Comparison of antibiotic complexes formed by cultures of Streptomyces griseoruber

Physico-chemical properties of antibiotic complexes formed by various strains of Streptomyces griseoruber: VNIIA 1195, VNIIA 1193-INA 2022/55 and IMiV 1618 were compared. It was shown that the antibiotic complex 1195 differed by the electron absorption spectrum, chromatographic mobility and indicator properties from lateriomycins A and B produced by the type culture of S. griseoruber, Jamaguchi et Saburi, 1955 and strain 70717, as well as from prodigiozin produced by S. griseoruber, VNIIA 1193. By the absorption spectra and chromatographic mobility the components of the antibiotic complex 1195 were identical with cinerubins A and B, pyrromycin and eta- and xi-pyrromycinones included in the composition of antibiotic 1618 produced by S. griseoruber 1618 and differed in their quantitative contents and absence of epsilon-pyrromycinones.     

Interactions and biocontrol of pathogenic Streptomyces strains co-occurring in potato scab lesions

Aims:  To test interactions between pathogenic strains of Streptomyces turgidiscabies , S. scabies and S. aureofaciens . To study biological control of S. turgidiscabies and S. scabies using the nonpathogenic Streptomyces strain (346) isolated from a scab lesion and a commercially available biocontrol agent ( S. griseoviridis strain K61; 'Mycostop').
Methods and Results:  Pathogenic strains of S. turgidiscabies and S. aureofaciens inhibited growth of S. scabies in vitro , whereas strain 346 and S. griseoviridis inhibited the pathogenic strains and were subsequently tested for control of scab in the greenhouse and field. Strains 346 and K61 suppressed development of common scab disease caused by S. turgidiscabies in the greenhouse. Strain 346 reduced incidence of S. turgidiscabies in scab lesions on potato tubers in the field.
Conclusions:  Streptomyces turgidiscabies shows antagonism against S. scabies that occurs in the same scab lesions and shares the ecological niche in the field. Biocontrol of S. turgidiscabies is possible with nonpathogenic Streptomyces strains but interactions may be complicated.
Significance and Impact of the Study:  Streptomyces turgidiscabies may have potential to displace S. scabies under the Scandinavian potato growing conditions. Biological control of the severe potato scab pathogen, S. turgidiscabies , is demonstrated for the first time. The results can be applied to enhance control of common scab.     

Phenotypic variation in Streptomyces sp. DSM 40537, a lipoteichoic acid producing actinomycete

Aims:  To reinvestigate the production of lipoteichoic acid (LTA) by the actinomycete strain Streptomyces sp. DSM 40537 (=ATCC 3351).
Methods and Results:  LTA was extracted and purified from strain Streptomyces sp. DSM 40537. The identification of the LTA was confirmed by Western blotting with a monoclonal antibody. During these studies, two stable phenotypic variants of DSM 40537 were obtained, one of which released a distinctive orange pigment. 16S rRNA gene sequencing of each variant yielded identical sequences and allowed phylogenetic analysis to be performed.
Conclusions:  Streptomyces sp. DSM 40537 was shown to exhibit stable morphological variation. The strain was confirmed to be a LTA-producing actinomycete and to belong to the Streptomyces albidoflavus cluster within the genus Streptomyces .
Significance and Impact of the Study:  These data provide important support for the hypothesis that the distribution of LTA is linked to that of wall teichoic acids and emphasizes the need to reinvestigate LTA distribution in actinomycetes.     

Production and structural elucidation of trehalose tetraesters (biosurfactants) from a novel alkanothrophic Rhodococcus wratislaviensis strain

Aims:  To isolate a biosurfactant-producing bacterial strain and to identify and characterize the chemical structure and properties of its biosurfactants.
Methods and Results:  The bacterium Rhodococcus wratislaviensis BN38, isolated from soil, was found to produce glycolipid biosurfactants when grown on 2% n -hexadecane. The glycolipids were isolated by chromatography on silica gel columns and their structures elucidated using a combination of multidimensional NMR and ESI-MS/MS techniques. The main product was identified as 2,3,4,2'-trehalose tetraester with molecular mass of 876 g mol⁻¹. It was also noted that the biosurfactant was produced under nitrogen-limiting conditions and could not be synthesized from water-soluble substrates. The purified product showed extremely high surface-active properties.
Conclusions:  The glycolipid biosurfactant produced by the alkanothrophic strain R. wratislaviensis BN38 was characterized to be 2,3,4,2'-trehalose tetraester which exhibited high surfactant activities.
Significance and Impact of the Study:  Strain BN38 of R. wratislaviensis is a potential candidate for use in bioremediation applications or in biosurfactant exploration.     

