Induction of Pleiotropic Mutation in Streptomyces griseus by Incubation under Stress Conditions for Mycelial Growth

When Streptomyces griseus strain 2247 was cultivated under stress conditions for growth, such as growth in media containing toxic compounds, pleiotropic mutants were obtained at a high frequency. These mutants have lost simultaneously streptomycin productivity, streptomycin resistance, spore-forming ability, and pigment productivity, although the genes for streptomycin biosynthesis and A-factor production are proficient.     

Effect of aminoglycoside antibiotics on the autolytic enzyme of Streptomyces griseus

The isolated cell wall of Streptomyces griseus 52–1 strain labelled with fluorescein isothiocyanate (FITC) and containing wall-bound autolytic enzyme was lysed as a function of different cations. The autolysis was accelerated by aminoglycoside antibiotics (streptomycin and the structurally closely related neomycin) which have a polycationic character. Since this strain is a streptomycin producer it is suggested that streptomycin may have a regulatory function on autolysis.     

Coevolution of antibiotic production and counter‐resistance in soil bacteria

We present evidence for the coexistence and coevolution of antibiotic resistance and biosynthesis genes in soil bacteria. The distribution of the streptomycin (strA) and viomycin (vph) resistance genes was examined in Streptomyces isolates. strA and vph were found either within a biosynthetic gene cluster or independently. Streptomyces griseus strains possessing the streptomycin cluster formed part of a clonal complex. All S. griseus strains possessing solely strA belonged to two clades; both were closely related to the streptomycin producers. Other more distantly related S. griseus strains did not contain strA. S. griseus strains with only vph also formed two clades, but they were more distantly related to the producers and to one another. The expression of the strA gene was constitutive in a resistance‐only strain whereas streptomycin producers showed peak strA expression in late log phase that correlates with the switch on of streptomycin biosynthesis. While there is evidence that antibiotics have diverse roles in nature, our data clearly support the coevolution of resistance in the presence of antibiotic biosynthetic capability within closely related soil dwelling bacteria. This reinforces the view that, for some antibiotics at least, the primary role is one of antibiosis during competition in soil for resources.     

Biosynthesis of Streptomycin

Investigation was carried out on demonstration of two substances constructing a precursor system located at a late stage of streptomycin biosynthesis by Streptomyces griseus. One of them is thought to be a natural precursor of Streptomycin(L) and the other is suggested as an enzymatic substance(H) transforming L to streptomycin. Both substances had no antibiotic potency and H was inactivated at low pH. L was obtained from a cell-free supernatant (active supernatant) prepared from suspension of young mycelium of Streptomyces griseus in glucose solution. H was obtained not only from active supernatant but also from cell-free extract of the organism.

Two ways of isolation were established for L. Active supernatant was adsorbed on a CM-cellulose column equilibrated with 0.05 m Tris-maleate buffer (pH 8.0). Elution of this column with the same buffer as was used for equilibration gave L-containing fraction separated from streptomycin which was eluted with the buffer including 1% of sodium chloride. L was adsorbed also on active carbon in aqueous solution at neutral pH and liberated from it at acidic pH with 95% methyl alcohol. The former method was useful to separate L from streptomycin, and the latter one was so to concentrate L.

H was isolated by using a column chromatography on DEAE-cellulose. After adsorbing active supernatant or cell-free extract of organism on a column equilibrated previously with the same buffer as above, H was eluted with the buffer including 1% of sodium chloride. Cell-free extract of S. griseus was a better source of H supply than the active supernatant.     

Spatial structure increases the benefits of antibiotic production in Streptomyces*

Bacteria in the soil compete for limited resources. One of the ways they might do this is by producing antibiotics, but the metabolic costs of antibiotics and their low concentrations have caused uncertainty about the ecological role of these products for the bacteria that produce them. Here, we examine the benefits of streptomycin production by the filamentous bacterium Streptomyces griseus. We first provide evidence that streptomycin production enables S. griseus to kill and invade the susceptible species, S. coelicolor, but not a streptomycin-resistant mutant of this species. Next, we show that the benefits of streptomycin production are density dependent, because production scales positively with cell number, and frequency dependent, with a threshold of invasion of S. griseus at around 1%. Finally, using serial transfer experiments where spatial structure is either maintained or destroyed, we show that spatial structure reduces the threshold frequency of invasion by more than 100-fold, indicating that antibiotic production can permit invasion from extreme rarity. Our results show that streptomycin is both an offensive and defensive weapon that facilitates invasion into occupied habitats and also protects against invasion by competitors. They also indicate that the benefits of antibiotic production rely on ecological interactions occurring at small local scales.     

