Protoplast fusion in Streptomyces fradiae strains producing neomycin and tylosin]

Interspecies fusion of protoplasts of the Streptomyces fradiae strains producing neomycin (an aminoglycoside antibiotic) and tylosin (a macrolide antibiotic) was performed with a view to isolate strains producing novel antibiotics. Fusion of the protoplasts of the neomycin- and tylosin-producing strains labelled by the resistance to monomycin and lincomycin, respectively, caused no formation of stable strains producing antibiotics differing in chromatographic mobility from the antibiotics produced by the initial strains. In fusion of the protoplasts of the unlabelled strains, heat-inactivated protoplasts of the active line of one strain (donor) and native protoplasts of the inactive line of the other strain (recipient) were used. When the neomycin-producing culture was used as a recipient the fusion led to formation of strain 195-34 producing antibiotics of the benzo(a)anthraquinone group. One of these antibiotics, i.e. antibiotic 34-I, proved to be a novel biologically active substance. After regeneration of the protoplasts of the initial strains, no stable strains producing antibiotics differing from neomycin and tylosin were isolated.     

Various principles of detecting the producers of beta-lactamase inhibitors among soil Actinomycetes

Principles of detecting organisms producing beta-lactamase inhibitors among soil actinomycetes were developed. For detecting such cultures it was recommended to use the Gauze agarized medium No. 1 supplemented with beta-lactam antibiotics. Benzylpenicillin proved to be the most efficient. Various liquid fermentation media for detecting the inhibitory activity of soil actinomycetes were compared. Two media were the most favourable i.e. the glucose-yeast medium No. 18/3 and the soybean-glucose medium with Na2SO4 and CoCl2 No. 20/3. The use of test cultures with relatively low resistance to benzylpenicillin was shown expedient in screening cultures producing beta-lactamase inhibitors. Test cultures with high resistance should be used in more detailed characterization of the selected cultures.     

富锌产γ-氨基丁酸乳酸菌的筛选及初步鉴定

用含0.2%-0.25%Zn^2＋的MRS培养基从发酵酸菜、酸奶中分离出6株具富锌能力细菌,经形态和生理生化特性鉴定,初步判断菌株为乳酸菌。采用纸层析检测MRSG培养基培养的6株菌上清液后,初步确定有4株能产γ-氨基丁酸。以此4菌株基因组为模板PCR扩增到540 bp的片段,经测序和序列分析,证明是谷氨酸脱羧酶基因高度保守区域,初步证明谷氨酸脱羧酶基因的存在。此4株菌发酵液经HPLC检测和分析后,证实2号菌株能转化谷氨酸钠生成GABA,产量为4.8 g/L,在应用方面具有很大的潜力。     

Cyclosporin biosynthesis and dynamics of bioenergetic processes in cyclosporin-producing strain

An attempt was made to show a correlation between definite bioenergetic parameters of the cells of the cyclosporine-producing culture and biosynthesis of cyclosporine. It was found that the three strains producing cyclosporine used in the study had an alternative cyanide-resistant pathway along with the classical cytochrome chain. In the strain forming only traces of the cyclopeptide during fermentation of the cyanide-resistant respiration constituted 60 to 80%. In the isogenic highly productive strains the cyanide-resistant respiration appeared to be markedly decreased beginning from the 1st day of fermentation and during the maximum biosynthesis of cyclosporine (on day 4 or 5 of fermentation) it reached zero. The ATP content in the cells of the highly productive strain, despite its decrease by the antibiotic biosynthesis peak, remained at a much higher level than that in the strain producing only traces of cyclosporine. A procedure for isolating functionally active mitochondria from the protoplasts was developed and a bioenergetic characterization of the mitochondria isolated from the strains with different antibiotic productions is presented.     

