Toxicity of cadmium to twoStreptomyces species as affected by clay minerals

Summary The effect of cadmium on the growth ofStreptomyces rimosus andS. bottropensis (both isolated from soil) was investigated. The modifying effect of the presence of the clay minerals kaolinite, bentonite and vermiculte on Cd toxicity was also included. After four days no growth was observed at 100 ppm CdCl₂ ofS. bottropensis and at 150 ppm in case ofS. rimosus. After six days some growth ofS. rimosus occurred at 150 ppm CdCl₂ and ofS. bottropensis at 100 ppm. Addition of the three clay minerals decreased the Cd toxicity considerably.     

Growth in acidic media increases production of phosphatidylinositol-specific phospholipase C byStaphylococcus aureus

Production of phosphatidylinositol-specific phospholipase C (PIPLC), an exoenzyme of some strains ofStaphylococcus aureus, has been epidemiologically associated with virulence. To investigate the elaboration ofS. aureus PIPLC, we have evaluated in vitro conditions that maximize production of enzymatically active PIPLC. PIPLC activity was assessed by measuring the release of³H-inositol-phosphate from the substrate³H-phosphatidylinositol. Lowering the pH ofS. aureus cultures from 7.0 to 5.4 progressively increased the yield of PIPLC. The final yield of PIPLC was at least five-fold greater when the initial culture pH was 5.4 compared with 7.0. Low pH enhanced PIPLC activity recovered from twoS. aureus strains capable of high PIPLC production, but not from a strain producing little PIPLC. At both pH 5.0 and 7.4, PIPLC production peaked during mid- to late-logarithmic phase. We conclude that an acidic starting pH of culture media increases the yield of PIPLC activity elaborated during active growth ofS. aureus.     

Associative effect of cellulolytic fungi andAzospirillum lipoferum on yield and nitrogen uptake by wheat

The associative effect of cellulolytic fungi, such asAspergillus awamori andA. niger, with the nitrogen fixer,Azospirillum lipoferum was studied in a soil amended with rice straw. All the inoculants gave significantly higher grain and straw yield and nitrogen uptake by wheat crop than did the uninoculated treatment. The doubling of chemical nitrogen dose significantly increased the yield and nitrogen uptake. It was observed thatA awamori performed significantly better followed byA. niger andA. lipoferum. The maximum benefit was obtained with combined inoculation ofA. awamori andA. lipoferum. Another experiment was conducted in the subsequent year in soil amended with and without rice straw using cellulolytic culture eitherA. awamori orSclerotium rolfsii, andA. lipoferum. Application of straw in soil significantly reduced the yield and N-uptake by wheat crop as compared to the controls. All the inoculants exceptS. rolfsii gave significantly higher grain yield. However, N-uptake by grain was significantly increased only by combined inoculation ofA. lipoferum and either one of the cellulolytic fungi. Similar trends on yield and N-uptake of straw due to inoculants were observed. The maximum benefit was obtained with combined inoculation ofA. awamori andA. lipoferum followed byA. awamori alone on grain yield and only combined inoculants on N-uptake by the crop.     

Molecular cloning of resistance genes and architecture of a linked gene cluster involved in biosynthesis of oxytetracycline by Streptomyces rimosus

Summary The isolation of mutants of Streptomyces rimosus which were blocked in oxytetracycline (OTC) production was described previously. The genes for the early steps of antibiotic biosynthesis mapped together. Genomic DNA fragments of S. rimosus which conferred resistance to OTC and complemented all of these non-producing mutants have been cloned. The cloned DNA is physically linked within approximately 30 kb of the genome of S. rimosus. The gene cluster is flanked at each end by a resistance gene each of which, independently, can confer resistance to the antibiotic. In OTC-sensitive strains of S. rimosus, the entire gene cluster including both resistance genes has been deleted. Complementation of blocked mutants by cloned DNA fragments in multi-copy vectors was often masked by a secondary effect of switching off antibiotic productions in strains othersise competent to produce OTC. This adverse effect on OTC production was not observed with recombinants using low copy-number vectors.     

