Optimized transformation of Streptomyces sp. ATCC 39366 producing leptomycin by electroporation

Streptomyces sp. ATCC 39366 produces leptomycin derivatives. Leptomycin B, a potent and specific inhibitor against the export of nuclear proteins, is the main product; however, the introduction of DNA into this strain is almost impossible, which has impeded its further use. We developed a Streptomyces sp. ATCC 39366 transformation protocol to introduce foreign DNA via electroporation. Various conditions were examined, including treatments of the cell wall with weakening agents, electroporation parameters, and DNA content. We found that only plasmid DNA isolated from a dam ^? ET12567 strain resulted in successful transformation. The mycelium growing in a yeast-peptone-dextrose medium supplemented with 1% glycine at 28°C on a rotary shaker (220 rpm) was more dispersed than those without supplementation and prone to electroporation. The maximum transformation efficiency of 8×10² CFU/μg plasmid DNA was obtained at a field strength of 13 kV/cm with a time constant of 13 ms (25-μF capacitor; parallel resistance, 600 Ω) using 1-mm electrocuvettes. The results of the transformations of two other Streptomyces species indicated that the optimized conditions established in this study might only be applicable to Streptomyces sp. ATCC 39366. However, this is the first report of successful transformation of Streptomyces sp. ATCC 39366, and will facilitate the construction of a gene knockout mutant in Streptomyces sp. ATCC 39366 to produce series of new leptomycin derivatives.     

Isolation of amylolytic,xylanolytic, and cellulolytic microorganisms extracted from the gut of the termite Reticulitermes santonensis by means of a micro-aerobic atmosphere

The aim of this work was to isolate enzyme-producing microorganisms from the tract of the termite Reticulitermes santonensis. The microorganisms were extracted from the guts and anaerobic (CO₂ or CO₂/H₂) and micro-aerobic atmospheres were used to stimulate growth. Three different strategies were tried out. First, the sample was spread on Petri dishes containing solid media with carboxymethylcellulose, microcrystalline cellulose or cellobiose. This technique allowed us to isolate two bacteria: Streptomyces sp. strain ABGxAviA1 and Pseudomonas sp. strain ABGxCellA. The second strategy consisted in inoculating a specific liquid medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. The samples were then spread on Petri dishes with the same specific medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. This led to the isolation of the mold Aspergillus sp. strain ABGxAviA2. Finally, the third strategy consisted in heating the first culture and spreading samples on agar plates containing rich medium. This led to the isolation of the bacterium Bacillus subtilis strain ABGx. All those steps were achieved in controlled atmospheres. The four enzyme-producing strains which were isolated were obtained by using a micro-aerobic atmosphere. Later, enzymatic assays were performed on the four strains. Streptomyces sp. strain ABGxAviA1 was found to produce only amylase, while Pseudomonas sp. strain ABGxCellA was found to produce β-glucosidase as well. Aspergillus sp. strain ABGxAviA2 showed β-glucosidase, amylase, cellulase, and xylanase activities. Finally, B. subtilis strain ABGx produced xylanase and amylase.     

Occurrence of a Lysogenic Streptomyces sp. on the Nodule Surface of Black Gram (Vigna mungo (L.) Hepper)

A lysogenic Streptomyces sp., strain NS.A4, which was isolated from the nodule surface of black gram (Vigna mungo (L.) Hepper), was found to inhibit rhizobia of fast-and slow-growing strains of cowpeas and soybeans. It exhibited plaques when there was a change in cultural conditions. Repeated culturing of the organism in nutrient agar and broth confirmed the infection of Streptomyces sp. strain NS.A4 by an actinophage. Addition of the culture filtrate of Streptomyces sp. strain NS.A4 to shaken broth cultures of three other Streptomyces spp. resulted in phage infection.     

Endophytic actinomycetes from spontaneous plants of Algerian Sahara: indole-3-acetic acid production and tomato plants growth promoting activity