Relationship between macrotetrolide production and specific activity of some hydrolases in a high and a low producing strain of Streptomyces griseus

The activities of five hydrolytic enzymes in the culture filtrate and in cell-free extracts from strains of Streptomyces griseus, differing in macrotetrolide production, have been determined over a fermentation period of 200 h. The specific activities of phosphatase, phosphodiesterase, and adenosine triphosphatase in the medium, and phosphatase and phosphodiesterase in the cell-free extract were lower in the low than in the high producing strain. No significant difference was found between the strains, for adenosine triphosphatase and protease activity in the cell-free extract or protease activity in the medium. The specific activity of esterase was higher in the low than in the high producing strain.     

Various methodological approaches in controlled screening of antibiotic producers among the group of Coryneform bacteria]

A methodical system for directed screening of cultures producing broad-spectrum antibiotics among soil saprophytic coryneform bacteria was developed. To isolate such cultures, it was recommended to use the glucose-yeast medium supplemented with malt extract (No. 18/3) and soybean-glucose medium with sodium sulfate and cobalt chloride (No. 20/3). The preliminary alkaline treatment of the soil substrates and the use of acidic soil samples were found to favour isolation of the Mycobacterium type cultures. It was recommended to use gram-negative tests microbes with relatively low antibiotic resistance for screening cultures producing broad spectrum antibiotics. Various agarized and liquid fermentation media were compared in regard to detection of antibiotic activity in the soil coryneform bacteria. The corn medium supplemented with protein-vitamin concentrate, glucose, lactose and starch (No. 116) proved to be the most efficient.     

Modulation of Biological Functions of Naegleria fowleri Amoebae by Growth Medium

Two strains of Naegleria fowleri amoebae were studied when the amoebae were maintained in the same growth medium or in two different media. A weakly pathogenic strain of N. fowleri , LEE, and a highly pathogenic strain, LEEmpCl, were compared for growth properties, the presence or absence of surface structures termed food cups, cytopathogenicity, cellular locomotion, susceptibility to complement-mediated lysis and immunological relatedness by western immunoblot analysis when grown in Nelson medium or in Cline medium. The two different strains of N. fowleri , LEE and LEEmpCl, were more similar in protein profiles and functional activity when both strains were grown in the same nutritional medium. Differences in growth, proteins synthesized, cytopathogenicity, susceptibility to complement lysis and rate of locomotion were noted when the same strain was grown in different media. Naegleria fowleri grown in Cline medium demonstrated an increased rate of growth, an increase in its rate of locomotion, an increased resistance to complement lysis, and destroyed target nerve cells by contact-dependent lysis. In contrast, the same strain of amoeba grown in Nelson medium showed slower growth, destroyed target cells by trogocytosis, and was less resistant to complement-mediated lysis.     

Mycotoxin production from fungi isolated from grapes

AIMS: In order to assess the potential for producing mycotoxins, fungi were isolated from wine producing grapes. METHODS AND RESULTS: The isolates were identified and Penicillium expansum, the most well recognized mycotoxin producer, was analysed for mycotoxin production by TLC. Many of the strains produced patulin and/or citrinin, often depending on whether they were grown on a grape or yeast extract sucrose media. CONCLUSION: Citrinin was produced by all strains grown in the yeast extract sucrose medium, but only one strain (from 51) was able to produce this compound in grape juice medium. Patulin was produced in the yeast extract medium by 20 strains and in grape juice medium by 33 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of mycotoxins in wine producing grapes is discussed. Grapes contamination with patulin seems not to contribute to wine contamination, and no ochratoxin producing fungi was identified.     

The action of its own antibiotic on the producer of imbricin growing on an agarized medium]

The action of imbricin on its own producer Streptomyces imbricatus grown on an agarized medium was studied. Comparatively low concentrations of the antibiotic were shown to have a high lethal action on the streptomycete. The morphological and cultural features of S. imbricatus did not change under the action of imbricin while the variation with respect to the antibiotic production property markedly increased. After the strain exposure to 200 micrograms/ml of imbricin, a stable variant with the antibiotic potency 20 per cent higher than that of the initial organism was isolated.     