Development of an intergeneric conjugal transfer system for rimocidin‐producing Streptomyces rimosus

Aims: To develop an intergeneric conjugation system for rimocidin‐producing Streptomyces rimosus. Methods and Results: High efficiencies of conjugation [10^?2–10^?3 transconjugants/recipient colony forming units (CFU)] were obtained when spores of S. rimosus were heat treated at 40°C for 10 min prior to mixing with E. coli ET12567(pUZ8002/pIJ8600) as donor. Mycelium from liquid grown cultures of S. rimosus could also be used as recipient instead of spores, with 24‐h cultures giving optimal results. TSA (Oxoid) medium containing 10 m mol l^?1 MgCl₂ was the preferred medium for conjugation. Southern hybridization was used to confirm that transconjugants of S. rimosus contained a single copy of pIJ8600 integrated at a unique chromosomal attachment site (attB). The transconjugants exhibited a high stability of plasmid integration and showed strong expression of green fluorescent protein when using pIJ8655 as the conjugative vector. Conclusion: Intergeneric conjugation between E. coli and S. rimosus was achieved at high efficiency using both spores and mycelium. Significance and Impact of the Study: The conjugation system developed provides a convenient gene expression system for S. rimosus R7 and will enable the genetic manipulation of the rimocidin gene cluster.     

Inhibition of fungi,actinomycetes and bacteria by Stachybotrys atra

The effect ofStachybotrys atra on fungi, actinomycetes and bacteria was studied in agar culture. Of the 27 genera and 52 species of fungi, 6 species of actinomycetes and 5 genera and 10 species of bacteria tested,S. atra was found to inhibit 95.7 % fungi, all the actinomycetes and 83.3 % of bacteria.Aspergillus ustus, 2 unidentified species ofFusarium, Rhizobium trifolii andPseudomonas spp. were however not inhibited. In experiments in soil,S. atra inhibited the growth ofTrichoderma viride. S atra was ineffective whereT. viride andS. atra were inoculated simultaneously or whereT. viride was added 7 days earlier. Besides,S. atra was not found to controlMacrophomina phaseoli infection on cotton. Soil culture ofS. atra showed phytotoxic effect on cotton seedlings.S. atra produced a dark brown to black thermostable toxic metabolite in the medium. The culture filtrate retarded the growth ofM. phaseoli in agar culture. Despite the importance ofS. atra as a fungistatic agent with wide antimicrobial spectrum, the use ofS. atra as a protectant against plant pathogens seems to be limited because of its phytotoxicity on cotton.     

Growth promotion ability of zoysiagrass rhizosphere fungi in consecutive plantings of wheat and soybean

Several isolates ofPhoma sp., certain nonsporulating fungi, as well asPenicillium andTrichoderma, all isolated from zoysiagrass rhizosphere, promoted growth of wheat and soybean under greenhouse conditions. However, the ability of these rhizosphere fungi to enhance plant growth varied with the crop tested. For example, most of the fungi effectively promoted the growth of wheat, whereas only a few fungi were effective on soybean. In consecutive plantings of wheat and soybean grown in soil previously infested with these zoysiagrass rhizosphere fungi, the growth promotion ability of the fungi was lowered. However, addition of fresh potting medium appeared to restore their growth-promotive effects. It appears that the activation of plant growth-promoting fungi in soil might depend on the availability of organic substrates to colonize, as evidenced by the promotion of plant growth.     

