The use of ethyl methanesulfonate for the induction of mutants inMycobacterium phlei PA

The mutagenic effect of ethyl methanesulfonate in a concentration of 0.2m on a prototrophic, acid-fast strainMycobacterium phlei PA was studied by following the induction of changes of three genetic markers: prototrophy to auxotrophy and sensitivity to two antituberculosis drugs (INH and STM) to resistence. Ethylmethanesulfonate was found to be a very effective mutagen in all three cases. Thirty auxotrophic strains were obtained, out of which eight exhibited a low frequency of spontaneous reversions and could hence be used for further studies. Of the phenotypes induced the glycine⁻ (serine⁻) type was most frequently isolated and represented more than half of all auxotrophs obtained. Requirements for lysine and purines were also observed. The EMS treatment (1% survival of the basic suspension) resulted in a 74-fold increase of the frequency of INH-resistant mutants and a frequency of STM-resistant mutants about 1.1/2 to almost 2 orders of magnitude higher than the spontaneous values     

The mutagenic effect of ethyl methanesulfonate on a non-acid-fast strain ofMycobacterium phlei

Ethyl methanesulfonate was used for the induction of three types of mutants in a non-acidfast strain ofMycobacterium phlei. A total of 20 auxotrophie mutants was isolated. The mutants were isolated mostly when using doses yielding higher survival of the cells of the basic suspension. The auxotrophic mutants isolated required mostly amino acids, two mutants required purines and three mutants required vitamins. By determining the frequency of spontaneous reversions, it was found that 9 auxotrophic mutants could be used for further genetic studies. These included the following phenotypes: isoleucine⁻, leucine⁻, lysine⁻, nicotinic acid⁻, pyridoxine⁻, and xanthine⁻. Seven scotochromogenic mutants were isolated after ethyl methanesulfonate treatment. One was ochre, the remaining six were orange. Six achromogenic mutants were detected. Spontaneous auxotrophic mutants, scotochromogenic and achromogenic mutants were not isolated. The treatment with 0.2m ethyl methanesulfonate resulted in an increase in the frequency of STM-resistant mutants, the maximum phenotypic expression taking place after 72 hours cultivation in a liquid medium without the drug. The frequency of induced STM-resistant mutants varied within the range of 8.6.10⁻⁵–1.0.10⁻⁴ as compared with the frequency of spontaneous mutants 5.8.10⁻⁶–8.8.10⁻⁶.     

The use of nitrosoguanidine in the study of mutagenesis in a non-acid-fast strain ofMycobacterium phlei

N-methyl-N-nitroso-N′-nitroguanidine was used for the induction of two types of mutations in the PN strain ofMycobacterium phlei. Nineteen auxotrophic, 136 scotochromogenic and 50 achromogenic mutants were isolated after of treatment with nitrosoguanidine at a concentration of 1,000 μg/ml. Auxotrophic mutants required primarily amino acids and vitamins and half of them may be used for further genetic work due to their low frequency of spontaneous reversions. Colonies of scotochromogenic mutants were orange with the exception of one, which was strawberry red. Most scotochromogenic mutants were detected on a streptomycin containing medium. Roughly two thirds of the scotochromogenic mutants and one half of achromogenic mutants did not revert to the original photochromogenic character.     

Mutagenesis byN-methyl-N-nitroso-N-nitroguanidine in synchronized cultures ofMycobacterium phlei

Conditions of maximum induction of back mutations byN-methyl-N-nitroso-N′-nitroguanidine (“nitrosoguanidine”) were studied in auxotrophic mutants ofMycobacterium phlei. In asynchronous cultures the effects of pH, buffer molarity and concentration and exposure time to nitrosoguanidine were studied. It was shown that between 6 and 10, pH does not affect the induction of back mutations but that with increasing pH up to 9 the lethal effect of nitrosoguanidine on cells is increased. Protracted treatment with nitrosoguanidine or buffer molarity did not affect the induction of back mutations. It was found with several strains ofMycobacterium phlei that it is most efficient to treat a culture with 0.5 mg or 1 mg nitrosoguanidine/ml for 20 min at pH 6. On the basis of these findings a method of induction of back mutations by nitrosoguanidine was developed for populations with synchronous cell division.     