The methods of protoplast formation and transformation of Streptomyces strains

Factors influencing formation, regeneration and transformation of protoplasts in streptomyces are described. Conditions for formation and regeneration of protoplasts in 4 industrial strains producing the macrolide antibiotic tylosin were studied. It was demonstrated possible to apply the method for transformation of the S. lividans type culture to 3 industrial strains of S. griseus producing grisin, an antibiotic used as a feed additive. Potential increasing of the efficiency of protoplast transformation and transfection in various actinomycetous strains including industrial ones is discussed. The stimulating effect of lyposomes on transformation of protoplasts in S. lividans 66 with DNA of plasmids pVG101 and pIJ350 as well as transfection with DNA of phages SH10 and KS404 was shown. The tylosin resistance genes in S. fradiae strain B45 were cloned which enabled isolating the cluster of the genes participating in tylosin biosynthesis.     

Synthesis of hydrolytic enzymes during production of tylosin byStreptomyces fradiae

Summary The exposure of a wild-type tylosin producing strain ofStreptomyces fradiae to mutagenic agents resulted in the isolation of several tylosin over-producing strains. Examination of three mutants, T4310, 612 and 3204 showed that improved tylosin production was associated with increased hydrolytic enzyme activity and cell growth. The wild-type strain showed lower levels of hydrolytic activity including, protease, amylase, lipase and esterase activities and attained a lower cell density than the mutants.     

Oxygen metabolism of catalase-negative and catalase-positive strains of Lactobacillus plantarum.

Two catalase-negative strains of Lactobacillus plantarum and a strain producing the atypical, nonheme catalase were studied to determine if the ability to produce the atypical catalase conferred any growth advantage upon the producing strain. Both catalase-negative strains grew more rapidly than the catalase-positive strain under aerobic or anaerobic conditions in a glucose-containing, complex medium. Upon exhaustion of glucose from the medium, all three strains continued growth under aerobic but not under anaerobic conditions. The continued aerobic growth was accompanied by production of acetic acid in addition to the lactic acid produced during growth on glucose. Oxygen was taken up by exponential phase-cell suspensions grown on glucose when glucose or glycerol were used as substrates. Cells harvested from glucose-exhausted medium oxidized glucose, glycerol, and pyruvate. Oxygen utilization by a catalase-negative strain increased as did the specific activity of reduced nicotinamide adenine dinucleotide peroxidase during late growth in the glucose-exhausted medium. The catalase-positive strain and the catalase-negative strain tested both possessed low but readily detectable levels of superoxide dismutase throughout growth. The growth responses are discussed in terms of the presence of enzymes which would allow the cells to remove potentially damaging reduction products of O2.     

Control of Pleuropneumonia-like Organisms in Cell Culture

Mammalian cell culture systems were maintained free of mycoplasmas by using a 3-day agar plate test as a weekly routine to monitor the conditions of the cells. If contaminated cell cultures were found, they were discarded and replaced from a pleuropneumonia-like organism (PPLO)-free cell bank. PPLO-free lines were established by treatment with various antibiotics. The KB cell line was freed of mycoplasmas by treatment for 1 week with a mixture of chlortetracycline, kanamycin, and chloramphenicol. L-929 cells were cleared of contamination with either spectinomycin or tylosin, and a synovial cell line was cleared with lincomycin or tylosin. Each cell line, after eradication of the contaminant, was stored in liquid nitrogen. A number of agents were tested to determine minimal inhibitory concentration against three known and three unidentified mycoplasmas. Chlortetracycline and tetracycline were found to be highly active against all strains, whereas tylosin, spectinomycin, and lincomycin, though less active, were equally useful because of their low toxicity against cells. Kanamycin was highly active against three strains, but inactive at high levels against the KB cell contaminants. A disc plate test was used to check isolated cell contaminants for sensitivity to various agents.     

