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The skyllamycins are a class of heavily modified, non-ribosomal peptides, first isolated from Streptomyces sp. KY11784. A Streptomyces strain with potent antibiotic activity against Bacillus subtilis was isolated from a sample of the New Zealand lichen Pseudocyphellaria dissimilis. Whole genome sequencing and biosynthetic gene cluster genetic analysis coupled with GNPS LCMS/MS molecular networking revealed that this strain had the capacity to produce skyllamycins, including previously undescribed congeners, and that these were likely the source of the observed biological activity. Guided by the results of the molecular networking, we isolated the previously reported skyllamycins A-C (1-3), along with two new congeners, skyllamycins D (4) and E (5). The structures of these compounds were elucidated using comprehensive 1D and 2D NMR analyses, along with HRESIMS fragmentation experiments. Antibacterial assays revealed that skyllamycin D possessed improved activity against B. subtilis E168 compared to previously reported congeners.

Streptomyces species have been an important source of bioactive secondary metabolites and clinically useful for antibiotic production, including fosfomycin, daptomycin, oxytetracycline, streptomycin, and chloramphenicol. The main objective of this study was to isolate actinomycetes, especially Streptomyces from 8 soil samples that collected from two different places, which are districts in Sulaimani governorate, Kurdistan Region of Iraq. Totally, 30 diagnostic tests were carried out for representative of isolated Streptomyces depending on the color groups. The 16Sr DNA gene was sequenced, for ten isolated test strains were amplified with 2 universal primers after extraction of genomic DNA, only 8 of them were successfully sequenced. Phylogenetic analysis of 8 test strains carried out using base sequences of 16Sr DNA genes in the core genome. Four of isolated test strains were identified as a Streptomyces fulvissimus, and others were nominated as a Streptomyces anulatus. The obtained sequence data were compared with the sequence data of the closest related species in the international databases using EzTaxon Server program and (%) similarities were determined. Finally, phylogenetic analysis indicated that isolated test strains were nominated as (H001, H002, H003, and H004), which recognized members of the S. anulatus and the similarity with their type strain is 99.93%. Whereas, other four isolated test strains, including D001, D002, D003, and D004 recognized as members of the S. fulvissimus and similarity with their type strain is 100%.

The properties of two-domain laccase of Streptomyces anulatus (SaSL) and its role in transformation of humic acids (HA) from chernozem, sod-podzolic soil and peat at alkaline pH values were studied. The SaSL was cloned, expressed in E. coli and obtained in an electrophoretically homogeneous state. SaSL is an oligomeric protein with a molecular weight of 235-300 kDa and six or eight subunits in the molecule. The molecular weight of the subunit is 37.7 kDa. Its catalytic properties are similar to those of the previously described two-domain laccase. The enzyme catalyzed oxidation of electron donors (K4[Fe(CN)6], ABTS) at acidic pH and phenolic substrates (2-methoxyphenol, 2,6-dimethixyphenol) at alkaline pH values. The efficiency of catalysis was higher in the case of electron donors than phenolic substrates. The enzyme showed a high thermal stability and was more stable at neutral and alkaline pH values. The enzyme effectively transformed humic acids at alkaline pH values. It was found that polymerization reactions took place during interaction of SaSL with HA, as well as with their high-molecular weight (>80 kDa) and low-molecular weight (

Silver nanoparticles (AgNPs) are well known for their broad spectrum of antimicrobial properties. Owing to its eco-friendly and cost-effective synthesis protocols, there is an increasing surge toward a green synthesis of AgNPs as alternatives to chemical and physical methods. Among the biogenic sources, bacteria are more preferred microorganisms to manipulate as they grow and form microbial colonies. Microbes are known to produce nanoparticles (NPs) with varied morphology and sizes; hence, they can provide a cost-effective large-scale production. In this regard, bacteria produce large quantities of extracellular as well as intracellular proteins and enzymes that can take part in the fast and sustainable synthesis of NPs. Bacteriogenic NPs possess an outer coating of bacterial biomolecules, which increases its efficiency of activity. Biomass or extracellular materials from microorganisms like Bacillus licheniformis, Pseudomonas deceptionensis, and Streptomyces anulatus are used for the synthesis of AgNPs. This chapter will focus predominantly on the synthesis of AgNPs from various types of bacteria, with their preferred physicochemical parameters and their applications in various fields.

Cholesterol oxidase biosensors have been used to determine the level of cholesterol in different serum and food samples. Due to a wide range of industrial and clinical applications of microbial cholesterol oxidase, isolation and identification of a new microbial source (s) of cholesterol oxidase are very important.The local isolate Streptomyces sp. strain NEAE-94 is a promising source of cholesterol oxidase. It was identified based on cultural, morphological and physiological characteristics; in addition to the 16S rRNA sequence. The sequencing product had been deposited in the GenBank database under the accession number KC354803. Cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 in shake flasks was optimized using surface response methodology. The different process parameters were first screened using a Plackett-Burman design and the parameters with significant effects on the production of cholesterol oxidase were identified. Out of the 15 factors screened, agitation speed, cholesterol and yeast extract concentrations had the most significant positive effects on the production of cholesterol oxidase. The optimal levels of these variables and the effects of their mutual interactions on cholesterol oxidase production were determined using Box-Behnken design. Cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 was 11.03, 27.31 U/mL after Plackett-Burman Design and Box-Behnken design; respectively, with a fold of increase of 6.06 times compared to the production before applying the Plackett-Burman design (4.51 U/mL).Maximum cholesterol oxidase activity was obtained at the following fermentation conditions: g/L (cholesterol 4, yeast extract 5, NaCl 0.5, K

