Streptomyces albus G produces an antibiotic complex identical to paulomycins A and B

An antibiotic complex active against multiply resistant strains of staphylococci and other Gram-positive bacteria was isolated from cultures of Streptomyces albus G. Silica gel and Sephadex LH-20 column chromatography gave two congeners with Mr values of 786 and 772, which differed by one -CH2-group. The two homologues contained an isothiocyanate group, and proved to be identical with paulomycins A and B produced by Streptomyces paulus; the FAB mass spectra, in addition, proved the same two congeners to be present in proceomycin obtained from Streptomyces alboniger.     

Cloning and nucleotide sequence of celA1, and endo-beta-1,4-glucanase-encoding gene from Streptomyces halstedii JM8.

The celA1 gene encoding an endo-beta-1,4-glucanase from a mesophilic actinomycete, strain JM8, identified as Streptomyces halstedii, was cloned and expressed in S. lividans JI66. From the nucleotide sequence of a 1.7-kb DNA fragment we identified an open reading frame of 963 nucleotides encoding a protein of 321 amino acids, starting at TTG (instead of ATG). The Cel1 mature enzyme is a protein of 294 amino acids (after signal peptide cleavage) and can be included in the beta-glycanase family B (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991). The Cel1 enzyme lacks a cellulose-binding domain as predicted by computer analysis of the sequence and confirmed by Avicel binding experiments. The promoter region of celA1 was identified by S1 mapping; the -35 region closely resembles those of housekeeping Streptomyces promoters. Three imperfectly repeated sequences of 15, 15, and 14 nucleotides were found upstream from celA1 [ATTGGGACCGCTTCC-(N85)-ATTGGGACCGCTTCC-(N2)-TGGGAGC GCTCCCA]; The 14-nucleotide sequence has a perfect palindrome identical to that found in several cellulase-encoding genes from Thermomonospora fusca, an alkalophilic Streptomyces strain, and Streptomyces lividans. This sequence has been implicated in the mechanism of induction exerted by cellobiose. Using an internal celA1 probe, we detected similar genes in several other Streptomyces species, most of them cellulase producers.     

Production of an antifungal antibiotic by Streptomyces aburaviensis 1DA-28

A broad-spectrum antifungal Streptomyces isolate, 1DA-28, from Indian soil has been characterized and identified as Streptomyces aburaviensis var. ablastmyceticus (MTCC 2469). Nutritional and cultural conditions for the production of antibiotic by this organism under shake-flask conditions have been determined. Antibiotic production in synthetic medium reached the maximum on the 5th day of incubation at 30 degreesC. Glucose and starch were found to be the best carbon sources while NH4NO3 was preferred as nitrogen source. Optimum temperature and pH for antibiotic production were 32 degreesC and 7.4, respectively. Phosphate at a concentration sub-optimal for growth enhanced antibiotic production. Supplementation of medium with casein hydrolysate improved both growth and antibiotic titre but yeast extract exhibited marked inhibition.     

Purification and characterization of Streptomyces albidoflavus antifungal components

Chitinolytic strain Streptomyces albidoflavus was isolated from soil of the central region of Poland. Its identification was based on analysis of 16S rRNA gene sequence. The colloidal chitin was revealed as the finest substrate for the production of chitinases by S. albidoflavus. The enzyme catalyzed the hydrolysis of the disaccharide 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotriose most efficiently and was, therefore, classified as an endochitinase. The chitinase of S. albidoflavus was purified by applying the two-step procedure: fractionation with ammonium sulphate and chitin affinity chromatography. The molecular weight of the purified enzyme determined by SDS-PAGE was approximately 50 kDa. The enzyme was characterised as thermostable during 180 min of preincubation at the temperature of 35°C and 40°C. The activity of the enzyme was strongly inhibited in the presence of Hg²⁺ and Mn²⁺ ions, SDS but stabilized by Ca²⁺ and Mg²⁺ ions. Both purified and crude chitinases from S. albidoflavus inhibited the development of fungal phytopathogens. Purified chitinase inhibited the growth of Alternaria alternata, Fusarium culmorum, Fusarium oxysporum and Botrytis cinerea. Additionally, the crude chitinase inhibited the growth of Fusarium solani.     

