella musicola. The isolate was identified as Pantoea ananatis 4G-9 by 16S rRNA sequence analysis. Secondary metabolite obtained from P. ananatis 4G-9 was found to have antifungal activity. The active compound was purified from crude extract using column chromatography. Purity of the active compound was assessed using high-performance liquid chromatography. Spectral analysis of compound using infrared, mass spectrometry and nuclear magnetic resonance indicated that the compound structure is an indole derivative. The compound showed strong and dose-dependent antifungal activity against M. musicola. This is the first report on P. ananatis isolated as an endophyte from banana leaves and its antifungal activity against M. musicola.     

Bioactivity‐Guided Isolation of Anti‐Inflammatory Constituents of the Rare Mushroom Calvatia nipponica in LPS‐Stimulated RAW264.7 Macrophages

Calvatia species, generally known as puffball mushrooms, are used both as sources of food and as traditional medicine. Among the Calvatia genus, Calvatia nipponica (Agaricaceae) is one of the rarest species. Using bioassay‐guided fractionation based on anti‐inflammatory effects, five alkaloids ( 1 – 5 ), two phenolics ( 6 and 7 ), and a fatty acid methyl ester ( 8 ) were isolated from the fruiting bodies of C. nipponica. Compound 8 was identified from C. nipponica for the first time, and all isolates ( 1 – 8 ) were tested for inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophages. Compound 7 showed mild inhibition while compound 8 significantly inhibited NO production with an IC₅₀ value of 27.50 ± 0.08 μm . The mechanism of NO inhibition of compound 7 was simulated by molecular docking analysis against nitric oxide synthase (iNOS), which revealed the interactions of 7 with the key amino acid residue and the heme in the active site. With the most potent inhibition against LPS‐induced inflammation, compound 8 was further investigated with respect to its mechanism of action, and the activity was found to be mediated through the inhibition of iNOS and COX‐2 expression.     

Identification and Antimicrobial Activity of Phenylacetic Acid Produced by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> Isolated from Fermented Soybean,Chungkook-Jang

A bacterial strain, B65-1, which showed strong antimicrobial activity, was isolated from Chungkook-Jang, a traditional Korean fermented-soybean food with antimicrobial properties. Based on carbon utilization pattern and partial 16S rRNA sequence analysis, the B65-1 strain was identified as Bacillus licheniformis. An antibiotic compound, active against bacteria and yeast such as Staphylococcus aureus, Escherichia coli, and Candida albicans, was isolated by various chromatographic procedures from culture filtrates of B. licheniformis B65-1. The purified antibiotic was identified to be phenylacetic acid, with the molecular formula C₈H₈O₂ by analyses of EI-MS and NMR. The phenylacetic acid was detected in fermented soybean made with the strain B65-1 as a starter, but was not present in extracts of nonfermented soybean. Our results indicated that the phenylacetic acid produced by B. licheniformis during fermentation of soybean is one of the main compounds of antimicrobial activity of Chungkook-Jang.     

Potential anti-leptospiral compound,leptomycin B from marine <Emphasis Type="Italic">Streptomyces indiaensis</Emphasis> MSU5: taxonomy,fermentation, compound isolation,in vitro and in vivo efficacy

Leptospirosis is a worldwide reemerging tropical zoonotic disease with symptoms of mild febrile illness to more severe multiple organ failure caused by pathogenic leptospiral strains. There was no effective antibiotic for treating leptospirosis. Here, the anti-leptospiral potential of marine actinobacterial compound from Streptomyces indiaensis MSU5 isolated from Manakudy marine sediment, Tamil Nadu, India was evaluated. The potential actinobacterial strain was identified by phenotypic, cell wall, 16S rRNA gene sequence and phylogenetic analysis. In vitro anti-leptospiral activity of the actinobacterial compound was determined using broth microdilution test against various serovars of Leptospira with different concentration ranging from 15.625 to 500 µg/ml. Mass production of anti-leptospiral compound was carried out in agar surface fermentation with optimized condition and purified by preparative TLC. The purified fraction of anti-leptospiral compound named as MSU5-1, and it was confirmed by microdilution test. Remarkably, the compound MSU5-1 showed minimum inhibitory concentration of 62.5 µg/ml and minimum bactericidal concentration of 125 µg/ml against human pathogenic leptospiral isolate strain N2. The structural elucidation of purified compound was carried out using UV, FT-IR, NMR and LC-MS analysis. The compound MSU5-1 was tentatively identified as leptomycin B (C₃₃H₄₈O₆) with molecular weight 541.1 g/mol. Anti-leptospiral activity of compound MSU5-1 exhibited 80% of survival rate in mice model, further it was confirmed by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) analysis. From the available literature, this is the first report on the marine actinobacterial compound for evaluating both in vitro and in vivo leptospiricidal activity.     