Exploration of the effect of soil bacteria on growth and metabolism of beneficial root endophytic fungi is relevant to promote favorable associations between microorganisms of the plant rhizosphere. Hence, the interaction between the plant-growth-promoting fungus Piriformospora indica and different soil bacteria was investigated. The parameters studied were fungal growth and its amino acid composition during the interaction. Fungus and bacteria were confronted in dual cultures in Petri dishes, either through agar or separated by a Perspex wall that only allowed the bacterial volatiles to be effective. Fungal growth was stimulated by Azotobacter chroococcum, whereas Streptomyces anulatus AcH 1003 inhibited it and Streptomyces sp. Nov AcH 505 had no effect. To analyze amino acid concentration data, targeted metabolomics was implemented under supervised analysis according to fungal-bacteria interaction and time. Orthogonal partial least squares-discriminant analysis (OPLS-DA) model clearly discriminated P. indica&ndash, A. chroococcum and P. indica&ndash, S. anulatus interactions, according to the respective score plot in comparison to the control. The most observable responses were in the glutamine and alanine size groups: While Streptomyces AcH 1003 increased the amount of glutamine, A. chroococcum decreased it. The fungal growth and the increase of alanine content might be associated with the assimilation of nitrogen in the presence of glucose as a carbon source. The N-fixing bacterium A. chroococcum should stimulate fungal amino acid metabolism via glutamine synthetase-glutamate synthase (GS-GOGAT). The data pointed to a stimulated glycolytic activity in the fungus observed by the accumulation of alanine, possibly via alanine aminotransferase. The responses toward the growth-inhibiting Streptomyces AcH 1003 suggest an (oxidative) stress response of the fungus.

Screening of different unexplored species of Streptomyces led to the identification of alanine dehydrogenase (AlaDH) from Streptomyces anulatus. This AlaDH was purified, characterized and was used as a bioreceptor for developing an ammonium biosensor that can detect ammonium ions in water samples. The AlaDH of S. anulatus was a dimer with each monomeric unit of Mol. Wt. 61 kDa. The optimum pH for AlaDH in oxidative deamination and reductive amination was 10 and 8.5 respectively with wide working pH range of 5–11. The optimum temperature was 40 °C in both the reactions with wide working temperature range of 20–50 °C. The enzyme retained more than 85% of its original activity after incubating at 60 °C for 30 min in the presence of DTT. Due to these properties of AlaDH, it was successfully used as a bioreceptor in the ammonium biosensor and the sensor showed linear response in the range of 0.1–300 mM NH4+ with the detection limit of 0.01 mM NH4+ and response time of 20 s. The sensor was showing good response at wide pH (5–11) and temperature range (20–50 °C) suggesting its usage at ambient and non-ambient conditions. The sensor was successfully validated with Nessler’s reagent method by using real water samples.

During the screening programme for antibiotic produced by actinomycetes, an antibiotic was isolated from the fermentation broth of Streptomyces anulatus NEAE-94. The ethyl acetate extract of S. anulatus NEAE-94 showed high biological activity against Staphylococcus aureus NRRL B-313, multidrug-resistant S. aureus and Bacillus subtilis NRRL B-543. Thin-layer chromatography of the ethyl acetate extract showed one yellowish orange spot with an Rf value of 0.83. The structural elucidation of the isolated compounds was carried out using ultraviolet–visible mass spectrometry (MS), Fourier transform-infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (1H-NMR) and gas chromatography–mass spectrometry (GC–MS) analyses. Characterization of the ethyl acetate extract revealed the presence of 22 compounds, including an unsaturated fatty acid, saturated fatty acids, fatty acid esters, alkanes, alkenes and a triterpene. The antimicrobial efficacy of S. anulatus NEAE-94 might be due to the synergistic effects of the identified compounds.

In an effort to identify a microbial enzyme that can be useful as a fungicide and biodegradation agent of chitinous wastes, a chitinase (Chi242) was purified from the culture supernatant of Streptomyces anulatus CS242 utilizing powder of shrimp shell wastes as a sole carbon source. It was purified employing ammonium sulfate precipitation and gel permeation chromatography techniques. The molecular weight of the purified chitinase was ~38 kDa by SDS-PAGE. The N-terminal amino acid sequence (A-P-G-A-P-G-T-G-A-L) showed close similarity to those of other Streptomyes chitinases. The purified enzyme displayed optimal activity at pH 6.0 and 50 °C respectively. It showed substantial thermal stability for 2 h at 30-60 °C, and exhibited broad pH stability in the range 5.0-13.0 for 48 h at 4 °C. Scanning electron microscopy confirmed the ability of this enzyme to adsorb onto solid shrimp bio-waste and to degrade chitin microfibers. Chi242 could proficiently convert colloidal chitin to N-acetyl glucosamine (GlcNAc) and N-acetyl chitobiose (GlcNAc)2 signifying that this enzyme is suitable for bioconversion of chitin waste. In addition, it exerted an effective antifungal activity towards fungal pathogen signifying its role as a biocontrol agent. Thus, a single microbial cell of Streptomyces anulatus CS242 justified its dual role.

An actinomyce (S07) isolated from a cyst of Heterodera filipjevi was characterised as Streptomyces anulatus by morphological, physiological and biochemical criteria and 16S rRNA analysis. The biocontrol potential of S07 was evaluated against both Heterodera avenae and H. filipjevi. After an initial assay of S07 culture filtrates on egg hatch in vitro and juvenile survival, the effect on these cereal cyst nematodes (CCN) was evaluated in the greenhouse using naturally infested soil and in the field over two consecutive years. The results showed that S07 significantly reduced the population densities of CCN females in wheat, concurrently increasing grain yield. It is concluded that S07 offers potential as a commercial biocontrol agent.

