The induction of mutants in a non-acid-fast strain ofMycobacterium phlei with nitrous acid

The inactivation and mutagenic effets of nitrous acid on a non-acid-fast strain ofMycobacterium phlei were studied. It was found that 0.017m NaNO₂ at pH 4.4 may be used for the induction of auxotrophic mutants, scotochromogenic and achromogenic mutants and STM-resistant mutants. Three doubly auxotrophic mutants, three mutants requiring amino acids and three mutants depending on vitamins were obtained. One mutant was not classified. Eighteen scotochromogenic mutants were isolated, seventeen of them were orange. Only ten achromogenic mutants were isolated. Twelve scotochromogenic and eight achromogenic mutants could be used in further genetic studies as they did not revert spontaneously to photochromogeny. Six auxotrophic mutants could be used due to their low frequency of spontaneous reversions. The frequency of STM-resistant mutants increased on an average seven-fold after the mutagenic treatment as compared with the spontaneous frequency.     

Inactivation of transforming DNA by ultraviolet light. 3. Further observations on the effects of 365 nm radiation

In the doubly auxotrophic strain ad 3A 38701 inos 37401, nitrosoethylurethane (NEU) produces a storage effect for adenine reversions but not for inositol reversions. Shaking treated spores in water for several hours destroys their response to storage. Short heat-treatment during or before plating increases the frequency of adenine reversions but not that of inositol reversions. Storage and heat-treatment decrease the inositol-specificity of NEU and may reverse it into adenine-specificity. Comparison with similar results obtained for diepoxybutane (DEB) shows that the cellular effects of NEU are more complex than those of DEB.     

Role of Escherichia coli DnaK and DnaJ chaperones in spontaneous and induced mutagenesis and their effect on UmuC stability

The frequency of spontaneous as well as induced reversions of auxotrophic mutations in Escherichia coli AB1157 and its DeltadnaK and DeltadnaKdnaJ derivatives was estimated. The obtained results demonstrate that both mutants tested are characterized by elevated frequency of spontaneous reversions compared to their AB1157 parent. In contrast, the frequency of reversions induced by UV and MMS, i.e. agents inducing the SOS response, is reduced in DeltadnaJ and DeltadnaKdnaJ mutants, pointing to the possible defect of these mutants in error prone repair. Due to the fact that UmuC protein is one of the main players executing the error prone repair, its stability in DeltadnaJ and DeltadnaKdnaJ mutants was also studied. Reduced UmuC stability was demonstrated only in the DeltadnaKdnaJ mutant.     

Mutagenic effect of ultraviolet radiation on the non-acid-fast strain ofMycobacterium phlei

The mutability of the PN strain ofMycobacterium phlei was examined after induction of auxotrophic mutants and of STM and VM-resistant mutants, by UV irradiation. A total of 30 auxotrophic mutants were isolated, most of them amino acid-dependent five purine-dependent, and one uracil-dependent. To induce the mutants higher UV doses had to be used so that the survival of cells in the original suspension would not exceed a few per cent. For further genetic work use can be made of 8 auxotrophic mutants (PN try^?ura^?, PN arg^?ura^?, PN ileu^?val^?, PN ileu^?, PN leu^?, PN lys^?, PN lys^?-VM^r, PN val^?), these showing a low frequency of spontaneous reversions. No spontaneous auxotrophic mutants have been found. The frequency of STM and VM-resistant mutants is increased upon UV irradiation, a post-irradiation incubation in a liquid medium without the drug being essential for their phenotypic expression. The highest increase of the number of these mutants is attained after 48 h of post-irradiation incubation and it has been found that, within a certain experimental scatter, the same frequency increase is found on using a complete or a minimal liquid medium. The frequency of spontaneous STM-resistant mutants lies within 5.8×10^?6–8.8×10^?6, of those VM-resistant between 3.1×10^?5 and 4.1×10^?5. The highest frequency of induced STM-resistant mutants lies between 3.0×10^?5 and 9.3×10^?5 and of VM-resistant mutants between 1.1×10^?4 and 2.2×10^?4     

The mutagenic effect of ultraviolet radiation and nitrous acid onMycobacterium phlei