Complementation analysis with new genetic markers in Phaffia rhodozyma

Isolation and characterization of auxotrophic mutants from wild-type and astaxanthin mutant strains of Phaffia rhodozyma is described. Differences in survival were observed when u.v. irradiation of P. rhodozyma wild-type and astaxanthin mutant strains were incubated in the dark or exposed to photoreactivating light. Ultra-violet mutagenesis was not effective to produce auxotrophic mutants in this yeast. Auxotrophic mutants were obtained with high efficiency through a nystatin enrichment procedure after a N-methyl-N-nitro-N-nitrosoguanidine (NTG) mutagenic treatment with a 0.12% survivor level. Stringent mutagenetic conditions were needed to obtain P. rhodozyma auxotrophs. The most frequent mutants were ade^- and met^- in a rather narrow auxotroph spectrum. These results may be associated with a possible diploid condition of this yeast. The high number of adenine auxotrophs obtained in relation to other auxotrophic mutants suggests the possibility of some degree of heterozygosity in the wild-type strain UCD 67-385.     

Yersinia intermedia: A new species of enterobacteriaceae composed of rhamnose-positive,melibiose-positive,raffinose-positive strains (formerly calledYersinia enterocolitica orYersinia enterocolitica-like)

The drugs griseofulvin (10 μg/ml), nalidixic acid (0.05 μg/ml), quinine dihydrochloride (50 μg/ml), quinine ethylcarbonate (50 μg/ml), quinine urea hydrochloride (50 μg/ml), quinine lactate (50 μg/ml), and pamaquine (50 μg/ml) were chosen for laboratory studies. The minimal inhibitory concentration of the drug was used for determining the range of drug concentration needed to produce “mutational synergism” with ultraviolet radiation. Forward mutation from streptomycin sensitivity to resistance was used as a marker for mutagenicity. No stimulatory or inhibitory effects were noted on viable counts and mutation frequency, when the drugs were added (20–60 μg/ml) to the growth medium of unirradiatedEscherichia coli HCR⁺, HCR⁻, and irradiated HCR⁻ strains. These drugs increased mutation frequency and lethality of irradiated HCR⁺ bacteria. Incorporation of adenine (6 μm) into the minimal expression medium reverses the mutagenic effect of chloroquine. Chloroquine (50 μg/ml) did not interfere with the photoactivation of irradiated HCR⁺ cells. Our findings suggest that these chemicals selectively interfere with excision-repair.     

Lethality and mutagenicity inHalobacterium mediterranei caused by N-methyl-N′-nitro-N-nitrosoguanidine

Compared withEscherichia coli, Halobacterium mediterranei was highly resistant to the lethal effect of N-methyl-N-nitro-N-nitrosoguanidine (nitrosoguanidine), but it was sensitive to the mutagenic action of this chemical agent. Nitrosoguanidine at 500 g ml^–1 gave a cell survival level between 1% and 10%, and this allowed us to obtain more Josamycin-resistant mutants compared with lower concentrations, which gave higher survival rates but fewer mutants. The efficiency of the mutagenicity obtained with the nitrosoguanidine treatment was examined under a variety of conditions. The optimal conditions for obtaining Josamycinresistant mutants were achieved by exposing, in darkness and without shaking, a suspension of about 10⁸ log-phase cells to 500 g nitrosoguanidine in 1 ml of 50 mM modified saline Tris-maleate buffer at pH 7.5, or in 1 ml of 5 mM modified saline Tris-citrate-maleate for 30 min at 37°C.     

A note on auxotrophic mutants of cowpea rhizobia

We have examined a variety of common mutagens in producing auxotrophic mutants in cowpea rhizobia strains JRC23 and IRC256. While NTG (N-methyl-N-nitro-N-nitrosoguanidine), EMS (ethylmethane sulfonate), NA (nitrous acid), and UV (ultraviolet) irradiation were mutagenic with the strain JRC23, these mutagenic agents did not mutate strain IRC256. On the contrary, transposon mutagenesis with Tn5 yielded auxotrophs in strain IRC256 but not in strain JRC23, while only methionine (Met^–) auxotrophs from strain JRC23, histidine (His^–), and adenine plus thiamine (Ade^–+Thi^–) auxotrophs from strain IRC256 were isolated.     

Mutagenesis in Streptomycete cultures exposed to bleomycin

Mutagenic properties of bleomycin, an antitumor antibiotic were studied with respect to 2 species of streptomycetes producing practically important antibiotics. A multifold increase in the frequency of prototrophic revertants among the survivors of strains His- and Met- of Actinomadura carminata exposed to bleomycin was observed. Bleomycin was effective in induction of various morphological mutants, and auxotrophs at a high survival rate of the spores of Str. cremeus var. tobramycini, a tobramycin-producing organism. It was shown with the method of subsequent mutagenesis that the efficacy of induction of morphological and auxotrophic mutants in germinating spores of Actinomadura carminata, a carminomycin-producing organism by bleomycin in a concentration of 100 micrograms/ml and an exposure time of 5 minutes was much higher that in the latent spores. The mutagenic effect of bleomycin is comparable with that of ionizing radiation.     