Twenty-seven endophytic actinomycete strains were isolated from five spontaneous plants well adapted to the poor sandy soil and arid climatic conditions of the Algerian Sahara. Morphological and chemotaxonomical analysis indicated that twenty-two isolates belonged to the Streptomyces genus and the remaining five were non-Streptomyces. All endophytic strains were screened for their ability to produce indole-3-acetic acid (IAA) in vitro on a chemically defined medium. Eighteen strains were able to produce IAA and the maximum production occurred with the Streptomyces sp. PT2 strain. The IAA produced was further extracted, partially purified and confirmed by thin layer chromatography (TLC) analysis. The 16S rDNA sequence analysis and phylogenetic studies indicated that strain PT2 was closely related to Streptomyces enissocaecilis NRRL B 16365^T, Streptomyces rochei NBRC 12908^T and Streptomyces plicatus NBRC 13071^T, with 99.52 % similarity. The production of IAA was affected by cultural conditions such as temperature, pH, incubation period and l-tryptophan concentration. The highest level of IAA production (127 μg/ml) was obtained by cultivating the Streptomyces sp. PT2 strain in yeast extract-tryptone broth supplemented with 5 mg l-tryptophan/ml at pH 7 and incubated on a rotary shaker (200 rpm) at 30 °C for 5 days. Twenty-four-hour treatment of tomato cv. Marmande seeds with the supernatant culture of Streptomyces sp. PT2 that contained the crude IAA showed the maximum effect in promoting seed germination and root elongation.     

Crude bacterial extracts of two new Streptomyces sp. isolates as bio-colorants for textile dyeing

Renewed demand for incorporation of natural dyes (bio-colorants) in textile industry could be met through biotechnological production of bacterial pigments. Two new Streptomyces strains (NP2 and NP4) were isolated for the remarkable ability to produce diffusible deep blue and deep red pigment into fermentation medium. Crude mycelial extracts of both strains were used as bio-colorants in conventional textile dyeing procedures avoiding downstream purification procedures. The yields of bio-colorants obtained in this way were 62 and 84 mg per g of mycelia for Streptomyces sp. NP2 and Streptomyces sp. NP4, respectively. Through nuclear magnetic resonance analysis of crude extracts before and after dyeing procedures, it was shown that both extracts contained prodigiosin-like family of compounds that exhibited different dyeing capabilities towards different textile fibers. Polyamide and acrylic fibers were colored to the deepest shade, polyester and triacetate fibers to a noticeable, but much lower shade depth, while cotton and cellulosic fibers stained weakly. These results confirmed that crude bacterial extracts had the characteristics similar to those of ionic and disperse dyes, which was consistent with the identified polypyrrolic prodigiosin-like structures.     

Isolation and characterization of soil Streptomyces species as potential biological control agents against fungal plant pathogens

The use of antagonist microorganisms against fungal plant pathogens is an attractive and ecologically alternative to the use of chemical pesticides. Streptomyces are beneficial soil bacteria and potential candidates for biocontrol agents. This study reports the isolation, characterization and antagonist activity of soil streptomycetes from the Los Petenes Biosphere Reserve, a Natural protected area in Campeche, Mexico. The results showed morphological, physiological and biochemical characterization of six actinomycetes and their inhibitory activity against Curvularia sp., Aspergillus niger, Helminthosporium sp. and Fusarium sp. One isolate, identified as Streptomyces sp. CACIS-1.16CA showed the potential to inhibit additional pathogens as Alternaria sp., Phytophthora capsici, Colletotrichum sp. and Rhizoctonia sp. with percentages ranging from 47 to 90 %. This study identified a streptomycete strain with a broad antagonist activity that could be used for biocontrol of plant pathogenic fungi.     

Characterization of a laccase-like multicopper oxidase from newly isolated Streptomyces sp. C1 in agricultural waste compost and enzymatic decolorization of azo dyes

Laccases or laccase-like multicopper oxidases (LMCOs) could catalyze the oxidation of various substrates coupled to the reduction of oxygen to water. In this study, eight strains with laccase activity were isolated from composting samples in different phases, among which strain C1 isolated from the thermophilic-phase sample presented the highest laccase activity. The purified LMCO of strain C1 showed a single protein band on SDS-PAGE gel with a molecular mass of about 38 kDa. The novel laccase showed alkaline resistance and moderate thermostability. The enzyme activity was activated by some metal ions such as Cu²⁺, Co²⁺ and Fe³⁺ at the concentration of 1 mM, while was strongly inhibited in the presence of Hg²⁺. The LMCO could efficiently decolorize the indigo carmine and diamond black PV with syringaldehyde as mediator, which suggested a great potential for dye decolorization in the textile industry. The novel strain was identified as Streptomyces sp. C1. The finding of new laccase-producing Streptomyces sp. C1 in this study will also contribute to the further explanation of the function of Actinomycetes in the thermophilic phase of composting.     

