Purification and structure elucidation of antifungal and antibacterial activities of newly isolated Streptomyces sp. strain US80

A new actinomycete strain, designated US80 and producing antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi, was isolated from Tunisian oasis soil. Cultural characteristic studies strongly suggested that this strain belongs to the genus Streptomyces. The nucleotide sequence of the 16S rRNA gene (1517 pb) of Streptomyces sp. strain US80 exhibited close similarity (97-98%) with other Streptomyces 16S rRNA genes. Similarity of 98% was obtained with the 16S rRNA gene of Streptomyces roseoflavus, which produces the aminoglycoside antibiotic flavomycin. Study of the influence of different nutritional compounds on production of bioactive molecules showed that the highest antimicrobial activities were obtained when glucose at 1% (w/v) was used as sole carbon source in the presence of magnesium. Extraction of fermentation broth of Streptomyces sp. strain US80 and various separation and purification steps led to isolation of three pure active molecules. The chemical structure of these three compounds, named irumamycin (1a), X-14952B (1b) and 17-hydroxy-venturicidin A (1c), was established on the basis on their IR, ESI-MS, 1H and 13C/APT NMR data and by comparison with reference data from the literature.     

Characterization of a red pigment-antibiotic produced by Streptomyces sp. strain NRC-C7

The streptomycete strain NRC-C 7 isolated from some Egyptian soil samples, produces a red-coloured pH indicator antibiotic active against gram positive bacteria. The producing organism belongs to the grey series of the genus Streptomyces and has grey coloured aerial mycelium with violet pigmented substrate mycelium, the violet pigment does not diffuse into the agar medium. Glucose sodium nitrate medium proved the most suitable medium for the production of this antibiotic. The pigment was extracted from the broth and purified. The solubility, Rf values, minimal inhibiting concentrations, optical rotation, UV, and IR spectra were studied. Melting point 180, elemental analysis: carbon 51.71%, hydrogen 6.25%, and oxygen 42.04%.     

Isolation, characterization and partial purification of a transferable membrane channel (amoebapore) produced by Entamoeba histolytica

Entamoeba histolytica kills cells by contact dependent cytolysis. The mechanism underlying this process must be of rapid onset because target cells round up and show marked zeiosis within 15 min of contact. In earlier work, we identified a remarkable ion-channel forming protein which we named amoebapore, that may contribute to the amoeba-induced target cell killing. Within the amoeba it exists as part of a supramolecular aggregate together with other proteins of unknown function. In this work we report the purification of a solubilized form of the amoebapore. Amoebapore was found to exist as an apparent dimer of the previously reported protein whose molecular weight had been determined under denaturing conditions. Two isoforms of this dimer, with pI values of 6.8 and 5.3 present at a ratio of 7 to 1, were identified and purified. Both isoforms demonstrate ion-channel forming activity in planar lipid membranes. These channels show a unit conductance of 5-20 pS and remain open for less than 1 s. Upon lateral aggregation, opening becomes concerted to a greater degree with channel conductance are observed. The isolated particulate form of amoebapore depolarizes cells.     

Isolation, purification, and partial characterization of a lipopolysaccharide from Haemophilus pleuropneumoniae.

Lipopolysaccharide (LPS) from Haemophilus pleuropneumoniae 1536, serotype 2, was isolated and purified by a procedure designed to be equally satisfactory for both smooth- and rough-type LPS. The LPS yield was 53%. Analysis of the preparations revealed that protein, nucleic acid, and cellular phospholipid contamination was negligible (less than 0.1%). Analysis of the sugar content of the LPS by gas-liquid chromatography and colorimetric analysis revealed the presence of rhamnose, mannose, galactose, glucose, heptose, glucosamine, galactosamine, and 3-deoxy-D-manno-2-octulosonic acid. The heptose and glucose contents appeared to be unusually high. The fatty acids of the LPS consisted of a mixture of C14:0 and C16:0 in a ratio of about 4.5:1 (50% of the total) and 3-hydroxy C14:0. When used as a preparatory dose for the dermal Shwartzman reaction, as little as 10 micrograms of the LPS injected intradermally in rabbits produced reddening and swelling. After intravenous injection of a 100-micrograms LPS provoking dose, necrosis was observed at all intradermal injection sites. Limulus amebocyte lysate gelation was observed with an LPS concentration as low as 0.5 ng/ml. A typical biphasic fever response was noted in rabbits injected with as little as 0.25 ng of LPS per kg of body weight.     