UV-mutagenesis in Bacillus subtilis. VII. Induction of auxotrophic mutants]

An experimental testing of material from thin-layered, transparent in passing light, colonies which appear with some frequency after plating Bacillus subtilis cells on agar medium with limited enrichment, has shown that such colonies are formed by auxotrophic mutants. The growth requirements for many of them has been identified. The most of mutants can be reversed to original phenotype by UV-irradiation. The frequency of auxotrophs increases after UV-irradiation of suspension of original cells. The sensitivity of auxotrophic mutants to inactivating action of UV-light is near to that of original cells, hence the increase of the frequency of mutants with dose is a result of induction, but not of selection of preexisting spontaneous auxotrophic mutants. The frequency of induced auxotrophs, in contrast to that of suppressor revertants, badly give way to declining in the time of temporary inhibition of postradiation growth. In the case of Bac, subtilis, the system of induced auxotrophic mutants on the medium with limited enrichment is rather comfortable in use and can be recommended for studying UV-induced mutagenesis in structural genes as well as for testing mutagenic activities.     

Heterokaryosis and the role of cytoplasmic inheritance in dark resting structure formation in Verticillium spp

Summary Auxotrophic and morphological mutants of Verticillium albo-atrum (producing darkly pigmented resting mycelium) and V. dahliae (forming dark microsclerotia) were isolated after treatment of conidia (haploid and uninucleate) with ultraviolet light. Hyphal tip and conidial analysis revealed that complementation between pairs of auxotrophs on minimal medium was due to a mosaic of homokaryotic and heterokaryotic regions with some hyphal tips growing syntrophically. A degree of incompatibility was observed in a few intraspecific, but in most of the interspecific, heterokaryon tests. Heterozygous diploid conidia (6–11 in length compared with 3–6 for haploids) were recovered at a frequency of 1 in 8x10⁶ by plating spores at high density on MM. Young diploid colonies segregated to give haploid and diploid sectors, some of which were recombinant types (parasexual cycle). Heterokaryons between complementary auxotrophs which were wild-type for dark pigmentation (hyl⁺) resembled wild-type and only darkly pigmented colonies were recovered by conidial analysis. Heterokaryons between hyl⁺ and hyaline (hyl) auxotrophs again resembled hyl⁺ morphology and usually only hyl⁺ colonies of both auxotrophic genotypes were recovered. Conidia from heterokaryons formed by stable hyl auxotrophs produced only hyl colonies of both auxotrophic genotypes. The important role played by cytoplasmic factors in the inheritance of darkly-pigmented resting structures in Verticillium was strongly confirmed by the present work.     

Isolation and preliminary characterization of auxotrophic and morphological mutants of the yeastlike form of Paracoccidioides brasiliensis.

N-methyl-N'-nitro-N-nitrosoguanidine, which is known to be a very effective mutagen in many systems, was used to induce mutants in the yeastlike form of Paracoccidioides brasiliensis strain IVIC Pb9, an imperfect fungus. Forty-three auxotrophic and 27 prototrophic morphological mutants were isolated after treatment with 50 mug of nitrosoguanidine per ml in 0.1 M citrate buffer, pH 5.0. Auxotrophic mutants required primarily either amino acids, purines, or pyrimidines. Some auxotrophs were also morphological mutants. The main morphological difference from the parental strain was the texture or the color of the yeast-like colonies. Only one prototrophic morphological mutant differed in the size and form of the yeastlike cells when compared with the parental strain. Suxotrophic mutants were used in pairwise combination to attempt heterokaryon formation without success.     

Physiology of acute silver toxicity in the starry flounder (Platichthys stellatus) in seawater