Soil chromium bioremediation: Synergic activity of actinobacteria and plants

The aim of this work was to evaluate a strategy to reduce the bioavailable chromium fraction in soil, using a Cr(VI) resistant microorganism, Streptomyces sp. MC1, under non sterile conditions, with maize plants as bioindicator and/or bioremediator.Soil samples were contaminated with 100, 200 and 400 mg kg⁻¹ of Cr(VI) or Cr(III). Bioavailable chromium (35%) was only detected in samples with Cr(VI). Soil samples with Cr(VI) 200 mg kg⁻¹ were inoculated with Streptomyces sp. MC1, and bioavailable chromium decreased up to 73%.Zea mays seedlings were planted in soil samples contaminated with chromium. Plantlets accumulated chromium mainly as Cr(III), and biomass decreased up to 88%. Streptomyces sp. MC1 was inoculated in soil samples contaminated with 200 mg kg⁻¹ of Cr(VI) and Z.mays seedlings were planted.Streptomyces sp. MC1 caused Z.mays biomass increase (57%), chromium accumulation and bioavailable chromium decreased up to 46% and 96%, respectively.This work constitutes the first contribution of cooperative action between actinobacteria and Z.mays in the bioremediation of Cr(VI) contaminated soil. The large removal capacity of bioavailable chromium by Streptomyces sp. MC1 and Z.mays infers that they could be successfully applied together in bioremediation of soils contaminated with Cr(VI).     

Biochemical detection of a metallo-β-lactamase in carbapenem resistant strain ofStreptomyces sp. CN229 isolated from soil

Streptomyces sp. CN229 was isolated from Tunisia soil. This strain displayed antimicrobial activity against Gram positive and Gram negative bacteria. In addition it is resistant to most β-lactam antibiotics including imipenem and meropenem (MIC imipenem >70 μg/ml). Metallo-β-lactamase (MβL) production was confirmed by either imipenem MIC decrease in the presence of ethylene diamine tetraactic acid (EDTA) or the inhibition zone enhancement around EDTA-impregnated imipenem, or meropenem discs. Isolectric focusing analysis demonstrated the production of β-lactamase with pI of 5.8 that is inhibited by EDTA.Streptomyces sp. CN229 was screened for the imipenem resistance genes,bla _VIM andbla _IMP previously identified inPseudomonas aeruginosa. The presence of these genes was not confirmed by specific PCR analysis. We concluded that carbapenem resistance inStreptomyces sp. CN229 strain is mainly due to production of a novel carbapenemase. Our data show for the first time that MβL is produced byStreptomyces sp. MβL-mediated imipenem and meropenem resistance inStreptomyces is a cause for concern in the study of resistance evolution and antibiotic cluster biosynthetic genes.     

Optimization,production and characterization of glycolipid biosurfactant from the marine actinobacterium,Streptomyces sp. MAB36

A potential glycolipid biosurfactant producer Streptomyces sp. MAB36 was isolated from marine sediment samples. Medium composition and culture conditions for the glycolipid biosurfactant production by Streptomyces sp. MAB36 were optimized, using two statistical methods: Plackett–Burman design was applied to find out the key ingredients and conditions for the best yield of glycolipid biosurfactant production and central composite design was used to optimize the concentration of the four significant variables, starch, casein, crude oil and incubation time. Fructose and yeast extract were the best carbon and nitrogen sources for the production of the glycolipid biosurfactant. Biochemical characterizations including FTIR and MS studies suggested the glycolipid nature of the biosurfactant. The isolated glycolipid biosurfactant reduced the surface tension of water from 73.2 to 32.4 mN/m. The purified glycolipid biosurfactant showed critical micelle concentrations of 36 mg/l. The glycolipid biosurfactant was effective at very low concentrations over a wide range of temperature, pH, and NaCl concentration. The purified glycolipid biosurfactant showed strong antimicrobial activity. Thus, the strain Streptomyces sp. MAB36 has proved to be a potential source of glycolipid biosurfactant that could be used for the bioremediation processes in the marine environment.     

<Emphasis Type="Italic">Streptomyces gamaensis</Emphasis> sp. nov., a novel actinomycete with antifungal activity isolated from soil in Gama,Chad

During an investigation exploring potential sources of novel species and natural products, a novel actinomycete with antifungal activity, designated strain NEAU-Gz11^T, was isolated from a soil sample, which was collected from Gama, Chad. The isolate was found to have morphological and chemotaxonomic characteristics typical of members of the genus Streptomyces. 16S rRNA gene sequence similarity studies showed that strain NEAU-Gz11^T belongs to the genus Streptomyces with high sequence similarity to Streptomyces hiroshimensis JCM 4098^T (98.0 %). Similarities to other type strains of the genus Streptomyces were lower than 98.0 %. However, the physiological and biochemical characteristics and low levels of DNA–DNA relatedness could differentiate the isolate genotypically and phenotypically from S. hiroshimensis JCM 4098^T. Therefore, the strain is concluded to represent a novel species of the genus Streptomyces, for which the name Streptomyces gamaensis sp. nov. is proposed. The type strain is NEAU-Gz11^T (=CGMCC 4.7304^T=DSM 101531^T).     