In vitro and in vivo antagonism of actinomycetes isolated from Moroccan rhizospherical soils against Sclerotium rolfsii: a causal agent of root rot on sugar beet (Beta vulgaris L.)

Aims:  To evaluate the ability of the isolated actinomycetes to inhibit in vitro plant pathogenic fungi and the efficacy of promising antagonistic isolates to reduce in vivo the incidence of root rot induced by Sclerotium rolfsii on sugar beet.
Methods and Results:  Actinomycetes isolated from rhizosphere soil of sugar beet were screened for antagonistic activity against a number of plant pathogens, including S.   rolfsii . Ten actinomycetes out of 195 screened in vitro were strongly inhibitory to S. rolfsii . These isolates were subsequently tested for their ability to inhibit sclerotial germination and hyphal growth of S. roflsii . The most important inhibitions were obtained by the culture filtrate from the isolates J-2 and B-11, including 100% inhibition of sclerotial germination and 80% inhibition of hyphal growth. These two isolates (J-2 and B-11) were then screened for their ability to protect sugar beet against infection of S. rolfsii induced root rot in a pot trial. The treatment of S. rolfsii infested soil with a biomass and culture filtrate mixture of the selected antagonists reduced significantly ( P  ≤ 0·05) the incidence of root rot on sugar beet. Isolate J-2 was most effective and allowed a high fresh weight of sugar beet roots to be obtained. Both antagonists J-2 and B-11 were classified as belonging to the genus Streptomyces species through morphological and chemical characteristics as well as 16S rDNA analysis.
Conclusion:  Streptomyces isolates J-2 and B-11 showed a potential for controlling root rot on sugar beet and could be useful in integrated control against diverse soil borne plant pathogens.
Significance and Impact of the Study:  This investigation showed the role, which actinomycete bacteria can play to control root rot caused by S.   rolfsii , in the objective to reduce treatments with chemical fungicides.     

Brewer's spent grain and corn steep liquor as substrates for cellulolytic enzymes production by Streptomyces malaysiensis

Aims:  To evaluate cellulase production by Streptomyces malaysiensis in submerged fermentation using brewer's spent grain (BSG) and wheat bran (WB) as carbon source, and corn steep liquor (CSL) as nitrogen source, as compared to yeast extract (YE), and partial characterization of the crude enzyme.
Methods and Results:  Maximum cellulase production by Streptomyces malaysiensis (720 U l⁻¹) occurred within 4 days incubation when using a growth medium containing BSG 0·5% (w/v) and CSL1·2% (w/v). CMCases activity showed to be stable over an acidic pH range (2·0–7·0) and in temperatures of 40–60°C. Zymogram indicated three bands of CMCase activity, with different molecular masses.
Conclusion:  S. malaysiensis was able to grow and produce good levels of CMCases using solely brewer's spent grain and corn steep liquor as low-cost substrates, making this strain and these low cost by-product worthy for further investigation, and potentially feasible for biotechnological applications in different areas.
Significance and Impact of the Study:  To our knowledge, this is the first study reporting the use of the low-cost by-products brewer's spent grain and corn steep liquor, as sole substrates for microbial enzyme production.     