Isolation and purification of a modified phenazine, griseoluteic acid, produced by Streptomyces griseoluteus P510

Antibiotic phenazine derivatives and their formation pathways were studied in a new Streptomyces strain P510. Culture characteristics and 16S rRNA nucleotide analysis confirmed strain P510 as Streptomyces griseoluteus. The culture medium of this strain showed strong antimicrobial and antifungal activities. Using organic solvent extraction, silica gel column chromatography and HPLC, a modified phenazine, griseoluteic acid, and a shikimic acid-derived metabolite, p-hydroxybenzaldehyde, were separated and purified. In addition, the biological activity of griseoluteic acid (GA), an important intermediate for biosynthesis of phenazine derivatives, was also investigated in this research. It significantly inhibited growth of Bacillus subtilis. The presence of GA and p-hydroxybenzaldehyde implied that the phenazine biosynthesis pathway in S. griseoluteus P510 might be initiated with shikimic acid, using phenazine-1, 6-dicarboxylic acid as the precursor. The discovery of a partial analogical sequence of phenazine biosynthetic genes, sgpC, sgpD and sgpE, in S. griseoluteus P510 further supported this hypothesis.     

Isolation, purification, and partial characterization of Brucella abortus matrix protein.

Peptidoglycan sacculi with peptidoglycan-associated proteins were prepared from cell envelopes of Brucella abortus by extraction with sodium dodecyl sulfate (SDS) at 50 degrees C. On extraction of these preparations with SDS at 100 degrees C, a protein was obtained whose removal from peptidoglycan was confirmed by electron microscopy. Incubation of the 50 degrees C SDS-extracted cell envelopes with 50 mM MgCl2 in SDS-2-beta-mercaptoethanol at 37 degrees C also extracted the protein, along with lipopolysaccharide. At temperatures below 60 degrees C, the protein did not bind SDS strongly and had an apparent molecular weight greater than 92,000 in SDS-polyacrylamide gel electrophoresis. At higher temperatures, SDS bound strongly, and the apparent molecular weight was 38,000. Urea at 5 M did not alter the electrophoretic mobility of this 38,000-molecular-weight form. Immunoelectrophoresis in detergents with antisera to cell envelopes, carbohydrate staining of SDS-polyacrylamide gels, and production of anti-lipopolysaccharide antibodies by mice immunized with the purified protein indicated that lipopolysaccharide was present in free and protein-bound forms. Sequential gel filtration in SDS-EDTA and SDS-NaCl removed most lipopolysaccharide. After further purification by preparative SDS-polyacrylamide gel electrophoresis, a gas-liquid chromatographic analysis showed residual lipid tightly associated with the protein. The results suggested that the interactions between matrix proteins and other outer membrane components are stronger in B. abortus than in Escherichia coli, which was used as a control throughout.     

Flocculation of influenza virus by a neuraminidase inhibitor, neuraminin, produced by Streptomyces sp.

Flocculation of virions by a viral neuraminidase inhibitor, neuraminin, was examined by low speed centrifugation and electron microscopy. The inhibitor could flocculate virions of influenza viruses but not those of Newcastle disease viruses. Virion-free neuraminidase of influenza virus was also flocculated by this inhibitor. The flocculation was temperature dependent and proceeded rapidly over a wide pH range. The amount of the inhibitor required for complete flocculation of virions was similar to that for the maximum inhibition of neuraminidase. Viral neuraminin receptors were stable to various enzymological, physical, and chemical treatments which caused a loss or reduction of the activity of neuraminidase.     

Antibiotics produced by Streptomyces olivaceus 142. II. Isolation, purification and activity spectrum of antibiotic WR 142-FPG.

A method of isolating antibiotic Wr 142-FPG from the fermentation broth and mycelium of mutant FPG of the strain Streptomyces olivaceus 142 has been developed. The method of purification of the active substance and activity spectrum agains microorganisms are described. Some of the basic properties of two preparations are given: a) the preparation designed I, partly purified, containing a peptide moetye and b) the preparation designed II, which is a homogeneous chromatographically preparation with mol. wt. about 510, obtained from preparation I by separation on silica gel. Both preparations are active against the same spectrum of microorganisms and are similarly cytotoxic for normal and neoplastic cells. They differ from each other in specific activity and toxicity. LD50 of preparation I for white mice i.p. is about 240 mg/kg and LD50 of preparation II about 5 mg/kg. Comparison of the activity of the purified preparation of WR 142-FPG with that of known anti-fungal antibiotics showed that Wr 142-FPG inhibits growth of Candida albicans and other pathogenic yeasts at concentrations 10--100 times lower than amphotericin B, antimycin A, nystatin and primaricin.     