The ‘gifted’ actinomycete <Emphasis Type="Italic">Streptomyces leeuwenhoekii</Emphasis>

Streptomyces leeuwenhoekii strains C34^T, C38, C58 and C79 were isolated from a soil sample collected from the Chaxa Lagoon, located in the Salar de Atacama in northern Chile. These streptomycetes produce a variety of new specialised metabolites with antibiotic, anti-cancer and anti-inflammatory activities. Moreover, genome mining performed on two of these strains has revealed the presence of biosynthetic gene clusters with the potential to produce new specialised metabolites. This review focusses on this new clade of Streptomyces strains, summarises the literature and presents new information on strain C34^T.     

A global positive regulator afsR2 stimulates tautomycetin production via pathway-specific regulatory gene over-expression in Streptomyces sp. CK4412

Tautomycetin (TMC) is a T cell-specific immunosuppressant with a unique ester bond linkage between a terminal cyclic anhydride and a linear polyketide chain. Since an afsR2 was proved to be a global antibiotics-stimulating regulatory gene in various Streptomyces species, an afsR2-induced TMC productivity stimulation was investigated in a TMC-producing Streptomyces sp. CK4412 strain. An afsR2 gene was cloned under the influence of a strong constitutive ermE* promoter in an integrative expression vector, followed by its conjugation into the Streptomyces sp. CK4412. Comparing TMC productivity and antifungal activity of the wild type and the afsR2-containing ex-conjugant revealed that afsR2 over-expression stimulated TMC production approximately 2.4-fold in Streptomyces sp. CK4412. Based on both RT-PCR and real-time RT-PCR analyses, an afsR2 over-expression significantly stimulated the expression of a TMC-specific positive regulatory gene, tmcN. This implies that the stimulatory effect of afsR2 functions in Streptomyces sp. CK4412 via up-regulation of a TMC pathway-specific positive regulatory gene over-expression.     

Streptomyces bullii sp. nov., isolated from a hyper-arid Atacama Desert soil

A Streptomyces strain isolated from a hyper-arid Atacama Desert soil was characterised using a polyphasic taxonomic approach. The strain, designated C2^T, had chemical and morphological properties typical of the genus Streptomyces. The isolate formed a branch in the Streptomyces 16S rRNA gene tree together with the type strain of Streptomyces chromofuscus and was also loosely related to Streptomyces fragilis NRRL 2424^T. DNA:DNA relatedness values between the isolate and its two phylogenetic neighbours showed that it formed a distinct genomic species. The strain was readily distinguished from these organisms using a combination of morphological and phenotypic data. Based on the genotypic and phenotypic results, isolate C2^T represents a novel species in the genus Streptomyces, for which the name Streptomyces bullii sp. nov. is proposed. The type strain is C2^T (=CGMCC 4.7019^T = KACC 15426^T).     

<Emphasis Type="Italic">Streptomyces thermoalkaliphilus</Emphasis> sp. nov., an alkaline cellulase producing thermophilic actinomycete isolated from tropical rainforest soil

During an investigation exploring potential sources of novel thermophilic species and natural products, a novel thermophilic and alkaliphilic actinomycete with alkaline cellulase producing ability, designated strain 4-2-13^T, was isolated from soil of a tropical rainforest in Xishuangbanna, Yunnan province, China. The morphological and chemotaxonomic characteristics of strain 4-2-13^T are consistent with those of the members of the genus Streptomyces. The strain forms extensively branched aerial mycelia and substrate mycelia. Spiral spore chains were observed on aerial mycelia; spores were oval to cylindrical, with smooth surfaces. The organism was found to contain ll-diaminopimelic acid as the diagnostic diamino acid in the cell wall peptidoglycan. The whole cell hydrolysates were found to contain glucose and ribose. The cellular fatty acid profile mainly consists of anteiso-C_17:0 and iso-C_16:0. The menaquinones were identified as MK-9(H₈), MK-10(H₆) and MK-9(H₆). The polar lipids profile were found to consist of diphosphatidylglycerol, phosphatidylmethylethanolamine, a ninhydrin-positive glycophospholipid, phosphatidylinositol, phosphatidylglycerol and unidentified glycolipids. The 16S rRNA gene sequence analysis showed that the organism belongs to the genus Streptomyces and in the 16S rRNA gene tree it formed a distinct phyletic line together with the closely related type strain Streptomyces burgazadensis Z1R7^T (95.2% sequence similarity). However, the phenotypic characteristics of strain 4-2-13^T are significantly different from those of S. burgazadensis Z1R7^T. Based on the phenotypic, chemotaxonomic and phylogenetic characteristics, strain 4-2-13^T represents a novel species in the genus Streptomyces, for which the name Streptomyces thermoalkaliphilus sp. nov. is proposed. The type strain is 4-2-13^T (= DSM 42159^T = CGMCC 4. 7205^T).     