Antagonistic action of Streptomyces pratensis S10 on Fusarium graminearum and its complete genome sequence

Wheat scab, mainly caused by Fusarium graminearum, can decrease wheat yield and grain quality. Chemical pesticides are currently the main control method but have an inevitable negative consequence on the environment and in food safety. This research studies a promising substitute, Streptomyces pratensis S10, which was isolated from tomato leaf mould and shows a significant inhibition effect on F. graminearum based on antagonism assays. The biocontrol mechanism is studied by enhanced green fluorescent protein labelling, quantitative real-time PCR, the Doskochilova 8 solvents system test and complete genome sequencing. Strain S10 can colonize in the wheat root, control wheat scab and decrease deoxynivalenol (DON) content. The control effects in vitro, planta and the plot experiments were 92.86%, 68.67% and 40.87% to 86.62%, respectively. S10 decreased DON content by inhibiting the mycelium growth and DON synthesis gene expression. The active substances of the S10 secondary metabolites had a high-temperature resistance and 29 putative biosynthetic gene clusters in its genome. The S10 control mechanism is multivariate, which shows potential in controlling wheat scab.     

PCR Detection of Oxytetracycline Resistance Genes otr(A) and otr(B) in Tetracycline-Resistant Streptomycete Isolates from Diverse Habitats

A range of European habitats was screened by PCR for detection of the oxytetracycline resistance genes otr(A) and otr(B), found in the oxytetracycline-producing strain Streptomyces rimosus. Primers were developed to detect these otr genes in tetracycline-resistant (Tc^R) streptomycete isolates from environmental samples. Samples were obtained from bulk and rhizosphere soil, manure, activated sludge and seawater. The majority of Tc^R streptomycetes originated from bulk and rhizosphere soil. Fewer Tc^R streptomycetes were isolated from manure and seawater and none from sewage. By PCR, three out of 217 isolates were shown to contain the otr(A) gene and 13 out of 217 the otr(B) gene. Surprisingly, these genes were detected in taxonomic groups not known as tetracycline-producing strains. The majority of the otr gene–carrying strains was assigned to S. exfoliatus or S. rochei and originated from all habitats from which Tc^R streptomycetes were obtained. Our results indicated that the occurrence of otr(A) and otr(B) genes in natural environments was limited and that otr(B), in comparison to otr(A), seemed to be more common.     

Studies on some fungi isolated from the rhizosphere of tomato plants and the consequent prospect for the control of Verticillium wilt

Summary Biological control of Verticillium wilt disease with antagonistic micro-organisms was studied. Antagonism of some fungi, isolated from tomato rhizosphere, toVerticillium albo-atrum R & B. was observedin vitro. A clearly defined zone, in which the growth of the pathogen was inhibited, was observed withPenicillium spp. (includingPenicillium chrysogenum Thom) andFusarium culmorum (S.G. Sm) Sacc., whileTrichoderma viride pers. ex Fries,Gliocladium spp. andPenicillium vermiculatum Dangeard, suppressed the growth ofV. albo-atrum by penetrating, and overgrowing it. OnlyT. viride andP. vermiculatum culture filtrate added to the Dox's agar, reduced the radial growth ofV. alboatrum. Root-dip application of culture filtrates ofT. viride andP. chrysogenum was found to be most effective in controlling the disease, followed by other species ofPenicillium andGliocladium spp. WhileFusarium culmorum provided no control. Improvement of plant height and vigour with a better yield due to culture filtrate treatment occurred. Root-dip application of antagonistic fungal propagules (T. viride, P. chrysogenum) to tomato seedlings was also very effective in controlling wilt in tomato plants grown inV. albo-atrum infested soil. Dedicated to the memory of the late Prof. Ivor Isaac with whom I had the pleasure of working     

Optimization of culture media for solid-state culture of<Emphasis Type="Italic">Pleurotus ferulae</Emphasis>