A comparative study of the wine fermentation performance of Saccharomyces paradoxus under different nitrogen concentrations and glucose/fructose ratios

Aims:  The main goal of the present study is to determine the effects of different nitrogen concentrations and glucose/fructose ratios on the fermentation performance of Saccharomyces paradoxus , a nonconventional species used for winemaking.
Methods and Results:  Ethanol yield, residual sugar concentration, as well as glycerol and acetic acid production were determined for diverse wine fermentations conducted by S. paradoxus . Experiments were also carried out with a commercial Saccharomyces cerevisiae wine strain used as control. The values obtained were compared to test significant differences by means of a factorial anova and the Scheffé test. Our results show that S. paradoxus strain was able to complete the fermentation even in the nonoptimal conditions of low nitrogen content and high fructose concentration. In addition, the S. paradoxus strain showed significant higher glycerol synthesis and lower acetic acid production than S. cerevisiae in media enriched with nitrogen, as well as a lower, but not significant, ethanol yield.
Conclusions:  The response of S. paradoxus was different with respect to the commercial S. cerevisiae strain, especially to glycerol and acetic acid synthesis.
Significance and Impact of the Study:  The present study has an important implication for the implementation of S. paradoxus strains as new wine yeast starters exhibiting interesting enological properties.     

Purification and structural characterization of bacillomycin F produced by a bacterial honey isolate active against Byssochlamys fulva H25

Aims:  Isolate and characterize antifungal peptides exhibiting activity against Byssochlamys fulva H25, a spoilage mould associated with juices and beverages.
Methods and Results:  A bacterium (H215) isolated from honey showed high antifungal activity against B. fulva H25. The antifungal producer strain was identified as Bacillus subtilis using 16S rDNA sequencing. The antifungal peptide was purified by 20% ammonium sulfate precipitation of the bacterial culture supernatant, followed by Octyl-Sepharose CL-4B and reverse phase-high performance liquid chromatography. The five active fractions were lyophilized and subjected to mass, tandem mass spectrometry and amino acid analysis to deduce their corresponding molecular masses and structural characteristics. The five peaks were determined to be identical to bacillomycin F, varying in the length of the fatty acid chain moiety from C14 to C16.
Conclusions:  The broad-spectrum antifungal activity produced by a bacterium from honey was determined to be due to the production of bacillomycin F.
Significance and Impact of the Study:  The antifungal compound produced by a bacterial strain isolated from honey was determined to be stable over a broad pH range and was stable to heat treatments up to 100°C. This is the first report of honey microflora producing bacillomycin F or any antifungal compound.     

Production and purification of agarase from a marine agarolytic bacterium Agarivorans sp. HZ105

Aims:  Isolation and characterization of an agarase-producing bacterium Agarivorans sp. HZ105.
Methods and Results:  An agarase-producing bacterium strain HZ105 had been isolated from marine sediment sample. Based on phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, as well as biochemical analyses, this strain was named Agarivorans sp. HZ105. Effect of pH, NaCl on the growth and agarase production of strain HZ105 was studied. Strain HZ105 produced three extracellular agarases which were purified to homogeneity from bands in the PAGE gel. Two agarases of these three had a molecular mass of 54, 58 kDa, respectively. And the MS and MS/MS spectra were used to identify the agarases.
Conclusions:  The MS spectra result showed that the agarases of strain HZ105 should be beta-agarase and belong to the family 50 of glycosyl hydrolases. The agarases could keep stable activity at room temperature.
Significance and Impact of the Study:  The strain HZ105 was useful to produce stable agarases. The solution produced by agar's degradation in the agar plates was first reported to be used for purification of agarase. Agarases were purified to homogeneity directly from the PAGE gel without stained by Coomassie brilliant blue.     

Biochemical characteristics and function of a threonine dehydrogenase encoded by ste11 in Ebosin biosynthesis of Streptomyces sp. 139

Aims:  Ebosin, a novel exopolysaccharide (EPS) produced by Streptomyces sp. 139 has antagonistic activity for interleukin-1 receptor (IL-1R) in vitro and remarkable anti-rheumatic arthritis activity in vivo. Ebosin biosynthesis gene ( ste ) cluster has been identified in our laboratory. This paper reports our effort to characterize the function of ste11 gene.
Methods and Results:  After the ste11 gene was cloned and expressed in Escherichia coli BL21, the recombinant Ste11 was purified and found capable of catalyzing NAD⁺ and l -threonine to NADH and 2-amino-3-ketobutyrate, hence identified as a threonine dehydrogenase (TDH). To investigate its function in the biosynthesis of Ebosin, the ste11 gene was knocked out with a double crossover via homologous recombination. The monosaccharide composition of EPS produced by the mutant strain (EPS-m) was altered from that of Ebosin. The analysis of IL-1R antagonist activity for EPSs showed that the bioactivity of EPS-m was lower than Ebosin.
Conclusions:  ste11 gene encoding a TDH may function as a modifier gene of Ebosin during its biosynthesis.
Significance and Impact of the Study:  TDH encoded by ste11 is functional in Ebosin biosynthesis. It is the first characterized TDH in Streptomyces .     