Production of cutinase by Thermomonospora fusca ATCC 27730

Ten strains belonging to various Thermomonospora species were tested for their ability to hydrolyse the insoluble plant polyester cutin. One strain, the thermophile T . fusca ATCC 27730, was found to produce a highly inducible cutinase when grown in broth medium containing purified apple cv. Golden Delicious cutin. Apple pomace, tomato peel, potato suberin and commercial cork were also shown to induce cutinase production. Addition of glucose to the culture medium either at the beginning of fermentation or after 2 days of incubation in the presence of apple cutin led to repression of cutinase production. The cutinase was active against a wide range of cutins, including those isolated from other apple cultivars as well as tomato, cucumber, grapefruit, and green pepper. Cutinase activity in the induced culture supernatant fluids exhibited a half-life of over 60 min at 70 °C and a pH optimum of 11·0. Some potential applications for cutinases are discussed.     

Increase in tylosin production by a commercial strain of Streptomyces fradiae

Conventional mutagenesis (UV irradiation and exposure to nitrosoguanidine) were used to produce and regenerate protoplasts, aiming at increasing the antibiotic activity of a Streptomycesfradiae strain producing tylosin. Variants exceeding the activity of the initial producer strain by 0.5-28.3% were obtained. The most active variants were produced by a combined exposure to UV and nitrosoguanidine, as well as upon regeneration of protoplasts formed from the cells of clones produced by UV irradiation. Unstable inheritance of the trait of increased tylosin production was demonstrated.     

Enhancement of antibiotic and secondary metabolite detection from filamentous fungi by growth on nutritional arrays

Aims: We asked to what extent does the application of the OSMAC (one strain, many compounds) approach lead to enhanced detection of antibiotics and secondary metabolites in fungi? Protocols for bacterial microfermentations were adapted to grow fungi in nutritional arrays. Methods and Results: Protocols for microfermentations of non‐sporulating fungi were validated using known antifungal‐producing fungi. Detection of antifungal activity was often medium dependent. The effects of medium arrays and numbers of strains on detection of antifungal signals were modelled by interpolation of rarefaction curves derived from matrices of positive and negative extracts. Increasing the number of fermentation media for any given strain increased the probability of detection of growth inhibition of Candida albicans. Increasing biodiversity increased detection of antifungal phenotypes, however, nutritional arrays could partly compensate for lost antibiotic phenotypes when biodiversity was limiting. Conclusions: Growth and extraction in microtiter plates can enable a discovery strategy emphasizing low‐cost medium arrays that can better exploit the metabolic potential of strains. Significance and Impact of the Study: Increasing fermentation parameters raise the probability of detecting bioactive metabolites from strains. The protocols can be used to pre‐select strains and their growth conditions for scale up that will most likely yield antibiotics and secondary metabolites.     

Characterization of several bovine rumen bacteria isolated with a xylan medium

Dehority, B. A. (Ohio Agricultural Research and Development Center, Wooster). Characterization of several bovine rumen bacteria isolated with a xylan medium. J. Bacteriol. 91:1724-1729. 1966.-Studies were conducted to characterize eight strains of bacteria isolated from bovine rumen contents, by use of a medium containing xylan as the only added carbohydrate source. Based on morphology, biochemical reactions, nutritional requirements, and fermentation products, five of the eight strains were identified as Butyrivibrio fibrisolvens. Many properties of the remaining three strains resembled Bacteroides ruminicola; however, propionic acid was consistently found as a fermentation product. When the type strains for B. ruminicola subsp. ruminicola and B. ruminicola subsp. brevis were compared with the present isolates, it was found that propionic acid was a normal fermentation product for the type strain B. ruminicola subsp. ruminicola when grown in a 40% rumen fluid-0.5% glucose broth. Production of propionic acid was markedly reduced for all strains when grown in a 20% rumen fluid-1% glucose broth. The three remaining strains were thus placed in the species B. ruminicola, and further classified into the subspecies ruminicola (one strain) and brevis (two strains) on the basis of their requirement for hemin. Although the type strain of B. ruminicola subsp. brevis did not produce propionic acid, both of the present isolates classified as this subspecies produced substantial amounts. One strain of B. ruminicola subsp. brevis had an absolute requirement for volatile fatty acids. Either isobutyric or dl-2-methylbutyric acid would satisfy this requirement, whereas isovaleric acid was ineffective. It is of interest that xylan-fermenting bacteria isolated from 10(-7) and 10(-8) dilutions of rumen contents by use of a xylan medium are similar to the xylan fermenters isolated at the same dilutions with a nonselective medium.     