Physiological effects of exposure to silver (AgCl_n ⁿ⁻¹; 250 μg Ag l⁻¹ or 1000 μg Ag l⁻¹) in seawater fish were investigated using adult starry flounders. While all fish survived up to 10 days in 250 μg Ag l⁻¹, flounders started to die after day 4 in 1000 μg l⁻¹. Dose-dependent increases in plasma and hepatic silver concentrations showed that silver was available for uptake. There were minimal negative effects on hematological parameters, acid-base status, and blood gases. Plasma ammonia showed a pronounced (three- to four-fold), but transient increase in flounders exposed to either 250 μg Ag l⁻¹ or 1000 μg Ag l⁻¹. Whole body ammonia and acid equivalent efflux measurements indicated that ammonia retention was due to a combination of stimulated production and inhibited excretion. In the 1000-μg Ag l⁻¹ group there was a similar transient increase in plasma [magnesium], which was restored by day 4. In contrast, plasma chloride and sodium levels increased gradually towards the point when fish began to die. At 250 μg Ag l⁻¹, the Na⁺/K⁺-ATPase activity of the intestine was unaffected but there was a two-fold increase in branchial Na⁺/K⁺-ATPase activity. The latter effect was interpreted as compensation for an elevated chloride and sodium load. The increases in plasma chloride and sodium concentrations were accompanied by a marked suppression of drinking, thereby indicating that acute silver toxicity was likely caused by a combination of elevated electrolyte concentrations and dehydration. Accepted: 9 June 1999     

Effects of heavy-metal additions on ammonification and nitrification in soils contaminated with cadmium,lead and zinc

Summary The sensitivity of the mineralization of nitrogen by a range of soils contaminated with heavy metals (up to 340 μg Cd g⁻¹, 7500 μg Pb g⁻¹ and 34000 μg Zn g⁻¹) to the addition of heavy metals in solution were studied using pot incubations (ammonification) and a soil perfusion technique (nitrification). The ammonification of peptone showed little correlation between treatments with Cd, Zn (1000 and 5000 μg g⁻¹) and Pb (10000 and 20000 μg g⁻¹) and origin of the soil. Nitrification was considerably more sensitive to heavy metals than ammonification. All the soils had active, often large, populations of ammonifying and nitrifying organisms which showed substantial similarities between the soils. The rate of nitrifying activity (NO₃−N production) was logrithmic in most cases. The presence of tolerant populations of nitrifying organisms in the contaminated soils was demonstrated. Tolerance was also eventually acquired after a longer lag phase, by the non-contaminated soil populations although the rate of activity was often reduced. Metals added in solution were adsorbed by the soil within 4 hours. Differences in toxicity between metal salts (chlorides, sulphates and acetate) were attributed to the amount left in solution. However, in many instances, acetate was found to stimulate all the stages in the mineralisation of nitrogen.     

Developmental,tissue culture,and genotypic factors affecting plant regeneration from shoot apical meristems of germinated <Emphasis Type="Italic">Zea mays</Emphasis> L. Seedlings

Summary Culture media, environmental and genotypic factors affecting regeneration from multi-shoot cultures derived from corn seedling apical explants were investigated. The frequency of shoot regeneration was highes for seedlings that were 4–5 cm in length. Flow cytometry was used to show that the most responsive culturs contained a high proportion of cells in the G1 phase. Proline in the multi-shoot induction medium (MSI) significantly increased the shoot induction frequency. Continuous low light (30–40 μEm⁻²s⁻¹) stimulated multi-shoot induction. The highest number of multi-shoots developed in medium containing 4 gl⁻¹ proline, 2 mgl⁻¹ (8.8 μM) 6-benzylaminopurine (BA), and 1 mgl⁻¹ (4.5 μM) 2,4-dichlorophenoxyacetic acid (2,4-D). Multi-shoots were induced in this culture system from 44 of 45 corn genotypes and approximately 70% of the genotypes exhibited a high to moderate response (greater than 20 shoots per explant in 4 wk of culture). This culture procedure is an efficient and widely applicable method for corn regeneration that may be a useful target for transformation.     

Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud

Summary As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N⁶-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l⁻¹ (4.52 μM) 2,4-D plus 0.5 mg l⁻¹ (2.22 μM) BA. and 2 mg l⁻¹ (8.88 μM) BA plus 1 mg l⁻¹ (4.14 μM) Picloram with or without 40 mg l⁻¹ (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l⁻¹ (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l⁻¹ (2.22 μM) BA, or 1 mg l⁻¹ (4.52 μM) 2,4-D with 0.50 mg l⁻¹ (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l⁻¹ (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l⁻¹ (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l⁻¹ (8,28 μM) Picloram, 1 mg l⁻¹ (4.44 μM) BA and 40 mg l⁻¹ (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.     

Antimutagenic effects of caffeine during nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium cells and phages

The effect of caffeine on nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium & nd its P22 and L phages was studied. The detected mutations included phage “clear” mutations, reversions of phage “amber” mutation, and prototrophic reversions of thehis ⁻ auxotroph ofSalmonella typhimurium. Neither therecA mutation of the host nor theerf mutation of the phage genome were found to affect the nitrosoguanidine-induced mutagenesis of the phage during vegetative growth. Beginning with a concentration of 0.2 mg/ml, caffeine decreased the frequency of mutants by 30–60%, attaining a maximum effect at 1.5 mg/ml and retaining this effect even at higher concentrations. A similar antimutagenic effeot was observed with the mutagenesis of the host cells. The nitrosoguanidine-induced mutagenesis does not seem to be related to the function of therecA cell gene or theerf phage gene. The mechanism of mutagenesis by nitrosoguanidine probably has two components, one of them caffeine sensitive, the other caffeine-resistant.     