Nematicidal activity of <Emphasis Type="Italic">Paecilomyces</Emphasis> spp. and isolation of a novel active compound

Many species of Paecilomyces are entomogenous fungi and several are efficacious toward nematodes. To study the potential of Paecilomyces species in controlling nematodes, fungal extracts of 40 Paecilomyces spp. were evaluated for their nematicidal activity against Bursaphelenchus xylophilus and Panagrellus redivivus. The extracts of six Paecilomyces spp. exhibited the nematicidal activity against P. redivivus, and 11 species exhibited the nematicidal activity against B. xylophilus. The methanol extract of strain 1.01761 incubating on Czapek solid medium killed more than 95% P. redivivus in 24 h at 5 mg/ml, and the filtrate of strain 1.01788 cultured in Sabouraud’s broth medium resulted in 90% mortality of B. xylophilus in 24 h at 5 mg/ml. A novel nematicidal compound 4-(4′-carboxy-2′-ethyl-hydroxypentyl)-5,6-dihydro-6-methylcyclobuta[b]pyridine-3,6-dicarboxylic acid, was isolated from Paecilomyces sp. YMF1.01761. The LD₅₀ value of the compound within 24 h against P. redivivus was 50.86 mg/L, against Meloidogyne incognita was 47.1 mg/L, and against B. xylophilus was 167.7 mg/L.     

Prodigiosin from <Emphasis Type="BoldItalic">Vibrio</Emphasis> sp. DSM 14379; A New UV-Protective Pigment

Pigments such as melanin, scytonemin and carotenoids protect microbial cells against the harmful effects of ultraviolet (UV) radiation. The role in UV protection has never been assigned to the prodigiosin pigment. In this work, we demonstrate that prodigiosin provides a significant level of protection against UV stress in Vibrio sp. DSM 14379. In the absence of pigment production, Vibrio sp. was significantly more susceptible to UV stress, and there was no difference in UV survival between the wild-type strain and non-pigmented mutant. The pigment’s protective role was more important at higher doses of UV irradiation and correlated with pigment concentration in the cell. Pigmented cells survived high UV exposure (324 J/m²) around 1,000-fold more successfully compared to the non-pigmented mutant cells. Resistance to UV stress was conferred to the non-pigmented mutant by addition of exogenous pigment extract to the growth medium. A level of UV protection equivalent to that exhibited by the wild-type strain was attained by the non-pigmented mutant once the prodigiosin concentration had reached comparable levels to those found in the wild-type strain. In co-culture experiments, prodigiosin acted as a UV screen, protecting both the wild-type and non-pigmented mutants. Our results suggest a new ecophysiological role for prodigiosin.     

Isolation and characterization of a marine algicidal bacterium against the harmful raphidophyceae <Emphasis Type="Italic">Chattonella marina</Emphasis>

A bacterial strain named AB-4 showing algicidal activity against Chattonella marina was isolated from coastal water of ULjin, Republic of Korea. The isolated strain was identified as Bacillus sp. by culture morphology, biochemical reactions, and homology research based on 16S rDNA. The bacterial culture led to the lysis of algal cells, suggesting that the isolated strain produced a latent algal-lytic compound. Amongst changes in algicidal activity by different culture filtrate volumes, the 10% (100 μl/ml) concentration showed the biggest change in algicidal activity; there, estimated algicidal activity was 95%. The swimming movements of Chattonella marina cells were inhibited because of treatment of the bacterial culture; subsequently, Chattonella marina cells became swollen and rounded. With longer exposure time, algal cells were disrupted and cellular components lost their integrity and decomposed. The released algicide(s) were heat-tolerant and stable in pH variations, except pH 3, 4, and 5. Culture filtrate of Bacillus sp. AB-4 was toxic against harmful algae bloom (HAB) species and nontoxic against livefood organisms. Bacillus sp. AB-4 showed comparatively strong activity against Akashiwo sanguinea, Fibriocapsa japonica, Heterosigma akashiwo, and Scrippsiella trochoidea. These results suggest that the algicidal activity of Bacillus sp. AB-4 is potentially useful for controlling outbreaks of Chattonella marina.     

Identification and characterization of a pigment-producing denitrifying bacterium

Herein, a denitrifying bacterium that produced greenish fluorescent pigment under aerobic conditions was accidentally isolated from municipal sewage sludge. Using 16S-rDNA sequence analysis, we identified the isolate as Pseudomonas aeruginosa R12, with 100% similarity. We achieved the highest pigment production rate (1.36 mg/L/h) in a 1-L bioreactor under aerobic conditions, using the optimal culture parameters determined in this study: 37°C, pH 8.0, 200 rpm, 5 wm aeration, and medium containing succinate and (NH₄)₂SO₄. The pigment was not a secondary metabolite and had no antibacterial activity on its co-isolates. Under anaerobic conditions, the isolate produced mainly N₂ and behaved as a strong denitrifier, displaying synergistic denitrification with co-isolated denitrifiers. To our knowledge, herein we have described the first instance in which P. aeruginosa R12 produces a fluorescent pigment under aerobic conditions. This newly-isolated strain therefore shows potential as a commercial resource for natural pigment.     