液体发酵法筛选樟芝优良诱变新菌株的研究

对3个樟芝野生菌株和5个物理诱变菌株进行液体发酵,通过测定发酵液黏度变化情况,观测培养性状、菌球及粗提物产量和多糖、蛋白质及三萜含量,从中筛选出适宜液体发酵的多糖、三萜及蛋白质高产优良菌株。试验结果显示：供试的8个樟芝菌株生长曲线基本一致,其中,菌株327和Gg生长速度较快,培养的第9–10天开始出现菌丝自溶现象;经过筛选,327菌株为多糖和蛋白质高产菌株,多糖和蛋白质的产量分别比出发菌株提高238.20%和10.33%;Gg为三萜高产菌株,三萜产量比出发菌株提高57.86%。野生樟芝菌株经物理诱变效果明显。     

16种茯苓菌株液体发酵产胞内多糖与三萜的比较研究

筛选茯苓高产胞内多糖和胞内三萜的优良液体发酵出发菌株。采用PDA富集固体平板培养与液体发酵培养测定菌丝体生长速率;采用液体发酵策略分析16种茯苓菌株产胞内多糖与胞内三萜的潜能。实验结果表明菌株生长于固体培养基与种子培养基的生长速率之间没有关联性;降低一级种子培养基初始pH值到4.0时能有效缓解茯苓菌株培养物褐化现象;AS5.137胞内多糖含量最高,达377.60±0.10 mg/g,而DB菌株显示出最高的胞内多糖产量,达1.01±0.13 g/L;Y1菌株胞内三萜含量最高,达83.89±4.28 mg/g,而Jingzhou28菌株胞内三萜产量最高,达136.63±26.66 mg/L。就生产茯苓胞内多糖与胞内三萜而言,AS5.137与DB菌株适合作为液体发酵产胞内多糖的出发菌株;Y1,Jingzhou28,Z(z)与Xingpinzhong菌株均较适合作为液体发酵产胞内三萜的出发菌株。     

Carbon monoxide cycling by Desulfovibrio vulgaris Hildenborough

Sulfate-reducing bacteria, like Desulfovibrio vulgaris Hildenborough, use the reduction of sulfate as a sink for electrons liberated in oxidation reactions of organic substrates. The rate of the latter exceeds that of sulfate reduction at the onset of growth, causing a temporary accumulation of hydrogen and other fermentation products (the hydrogen or fermentation burst). In addition to hydrogen, D. vulgaris was found to produce significant amounts of carbon monoxide during the fermentation burst. With excess sulfate, the hyd mutant (lacking periplasmic Fe-only hydrogenase) and hmc mutant (lacking the membrane-bound, electron-transporting Hmc complex) strains produced increased amounts of hydrogen from lactate and formate compared to wild-type D. vulgaris during the fermentation burst. Both hydrogen and CO were produced from pyruvate, with the hyd mutant producing the largest transient amounts of CO. When grown with lactate and excess sulfate, the hyd mutant also exhibited a temporary pause in sulfate reduction at the start of stationary phase, resulting in production of 600 ppm of headspace hydrogen and 6,000 ppm of CO, which disappeared when sulfate reduction resumed. Cultures with an excess of the organic electron donor showed production of large amounts of hydrogen, but no CO, from lactate. Pyruvate fermentation was diverse, with the hmc mutant producing 75,000 ppm of hydrogen, the hyd mutant producing 4,000 ppm of CO, and the wild-type strain producing no significant amount of either as a fermentation end product. The wild type was most active in transient production of an organic acid intermediate, tentatively identified as fumarate, indicating increased formation of organic fermentation end products in the wild-type strain. These results suggest that alternative routes for pyruvate fermentation resulting in production of hydrogen or CO exist in D. vulgaris. The CO produced can be reoxidized through a CO dehydrogenase, the presence of which is indicated in the genome sequence.