Overproduction, purification, and biochemical characterization of a xylanase (Xys1) from Streptomyces halstedii JM8.

Streptomyces halstedii JM8, isolated from straw, produces and secretes into the culture supernatant at least two proteins with hydrolytic activity towards xylan. The cloning of a DNA fragment of this microorganism in several Streptomyces strains permitted us to overproduce both proteins. N-terminal sequence analyses, immunoblot assays, and time course overproduction experiments allowed us to ensure that both xylanases were encoded by the same gene and that the smallest form (35 kDa) originated from the large one (45 kDa) by proteolytic cleavage on the C terminus. The production of both forms was studied in different strains carrying the gene in a multicopy plasmid. The best production was obtained with Streptomyces parvulus transformed with the plasmid pJM9, a pIJ702 derivative, which yielded 144 U/ml. Both forms of the xylanase were purified with a fast-performance liquid chromatography system and characterized biochemically. The optimal pH and temperature, for both, were 6.3 and 60 degrees C, respectively, in 7.5-min assays. Both proteins were highly stable in a wide range of pHs (4 to 10) and temperatures (4 to 50 degrees C); nevertheless, after 1-h incubations, both enzymes lost most of their activity at temperatures over 55 to 60 degrees C. Endoxylanolytic activity was demonstrated in both enzymes, but no beta-xylosidase activity was detected.     

Purification and characterization of novel antifungal compounds from the sourdough Lactobacillus plantarum strain 21B

Sourdough lactic acid bacteria were selected for antifungal activity by a conidial germination assay. The 10-fold-concentrated culture filtrate of Lactobacillus plantarum 21B grown in wheat flour hydrolysate almost completely inhibited Eurotium repens IBT18000, Eurotium rubrum FTDC3228, Penicillium corylophilum IBT6978, Penicillium roqueforti IBT18687, Penicillium expansum IDM/FS2, Endomyces fibuliger IBT605 and IDM3812, Aspergillus niger FTDC3227 and IDM1, Aspergillus flavus FTDC3226, Monilia sitophila IDM/FS5, and Fusarium graminearum IDM623. The nonconcentrated culture filtrate of L. plantarum 21B grown in whole wheat flour hydrolysate had similar inhibitory activity. The activity was fungicidal. Calcium propionate at 3 mg ml(-1) was not effective under the same assay conditions, while sodium benzoate caused inhibition similar to L. plantarum 21B. After extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and chromatographic and spectroscopic analyses, novel antifungal compounds such as phenyllactic and 4-hydroxy-phenyllactic acids were identified in the culture filtrate of L. plantarum 21B. Phenyllactic acid was contained at the highest concentration in the bacterial culture filtrate and had the highest activity. It inhibited all the fungi tested at a concentration of 50 mg ml(-1) except for P. roqueforti IBT18687 and P. corylophilum IBT6978 (inhibitory concentration, 166 mg ml(-1)). L. plantarum 20B, which showed high antimold activity, was also selected. Preliminary studies showed that phenyllactic and 4-hydroxy-phenyllactic acids were also contained in the bacterial culture filtrate of strain 20B. Growth of A. niger FTDC3227 occurred after 2 days in breads started with Saccharomyces cerevisiae 141 alone or with S. cerevisiae and Lactobacillus brevis 1D, an unselected but acidifying lactic acid bacterium, while the onset of fungal growth was delayed for 7 days in bread started with S. cerevisiae and selected L. plantarum 21B.     