Lethal and mutagenic effects of nitrous acid and UV radiation onMycobacterium phlei were studied Three auxotrophic strains of the PA strain ofMycobacterium phlei were obtained: ala^-, his^-, and gly^- (ser^-) INH^r Bods of the his^- strain grown in liquid media are longer to filamentous as compared with cells of the prototrophic PA strain grown in the same media, whereas cells of the gly^- (ser^-) INH^r mutant are shorter to coccobacillary. Cells of the ala^- strain are characterized by their various length from normal to coccobacillary. The auxotrophic strains obtained differ from each other by a frequency of spontaneous reversions to prototrophy. The his^- strain is the most stable, a frequency of spontaneous reversions to prototrophy being 10^-9. The gly^- (ser^-) INH^r strain reverts spontaneously to prototrophy with a frequency of 10^-8 to 10^-7. The ala^- strain spontaneously reverting with a frequency of 10^-5 is the most labile. The auxotrophic mutants obtained do not differ from the original prototrophic strain in the other properties studied. A change in a frequency of INH and STM-resistant mutants was also studied. It was found that under the influence of UV radiation a frequency of INH-resistant mutants increases 43 to 80 fold as compared with a frequency of spontaneous mutations, this latter being about 2.6 × 10^-6. No substantial increase in a frequency of STM-resistant mutants was found using UV irradiation under the given experimental conditions; their spontaneous frequency equals to 9.0 × 10^-9 to 2.0 X 10^-8.     

Insufficient uracil supply in fully aerobic chemostat cultures of Saccharomyces cerevisiae leads to respiro-fermentative metabolism and double nutrient-limitation

A combination of chemostat cultivation and a defined medium was used to demonstrate that uracil limitation leads to a drastic alteration in the physiology of auxotrophic cells of Saccharomyces cerevisiae. Under this condition, the carbon source is dissimilated mainly to ethanol and acetate, even in fully aerobic cultures grown at 0.1 h^?1, which is far below the critical dilution rate. Differently from nitrogen-, sulphur-, or phosphate-limited cultures, uracil limitation leads to residual sugar (either glucose or sucrose) concentrations below 2 mM, which characterizes a situation of double-limitation: by the carbon source and by uracil. Furthermore, the specific rates of CO₂ production and O₂ consumption are increased when compared to the corresponding prototrophic strain. We conclude that when auxotrophic strains are to be used for quantitative physiological studies, special attention must be paid to the cultivation conditions, mainly regarding medium formulation, in order to avoid limitation of growth by the auxotrophic nutrient.     

Phenylalanine production from a rec Escherichia coli-strain in fed-batch culture

The production of phenylalanine from a plasmid-harboring auxotrophic Escherichia coli mutant (E. coli W3110 Δtyr, Δtrp, Δphe/pJN6) was studied in two types of constantly-fed-batch cultures. The plasmid contains genes essential for phenylalanine production. In tyrosine fed-batch cultures the cell mass was increased and the strong inhibition and repression of phenylalanine synthesis by tyrosine was avoided. In this way r_p can be increased since production also occurred during the growth phase. Experiments with different feed rates of tyrosine, corresponding to different growth rates, showed that a high μ during the feeding period was necessary for obtaining a high q_p in the non-growth period.Glucose fed-batch cultures were employed to reduce the byproduct formation that occurred if excess of glucose was present in the culture liquid. By choosing a proper feed rate q_s in the non-growing cells could be controlled at a reduced level suitable for obtaining a high Y_p/s. The byproduct formation was thereby reduced and an average Y_p/s of 0.20 was obtained from the non-growing cells.     

A case of mutagen specificity attributable to a plating medium effect

Summary In anadn ^– met ^– di-auxotrophic strain ofSchizosaccharomyces pombe met ⁺ reversions are several hundred times more frequent thanadn ⁺ reversions after treatment with ultra-violet light. They are only slightly more frequent thanadn ⁺ reversions when HNO₂ is used as a mutagen (mutagen specificity). The poor response of theadn-1,199 allele to the mutagenic action of U.V. can be largely overcome by replacing themet-4,D19 allele with its normalmet ⁺ allele (influence of the genetic background). It was shown that both the mutagen specificity and its dependence on the genetic background are due, largely at least, to the inhibition ofadn ⁺ reversions on a plating medium containingl-methionine. This inhibition is very strong for U.V.-induced reversions but only weak for HNO₂-induced ones. It would be wrong to assume that other mutants at theadenine-1 locus behave in the same manner.With 1 Figure in the Text     

Fermentative Production of l-Leucine with Auxotrophic Mutants of Corynebaeterium glutamicum

An auxotrophic mutant of Corynebaeterium glutamicum was found to accumulate a large amount of l-leucine in the culture medium. The nutritional requirement of the mutant is rather complex but it’s growth was most remarkably stimulated by l-phenylalanine. Acetate (1.5~3.0%) or pyruvate (3%) stimulated the l-leucine production. By a further mutagenic treatment, 329 mutants earring some defect in addition to phenylalanine auxotrophy were derived from the mutant No. 190. Among them, a histidine auxotrophic derivative produced twice as much l-leucine as the parent strain, i.e., the level of l-leucine production by this derivative reached 16 mg/ml in a medium containing 12% glucose, 1 % (NH₄)₂SO₄ and 2.5% CH₃COONH₄ as carbon and nitrogen sources. Some other auxotrophic markers such as isoleucine- (or threonine-), threonine-, purine(s)-, homoserine-, or methionine- auxotrophy also improved the L-leucine production by No, 190.     