Isolation, partial purification and in vitro characterization of osteogenic inhibitory protein

A noncollagenous protein has been extracted and partially purified from adult cortical bone. This protein copurifies with another bone matrix protein, bone morphogenetic protein, until treatment with nonionic detergents. Characterization of the biological activity of this new protein has demonstrated it to be a potent osteogenic inhibitor in vitro. The inhibitor antagonizes the chondrogenic activity of devitalized, demineralized bone matrix as well as the activity of soluble bone morphogenetic protein. Bone matrix induced collagen and glycosaminoglycan synthesis are both inhibited in the presence of various concentrations of the osteogenic inhibitory protein. Inhibition of collagen synthesis required the presence of osteogenic inhibitory protein from the initiation of the tissue culture while glycosaminoglycan synthesis could be inhibited at any stage of differentiation. We postulate that this osteogenic inhibitory protein is essential in normal homeostatic bone metabolism, perhaps acting directly on bone morphogenetic protein.     

丰肉结海绵相关链霉菌LS298活性代谢产物研究

目的对我国南海丰肉结海绵相关链霉菌LS298的活性代谢产物进行研究。方法采用硅胶柱、凝胶柱及HPLC等色谱方法对LS298发酵产物进行分离纯化;通过核磁共振、质谱等波谱分析手段对分离得到的化合物进行结构鉴定;以滤纸片扩散法及MTT法分别检测其抗菌和抗肿瘤活性。结果分离得到12个化合物。分别为尿嘧啶核苷、2'-脱氧尿嘧啶核苷、邻苯二甲酸正丁二酯、邻苯二甲酸二(2-乙基己)酯、3-甲酰胺-吲哚、环(脯氨酸-缬氨酸)、环(脯氨酸-苯丙氨酸)、环(脯氨酸-酪氨酸)、环(脯氨酸-亮氨酸)、meleagrin、5-hydroxyectoine和echinomycin。其中,3-甲酰胺-吲哚系首次从微生物中分离得到;meleagrin系首次从放线菌中分离得到。初步的药理研究表明,化合物echinomycin不仅具有较强的抗菌活性,亦具有很强的体外抗肿瘤活性。结论化合物echinomycin是链霉菌LS298的主要抗菌和抗肿瘤活性成分之一。     

Isolation and characterization of Pseudomonas sp. strain HF-1, capable of degrading nicotine

A nicotine-degrading bacterium, strain HF-1, was isolated from tobacco waste-contaminated soil and identified as a member of Pseudomonas sp. based on morphology, physiological tests, Vitek system (GN+), Biolog GN, 16S rDNA sequence and phylogenetic characteristics. The thermal denaturation test indicated that the G+C content of the DNA of HF-1 was 62.7 mol%. The relationship between growth and nicotine degradation of the isolate suggested that strain HF-1 could utilize nicotine as sole source of carbon, nitrogen and energy. Viridescent pigment was observed during nicotine degradation by strain HF-1. The isolate grew optimally at 30 degrees C, initial pH 6.5-7.5 and 1.3 g l(-1) of nicotine concentration in the nicotine inorganic salt medium, which is basically consistent with degradation of nicotine by the isolate. Strain HF-1 could degrade 99.6% of nicotine under the optimized incubation conditions for 25 h monitored by high-performance liquid chromatography. This study demonstrates that Pseudomonas sp. strain HF-1 had strong ability to degrade nicotine, and may be useful for bioremediation of environments contaminated by tobacco waste.     

Optimization of indigo production by a newly isolated Pseudomonas sp. QM

Optimization of indigo production process from indole using a newly isolated phenol‐degrading bacterial strain was performed by Plackett‐Burman design and response surface methodology. The strain designated as QM was identified as Pseudomonas sp. according to 16S rDNA analysis. Spectrum analysis of indole biotransformation products revealed the presence of indigo and a by‐product indirubin. To improve indigo yield, Plackett‐Burman design was used to select significant factors from 8 viriables. Then response surface methodology based on a 2³ central composite design was used to further optimize the transformation process. Under the optimal conditons, strain QM can produce 27.20 mg/l indigo after 24 h cultivation at 30 °C, which was 151.3% higher than that from the initial conversion condition. The results indicated that Pseudomonas sp. QM should be a potential candidate for indigo industial production. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Specific inhibition of viral neuraminidases by an inhibitor, neuraminin, produced by Streptomyces sp.

