Biochemical mutants of Actinomyces griseus, a producer of the antibiotic grisin. The isolation of the mutants and a study of the level of grisin antibiotic synthesis]

Biochemical mutants of Actinomyces griseus producing grisin were obtained under the action of chemical mutagens. The mutants were divided into 2 groups. The mutants with impaired synthesis of amino acids of the aspartic acid family, i.e. lysine, homoserine and methionine were included into the 1st group. The 2nd group included the mutants with impaired synthesis of the other amino acids, i.e. histidine, arginine, tyrosine, phenylalanine and valine. The antibiotic production level in the biochemical mutants was studied. It was found that the level of the antibiotic production was decreased in most of the biochemical mutants. A necessity for increased fonds of lysine and arginine for biosynthesis of grisin was shown.     

Effect of mutagenic factors on the variability of cultures of Actinomyces griseus--preducer of the antibiotic grisin

The study of the lethal and mutagenic effect of ethylenimine, nitrozoguanidine, nitrozomethylurea and nitrozoethylurea on Act. griseus Krainsky 15, producing grisin, an antibiotic widely used in agriculture as a stimulator of domestic animal growth showed that induction of mutants with increased antibiotic production levels was most favourable under the effect of ethylenimine. The above mutagens were highly active with respect to induction of morphological mutants. No clear correlation between the colony morphology and antibiotic production property was observed. However, it was noted that the dwarf colonies had a significantly decreased antibiotic activity.     

Selection of strains of Streptomyces griseus Kr., the producer of a streptothricin antibiotic grisin, using the method of protoplast fusion

The effect of protoplasting on antibiotic activity of the grisin-producing organism was shown. High frequency of Grn- mutants after strain VG307f protoplasting and no capacity in these mutants for reversion to the initial Grn+ phenotype were shown. The reversion frequency was less than 10(-8). Moreover, it was shown that all the Grn- mutants lost their stability (GrnR) to the effect of their own antibiotic. With respect to strain VG212 there was noted a significant increase in the number of both the minus and the plus variants after the protoplast formation and regeneration. Fusing of protoplasts of strains VG307f and VG212 belonging to the divergent lines in selection of S. griseus Kr. yielded the phage stable strain VG7849 with high levels of the antibiotic production and improved technological properties.     

Use of the protoplast fusion method in the selection of Streptomyces griseus--the producer of the streptothricin antibiotic grisin

An effective method for protoplast fusion in S. griseus producing grisin was developed. The method requires the use of polyethylene glycol with a molecular weight of 1000. It was demonstrated that protoplasts formed most effectively in this organism, when the mycelium of the streptomycete previously treated with ultrasound in the process of its growth was used for the treatment with lysozyme. The efficacy of protoplast regeneration in the strains with the use of the modified hypertonic medium R2MD was 25-75 per cent. The possibility of using the protoplast fusion method for constructing phage resistant strains producing kormogrisin was shown.     

Arsenate as a potential negative selection agent for deficiency variants in cultured plant cells

Sodium arsenate is toxic to cultured soybean [Glycine max (L.) Merr.] cells, killing virtually 100% of the cells during a 24-h exposure at a 1–2 mM concentration. However, when growth is previously halted by nitrogen deprivation 50–100% of the cells survive arsenate treatment. Because of this growthdependent toxicity, arsenate has promise as a negative selection agent for cultured plant cells. Using arsenate (2 mM) I was able to select from among 2×10⁷ cells a cell line with a growth requirement for an amino acid mixture. This trait was maintained through 9 months of passage but then was lost.     

A-Factor as a selective agent for isolation of the soil Gram-negative bacterium strain--the producent of an antibacterial antibiotic]

It was shown the stimulating role of A-factor on soil prokaryotes growth. Soil sample culturing on agar medium, containing A-factor, resulted in the colony forming units (CFU) increasing in comparison with culturing on the medium without this regulator. Gram-negative bacteria were the reason of CFU increasing; previously the effect of A-factor on bacteria of this group was not shown. Gram-negative rod bacterial strain No. 35 was isolated and shown that CFU number was approximately twice increased at A-factor concentration in agar medium 2 and 7 mcg/ml; high A-factor concentration up to 28 mcg/ml was not effective. Isolated strain No. 35 is the producer of antibiotic, effective against gram-positive bacteria.     