Regulation of tacrolimus production by altering primary source of carbons and amino acids

Aims:  In the present communication, attempts have been made to regulate the tacrolimus production by supplementing commercial source of carbons and amino acids timely.
Methods and Results:  Tacrolimus production was regulated by supplying vegetable oils and amino acids, individually and in combination. Tacrolimus quantification was done by HPLC. Streptomyces spp. MA6858 B3178 was found to produce 115·3 mg l⁻¹ of tacrolimus. The rotation speed of shake flask, pH of the broth and supply of air were maintained at 7·1, 230 rev min⁻¹ and 2·0 vvm air respectively.
Conclusions:  The effect of carbons on tacrolimus production was noticed to be of diphasic manner. During the first 24 h of culture, monosaccharide is used for the growth of microbe. However, after the lapse of 36 h, addition of soya oil and l -lysine in combination enhanced the tacrolimus production to 115·3 mg l⁻¹. Besides this, pH of broth was also noticed as a critical factor in monitoring tacrolimus biosynthesis.
Significance and Impact of the Study:  The newly isolated mutant Streptomyces spp. MA6858 B3178 having high potential for tacrolimus production as compared to existing data can be well used for the commercialization of tacrolimus.     

Characterization of a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39

Aims:  The aims of this study were to identify and characterize the novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39.
Methods and Results:  Strain B39 was closely related to Paenibacillus cookii in 16S rRNA gene sequence. Nonetheless, this isolate can be identified as a novel Paenibacillus sp. with respect to its physiological characteristics, biochemical reactions, and profiles of fatty acid compositions. A cellulase with both CMCase and avicelase activities was secreted from strain B39 and purified by ion-exchange chromatography. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis, the molecular weight of B39 cellulase was determined as 148 kDa, which was much higher than other cellulases currently reported from Paenibacillus species. The enzyme showed a maximum CMCase activity at 60°C and pH 6·5. Addition of 1 mmol l⁻¹ of Ca²⁺ markedly enhanced both CMCase and avicelase activities of the enzyme.
Conclusions:  We have identified and characterized a novel thermophilic Paenibacillus sp. strain B39 which produced a high-molecular weight cellulase with both CMCase and avicelase activities.
Significance and Impact of the Study:  Based on the ability to hydrolyse CMC and avicel, the cellulase produced by Paenibacillus sp. strain B39 would have potential applications in cellulose biodegradation.     

Characterization of Streptomyces D3 derived from protoplast fusion between Streptomyces cyaneus 190-1 and Streptomyces griseoruber 42-9

Streptomyces D3, derived from protoplast fusion between Streptomyces cyaneus 190-1 and Streptomyces griseoruber 42-9, has the ability to produce high levels of xylose isomerase when grown on hemicellulosic materials such as xylan as the carbon source. Comparison between the partial nucleotide sequences of the 16S ribosomal RNA genes from S. cyaneus 190-1, S. griseoruber 42-9, and fusant D3 showed that the 16S rRNA gene of fusant D3 was identical to that of S. cyaneus 190-1. Partial sequence analysis of the xylose isomerase genes also indicated that the gene of fusant D3 was identical to that of S. cyaneus 190-1. The partial DNA fragments for the xylanase genes (xlnA and xlnB) of fusant D3 were amplified by PCR, and subjected to Southern hybridization analysis. The results revealed that the xlnB gene of fusant D3 was similar to that of S. cyaneus 190-1, but that the xlnA gene of fusant D3 was similar to that of S. griseoruber 42-9. These results suggest that the majority of the genome of fusant D3 may be derived from S. cyaneus 190-1.     