International audience; Gray mold, caused by Botrytis cinerea, is one of the most destructive diseases of grapevine and is controlled with an intense application of fungicides. As alternatives to chemicals, beneficial microbes may promote plant health by stimulating the plant's immune system. An actinomycete, Streptomyces anulatus S37, has been screened from the rhizosphere microbiome of healthy Vitis vinifera on the basis of its ability to promote grapevine growth and to induce resistance against various phytopathogens, including B. cinerea. However, molecular mechanisms involved locally after direct perception of these bacteria by plant cells still remain unknown. This study focuses on local defense events induced in grapevine cells during interactions with S. anulatus S37 before and after pathogen challenge. We demonstrated that S. anulatus S37 induced early responses including oxidative burst, extracellular alkalinization, activation of protein kinases, induction of defense gene expression and phytoalexin accumulation, but not the programmed cell death. Interestingly, upon challenge with the B. cinerea, the S. anulatus S37 primed grapevine cells for enhanced defense reactions with a decline in cell death. In the presence of the EGTA, a calcium channel inhibitor, the induced oxidative burst, and the protein kinase activity were inhibited, but not the extracellular alkalinization, suggesting that Ca2+ may also contribute upstream to the induced defenses. Moreover, desensitization assays using extracellular pH showed that once increased by S. anulatus S37, cells became refractory to further stimulation by B. cinerea, suggesting that grapevine cells perceive distinctly beneficial and pathogenic microbes.

Four glutarimide-derived compounds including a new 3-[2-[2-hydroxy-3-methylphenyl-5-(hydroxymethyl)]-2-oxoethyl] glutarimide (1) and three known 3-[2-(2-hyroxy-3,5- dimethylphenyl)-2-oxoethyl] glutarimide (2, actiphenol), 3-hydroxy-3-[2-(2-hydroxy-3,5-dimethylphenyl)-2-oxoethyl] glutarimide (3) and 3-[2-[2-hydroxy-3-(hydroxymethyl)-5-methylphenyl]-2-oxoethyl] glutarimide (4), along with a known indole alkaloid 3-(hydroxyacetyl) indole (5), were isolated from ethyl acetate extract of the fermentation broth of the marine sponge-derived Streptomyces anulatus S71. Their structures were deduced by extensive studies of NMR and mass spectra.

The need for new antibiotics has been highlighted recently with the increasing pace of emergence of drug resistance pathogens. Emerging strains of bacteria resistant to most advanced antibiotics have become issues of very important public health concern. Modification of existing antibiotics with the addition of side chains or other chemical group and genomics based drug targeting have been the preferred method of drug development at the corporate level in recent years. In this regard, soil samples were collected from farmlands located in Ibadan in Oyo state, Ago – Iwoye, and Ikenne in Ogun state, Nigeria. Two putative Streptomyces strains and Bacillus strains isolated from the 16 selected farmland soils were characterized and assessed for antibiotic production and activity against a wide range of bacteria including Klebsiella pneumonia, Serratia marscens, Proteus vulgaris, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis. The extracts of the putative obtained from Streptomyces somaliensis, Streptomyces anulatus, Bacillus megaterium, and Bacillus subtilis showed activities against minimum of 3 and maximum of the 4 of the 7 tested bacteria. Inhibition zones were found to range between 2.0 - 25.0 mm diameters at a concentration of 1ml. The minimum inhibitory concentrations (MICs) of the crude extracts against the tested organisms ranged from 50% and above. Bacillus megaterium, and Streptomyces somaliensis were found to inhibit all the pathogenic bacteria, while S. anulatus was unable to inhibit Proteus vulgaris and Staphylococcus aureus, and B. Subtilis was unable to inhibit Enterococcus faecalis.

Bioactive herbicidal compounds produced by soil microorganisms might be used to creating a bioherbicide for biological weed control. A total of 1,300 bacterial strains were isolated and screened for herbicidal activity against grass and broadleaf weeds. Among primarily selected 102 strains, the herbicidal activity of bacterial fermentation broths from the following three isolates strain-101, strain-128, and strain-329 reduced the growth of D. sanguinalis by 66.7%, 78.3%, and 100%, respectively as compared with control. Phylogenetic analysis of 16S rRNA gene sequencing determined that the strain-329 has 99% similarity to Streptomyces anulatus (HBUM 174206). The potential bioherbicidal efficacy of Streptomyces strain-329 was tested on grass and broadleaf weeds for phytotoxic activity through pre- and post-emergence applications. At pre-emergence application, the phytotoxic efficacy to D. sanguinalis and S. bicolor on seed germination were 90.4% and 81.3%, respectively at the 2x concentration, whereas in the case of Solanum nigrum, 85.2% phytotoxic efficacy was observed at the 4x concentration. The efficacy of Streptomyces strain-329 was substantially higher at post-emergence application, presenting 100% control of grass and broadleaf weeds at the 1x concentration. Two herbicidal compounds coded as 329-C1 and 329-C3 were extracted and purified by column chromatography and high-performance liquid chromatography methods. The active compound 329-C3 slightly increased leaf electrolytic leakage and MDA production as concentration-dependent manner. These results suggest that new Streptomyces sp. strain-329 produced bioherbicidal metabolites and may provide a new lead molecule for production an efficient bioherbicide to regulate grass and broadleaf weeds.