In order to elucidate the possibility of artificial production ofP. ferulae by solid-state culture, the optimization of culture conditions was carried out. When NH₄H₂PO₄ and CaCO₃ were used in the cultures using test tube with 30 g ofPopulus sawdust at 25°C±1 in the dark, the favored mycelial growth was observed with 1% of NH₄H₂PO₄ and the production of polysaccharide was 7.85 mg/100 mg of mycelium with 1% of CaCO₃. The mixtures of 80% ofPopulus sawdust and 20% of rice bran at 60% of water content were determined to be optimal for the production of fruiting bodies in the sawdust culture. When three treatments containing various ratios of garlic powder were conducted, yields of fruiting bodies were drastically higher than those of synthetic mixture without garlic powder. The highest yield (143 g/bag) was obtained with 7% garlic powder. The yield of synthetic mixture containing 7% of garlic powder was 83% higher than that of sawdust culture. The reason why garlic powder did support growth was not clear but it is possible that garlic powder might contain effective components for the formation of fruiting body. The optimal synthetic mixture composition consisted of cotton seed 77%, lime 6.4%, K₂HPO₄ 0.2%, KH₂PO₄ 0.2%, CaHPO₄, 0.2%, corn flour 4%, wheat flour 5%, and garlic powder 7%.     

Formation of biologically active substances by rhizosphere bacteria and their effect on plant growth

Nine out of seventeen strains of bacteria with a pronounced effect on seed germination and on seedling growth, isolated from root surfaces and rhizosphere soil of maize, were selected for a study on the formation of biologically active substances. β-Indole acetic acid (45–72 μg/1.000 ml) was produced by four strains, gibberelline-like substances (1.0–60.0 μg/1.000ml) by all strains, biotin and pantothenic acid by the majority of strains and nicotinic acid by five strains. Amino acids were formed by all strains but in low amounts. Four strains produced growth inhibitors. The highest amounts of biologically active substances were found in cultures ofPseudomonas fluorescens andBacillus brevis. The various cultures ofPseudomonas fluorescens differed in their capability to produce biologically active substances. The majority of bacterial cultures or their supernatants significantly stimulated the germination of seeds and some of them significantly affected the growth of plants. Inoculation of maize seeds with strainsPseudomonas fluorescens andChromobacterium violaceum significantly increased the yield of dry matter of plants.     

Effect of various inhibitors on the multiplication of BLE bacteriophage inBacillus licheniformis

A total of 40 substances were tested for their inhibitory effect on the multiplication of a bacteriophage in a growing culture ofBacillus licheniformis and their influence on bacitracin production. Acriflavine was the only substance which, at a concentration of 3 μg ml^-1, completely suppressed phage multiplication while having no effect on the growth ofBacillus licheniformis and on the production of the antibiotic.     

The fermentative production of oxytetracycline on industrial by-products byStreptomyces rimosus 12907

Three strains ofStreptomyces rimosus were grown on four different media. The one suitable for the production of oxytetracycline byStreptomyces rimosus 12907 was modified by black strap molasses, fodder yeast (40% total protein) and rice bran. The volume of the fermentation medium was scaled up in a 1200-litre fermentor aerated with sterile air obtained from a system used in the purification of air. 850 g crude oxytetracyeline was obtained when the fermented medium (700 litres) was extracted with 1-butanol. This work was carried out in the Laboratories of the Chemical Factories of the Egyptian Sugar anio Distillation Company with the cooperation of the National Research Centre, Dokki, Cairo, Arab Republd of Egypt (A.R.E.).     

Social organization in two primitive attine ants,cyphomyrmex rimosus and myrmicocrypta ednaella, with reference to their fungus substrates and food sources

In the Neotropical rainforest of Barro Colorado Island, Panama, social organization and behavior were observed in 2 primitive attine ant species,Myrmicocrypta ednaella andCyphomyrmex rimosus. Both species took nutrients from mycelia on fungus (i.e. mycophagy), and from plant nectar and sap which they collected outside the nest (i.e. phytophagy). They also obtained alimentary liquid by soliciting nestmates (i.e. stomodeal trophallaxis). Queens and larvae were wholly mycophagous, while older workers were much dependent on nectar, sap and alimentary liquid and younger workers were mostly mycophagous but only partly phytophagous.M. ednaella used wood chips as substrate for the fungus garden. Its fungus-growing behavior was similar to those hitherto observed in other primitive attine species. In contrast, the behavior ofC. rimosus was unique in its utilization of crop liquid as a substrate. In the rainforest,C. rimosus workers frequently forage outside the nests to collect nectar and sap, most of which is probably regurgitated for fungus cultivation.     