Single mutations of residues outside the active center of the xylanase Xys1 Delta from Streptomyces halstedii JM8 affect its activity

Mutagenesis of the xylanase Xys1 of Streptomyces halstedii JM8 has been done by error prone PCR. Mutants with modified hydrolytic activity were isolated, the recombinant variant proteins purified and the catalytic activities of each one determined and compared with the wild type enzyme. Two of the isolated single point mutants, m1 (G133D) and m8 (N148D), showed 22-25% increase in specific activity towards xylan compared to wild type xylanase. Two other mutants, m5a (D175A) and m7 (T160A), showed a significant reduction in specific activity of 40-50% with respect to the wild type enzyme. These residues are mainly located in the beta alpha-loops of the xylanase, the region showing the main structural divergences within family 10 of xylanases. This study shows the usefulness of random mutagenesis to point out some key residues not directly involved in the active center, but in which mutation produces subtle structural rearrangements affecting the enzymatic function.     

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.     

Inhibition of the accumulation of lipid droplets in macrophage J774 by bafilomycin B1 and destruxin E.

Two microbial metabolites, bafilomycin B1 and destruxin E, have been found to inhibit significantly the oxidized low density lipoprotein (LDL)-induced accumulation of lipid droplets at 3 nM and 0.5 microM, respectively, in macrophage J774. The incorporation of [14C]oleate into cholesteryl esters in the cells incubated with oxidized LDL was inhibited to the same extent by the two compounds. Both compounds had no effect on the cell surface binding at 4 degrees C and the internalization of oxidized 125I-LDL as well as on the activity of acyl-CoA:cholesterol acyltransferase. However, when incubated with these compounds at 37 degrees C, receptors for oxidized LDL were partially trapped within the cell. In accordance with receptor accumulation, ATP-dependent acidification of endosomes and lysosomes was significantly inhibited by 50 nM bafilomycin B1 and 1 microM destruxin E, respectively. From these results it was concluded that the inhibition of ATP-dependent acidification of endosomes and lysosomes by bafilomycin B1 and destruxin E resulted in the reduction of oxidized LDL-induced synthesis of cholesteryl ester and thereby caused a reduced accumulation of lipid droplets in macrophage J774.     

Factors affecting antifungal activity of Streptomyces philanthi RM-1-138 against Rhizoctonia solani

Sheath blight disease of rice caused by Rhizoctonia solani Kühn is economically important disease in most of the world’s rice growing areas. The disease causes severe yield losses of >20 % of rice in Thailand. Our previous investigation reported the antifungal activity of Streptomyces philanthi RM-1-138 against R. solani PTRRC-9. In this study, glucose yeast-malt extract medium, initial pH of 7.5 and a temperature of 30 °C were found to be optimum for both cell growth and antifungal activity of S. philanthi RM-1-138. The inhibition of 94 and 100 % on the growth of R. solani PTRRC-9 were achieved from the antifungal metabolites of the 6 and 9-days-old culture filtrates of S. philanthi RM-1-138, respectively. Heat treatment on the culture filtrate had slight effect on its antifungal activity. The culture broth demonstrated higher antifungal activity on growth of R. solani PTRRC-9 (90.4 %) than the culture filtrate (31.5 %) and its effective dose was at 0.1 % (v/v). The present results indicated the possibilities of using either the culture broth or culture filtrate of S. philanthi RM-1-138 to inhibit growth of R. solani PTRRC-9.     

In bafilomycin A1-resistant cells,bafilomycin A1 raised lysosomal pH and both prodigiosins and concanamycin A inhibited growth through apoptosis

In bafilomycin A(1)-resistant cells (Vero-317 and MC-3T3-E1), bafilomycin A(1) neither inhibited cell growth, induced cell death, nor activated caspase-3. However, 100 nM bafilomycin A(1) did raise the lysosomal pH similar to 10 mM NH(4)Cl. Prodigiosins, H(+)/Cl(-) symporters that raise the lysosomal pH, inhibited cell growth through apoptosis and caused the activation of caspase-3. Concanamycin A also inhibited the growth of these cells through apoptosis. 10 mM NH(4)Cl inhibited the growth of these cells as well, but cytostatically. These results suggest that plecomacrolides inhibited cell growth apoptotically through specific site(s), in contrast to the cytostatic effect of 10 mM NH(4)Cl, besides raising the lysosomal pH.     