Marine actinomycetes isolated from seaweed were screened for growth activation effects on the biodiesel-producing microalgae Dunaliella salina. Of the 98 actinomycetes studied, strain 288-11 isolated from the rhizosphere of Undaria pinnatifida showed potent growth activation. The strain was identified as Streptomyces anulatus based on 16S rDNA sequence analysis. The cell density increased up to 2.1-fold with the addition of 1 mg/mL of extract to the medium. To understand the effect of adding S. anulatus extract, the gross biochemical composition and fatty acids of D. salina were determined.

Thirty bacterial isolates were obtained from different sources and sites at Jeddah, Saudi Arabia, on chitin agar medium; 9 of the 30 isolates were cultured in liquid medium containing chitin as sole carbon and nitrogen sources. Isolate SM21, which was isolated from shrimp shells, showed the best growth and chitinase production in liquid medium. According to its morphological, physiological and biochemical characteristics, SM21 belongs to the genus Streptomyces and was identified as Streptomyces anulatus SM21. Identification was confirmed using 16S rDNA analysis. The chitinase enzyme was precipitated with 80% NH4SO4 and purified using DEAE-cellulose ion exchange chromatography followed by Sephadex G-100 gel filtration. The molecular weight determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis was 28 kDa. Genetic improvement using the protoplast fusion technique was carried out between the identified Streptomyces isolate and Streptomyces coelicolor SM1. These two species, which have different resistance profiles to streptomycin and tetracycline (400 μg/ml and 10 μg/ml, respectively), were used in an intraspecific protoplast fusion using PEG 6000. The percentage of real protoplasts that could regenerate successfully was 71% for S. coelicolor SM1 and 80% for S. anulatus SM21. Out of three recombinant fusants obtained, one (named Fu3) showed higher chitinase production compared to both parents (5 fold increase).

Streptomyces griseus and related species form the biggest but least well-defined clade in the whole Streptomyces 16S rRNA gene tree. Multilocus sequence analysis (MLSA) has shown promising potential for refining Streptomyces systematics. In this investigation, strains of 18 additional S. griseus clade species were analysed and data from a previous pilot study were integrated in a larger MLSA phylogeny. The results demonstrated that MLSA of five housekeeping genes (atpD, gyrB, recA, rpoB and trpB) is better than the previous six-gene scheme, as it provides equally good resolution and stability and is more cost-effective; MLSA using three or four of the genes also shows good resolution and robustness for differentiating most of the strains and is therefore of value for everyday use. MLSA is more suitable for discriminating strains that show >99 % 16S rRNA gene sequence similarity. DNA–DNA hybridization (DDH) between strains with representative MLSA distances revealed a strong correlation between the data of MLSA and DDH. The 70 % DDH value for current species definition corresponds to a five-gene MLSA distance of 0.007, which could be considered as the species cut-off for the S. griseus clade. It is concluded that the MLSA procedure can be a practical, reliable and robust alternative to DDH for the identification and classification of streptomycetes at the species and intraspecies levels. Based on the data from MLSA and DDH, as well as cultural and morphological characteristics, 18 species and three subspecies of the S. griseus clade are considered to be later heterotypic synonyms of 11 genomic species: Streptomyces griseinus and Streptomyces mediolani as synonyms of Streptomyces albovinaceus; Streptomyces praecox as a synonym of Streptomyces anulatus; Streptomyces olivoviridis as a synonym of Streptomyces atroolivaceus; Streptomyces griseobrunneus as a synonym of Streptomyces bacillaris; Streptomyces cavourensis subsp. washingtonensis as a synonym of Streptomyces cyaneofuscatus; Streptomyces acrimycini, Streptomyces baarnensis, Streptomyces caviscabies and Streptomyces flavofuscus as synonyms of Streptomyces fimicarius; Streptomyces flavogriseus as a synonym of Streptomyces flavovirens; Streptomyces erumpens, ‘Streptomyces ornatus’ and Streptomyces setonii as synonyms of Streptomyces griseus; Streptomyces graminofaciens as a synonym of Streptomyces halstedii; Streptomyces alboviridis, Streptomyces griseus subsp. alpha, Streptomyces griseus subsp. cretosus and Streptomyces luridiscabiei as synonyms of Streptomyces microflavus; and Streptomyces californicus and Streptomyces floridae as synonyms of Streptomyces puniceus.

The bacterium Streptomyces anulatus 9663, isolated from the intestine of different arthropods, produces prenylated derivatives of phenazine 1-carboxylic acid. From this organism, we have identified the prenyltransferase gene ppzP. ppzP resides in a gene cluster containing orthologs of all genes known to be involved in phenazine 1-carboxylic acid biosynthesis in Pseudomonas strains as well as genes for the six enzymes required to generate dimethylallyl diphosphate via the mevalonate pathway. This is the first complete gene cluster of a phenazine natural compound from streptomycetes. Heterologous expression of this cluster in Streptomyces coelicolor M512 resulted in the formation of prenylated derivatives of phenazine 1-carboxylic acid. After inactivation of ppzP, only nonprenylated phenazine 1-carboxylic acid was formed. Cloning, overexpression, and purification of PpzP resulted in a 37-kDa soluble protein, which was identified as a 5,10-dihydrophenazine 1-carboxylate dimethylallyltransferase, forming a C–C bond between C-1 of the isoprenoid substrate and C-9 of the aromatic substrate. In contrast to many other prenyltransferases, the reaction of PpzP is independent of the presence of magnesium or other divalent cations. The Km value for dimethylallyl diphosphate was determined as 116 μm. For dihydro-PCA, half-maximal velocity was observed at 35 μm. Kcat was calculated as 0.435 s-1. PpzP shows obvious sequence similarity to a recently discovered family of prenyltransferases with aromatic substrates, the ABBA prenyltransferases. The present finding extends the substrate range of this family, previously limited to phenolic compounds, to include also phenazine derivatives.