Isolation and identification of lipopeptide antibiotics from <Emphasis Type="Italic">Paenibacillus elgii</Emphasis> B69 with inhibitory activity against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Two lipopeptide antibiotics, pelgipeptins C and D, were isolated from Paenibacillus elgii B69 strain. The molecular masses of the two compounds were both determined to be 1,086 Da. Mass-spectrometry, amino acid analysis and NMR spectroscopy indicated that pelgipeptin C was the same compound as BMY-28160, while pelgipeptin D was identified as a new antibiotic of the polypeptin family. These two peptides were active against all the tested microorganisms, including antibiotic-resistant pathogenic bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA). Time-kill assays demonstrated that pelgipeptin D exhibited rapid and effective bactericidal action against MRSA at 4×MIC. Based on acute toxicity test, the intraperitoneal LD50 value of pelgipeptin D was slightly higher than that of the structurally related antimicrobial agent polymyxin B. Pelgipeptins are highly potent antibacterial and antifungal agents, particularly against MRSA, and warrant further investigation as possible therapeutic agents for bacteria infections resistant to currently available antibiotics.     

Physicochemical characterization and antioxidant activity of melanin from a novel strain of Aspergillus bridgeri ICTF-201

Aims: The aim of the study is to isolate and characterize a melanin pigment from a new strain of Aspergillus bridgeri isolated from rhizosphere soil of Eucalyptus tree and to investigate its antioxidant activity. Methods and Results: The extracellular pigment was alkali soluble, acid‐resistant and insoluble in organic solvents and water. The pigment was precipitated on treatment with FeCl₃, ammoniacal AgNO₃ and potassium ferricyanide and was bleached in the presence of oxidants and reductants. It was confirmed as melanin based on the Fourier transform infrared and electron paramagnetic resonance spectroscopy techniques apart from chemical analysis. Inhibition of melanin production by inhibitors like tricyclazole, 6‐hydroxyflavanone, 4‐hydroxy‐7‐methoxy‐3‐phenyl‐coumarin, 7‐hydroxy‐4‐phenyl‐coumarin and 7‐hydroxy‐3,4,8‐trimethylcoumarin confirmed that melanin produced by A. bridgeri is synthesized by 1,8‐dihydroxynaphthalene (DHN)‐melanin pathway. The melanin showed good free radical scavenging activity by DPPH method with an EC₅₀ of 54·12 μg ml^?1. Conclusions: The results of the study indicate that the melanin produced by the newly isolated A. bridgeri strain is a member of DHN melanin family and exhibited significant free radical scavenging activity. Significance and Impact of the Study: This is the first report on characterization of DHN melanin produced by a novel strain of A. bridgeri and may find potential application as a natural antioxidant in the cosmetic and pharmaceutical industries.     

ANTIMICROBIAL AND ANTIHYPERLIPIDEMIC ACTIVITIES OF ISOLATED QUERCETIN FROM ANABAENA AEQUALIS1

This study aimed to isolate quercetin (for the first time) from Anabaena aequalis Borge, which inhabits soil surface of Wadi El‐Alaqui Protectorate located in Aswan city, Egypt. The isolated compound showed significant antibacterial activity against the gram‐positive bacteria Sarcina maxima and Micrococcus kristinae, the gram‐negative bacterium Klebsiella pneumoniae, as well as against the filamentous fungus Aspergillus flavus. The isolated compound was identified as quercetin using the structure elucidation based on UV, electrospray ionization mass spectrometry (ESIMS), ¹H and ¹³C NMR, proton–proton correlation spectroscopy (¹H‐¹H COSY), distortionless enhancement by polarization transfer (DEPT), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlations spectrum (HMBC). Medium lethal dose (LD₅₀) of the isolated compound and its side effects against hyperlipidemia induced by ethanol intake in albino rats were carried out. No deaths were reported in rats within 72 h, which suggests that the isolated compound plays a beneficial role as an antihyperlipidemic agent in the treatment of alcohol‐induced hepatic tissue damage, which can be described as one of the therapeutic values.     

A New Amino Acid Antibiotic KT–151, Produced by Streptomyces luteogriseus,Strain No. KT–151

From the results of taxonomic studies, Streptomyces sp. strain No. KT–151 isolated from a soil sample collected in Kumamoto City, was identified as a strain belonging to Streptomyces luteogriseus Schmitz, Deak, Crook and Hooper 1964. A new antibiotic, produced by this strain, was isolated as a leaflet crystal by ion-exchange chromatography and found to be an amino acid with the molecular formula, C₅H₁₂N₂O₂, and named antibiotic KT–151 (refered to as KT–151 hereinafter). The antibiotic showed antimicrobial activity against various Gram-positive and Gram-negative bacteria in a chemically defined medium but it was antagonized by several amino acids such as valine, leucine, isoleucine and threonine.