Expression of the genes coding for the xylanase Xys1 and the cellulase Cel1 from the straw-decomposing Streptomyces halstedii JM8 cloned into the amino-acid producer Brevibacterium lactofermentum ATCC13869

The xylanase ( xysA) and the cellulase ( celA1) genes from Streptomyces halstedii JM8 were cloned into Escherichia coli/ Brevibacterium lactofermentum shuttle vectors and successfully expressed in both hosts when placed downstream from the kanamycin resistance promoter (Pkan) from Tn 5 but not when under the control of their own promoters. Xylanase was secreted into the culture media of B. lactofermentum by removal of the same leader peptide as is removed in S. halstedii. The main difference between the production of xylanase by Streptomyces and corynebacteria was the low level of processing of the mature extracellular xylanase by B. lactofermentum, probably due to the lack of protease activity in this microorganism.     

Piericidins C5 and C6: new 4-pyridinol compounds produced by Streptomyces sp. and Nocardioides sp

Piericidins C5 (1) and C6 (2), two new members of the piericidin family, were isolated from a Streptomyces sp. and a Nocardioides sp., together with known piericidins C1 (3), C2 (4), C3 (5), C4 (6), D1 (7), and A3 (8). The structures were determined on the basis of their spectroscopic data. Both new compounds inhibited cell division of fertilized starfish (Asterina pectinifera) eggs at the minimum inhibitory concentration of 0.09 microg/mL.     

C1-cycle of sulfur compounds

C₁ organic sulfides are part of many ecosystems and play an important role in the global sulfur budget and climate regulation. At this point, fluxes and conversions of these compounds are only superficially understood. Understanding of the regulating mechanisms will be necessary to quantify the role of these compounds in the global sulfur budget at their climatic role. In this review, the current knowledge of fluxes and conversions of C₁ organic sulfides in different ecosystems is presented.Abbreviations CCN cloud condensation nuclei - COS carbonylsulfide - DMS dimethylsulfide - DMDS dimethyldisulfide - DMSO dimethylsulfoxide - DMSO₂ dimethylsulfurdioxide - DMSP dimethylsulfoniopropionate - MA methylamine - 3-MPA 3-mercaptopropionate - MPPA 3-methiolpropionate - MT methanethiol     

Analysis of xysA, a gene from Streptomyces halstedii JM8 that encodes a 45-kilodalton modular xylanase, Xys1.

The gene xysA from Streptomyces halstedii JM8 encodes a protein of 461 amino acids (Xys1) which is secreted into the culture supernatant as a protein of 45 kDa (Xys1L). Later, this form is proteolytically processed after residue D-362 to produce the protein Xys1S, which conserves the same xylanolytic activity. The cleavage removes a domain of 99 amino acids that shows similarity to bacterial cellulose binding domains and that allows the protein Xys1L to bind to crystalline cellulose (Avicel). Expression of this monocistronic gene is affected by the carbon source present in the culture medium, xylan being the best inducer. By using an anti-Xys1L serum, we have been able to detect xylanases similar in size to Xys1L and Xys1S in most of the different Streptomyces species analyzed, suggesting the ubiquity of these types of xylanases and their processing mechanism.     

利迪链霉菌A02抗真菌活性产物的分离和结构鉴定

利迪链霉菌A02是从京郊森林土壤中分离筛选出的植物病原真菌高效拮抗菌株。为了明确其抑菌活性的物质基础,利用大孔树脂和硅胶吸附柱层析、HPLC循环制备分离等方法,从菌株A02发酵液中分离获得了纯度达99.845%以上的单一组分活性化合物。经紫外光谱、高分辨质谱、红外光谱和核磁共振谱的测定和解析,确定了该活性化合物的分子量为665,分子式为C33H47NO13,化学结构与四烯大环内酯类抗生素纳他霉素相同。这一结果揭示了利迪链霉菌产生抗真菌天然产物的新功能,并为纳他霉素在植物病害生物防治中的应用开拓了新的途径。