Endophytic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. AC35, a producer of bioactive isoflavone aglycones and antimycins

In this research, a microbial endophytic strain obtained from the rhizosphere of the conifer Taxus baccata and designated as Streptomyces sp. AC35 (FJ001754.1 Streptomyces, GenBank) was investigated. High 16S rDNA gene sequence similarity suggests that this strain is closely related to S. odorifer. The major fatty acid profile of intracellular lipids was also carried out to further identify this strain. Atomic force microscopy and scanning acoustic microscopy were used to image our strain. Its major excreted substances were extracted, evaluated for antimicrobial activity, purified, and identified by ultraviolet–visible spectroscopy (UV–vis), liquid chromatography–mass spectrometry (LC–MS/MS) and nuclear magnetic resonance as the bioactive isoflavone aglycones—daidzein, glycitein and genistein. Batch cultivation, performed under different pH conditions, revealed enhanced production of antimycin components when the pH was stable at 7.0. Antimycins were detected by HPLC and identified by UV–vis and LC–MS/MS combined with the multiple reaction monitoring. Our results demonstrate that Streptomyces sp. AC35 might be used as a potential source of effective, pharmaceutically active compounds.     

Ribosome engineering and fermentation optimization leads to overproduction of tiancimycin A,a new enediyne natural product from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CB03234

Tiancimycin (TNM) A, a recently discovered enediyne natural product from Streptomyces sp. CB03234, showed rapid and complete killing of cancer cells and could be used as a payload in antibody drug conjugates. The low yield of TNM A in the wild-type strain promoted us to use ribosome engineering and fermentation optimization for its yield improvement. The Streptomyces sp. CB03234-R-16 mutant strain with a L422P mutation in RpoB, the RNA polymerase β-subunit, was obtained from the rifamycin-resistant screening. After fermentation optimization, the titers of TNM A in Streptomyces sp. CB03234-R-16 reached to 22.5 ± 3.1 mg L^?1 in shaking flasks, and 13 ± 1 mg L^?1 in 15 L fermentors, which were at least 40-fold higher than that in the wild-type strain (~ 0.3 mg L^?1). Quantitative real-time RT-PCR revealed markedly enhanced expression of key genes encoding TNM A biosynthetic enzymes and regulators in Streptomyces sp. CB03234-R-16. Our study should greatly facilitate the future efforts to develop TNM A into a clinical anticancer drug.     

Streptomyces leeuwenhoekii sp. nov., the producer of chaxalactins and chaxamycins,forms a distinct branch in Streptomyces gene trees

A polyphasic study was carried out to establish the taxonomic status of an Atacama Desert isolate, Streptomyces strain C34^T, which synthesises novel antibiotics, the chaxalactins and chaxamycins. The organism was shown to have chemotaxonomic, cultural and morphological properties consistent with its classification in the genus Streptomyces. Analysis of 16S rRNA gene sequences showed that strain C34^T formed a distinct phyletic line in the Streptomyces gene tree that was very loosely associated with the type strains of several Streptomyces species. Multilocus sequence analysis based on five house-keeping gene alleles underpinned the separation of strain C34^T from all of its nearest phylogenetic neighbours, apart from Streptomyces chiangmaiensis TA-1^T and Streptomyces hyderabadensis OU-40^T which are not currently in the MLSA database. Strain C34^T was distinguished readily from the S. chiangmaiensis and S. hyderabadensis strains by using a combination of cultural and phenotypic data. Consequently, strain C34^T is considered to represent a new species of the genus Streptomyces for which the name Streptomyces leeuwenhoekii sp. nov. is proposed. The type strain is C34^T (= DSM 42122^T = NRRL B-24963^T). Analysis of the whole-genome sequence of S. leeuwenhoekii, with 6,780 predicted open reading frames and a total genome size of around 7.86 Mb, revealed a high potential for natural product biosynthesis.