Cloning of grisin resistance gene gsr and the study of its functioning in Streptomyces griseus Kr. strain

S. griseus Kr. is a commercial strain producing grisin, an antibiotic of the streptothricin group used as a feed additive. It was shown earlier that genetic instability of the strain was very high which was evident from a high frequency of nonreverting Grn- Grns mutants. With densitographic analysis of chromosomal DNA electrophoregrams and DNA-DNA hybridization it was revealed that the molecular basis of the genetic instability of the S. griseus strain was deletion of a DNA fragment about 20 kb in size containing a grisin resistance gene. The resistance gene designated as gsr was cloned to S. lividans TK 64 within the plasmid vector pIJ699. The restriction map of a cloned DNA fragment with a gsr gene was constructed and its similarity to that of a nat gene resistant to norseothricin, another streptothricin was observed. Introduction of a gsr gene within the multicopy plasmid pIJ699 into S. griseus 212, a highly productive strain synthesiing the antibiotic, led to an increase in its resistance and productivity. Proceeding from the preliminary data on possible linkage of a gsr gene and grisin biosynthesis genes, it appeared possible to use the cloned gene as a molecular probe in cloning the biosynthesis genes.     

Action of mitomycin C on the variability of Actinomyces hygroscopicus in forming a proteolytic complex of hygrolytin enzymes and the antibiotic, hygromycin B]

The lethal and mutagenic effect of mitomycin C in doses of 10 and 15 micrograms/ml on the spores and 24-hour culture of Act. hygroscopicus, strain O878 producing hygrolytin, a proteolytic enzyme and hygromycin B, an antibiotic was studied. It was found that mitomycin C had a high lethal effect on the organism. The lethal effect of the antibiotic depended on the stage of the culture development, mitomycin C dose and exposure time. The 24-hour culture was most sensitive to the effect of mitomycin in a dose of 50 micrograms/ml. Exposure to mitomycin increased the actinomycete variation with respect to the colony morphology and induction of new morphological mutations. Exposure of strain O878 to mitomycin C significantly increased the culture variation with respect to the quantitative features of production of the hygrolytin proteolytic enzyme complex and hygromycin B. The character of the strain induced variation with respect to the features studied was different which indicated the absence of correlation between them. The use of mitomycin C proved to be promising in selection of Act. hygroscopicus with a purpose of increasing the culture proteolytic and antibiotic activity.     

Action of an antiestrogen agent, tamoxifen on the mammae of ovariectomized rats]

We have been studying the estrogen and antiestrogen action of tamoxifen and of clomifen on the development and secretion of acini and of the galactophore canals of the mammary gland. The ICI (50 or 200 microgram) has no effect : the very strong dose of 1 mg gives a light stimulation of the acini secretion which can be compared to that of light doses of clomifen (20 or 50 microgram) or of estradiol (0.25 microgram). Only 10 microgram of estradiol alone allow the development, the opening and secretion of acini. With 20 microgram, this action is more apparent, not only on the often dilated acini, but also on the canals. Administered along with 20 microgram of estradiol, tamoxifen is slightly antagonist with 50 microgram, strongly antagonist with 200 microgram, and completely antagonist with 1 mg. 50 microgram of clomifen are inefficient ; it is synergetic if the treatment starts and is over 4 days before the estradiol treatment.     

Male mate preference as an agent of fecundity selection in a polymorphic salamander

Color polymorphisms are associated with variation in other traits which may affect individual fitness, and these color‐trait associations are expected to contribute to nonrandom mating in polymorphic species. The red‐backed salamander (Plethodon cinereus) exhibits a polymorphism in dorsal pattern: striped and unstriped, and previous studies have suggested that they may mate nonrandomly. However, the mechanism(s) contributing to this behavior remain unclear. Here we consider the role that male preference may have in driving mating behavior in P. cinereus. We limit our focus to striped individuals because this morph is most likely to be choosy given their dominant, aggressive behavioral profiles relative to unstriped males. Specifically, we evaluated (a) whether striped males preferentially associate with females with respect to her dorsum color, size, and body condition and (b) if so, whether female traits are evaluated via visual or chemical cues. We also considered whether the frequency of another male social behavior, nose taps, was associated with mate preferences. We found that striped male P. cinereus nose tapped more often to preferred females. However, males only assessed potential mates via chemical cues, preferring larger females overall. Reproductive phenology data on a sample of gravid females drawn from the same population indicated that the color morphs do not differ in reproductive traits, but larger females have greater fecundity. Given our findings, we conclude that female P. cinereus are under fecundity selection, mediated by male preference. In this manner, male mating behavior contributes to observations of nonrandom mate associations in this population of P. cinereus.     