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.     

Stable polyphosphate accumulation by a pseudo-revertant of an Escherichia coli phoU mutant

phoU mutants of bacteria are potentially useful for the removal of inorganic phosphate (P_i) from sewage because they can accumulate a large amounts of polyphosphate (polyP). However, the growth of phoU mutants is severely defective and is easily outgrown by revertant(s) that have lost the ability to accumulate polyP during growth in a nutrient-rich medium. We found that a pseudo-revertant, designated LAP[+], that appeared in a culture of an Escherichia coli phoU mutant that could accumulate polyP even after ten serial passages. Reduction in the expression of the P_i-specific transporter Pst in LAP[+] may contribute to relieving stresses such as excess P_i incorporation that could stimulate reversions. The discovery of a LAP[+] provides a clue to generate phoU mutants that accumulate polyP in a stable manner.     

Importance of polyamines in cell cycle kinetics as studied in a transgenic system

Polyamines are organic cations, which are considered essential for normal cell cycle progression. This view is based on results from numerous studies using a variety of enzyme inhibitors or polyamine analogues interfering with either the metabolism or the physiological functions of the polyamines. However, the presence of non-specific effects may be hard to rule out in such studies. In the present study, we have for the first time used a transgenic cell system to analyze the importance of polyamines in cell growth. We have earlier shown that expression of trypanosomal ODC in an ODC-deficient variant of CHO cells (C55.7) supported growth of these otherwise polyamine auxotrophic cells. However, one of the transgenic cell lines grew much slower than the others. As shown in the present study, the level of ODC activity was much lower in these cells, and that was reflected in a reduction of cellular polyamine levels. Analysis of cell cycle kinetics revealed that reduction of growth was correlated to prolongation of the G₁, S, and G₂ + M phases in the cells. Providing exogenous putrescine to the cells resulted in a normalization of polyamine levels as well as cell cycle kinetics indicating a causal relationship.     

Development of papillae on colonies of two isopoly-auxotrophic strains ofSaccharomyces cerevisiae allelic inRAD6 during adenine starvation

Papilla formation on colonies of two isopolyauxotrophic strains (ade 2 his3 leu2 trp1 ura3) allelic inRAD6 was compared in order to find proper conditions for selecting mutants ofSaccharomyces cerevisiae with altered starvation-induced mutability. The most promising for this purpose appeared to be culturing low numbers of colonies on suboptimal plates with a growth-limiting amount of adenine at 28 °C for 20 d. Inactivation of theRAD6 gene which suppresses the level of starvation-associated mutagenesis markedly enhanced papilla formation under these conditions. Formation of almost all papillae on 20-d-old colonies of BJC3 was caused by mutation. Most of the papillae (75%) were white Ade⁺ revertants. Three groups of these papillae were distinguished (Ade⁺, Ade⁺ Rad6⁺ and Ade⁺ Trp⁺). Both, Ade⁺ Rad6⁺ and Ade⁺ Trp⁺ double reversions were very probably caused by a suppressor mutation. The less frequent red papillae had the same auxotrophic markers and UV sensitivity as BJC3 but their outgrowth in liquid media was greater. It appears that creation of these papillae is caused by mutation affecting the cell response to growth limitation by low concenttations of adenine.     

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.     

Phase Change in Hedera helix: Stabilization of the Mature Form with Abscisic Acid and Growth Retardants

Applications of ABA to the mature form of Hedera helix stabilize its morphological characteristics and prevent GA₃ induced reversion to the juvenile form. Plants treated with GA₃ reverted to the juvenile form whereas those supplied with ABA in conjunction with GA₃ remained mature. When mature plants were treated with 5 nanomoles of GA₃ and 5 micromoles of ABA, reversion did not occur, but when the GA₃ dose was raised to 25 nanomoles with the same level of ABA, reversion did occur. This implies that the relative amounts of GA₃ and ABA applied are important in controlling growth form and not the absolute levels of these hormones. Applications of growth retardants (Chlormequat, Ancymidol, and SADH) stabilize the mature form by preventing spontaneous reversions induced under low light intensity. These two lines of evidence support the hypothesis that the mature morphological form can be stabilized by regulating the effective level of gibberellins in the plant and this can be accomplished by inhibition of gibberellin action or gibberellin biosynthesis.     

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     

Indirect selection for plasmid mutants: isolation of ColVBtrp mutants defective in self-maintenance in Escherichia coli.