A novel beta-N-acetylglucosaminidase (GlcNAcase) inhibitor named TMG-chitotriomycin (1) was isolated from the culture filtrate of Streptomyces anulatus NBRC13369. The strain produced 1 only when colloidal chitin was used as the sole carbon source in the production medium. The structure of 1 was determined by spectral and constitutive sugar analyses of the corresponding alditol derivatives to be an equilibrated mixture of alpha-d-N,N,N-triMeGlcNH2-(1,4)-beta-d-GlcNAc-(1,4)-beta-d-GlcNAc-(1,4)-d-GlcNAc and its C-2 epimer of the reducing end residue. TMG-chitotriomycin (1) showed potent and selective inhibition of insect and fungal GlcNAcases with no inhibition of mammalian and plant GlcNAcases. In contrast, the known GlcNAcase inhibitor nagstatin potently inhibited all GlcNAcases. It should be emphasized that synthesized d-N,N,N-triMeGlcNH2, which is the component sugar of 1, showed no inhibition of the insect Spodoptera litura GlcNAcase. These results suggest that the (GlcNAc)3 unit positioned at the reducing end of 1 is essential for its enzyme inhibitory activity. The unique inhibitory spectrum of 1 will be useful to study chitinolytic systems and to develop selective fungicides or pesticides.

Two biologically active microbial strains were isolated from soil samples collected from different geographical areas of India. These strains were characterized as Streptomyces halstedii MTCC 6817 and Streptomyces anulatus MTCC 6818. Active compounds were extracted from the purified fermented broth and one of them was chemically characterized as actinomycin-D. Though these two strains produced the same antibiotic, their nutritional requirements vary substantially. For S. halstedii, a medium containing 1.5% fructose, 0.05% N-acetylglucosamine, and basal production medium, gave the maximum titre, 754% higher, as compared to production medium alone. In the case of Streptomyces anulatus, maximum antibiotic production was attained with a production medium containing 1.5% galactose and 0.05% L-asparagine monohydrate.

The effect of cell storage at −18°C for 18–24 months on reproductive capacity was investigated for various microorganisms (gram-positive and gram-negative bacteria, yeasts, and filamentous fungi) immobilized in poly(vinyl alcohol) cryogel. To examine the viability of immobilized cells after defrosting, the bioluminescent method of intracellular ATP determination was used. A high level of metabolic activity of immobilized cells after various periods of storage was recorded for Streptomyces anulatus, Rhizopus oryzae, and Escherichia coli, which are producers of the antibiotic aurantin, L(+)-lactic acid, and the recombinant enzyme organophosphate hydrolase, respectively. It was shown that the initial concentration of immobilized cells in cryogel granules plays an important role in the survival of Str. anulatus and Pseudomonas putida after 1.5 years of storage. It was found that, after slow defrosting in the storage medium at 5°C for 18 h of immobilized cells of the yeast Saccharomyces cerevisiae that had been stored for nine months, the number of reproductive cells increased due to the formation of ascospores.

Marine sponges often harbor dense and diverse microbial communities including actinobacteria. To date no comprehensive investigation has been performed on the culturable diversity of the actinomycetes associated with South China Sea sponges. Structurally novel aromatic polyketides were recently discovered from marine sponge-derived Streptomyces and Saccharopolyspora strains, suggesting that sponge-associated actinomycetes can serve as a new source of aromatic polyketides. In this study, a total of 77 actinomycete strains were isolated from 15 South China Sea sponge species. Phylogenetic characterization of the isolates based on 16S rRNA gene sequencing supported their assignment to 12 families and 20 genera, among which three rare genera (Marihabitans, Polymorphospora, and Streptomonospora) were isolated from marine sponges for the first time. Subsequently, β-ketoacyl synthase (KSα) gene was used as marker for evaluating the potential of the actinomycete strains to produce aromatic polyketides. As a result, KSα gene was detected in 35 isolates related to seven genera (Kocuria, Micromonospora, Nocardia, Nocardiopsis, Saccharopolyspora, Salinispora, and Streptomyces). Finally, 10 strains were selected for small-scale fermentation, and one angucycline compound was detected from the culture extract of Streptomyces anulatus strain S71. This study advanced our knowledge of the sponge-associated actinomycetes regarding their diversity and potential in producing aromatic polyketides.

Pyrolysis/gas chromatography/mass spectrometry was used to elucidate the ability of streptomycetes to modify pine wood under solid-state fermentation. The pyrograms of sound and decayed wood showed the typical pyrolysis products derived from carbohydrates and lignin. In general, the relative abundance of the former was lower in treated woods than in sound wood. Several major peaks released from guaiacyl units of lignin were also revealed. Although some differences could be observed in the relative abundance of the released guaiacyl units from lignin between sound and treated woods, a clear decrease in the lignin-derived products by the action of the microorganisms was not observed. Nevertheless, an increase in the relative abundance of the most of guaiacyl units with a higher oxidation degree was mainly detected in woods treated by the strains Streptomyces UAH 7, Streptomyces anulatus , Streptomyces UAH 48, Streptomyces avermitilis and Streptomyces badius . These results suggest an oxidative action of these strains on the lignin of pine wood, which was corroborated by a decrease in the (aldehyde/ketone + acid) ratio. In addition, the breakdown of the alkyl chain linkages by the strain Streptomyces UAH 7 could be also observed. Analytical pyrolysis allow to demonstrate for first time the oxidative attack of selected Streptomyces strains on lignin moiety of pine wood, which is an important requirement for biomechanical pulping purposes.