Neuraminin was characterized with respect to its inhibition of viral and bacterial neuraminidases. It inhibits the viral but not the bacterial enzymes. In addition, neuraminin distinguishes influenza from Newcastle disease virus. Neuraminin inhibits hydrolysis of fetuin, orosomucoid, and human red blood cell surface glycoproteins by influenza virus or its isolated neuraminidase. The inhibition does not occur when neuraminlactose or colominic acid (a homopolymer of N-acetylneuraminic acid) is used as the substrate. In contrast, neuraminin equally effectively inhibits the action of the Newcastle disease virus toward these substrates. The inhibition was noncompetitive and saturated at a very low concentration of the inhibitor irrespective of the kind and concentration of the substrates.     

Isolation, purification, and partial characterization of an enterotoxin from extracts of Entamoeba histolytica trophozoites.

Soluble cell-free extracts of pathogenic Entamoeba histolytica, as well as serum-free minimal media in which trophozoites are incubated, contain substances that cause the rapid rounding up and detachment of tissue-cultured monolayers of mammalian cells (cytopathic activity) and induce fluid secretion in ligated intestinal loops of indomethacin-pretreated rats (enterotoxic activity). A semiquantitative assay for the determination of the cytopathic activity based on the rate of detachment of tissue-cultured baby hamster kidney cells was developed. Two peaks containing cytopathic activity were obtained upon gel filtration of the soluble extracts: peak I, with over 60% of the activity, emerged in the 30,000 to 50,000 molecular weight region, and peak II, containing the remaining activity, was in the 15,000 to 25,000 molecular weight region. The activity of peak I was found to be heat labile and inhibited by sialoglycoproteins such as fetuin and mucin (5 mg/ml), as well as by sialic acid. Protease inhibitors such as antitrypsin, pepstatin, phenylmethylsulfonyl fluoride, metaloprotease inhibitors, and bacitracin had no effect on the cytopathic activity. Marked inhibition of cytopathic activity was observed, however, with iodoacetamide and p-chloromercuribenzoate, which affect sulfhydryl groups. The toxic material in peak II was found to have ionophoric activity and was not inhibited by sialic acid-containing compounds. The materials from both peaks had enterotoxic activity in intestinal ligated loops. The active substance from peak I was further purified (200X) on an agarose-fetuin affinity column, yielding one major protein band with an apparent molecular weight of ca. 30,000 on sodium dodecyl sulfate. Amino acid analysis revealed that the protein was very poor in sulfur amino acids. The sialic acid-sensitive toxic activity was higher in known virulent strains such as HM-1:IMSS and could be markedly augmented after preincubation of the trophozoites with certain Escherichia coli strains.     

Isolation of manumycin-type derivatives and genome characterization of a marine Streptomyces sp. C1-2

A marine actinomycete strain C1-2 was taxonomically characterized as the genus Streptomyces, based on whole-genome sequence analysis. The highest average nucleotide identity (ANI) value (98.96%) and digital DNA–DNA hybridization (DDH) value (90.4%) were observed between Streptomyces sp. C1-2 and Streptomyces griseoaurantiacus. Thus, Streptomyces sp. C1-2 could be identified as S. griseoaurantiacus. Genome analysis revealed that Streptomyces sp. C1-2 contained 22 biosynthetic gene clusters (BGCs) for secondary metabolites, where among them, 54% have low similarities with known BGCs. The chemical investigation led to the isolation of three new manumycin-type derivatives and two known analog antibiotics named SW-B and cornifronin B. All compounds showed antioxidant activity with the half-maximal inhibitory concentration (IC₅₀) values in a range of 50.82 ± 0.8–112.04 ± 1.0 μg/mL with no cytotoxicity against Vero cells. This is the first report of the antioxidant property of manumycin-type derivatives. Moreover, two known compounds exhibited antifungal activity against Phytophthora capsici, Fusarium oxysporum f. sp. cucumerinum, and Magnaporthe grisea, with the minimum inhibitory concentration (MIC) values in a range of 125–500 μg/mL.     

A newly isolated Streptomyces rimosus strain capable of degrading deltamethrin as a pesticide in agricultural soil

Chemical pesticides or insecticides with complex structures are highly abundant in the biosphere and have inevitable side effects on farmland, natural resources, and human health. Deltamethrin is the most popular and widely used pesticide that disrupts the cellular calcium channels. In the present study, isolated strains of bacteria were examined to determine the ones that were capable of degrading deltamethrin. Different species of bacteria were evaluated in terms of the capability to degrade deltamethrin. It is important to note that Streptomyces rimosus was able to degrade up to 200 mg/L deltamethrin concentration and could be grown in mineral salt medium agar containing deltamethrin to be used as a source of carbon and energy. The results demonstrated that there is a diversity of deltamethrin-degrading bacteria in agricultural soil ecosystems. The application of these bacteria, especially S. rimosus, might be used as a bioremediation technique to decrease pesticide contamination of the ecosystem.