Isolation and identification of an antibiotic from culture 8-86]

An antibiotic complex was isolated from culture 8-86 referred to Bacillus. The complex consisted of components 8-86A and 8-86B active against gram-negative organisms. By its physico-chemical properties such as IR and UV spectra, amino acid composition, specific rotation and fatty acid composition component 8-86B was shown to be close to polymyxin F.     

Action of an antiestrogen agent, tamoxifen on the uterus and vagina of the ovariectomized rat]

Tamoxifen has more or less strong estrogen influence according to the targets : a light one on the uterus (1 mg being much less strong than 0.25 microgram of estradiol), a dynamic one on the vagina (50 microgram of tamoxifen make the vagina open in as short a time as 0.25 microgram of estradiol do but the keratinisation is still not completed even with 1 mg of tamoxifen). We can still see this influence four days after the end of the treatment. This influence is weak on the uterus until the 11 th day and it is much stronger on the vagina until about the 8 th day. Tamoxifen has an antiestrogenic action when opposed to 20 microgram of estradiol : this action is limited as soon as you give a dosis of 50 microgram on the uterus and it is nearly total with a dose of 1 mg ; we can notice it on the vaginal only from 200 microgram on.     

Action of penicillin on the causative agent of paratrachoma in the dynamics of an in vitro infection]

Monolayer cultures of L-cells (mouse fibroblasts) were inoculated with the causative agent of paratrachoma (strain LB-I). Simultaneously penicillin in doses of 0.01, 0.1, 1, 10, 25, 50, 100, 150 and 200 microgram/ml was added and its effect on the causative agent in the infection dynamics (18, 24, 48 and 72 hours after the inoculation) was studied with the light and electron microscopes. Gradual changes in the ultrastructure of the vegetative forms were observed by the 24th hour with the use of penicillin in a dose of 0.01 microgram/ml: the size of the vegetative forms increased, the cell wall membranes separated and periplasmic space significantly enlarged, the protoplast fragments disjoined into it, large forms vacuolized and were fragment with membranes, sometimes multilayer ones. When the culture was exposed to large doses of penicillin, the rate of the changes in the structure was higher and they were simultaneously of several types. Various types of the changes and possible modes of their formation were analyzed. Morphologically they are similar to the processes observed in L-transformation of bacteria. However, these structures were not infectious.     

The use of psyllium (isubgol) as an alternative gelling agent for microbial culture media

Psyllium (Isubgol), the mucilage husk of Plantago ovata, was successfully used as an alternative gelling agent (5% w/v as ground husk for pouring medium and 4% w/v as ground husk in combination with 0.5% w/v agar for slant) for microbial culture. Most of the undesirable features of psyllium-gelled media (slant as well as pouring) were removed by adding the u.v.-treated (20min) or oven-sterilized (120^°C for 1h) psyllium as ground husk to autoclaved medium at 50–60^°C under aseptic condition just before pouring.     

Direct selection of Aspergillus terricola variants having different toxicity on a culture of fibroblasts

A direct method is proposed to select less toxic mutants of Aspergillus terricola on a culture of fibroblasts. The conidia are first irradiated with UV, and then are used to grow colonies on the glass surface of tubes or flasks containing medium 199. The cells of fibroblasts are added thereupon and the two cultures are being grown together for 24--48 hours. The colonies which inhibit the growth of fibroblasts to a less extent are selected using microscopy. The method can be used for primarily selection of experimentally produced mutants of Aspergillus which form fibrinolytic enzymes possessing low toxicity.     

Cloning a mutatedtrp operon for the biosynthesis of an antibiotic agent

Summary Plasmid pCG8 containing a mutatedtrp operon was constructed and introduced into anEscherichia coli host for increasing tryptophan biosynthesis. Since tryptophan is a biosynthetic precursor of pyrrolnitrin, the mutated trp operon was inserted into aPseusdomonas vector pME290 to obtain the plasmid pCG12, and then was introduced intoPseudomonas pyrrocinia for enhancing the pyrrolnitrin synthesis. Data showed that the production of pyrrolnitrin of the transformed strain was five-times greater than that of the parental strain.