Streptomycin

Summary Streptomyces griseus DTH-2 was grown in 5 1 fermentors on complex media containing calcium carbonate as a buffering agent. It was shown that automatic pH control (4N KOH) could substitute the calcium carbonate giving higher yields of streptomycin. The yield was further increased by omitting inorganic phosphate from the medium and by differential addition of glucose/ammonium sulphate during the fermentation. The maximal yield obtained was 8.5 g of streptomycin per liter.     

MORPHOGENESIS IN VERTICILLIUM: A SELF-PRODUCED,DIFFUSIBLE MORPHOGENETIC FACTOR

A microsclerotial isolate of Verticillium albo-atrum produces a diffusible morphogenetic factor (DMF). At certain levels, DMF stimulates production of microsclerotia and melanin and inhibits hyphal elongation and sporulation. At higher concentrations it can inhibit production of microsclerotia and melanin as well as germination of Verticillium spores. Near-ultraviolet radiation (mostly 3200–4000 A, emission peak 3650 A), which inhibits production of microsclerotia and melanin, appears to act by suppressing synthesis of DMF. Once formed, DMF withstood 5 days’ exposure to near-UV. Dark-reared liquid cultures (shake or still) produced DMF while those reared under near-UV did not. DMF is active from pH 4 to pH 10, seems to be non-volatile, is dialyzable and water-soluble. In preliminary tests, it was insoluble in ether, ethanol and methylene dichloride. Acetone inactivated it. It may be 1 substance or a group of substances. We have developed assays for DMF in agar and liquid media employing the germination of Verticillium spores.     

Variety of Hybrid Characters among Recombinants Obtained by Interspecific Protoplast Fusion in Streptomycetes

To discover whether the protoplast fusion method is useful or not for interspecific breeding, some methods were devised, and the appearance of various hybrids with different characters and the change of antibiotic activities in the recombinants obtained by the protoplast fusion were investigated. The purification of protoplasts, the choice of parental natural characters as selection markers, and the adoption of a replica method for selecting all types of recombinants were devised and used for these experiments. Protoplast fusion was done between S. griseus KCC S-0644 and each strain of 5 species that were clearly different species from S. griseus, in addition to being streptomycin sensitive (SM^s) and capable of L-arabinose utilization for growth (Ara⁺). Recombinants (SM^r, Ara⁺) obtained by protoplast fusion displayed a great variety of hybrids in their taxonomic characters, e.g., 21 recombinant strains obtained by the cross between S. griseus and S. griseoruber consisted of 14 types of hybrids. Antibiotic productivity was examined in all recombinants obtained. Although both parental species produced their respective antibiotics, 60% of the recombinants did not produce any antibiotic and 24% produced different antibiotics from those of their parents. Among those recombinants, it was also found that the distribution of the productivity of each antibiotic among the recombinants was entirely different from that of the allelo-character in each taxonomic feature.     

Myo-inositol-1-phosphate synthase from streptomyces griseus (Studies on the biosynthesis of cyclitols,XXXVIII1)

Summary It could be shown that Streptomyces griseus, the microorganism producing the antibiotic streptomycin and also mutant strains of this species that cannot synthesize streptomycin, possess myo-inositol-1-phosphate synthase (EC 5.5.1.4), the enzyme cyclizing D-glucose 6-phosphate. The enzyme isolated from that organism is extremely instable, its molecular weight is approximately 260,000, and it requires a divalent metal ion for its activity. This is the first instance that an enzyme of this specificity has been found in a prokaryotic organism.     

Digest: Structuring interactions in Streptomyces*

For bacteria growing in colonies, spatial structure can allow maintenance of costly traits such as the production of antibiotics. Using spatially structured environments, Westhoff et al. examined the benefits of streptomycin production for the bacterium Streptomyces griseus in competition with a streptomycin-susceptible strain. Streptomyces griseus outcompeted susceptible competitors, but the benefit of its antibiotic decreased as competitor resistance to streptomycin increased. Spatial structure also increased the ability of S. griseus to invade susceptible competitor populations from low starting densities. These results demonstrate that spatially structured environments can both provide and amplify benefits of antibiotics to antibiotic-producing bacteria on a microbial scale.     

Fermentation Studies with Streptomyces griseus: II. Synthetic Media for the Production of Streptomycin

Synthetic media for streptomycin fermentation were studied to determine which media gave highest yields of streptomycin. The effect of salts on streptomycin production by Streptomyces griseus was examined, and a suitable combination of salts was established in a glucose-casein medium. This medium yielded 3,000 μg/ml of the antibiotic with an inoculum of 1.6%. Substitution of amino acids for casein was examined. Of 17 amino acids tested, best results were obtaind with sodium aspartate. Substitution of ammonium salts was tried, and an excellent streptomycin yield was obtained with a medium containing ammonium citrate.     