Wetlands have important global ecological functions, which include carbon storage and water interception. Wetland contributes to the maintenance of regional and global biodiversity. Though many important wetland ecological functions are based on microbial metabolism, we have scanty knowledge on microbial diversity in wetlands. Plant rhizoplane habitats are considered to harbor highly diverse bacterial communities. Most of the floating mats on river Nile are dominated by papyrus (Cyperus papyrus). Papyrus root samples were collected from a floating mat at the "Gold Island" inside the Nile River at Cairo, Egypt in February 1996 and May 1997 in order to investigate the rhizoplane actinobacteria communities. The root-tip regions were cut off, repeatedly washed, macerated and plated. Using the plate-count technique with three actinobacteria media, an average of 2.1 x 10(4) CFUg-1 root actinobacteria were obtained. All actinobacteria colonies were isolated, purified and investigated by classical and molecular methods. In the papyrus rhizoplane Streptomyces anulatus, Micromonospora sp., Rhodococcus luteus, Verrucosispora gifhornensis and Aureobacterium liquefaciens dominated, moreover Actinoplanes utahensis, and Str. diastaticus were also present. The physiological traits of the members of dominant groups revealed that these bacteria might be active in the rhizoplane and can be present there is their vegetative forms.

A detailed screening of the secondary metabolite pattern produced by different athropod associated strains of the species Streptomyces anulatus resulted in the isolation and structure elucidation of the endophenazines A-D (2, 4-6). The structures were assigned by spectroscopic methods and chemical transformations. 4 represents a chromophoric system based on a phenazin-7-one, 5 and 6 are new 5,10-dihydrophenazine derivatives.

Four new members of the phenazine family, endophenazines A-D, and the already known phenazine-1-carboxylic acid (tubermycin B) were detected in the culture broth of various endosymbiotic Streptomyces anulatus strains by chemical screening in a combination of TLC-staining reagents and HPLC-diode array analysis. The endosymbiotic strains were isolated from four different arthropod hosts at various sites. The new phenazine compounds showed antimicrobial activities against Gram-positive bacteria and some filamentous fungi, and herbicidal activity against Lemna minor (duckweed).

Microbial synthesis of nanoparticles is a green approach that interconnects nanotechnology and microbial biotechnology. Here, we synthesized the silver nanoparticles (AgNPs) using bacterial strains of Listeria monocytogenes, Bacillus subtilius and Streptomyces anulatus. We tested the efficacy of AgNPs against the larvae, pupae and adults of Anopheles stephensi and Culex quinquefasciatus. We have also investigated the antifungal activity of AgNPs against the soil keratinophilic fungus of Chrysosporium keratinophilum. The efficacy tests were then performed at different concentrations and varying numbers of hours by probit analysis. The results were obtained using a UV-visible spectrophotometer, and the images were recorded with a transmission electron microscope (TEM). The synthesized AgNPs were in varied shape and sizes. The larvae and pupae of Cx. quinquefasciatus were found highly susceptible to AgNPs synthesized using the L. monocytogenes, B. subtilius and S. anulatus than the An. stephensi, while the adults of An. stephensi were found more susceptible to the AgNPs synthesized using the L. monocytogenes, B. subtilius and S. anulatus the Cx. quinquefasciatus. Further, these nanoparticles have also been tested as antifungal activity against the entomopathogenic fungus C. keratinophilum. The higher zone of inhibition occurred at the concentration level of 50 μl. This study gives an innovative approach to develop eco-friendly AgNPs which act as an effective antifungal agent/fungicide and insecticide.

A novel actinomycete strain, Streptomyces anulatus S37, has been isolated from the rhizosphere of healthy Moroccan Vitis vinifera on the basis on its ability to promote grapevine growth and to induce natural defences against various phytopathogens. In the present work, the main bioactive metabolites produced by S. anulatus S37 were isolated. A crude n-BuOH extract of the S37 fermentation broth was firstly partitioned in a biphasic solvent system composed of n-heptane, methanol, and water (5:1.5:3.5, v/v). The most active organic fraction (1.1g) as revealed by TLC-bioautography was subsequently separated by a two-step centrifugal partition chromatography procedure. The first separation was performed in the ascending mode at 6mL/min with the biphasic solvent system n-heptane, ethyl acetate, methanol and water (2:1:2:1, v/v), to finally recover 40mg of a pure compound identified as streptochlorin by NMR spectroscopy. In a second separation, the solvent system n-heptane, acetonitrile, and water (5:5:4, v/v) was used in the ascending mode at 3mL/min to purify 135mg of nigericin and 53mg of piericidin A1. Assays performed with the three compounds have confirmed their inhibitory impact on the growth of Botryris cinerea in dual confrontation and also on V. vinifera L. plantlets.