Isolation of an antifungal Paenibacillus strain HT16 from locusts and purification of its medium-dependent antagonistic component

Aims:  To isolate an antagonist for use in the biological control of the phytopathogenic fungus Penicillium expansum and purify the antifungal component produced by the antagonist.
Methods and Results:  An antifungal strain HT16 was isolated from locusts, showing strong inhibition to Pen. expansum . Based on its in vitro effectiveness, HT16 was characterized as a strain of Paenibacillus polymyxa by phenotypic tests and 16S rDNA sequence analysis. It was found that the antifungal component HT16 secreted was only induced by Poria cocos sclerotium (PCS), and it remained active after sterilization at 121°C for 15 min. The protein was purified by ammonium sulfate precipitation, heating process, and ultrafiltration using a 10 kDa cut-off membrane. The molecular weight of the purified antifungal protein, which was determined by mass spectrometry, was 4517 Da.
Conclusions:  A novel bacterial strain HT16 antagonistic to Pen. expansum was isolated from locusts and identified as Pae. polymyxa . The antifungal protein of 4517 Da was purified, and its production needed the inducer PCS in the fermentation medium.
Significance and Impact of the Study:  The antagonistic protein from Pae. polymyxa showed strong antifungal activity against phytopathogenic fungus Pen. expansum . This strain HT16 and the antifungal metabolite are therefore strong candidates for the biocontrol of phytopathogens in agriculture.     

Taxonomy of the Streptomyces strain ZG0656 that produces acarviostatin α-amylase inhibitors and analysis of their effects on blood glucose levels in mammalian systems

Aims:  To clarify the taxonomic status of strain ZG0656 and analyse the effects of its acarviostatin products on blood glucose levels in mammalian systems.
Methods and Results:  Our program to screen for new α-amylase inhibitors led to the isolation of strain ZG0656. The polyphasic taxonomic study revealed that strain ZG0656 represents a novel variation of Streptomyces coelicoflavus , for which we propose the name S. coelicoflavus var. nankaiensis . Four chemically distinct α-amylase inhibitors, acarviostatins I03, II03, III03 and IV03, were isolated from strain ZG0656. Acarviostatins III03 and IV03 are both novel oligomers. All four acarviostatins are mixed noncompetitive porcine pancreas α-amylase inhibitors. Acarviostatin III03 is the most potent α-amylase inhibitor known to date. Moreover, in the in vitro and in vivo experiments, acarviostatins III03 showed significant inhibition of starch hydrolysis and glucose transfer to blood.
Conclusions:  Strain ZG0656 is a novel variation of S. coelicoflavus, whose products are novel effective α-amylase inhibitors. Among the products, acarviostatins III03 could significantly depress blood glucose levels in mammalian systems and be developed towards a possible therapeutic agent for diabetes.
Significance and Impact of the Study:  Acarviostatin III03 is the most potent α-amylase inhibitor known to date. The oligomer will benefit the research on the relationship between α-amylase and various inhibitors and will offer more choices in diabetes treatments.     

Diversity analysis of antagonists from rice-associated bacteria and their application in biocontrol of rice diseases

Aims:  To understand the diversity, taxonomy and antagonistic potential of rice-associated bacteria, and to discover new bacteria for biocontrol of rice foliar pathogens.
Methods and Results:  Amplified ribosomal DNA restriction analysis (ARDRA), BOX-PCR and 16S rRNA gene sequence analysis were used to identify the diversity of 203 rice-associated antagonistic bacteria. Eleven potential biocontrol bacteria were used to test their biological control of rice blast in a natural field experiment. Eleven different genera were encountered in five divisions, including Bacilli , Alphaproteobacteria , Betaproteobacteria , Gammaproteobacteria and Deinococci . The most prominent genus in all microenvironments was Bacillus (68·5%). The efficacy of rice leaf blast biocontrol was 64·35% for strain 1Pe2, 57·86% for strain 2R37 and 56·44% for strain 1Re14.
Conclusions:  Biocontrol data from the field experiments demonstrated no positive correlation between antagonism, physiological characteristics and biocontrol efficacy. There was significant diversity among the rice-associated bacteria isolated from different microenvironments. The most prominent genus of all microenvironments was Bacillus . Brevibacillus brevis strain 1Pe2 and Deinococcus aquaticus strain 1Re14 have good potential for field application and commercial use.
Significance and Impact of the Study:  This is the first attempt to study the diversity and identification of rice-associated antagonistic bacteria from different microenvironments, and endophytic bacteria Deinococcus aquaticus strain 1Re14, Acidovorax sp. isolate 3Re21 and Brevibacillus brevis strain 1Pe2 are first reported as rice-associated bacteria.     