An efficient method for isolation of a large number of plasmid mutants is described. It is based on the fact that N-methyl-N'-nitro-N-nitrosoguanidine induces a number of closely linked mutations within a short segment of the bacterial chromosome. Thus, selection for reversions of an auxotrophic marker located on the ColVBtrp plasmid yielded a large fraction (more than 50 percent) of mutants defective in some plasmid functions, including its own maintenance in the host bacteria. The results of preliminary characterization of strains carrying these mutated plasmids are presented.     

The influence of amino acid starvation on the UV reversibility of the strains ofEscherichia coli B/rthy − trp − Hcr + andEscherichia coli B/rthy − trp − Hcr −

The fraction of inducedtrp ⁺ reversions in the strains ofEscherichia coli B/rthy ⁻ trp ⁻ Hcr ⁺ andEscherichia coli B/rthy ⁻ trp ⁻ Hcr ⁻ was studied in the course of starvation for an essential amino acid. UV light as a mutagenic factor was used. It was found that there is a decrease in the proportion of inducedtrp ⁺ reversions in the strain ofHcr ⁺ type during starvation. Such a decrease was however observed only with that fraction oftrp ⁺ reversions which is expressed in selective plates where several divisions of irradiated cells are caused. The proportion oftrp ⁺ reversions expressed on minimal plates does not change during starvation. With the strain ofHcr ⁻ type the proportion of inducedtrp ⁺ mutations remains unaltered irrespective of the nature of the selective plates.     

Isolation of auxotrophic mutants in support of ammonia assimilation via glutamine synthetase in Methanobacterium ivanovii

Various enzymes involved in the initial metabolic pathway for ammonia assimilation by Methanobacterium ivanovii were examined. M. ivanovii showed significant activity of glutamine synthetase (GS). Glutamate synthase (GOGAT) and alanine dehydrogenase (ADH) were present, wheras, glutamate dehydrogenase (GDH) was not detected. When M. ivanovii was grown with different levels of NH ₊ ⁴ (i.e. 2, 20 or 200 mM), GS, GOGAT and ADH activities varied in response to NH ₊ ⁴ concentration. ADH was not detected at 2 mM level, but its activity increased with increased levels of NH ₊ ⁴ in the medium. Both GS and GOGAT activities increased with decreasing concentrations of NH ₊ ⁴ and were maximum when ammonia was limiting, suggesting that at low NH ₊ ⁴ levels, GS and GOGAT are responsible for ammonia assimilation and at higher NH ₊ ⁴ levels, ADH might play a role. Metabolic mutants of M. ivanovii that were auxotrophic for glutamine were obtained and analyzed for GS activity. Results indicate two categories of mutants: i) GS-deficient auxotrophic mutants and ii) GS-impaired auxotrophic mutants.Abbreviations GS Glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase     

Characterization of the Link between Ornithine,Arginine, Polyamine and Siderophore Metabolism in Aspergillus fumigatus

The opportunistic fungal pathogen Aspergillus fumigatus produces siderophores for uptake and storage of iron, which is essential for its virulence. The main precursor of siderophore biosynthesis (SB), ornithine, can be produced from glutamate in the mitochondria or by cytosolic hydrolysis of ornithine-derived arginine. Here, we studied the impact of mitochondrial versus cytosolic ornithine biosynthesis on SB by comparison of the arginine auxotrophic mutants ΔargEF and ΔargB, which lack and possess mitochondrial ornithine production, respectively. Deficiency in argEF (encoding acetylglutamate kinase and acetylglutamyl-phosphate-reductase), but not argB (encoding ornithine transcarbamoyl transferase) decreased (i) the cellular ornithine content, (ii) extra- and intracellular SB, (iii) growth under harsh iron starvation, (iv) resistance to the ornithine decarboxylase inhibitor eflornithine, and (v) virulence in the Galleria mellonella larvae model. These lines of evidence indicate that SB is mainly fueled by mitochondrial rather than cytosolic ornithine production and underline the role of SB in virulence. Ornithine content and SB of ΔargB increased with declining arginine supplementation indicating feedback-inhibition of mitochondrial ornithine biosynthesis by arginine. In contrast to SB, the arginine and polyamine contents were only mildly affected in ΔargEF, indicating prioritization of the latter two ornithine-consuming pathways over SB. These data highlight the metabolic differences between the two arginine auxotrophic mutants ΔargEF and ΔargB and demonstrate that supplementation of an auxotrophic mutant does not restore the wild type metabolism at the molecular level, a fact to be considered when working with auxotrophic mutants. Moreover, cross pathway control-mediating CpcA was found to influence the ornithine pool as well as biosynthesis of siderophores and polyamines.