Streptomyces anulatus, an indicator microbe of mold in buildings, was grown on different building materials in order to study the impact of growth conditions on the ability of the spores of this microbe to induce toxicity and inflammatory responses. The microbes were grown for 2 months on sterilized and unsterilized wood, chipboard, concrete, plaster board and mineral wool in tight glass vessels under humid conditions. The highest microbial spore concentration was detected on the sterilized mineral wool followed by the sterilized plaster board and the unsterilized mineral wool. Mouse RAW264.7 macrophages were exposed in vitro for 24 h to the spores of S. anulatus and the production of the inflammatory mediators, nitric oxide (NO), tumor necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), interleukin-10 (IL-10) and cytotoxicity, were measured. The dose equivalent to 5 x 10(5) spores/ml of medium was used to compare the different materials. The most intense production of NO (11.6 microM), TNF alpha (560 pg/ml) and IL-6 (2800 pg/ml) in macrophages was induced by the spores grown on sterilized plaster board. They also caused the greatest loss of cell viability (39%). The spores grown on sterilized concrete induced significant production of NO (1.5 microM) and decreased cell viability (22%), and the spores grown on unsterilized and sterilized mineral wool increased production of NO (4.1 microM and 0.8 microM, respectively). The spores did not stimulate production of the anti-inflammatory cytokine IL-10. These results indicate that the ability of S. anulatus to induce inflammatory responses and cytotoxicity in macrophages is dependent on the growth conditions provided by different building materials.

Moisture associated microbial growth in buildings may cause respiratory symptoms such as pulmonary inflammation. We studied the effects of spores of Streptomyces anulatus, commonly found in moldy buildings, on the production of nitric oxide (NO), interleukin-6 (IL-6), interleukin-5 (IL-5), interleukin-4 (IL-4), and tumour necrosis factor alpha (TNFα), as well as cell viability in human alveolar II type epithelial cell line (A549). Cells were exposed in vitro to S. anulatus spores with and without interferon-γ (IFNγ) in vitro. Lipopolysaccharide (LPS) was used as a reference substance. S. anulatus alone, and in combination with IFNγ induced NO and IL-6 production and decreased cell viability whereas IL-4, IL-5 or TNFα production were not affected. IFNγ alone had a weaker but otherwise similar effect as S. anulatus on NO and IL-6 production and it potentiated the effects of S. anulatus. LPS did not induce NO or cytokine production, or affect cell viability in A549 cells. These data indicate that spores of S. anulatus induce the excretion of inflammatory mediators in respiratory epithelial cells, which may partly explain the adverse respiratory health effects experienced by individuals exposed to the indoor air of moldy houses.

A Montana soil actinomycete, Streptomyces anulatus, produced (1 x 10(-2)% yield) a new cancer cell growth inhibitory cyclooctadepsipeptide named montanastatin (1) accompanied by the potent anticancer antibiotic valinomycin (2) in very high (5.1%) yields. Valinomycin but not montanastatin inhibited growth of a number of pathogenic bacteria and fungi. Interpretation of high-field (500 MHz) NMR and high-resolution FAB mass spectral data allowed assignment of the structure cyclo-(D-Val-L-Lac-L-Val-D-Hiv) to montanastatin. Valinomycin (2) was also isolated from actinomycetes cultured from a tree branch and animal feces collected in Malaysia. Streptomyces exfoliatus, isolated from the tree branch, was found to contain valinomycin in 1.6% yield, while the fecal isolate, S. anulatus, gave valinomycin in 0.9% yield.

The Streptomyces strain Streptomyces anulatus JH801 showed submerged sporulation and production of a genotoxic activity. The genotoxin production of this strain was found to be correlated to morphology and is likely to be related to sporulation. The influence of the medium on genotoxin production was investigated. Genotoxin production and sporulation were not inhibited by glucose, but were negatively affected by low ammonia concentrations. No correlation was found between genotoxin production and other morphological parameters such as total hyphal length and total average tip number. Bioassay-directed fractionation of the chloroform extract of S. anulatus JH801 resulted in the isolation of dihydroabikoviromycin. This compound shows selective activity towards DNA repair-deficient Escherichia coli. The isolation and structure of this genotoxin are reported.

The potential genotoxicity of dihydroabikoviromycin was assessed in bacterial and sister-chromatid exchange (SCE) test systems. Direct cytotoxicity was also assayed using bioluminescence methods to screen for differences in cell viability among different tumour cell lines following exposure to the drug. Differential killing tests with Escherichia coli WP2 and its repair-deficient derivative CM871 indicated that a functional DNA repair system was protective against the action of dihydroabikoviromycin, implying that this compound causes some form of DNA damage and is almost certainly therefore genotoxic. Dose-dependent reversion from His- to His+ with dihydroabikoviromycin was observed in the Ames test with Salmonella typhimurium TA100, but not in frameshift tester strain TA98. Dihydroabikoviromycin also induced the sfiA gene, as indicated by beta-galactosidase induction in an SOS-chromotest with E. coli PQ37 strain. A dose-related increase in SCEs by dihydroabikoviromycin was observed in CHO cells. Growth of tumour cells was also suppressed by dihydroabikoviromycin at a dose of 10 micrograms/ml.

Summary One hundred and twenty three poly(3-hydroxyalkanoates) degrading streptomycetes (referred to as PHA strains) from soils, composts, natural waters, and some other sources were characterized by gas chromatographic analysis of their methylated cellular fatty acids. They were compared to reference strains of the genera Streptomyces (46 strains) and Streptoverticillium (9 strains) by numerical analysis. Saturated iso- and anteiso-branched fatty acids of between 14 and 18 carbons constituted the major components in the fatty acid profiles of most of the strains. In the dendrogram, obtained by unweighted average linkage clustering of the Euclidean distances Δ, calculated between each pair of fatty acid profiles, all strains were linked at Δ = 43. At Δ = 11, 18 clusters, consisting each of 2 to 63 strains, could be delineated, and 16 strains remained unclustered. The clusters could also be recognized by principal-component analysis, although small overlaps between some clusters were evident. Most clusters could be differentiated from each other by their fatty acid compositions. The PHA strains were scattered over 14 different clusters, 11 of which also contained reference strains, and 9 PHA strains remained unclustered. In addition to the PHA strains, 29 of the 46 reference strains of Streptomyces were also shown to degrade poly(3-hydroxybutyrate) in vitro. Poly(3-hydroxybutyrate-co-10%3-hydroxyvalerate) was degraded in vitro by all 56 PHA strains tested. The study demonstrates the biodiversity of streptomycetes involved in the degradation of poly(3-hydroxyalkanoates) in different habitats. The numerical analysis of fatty acid profiles confirms the homogeneity of the Streptomyces albidoflavus group, and the heterogeneity of the Streptomyces cyaneus and Streptomyces anulatus groups, and supports the reunification of the genera Streptomyces and Streptoverticillium.