一株产蓝色素链霉菌18-A-5的鉴定与系统发育分析

从原始热带雨林土壤中,分离到一株产蓝色色素菌株18-A-5,对其进行了系统分类学研究。形态学特征观察表明,在高氏合成一号培养基上初产蓝绿色色素,日久为深蓝色,基内菌丝蓝色,气生菌丝灰白色,产灰色孢子,孢子丝直或柔曲,形成长孢子链,孢子圆柱形。其DNA的G＋C摩尔分数为62.4%,16S rDNA序列分析结果(GenBank登陆号为EU054353),18-A-5与生靛链霉菌Streptomyces indigoferus ATCC23924^T 、草绿色链霉菌Streptomyces herbaricolor ATCC23924^T具有极高的同源性,达100％,聚类分析表明,18-A-5与生靛链霉菌Streptomyces indigoferus、草绿色链霉菌Streptomyces herbaricolor两株菌聚类在一起,分支置信度为74%。结合生理生化特性、细胞壁化学组成分析、脂肪酸分析等将菌株18-A-5定名为草绿色链霉菌Streptomyces herbaricolor。并对该蓝绿色可溶性色素性质进行了耐酸碱性、热稳定性、抗菌谱等初步分析。     

A spontaneous lactate dehydrogenase deficient mutant of Streptococcus rattus for use as a probiotic in the prevention of dental caries

Aims:  To study the ability of daily applications of Streptococcus rattus strain JH145 to affect the numbers of an implanted Streptococcus mutans strain in a rat model.
Methods and Results:  A spontaneous L(+)-lactate dehydrogenase (LDH)-deficient mutant of Streptococcus rattus , JH146, was isolated by screening on selective medium and compared with a previously isolated spontaneous LDH deficient strain, JH145. Both strains were shown to have single base pair deletion mutations in the structural gene ( ldh ) for LDH, and reversion frequencies were approximately the same. Animals treated once daily with ≥10⁶ CFU (colony forming units) of JH145 showed a statistically significant decrease in the proportion of implanted S. mutans to total cultivable bacteria in oral swab samples. The rate of decrease in S. mutans levels was dose-dependent. No adverse effects were observed by in-life observation of treated animals, and histopathological, haematological and blood chemistry analyses were unremarkable.
Conclusions:  The results presented indicate that daily application of JH145, a naturally occurring LDH-deficient variant of S. rattus , can compete with S. mutans for its habitat on the tooth surface.
Significance and Impact of the Study:  S. rattus JH145 has potential as a probiotic for use in the prevention of dental caries.     

Production of tylosin in solid-state fermentation by Streptomyces fradiae NRRL-2702 and its gamma-irradiated mutant (γ-1)

Aims:  To develop solid-state fermentation system (SSF) for hyper production of tylosin from a mutant γ-1 of Streptomyces fradiae NRRL-2702 and its parent strain.
Methods and Results:  Various agro-industrial wastes were screened to study their effect on tylosin production in SSF. Wheat bran as solid substrate gave the highest production of 2500 μg of tylosin g⁻¹ substrate by mutant γ-1 against parent strain (300 μg tylosin g⁻¹ substrate). The tylosin yield was further improved to 4500 μg g⁻¹ substrate [70% moisture, 10% inoculum (v/w), pH 9·2, 30°C, supplemental lactose and sodium glutamate on day 9]. Wild-type strain displayed less production of tylosin (655 μg of tylosin g⁻¹ substrate) in SSF even after optimization of process parameters.
Conclusion:  The study has shown that solid-state fermentation system significantly enhanced the tylosin yield by mutant γ-1.
Significance and Impact of the Study:  This study proved to be very useful and resulted in 6·87 ± 0·30-fold increase in tylosin yield by this mutant when compared to that of wild-type strain.