The influence of purine bases upon the growth of Streptomyces rimosus

Summary The BS-21 strain of Streptomyces rimosus can utilize adenine, and guanine, as sole nitrogen sources, and in small concentrations, can increase the mycelium production in media containing threonine, ornithine and cysteine, further, it initiates growth in media containing valine, isoleucine, leucine and oxyproline. Pyrimidine bases are unsuitable as exclusive nitrogen sources, and unlike purine bases, do not increase growth in the presence of the nitrogen sources listed before. The yield of oxytetracycline follows the production rate of mycelium production.     

The influence of the oxidation-reduction potential of the medium upon the growth ofAzotobacter chroococcum

Summary 1. Under aerobic conditions the development ofAzotobacter is suppressed by the presence of reducing substances in concentrations lowering the aerobic potential more than ±120 mV.2. The development ofAzotobacter is not only retarded by the presence of these reducing substances, but the cells are killed.3. Under anaerobic conditions no development ofAzotobacter could be observed even when the solution was poised at a high redox potential. The cells were not killed by the anaerobic conditions; as soon as aerobic conditions were restored, a rapid development ofAzotobacter was observed.4. When the anaerobic conditions coincide with a low redox potentiale.g. in culture solutions containing glucose, the cells ofAzotobacter were killed.5. The limits of potential endured byAzotobacter appeared to be dependent on the relations between the ions in the medium.6. The cells ofAzotobacter secrete certain substances enabling them to develop at low potentials.7. The conclusion is drawn that the development ofAzotobacter is in general influenced by the redox potential of the medium. However, this influence may be very complex, as the potential of the medium is only important in so far as the redox potential(s) in the cell are changed. The factors influencing the relation between the redox potential of the medium and the redox potentials of the cell are discussed.8. ForAzotobacter, oxygen cannot be replaced by other redox systems with a suitable redox potential, most probably because these redox systems have to react with the respiratory enzymes of the cell.     

Microbial conversion of tamoxifen

Summary Screening studies of tamoxifen (TAM) have shown that Streptomyces rimosus ATCC 2234 was able to metabolize TAM to 4-hydroxytamoxifen, which was obtained in a 10% yield and identified by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Several other microorganisms were capable of biotransforming TAM to tamoxifen-N-oxide and desmethyltamoxifen. The isolated metabolites were identified by gas chromatography/mass spectrometry and by two-dimensional NMR spectroscopic techniques. This is the first report of microbial hydroxylation of TAM. Offprint requests to: S. H. El-Sharkawy     

Production of Alkaline Protease from n-Paraffins by a Kabicidin Resistant Mutant Strain of Fusarium sp.

An antagonistic fungus to Valsa ceratosperma was isolated from soil, and identified as Fusarium solani. The fungus was found to produce at least two antifungal substances in stationary culture. These two substances were isolated from their culture filtrate as chromatographically homogeneous, amorphous solids. Their examined physico-chemical properties appeared to be identical with cyclosporin A and C, obtained from the fermentation broth of Trichoderma polysporum, and they showed a pronounced inhibitory effect on growth of V. ceratosperma.     

Studies on Streptomycetes

Investigation was made on the mycological properties of a strain No. 45449 isolated from a sample of soil collected in Fukuchiyama. Since the antibiotics produced by the strain resembled hydroxymycin and paromomycin, the strain was compared with the hydroxymycin and paromomycin-producing strains, S. paucisporogenes and S. rimosus forma paromomycinus, and as a result the strain No. 45449 was found to be different from the latter two strains. Among known strains, S. flavogriseus resembles the present strain, but they are different morphologically and in the kind of the antibiotics they produce. Thus, as the strain No. 45449 was found to be a new strain, it was named S. pulveraceus nov. sp. The antibiotics produced by the present strain are physiologically basic substances active against Gram-positive and negative bacteria and acid fast bacteria, and they are considered to belong to the neomycin-kanamycin group.