A New Regulatory Function of A-Factor: Stimulation of the Germination of Streptomycete Spores

Spore germination in streptomycetes was shown to be stimulated by exogenously added A-factor. Agar medium either containing or not containing A-factor was inoculated with spore suspensions of three strains differing in their ability to produce regulators of the A-factor group: Streptomyces griseus 773, which produces A-factor and two its lower homologs; S. coelicolor A3(2), which forms six Acl-factors (A-factor analogues); and S. avermitilis JCM5070, which fails to form regulators of this group. A count of the grown colonies showed that exogenous A-factor stimulated spore germination in strains that were themselves able to synthesize regulators of the A-factor group. In S. griseus 773, the number of germinated spores increased by 67% on average after the addition of A-factor to the medium in an amount of 10 g/ml. In strain S. coelicolor A3 (2), the number of germinated spores increased by 75% after the addition of 1 g/ml of A-factor. During germination of the S. avermitilis JCM5070 spores, no changes in the CFU number was observed after the addition of A-factor.     

Comparison of Str. griseus strains which produce streptomycin and those which do not

Summary Sreptomyces griseus strains which produce streptomycin and which do not were compared by their cultural-physiological behaviour, their capacity of utilizing citrate, micromorphology and phage sensitivity.No correlation is found between any of the examined characteristics and capacity to produce streptomycin.There is one common characteristic among all three strains which do not produce streptomycin i. e., they have a short life cycle in submerged culture.     

Antibiosis of Streptomyces griseus Against Colletotrichum lindemuthianum

Streptomyces griseus produces an antibiotic substance against Colletotrichum lindemuthianum because, in dual culture on potato dextrose agar (PDA), a large inhibitory zone was formed between the colonies of S. griseus and C.lindemuthianum. When S. griseus was placed adjacent to a colony of C. lindemuthianum in PDA, S.griseus grew poorly in PDA but it thrived and sporulated profusely when it came in contact with a colony of C. lindemuthianum indicating that S.griseus might require some essential nutrients from C.lindemuthianum. Scanning electron microscopy showed that hyphae of S.griseus in contact with C.lindemuthianum produced appressorium-like swellings or simply grew on the hyphal surface of C.lindemuthianum. Internal parasitism was evidenced by the presence of hyphae and conidia of the mycoparasite inside the host hyphae. Internal parasitism resulted in formation of several blebs. Cell walls of the parasitized hyphae degenerated having a sponge-like texture. The blebs broke off and left rounded holes on the hyphae.     

Media for the detection of Bacillus larvae spores in honey

Bacillus larvae, the causative agent of American foulbrood in honey bees completes its life cycle of germination, outgrowth and sporulation in young honey bee larvae by killing them and often bringing about the destruction of the entire hive. While B. larvae germinates and outgrows on complex organic media in vitro, the literature suggests, for reasons that are not at all clear, that a relatively large number of spores of B. larvae are required to yield each visible colony (colony forming units, CFU) on media. Various researchers have reported that from 16 to 3,000 or more spores of B. larvae are required to yield a single colony on an agar plate. HANSEN in Denmark designed a useful method of spreading approximately 80 mg of honey directly on the surface of a PETRI plate containing “J” agar medium to determine if B. larvae spores are present in the honey. In the present study, selected media were tested for the ability to recover B. larvae spores in honeys in the form of visible colonies (CFU) using HANSEN's strek method. A modification of a medium (TMYGP) developed by DINGMAN and STAHLY, (T-HCL-YGP agar), recovered considerably more viable B. larvae spores in the form of visible colonies (CFU) than HANSEN's “J” medium. When “J” medium was fortified with 0.1% sodium pyruvate, it was comparable to modified T-HCL-YGP medium in its recovery of B. larvae spores. Brain heart infusion agar (BHIA) with the addition of thiamine recovered more spores in the form of viable colonies than did “J” medium but it was not as efficient as T-HCL-YGP medium. Serial dilution from 100 to 10,000 times of weighed samples of honey with deionized water led to higher spore counts (CFU per g honey) than that indicated by undiluted honeys plated at 80 mg levels directly onto the surface of media by the HANSEN procedure.     