An antagonistic rhizoplane bacterium (Serratia Marcescens) and a chitin-degrading microbe (Streptomyces anulatus) were used for establishing a binary microbe system for biological control of Fusarium wilt disease of tomato. For effectively tracing root-colonization, the rhizoplane bacterium was genetically marked with the lux genes and the tetracycline resistance gene through bacterial conjugation. Successful colonization by the bacterium on roots from bacterized seeds was visualized by bioluminescence of the transconjugant into which the lux gene operon had been integrated. Antifungal activity of the transconjugant was stable and effectire even when the bacterium was colonized on tomato roots. Multiplication of the transconjugant in soil was supported by the chitin-degrading microbe which had been previously added in chitin-supplemented soil. The chitin-degrading microbe produced extracellular endochitinases and hydrolyzed chitin which was added to soil. Chitohydrolytic products supported the multiplication and root-colonization of the rhizoplane bacterium in soil. The binary microbe system comprising of chitin-degrading microbe and gene-marked transconjugant was effective in controlling of tomato wilt disease caused by Fusarium oxysporum f. sp. lycopersici.

Summary Cellular proteins of 32 strains of Streptomyces and 5 of Streptoverticillium were examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The protein profiles obtained were analysed by computer-assisted techniques and the results compared with those of a previous numerical taxonomic study of phenotypic characteristics. Valuable taxonomic correlations were found between the protein electrophoretic and phenotypic data though it was evident that certain numerically circumscribed taxa, notably Streptoverticillium griseocarneum, were electrophoretically heterogeneous. Species specific profiles were observed for Streptomyces albidoflavus, Streptomyces anulatus and Streptomyces cyaneus. It can be concluded that numerical analysis of SDS-PAGE patterns of cellular proteins provides a rapid way of clarifying and refining subgeneric relationships established within the genera Streptomyces and Streptoverticillium.

A DNA-DNA hybridization survey was performed on 13 Streptomyces species and two subspecies, dispersed over five genotypically defined clusters as delineated by Lanoot et al. [Syst Appl Microbiol 27 (2004), 84-92]. Within each of the latter clusters, strains shared DNA-DNA relatedness values above 70 %. On the basis of published recommendations, the following eight Streptomyces species with validly published names are considered as later synonyms: Streptomyces nigrifaciens as a synonym of Streptomyces flavovirens; Streptomyces citreofluorescens, Streptomyces chrysomallus subsp. chrysomallus and Streptomyces fluorescens as synonyms of Streptomyces anulatus; Streptomyces chibaensis as a synonym of Streptomyces corchorusii; Streptomyces flaviscleroticus as a synonym of Streptomyces minutiscleroticus; and Streptomyces lipmanii, Streptomyces griseus subsp. alpha, Streptomyces griseus subsp. cretosus and Streptomyces willmorei as synonyms of Streptomyces microflavus. Emended descriptions are proposed.

Telomestatin is a natural product isolated from Streptomyces anulatus 3533-SV4 and has been shown to be a very potent telomerase inhibitor. The structural similarity between telomestatin and a G-tetrad suggested to us that the telomerase inhibition might be due to its ability either to facilitate the formation of or trap out preformed G-quadruplex structures, and thereby sequester single-stranded d[T(2)AG(3)](n) primer molecules required for telomerase activity. Significantly, telomestatin appears to be a more potent inhibitor of telomerase (5 nM) than any of the previously described G-quadruplex-interactive molecules. In this communication we provide the first experimental evidence that telomestatin selectively facilitates the formation of or stabilizes intramolecular G-quadruplexes, in particular, that produced from the human telomeric sequence d[T(2)AG(3)](4). A simulated annealing (SA) docking approach was used to study the binding interactions of telomestatin with the intramolecular antiparallel G-quadruplex structure. Each intramolecular G-quadruplex molecule was found to bind two telomestatin molecules (unpublished results). A 2:1 model for the telomestatin bound in the external stacking mode in an energy minimized complex with the human telomeric basket-type G-quadruplex was constructed. Our observation that a G-quadruplex-interactive molecule without significant groove interactions is able to reorient in a G-quadruplex structure proints to the importance of core interaction with an asymmetric G-quadruplex structure in producing selective binding. Furthermore, the G-quadruplex interactions of telomestatin are more selective for the intramolecular structure in contrast to other G-quadruplex-interactive agents, such as TMPyP4.

New 5-lipoxygenase inhibitors, designated epocarbazolins A and B, were isolated from the culture broth of Streptomyces anulatus T688-8. These compounds showed potent rat 5-lipoxygenase inhibitory activity with weak antibacterial activity. Structural studies revealed that epocarbazolins are new carbazole antibiotics having a novel substitution pattern and an epoxide in the side chain.