Different sealing materials for petri dishes strongly affect shoot regeneration and development from leaf explants of quince ‘BA 29’

Summary The effect of different sealing materials [i.e., polyvinyl chloride (PVC) transparent film, and Parafilm (PARA) for Petri dishes was investigated on shoot regeneration from quince (Cydonia oblonga L.) ‘BA 29’ leaf explants. Leaves were excised from proliferating shoot cultures, transversally scored, and placed with the abaxial side down in 60-mm Petri dishes containing 10 ml of Murashige and Skoog modified medium, with 5.4 μM α-naphthaleneacetic acid, 4.5 μM thidiazuron, 200 mg l⁻¹ cefotaxime, and 0.25% (w/v) Phytagel (IM medium) for shoot bud induction, and cultured in darkness at 22±2°C for 28 d. Then the explants were transferred to standard conditions (16-h photoperiod at 30 μmol m⁻² s⁻¹ photosynthetically active radiation) on a medium similar to IM, except for lack of NAA, and with 0.65% (w/v) agar instead of Phytagel, for an additional 15–28 d. The sealing combinations PARA-PARA, PARA-PVC, PVC-PARA, and PVC-PVC (in the induction-expression phases) were compared during regeneration and for their carry-over effect on shoot development after transfer of explants to an elongation medium (0.9 μM 6-benzyladenine). Carbon dioxide accumulated at 27.2 mmol mol⁻¹ at the end of induction, and gradually decreased from 35.4 mmol mol⁻¹ on day 9 to 22.5 mmol mol⁻¹ on day 28 of the expression phase in PARA-sealed Petri dishes, being always much higher than after sealing with PVC (1–2 mmol mol⁻¹). Ethylene concentration was 0.1 and 0.04 μmol mol⁻¹ in the first part of the induction and expression phase, respectively, in PARA-sealed Petri dishes, and slightly decreased with duration of exposure to light during expression; while it was absent in most PVC-sealed dishes. The PARA-PARA and PVC-PVC (induction-expression) combinations gave, respectively, the worst and best results of regeneration and successive shoot development.     

Development of a colorimetric assay for rapid quantitative measurement of clavulanic acid in microbial samples

We developed a colorimetric assay to quantify clavulanic acid (CA) in culture broth of Streptomyces clavuligerus, to facilitate screening of a large number of S. clavuligerus mutants. The assay is based on a β-lactamase-catalyzed reaction, in which the yellow substrate nitrocefin (λ _max=390 nm) is converted to a red product (λ _max=486 nm). Since CA can irreversibly inhibit β-lactamase activity, the level of CA in a sample can be measured as a function of the A ₃₉₀/A ₄₈₆ ratio in the assay mixture. The sensitivity and detection window of the assay were determined to be 50 μg L⁻¹ and 50 μg L⁻¹ to 10 mg L⁻¹, respectively. The reliability of the assay was confirmed by comparing assay results with those obtained by HPLC. The assay was used to screen a pool of 65 S. clavuligerus mutants and was reliable for identifying CA over-producing mutants. Therefore, the assay saves time and labor in large-scale mutant screening and evaluation tasks. The detection window and the reliability of this assay are markedly better than those of previously reported CA assays. This assay method is suitable for high throughput screening of microbial samples and allows direct visual observation of CA levels on agar plates.     

Optimization of Sterilization of <Emphasis Type="Italic">Escherichia coli</Emphasis> in Milk by Surfactin and Fengycin Using a Response Surface Method

In this paper, the sensitivity of Escherichia coli to surfactin and fengycin was observed, and the optimization of the antimicrobial activity of surfactin and fengycin to E. coli in milk by a response surface methodology was studied. Results showed that E. coli had high sensitivity to these antibiotics, whose minimal inhibitory concentrations were 15.625 μg·mL⁻¹ and 31.25 μg·mL⁻¹, respectively. The optimization result indicated that E. coli could be sterilized by 5 orders of magnitude when the temperature was 5.5°C, the action time was 15.8 h, and the concentration (surfactin/fengycin weight ratio 1:1) was 14.63 μg·mL⁻¹.