微波诱变结合抗性突变筛选放线菌素D高产菌株

采用微波结合链霉素抗性筛选法选育放线菌素D的高产菌株。通过考察链霉素对Streptomyces rubiginosohelvolus FIM-N31菌株孢子生长情况的影响确定链霉素致死浓度,出发菌株FIM-N31的孢子经微波辐照处理后,涂布在含链霉素致死浓度（50 μg/mL）的培养基平板上培养,获得了大量的链霉素抗性基因突变株。摇瓶发酵筛选突变株,结果获得一株遗传性状稳定的放线菌素D高产菌Str186,其产放线菌素D的能力比出发菌株提高了8倍以上。     

Biotransformation of antibiotics

Summary The absence of a lag period in the bioconversion of chloramphenicol by spores and the pronounced influence of the pH of the medium on this reaction strongly suggested that chloramphenicol acetyltransferase (CAT) is located at or near the surface of the spores of Streptomyces griseus. A two-hour exposure of spores to dilute solutions of -mercaptoethanol or surfactants resulted in significant decrease in activity even in the presence of glucose as an energy source. However, the inclusion of any of the reagents in the reaction mixture neither influenced the conversion activity nor the spore viability. These treatments did not reveal any cryptic activity for CAT in the spores. In addition, more drastic treatment of the spores with ethylenediaminetetraacetate (EDTA) or with concentrated salt solutions did not reduce the activity nor significantly affect the spore viability. Considering the modes of action of -mercaptoethanol and the surfactants, a combination of disulfide bridges and lipoprotein interactions may be responsible for the binding of CAT to the surface of the spore. Moreover, results of acid treatment of intact spores indicated that most of CAT activity, if not all, is located at the spore surface. Incorporation of ¹⁴C-acetate by cell-free extracts of Streptomyces griseus clearly showed that CAT selectively catalyzed the formation of chloramphenicol-3-acetate at an optimum pH of 6.5. The shape of the pH-activity curve in cell-free extracts is essentially identical to that of the enzyme in intact spores and is additional evidence that the enzyme is located at the spore surface.     

Beauveria bassiana submerged conidia production in a defined medium containing chitin,two hexosamines or glucose

Summary Beauveria bassiana in liquid culture can produce blastospores and occasionally submerged conidia. For use as a bioinsecticide, conidia have definite advantages. Numerous studies have investigated conidia production in liquid cultures using synthetic and industrial grade media supplemented with glucose. We have studied growth, development and sporulation in microcultures using growth media containing chitin monomers. For the production of submerged conidia growth media containing N-acetyl-d-glucosamine (GlcNAc) proved to be better than yeast extract-peptone-glucose (YPG), glucose plus ammonium salts (Glc+NH₄Cl) or N-acetyl-d-galactosamine (GalNAc). Sixty-one percent of the spores in the GlcNAc medium were submerged conidia with the remainder being blastospores. The concentration of submerged conidia reached 8.0 × 10⁵/ ml after two days in GlcNAc medium as compared to 8.9 × 10⁵/ml in YPG medium. Therefore, in terms of percentage of submerged conidia produced, GlcNAc medium generated more submerged conidia in spite of its lower cell yields. Growth in a medium containing chitin, a polymer of GlcNAc, resulted in 86.3% of the spores as submerged conidia exceeding 10⁶/ml after 48 h. Growth under phosphate limitation resulted in an increased percentage of submerged conidia for all media tested. Electron microscopy and spore protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that structural and compositional differences exist between the spore types.     

Effect of culture medium on the production and virulence of submerged spores of Metarhizium anisopliae and Beauveria bassiana against larvae and adults of Aedes aegypti (Diptera: Culicidae)

Three Metarhizium anisopliae and three Beauveria bassiana isolates were cultivated in media containing casamino acids, soybean flour or sunflower seed flour and were shaken for three days. M. anisopliae presented similar yields of around 10⁶ submerged spores/ml without significant differences among them, whereas B. bassiana produced yields of around 10⁸ spores/ml, of which GHA strain produced more submerged spores in the casamino acids medium. The other two strains showed no significant difference in the production of submerged spores in the three media used. Differences in mortality on Aedes aegypti larvae were observed with the submerged spores of Metarhizium depending on isolate and medium used. M. anisopliae 2157 caused significantly higher mortality (40%) when cultivated in casamino acids medium. It presented an LC₅₀ of 8.93 × 10⁵ submerged spores/ml water against mosquito larvae five days after application, whereas it caused 27% mortality in Ae. aegypti adults 10 days after application. In conclusion, fungal nutrition affected virulence of some isolates of M. anisopliae against Ae. aegypti larvae while such an effect was not noted for B. bassiana isolates.