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.     

Isolation of auxotrophic mutants ofAspergillus niger by ethylmethane sulphonate treatment

The alkylating agent EMS can be employed for the isolation of auxotrophic mutants of theAspergillus niger strain K 10. The maximum number of auxotrophic mutants (2.9–5.1%) corresponded approximately to the number of mutants obtained by EMS treatment of yeasts, but it was by order lower than the number of mutants generally obtained by EMS treatment in bacteria. The majority of isolated mutants grew worse than the parent strain in the liquid medium and also formed lower amount of organic acids. The organic acid which was most frequently accumulated by mutants was citric acid.     

Ethyl methanesulfonate mutagenesis in extremely halophilic archaebacteria: Isolation of auxotrophic mutants ofHaloferax mediterranei andHaloferax gibbonsii

The lethality and mutagenicity in ethyl methanesulfonate (EMS)-treated cells of five archaebacterial strains belonging to each of the three described genera of non-alkaliphilic halobacteria were investigated. In order to test the efficiency of the mutagenesis under a variety of experimental conditions, we chose the fast-growing halobacteriumHaloferax mediterranei as a model strain. A strong induced mutagenicity was found, since the spontaneous mutation rate (expressed as the rate of resistance to the antibiotic josamycin) increased up to 500-fold after mutagen exposure. The mutagenesis was also successfully used in obtaining auxotrophic mutants. Although a heterogeneous response to the induced effects caused after EMS exposure was detected for the other halophilic archaebacteria tested, a clear, efficient mutagenicity ofHalobacterium halobium andHaloferax gibbonsii was observed; auxotrophic mutants of this halobacterium were also produced. Optimal experimental conditions for EMS mutagenesis of some halobacteria were determined.     

Genetic analysis of methylotrophic yeastCandida boidinii PLD1

This paper reports the initial experiments for genetic analysis of the haploid methylotrophic yeastCandida boidinii PLD1. The collection of multiply marked auxotrophic mutants was obtained after treatment with UV-light or X-rays. Protoplasts from several mutants were fused by the PEG-CA²⁺ technique and five prototrophic hybrids were isolated. The genetic structure of the hybrids was studied by means of spontaneous and induced mitotic segregation. Our data suggest that hybrids are diploids, heterozygous by parental auxotrophic markers. We obtained genetic linkage between mutationslys2-8-met-3 from one hand andade-17-arg-24 from the other. The genetic maps constructed showed similar characteristics concerning both the order of the markers and their map distances.     

Mapping of the chromosome ofMycobacterium phlei by means of mutagenesis of the replication point

The aim of the present work was to construct a replication map of the chromosome ofMycobacterium phlei. The method of mutagenesis of the replication point by means of nitrosoguanidine was applied to synchronously multiplying populations. Back mutations and forward mutations were induced m auxotrophic mutants PAmet and PAleu as well as in double auxotrophic mutants with methlomne as the reference marker and the following order of replication of eleven genes on the chromosome was thus established:leu-Eth, Res-Stm, Oyk-pur-met, arg, Cyk-Bac-inl     

The replication map of the chromosome ofMycobacterium phlei

It was the aim of the present work to construct the replication map of the chromosome ofMycobacterium phlei. The method of mutagenesis of the replication point by N-methyl-N-nitroso-N’-nitroguanidine in synchronously dividing populations and the method of analysis of gene frequency were applied. The order of replication of 19 genes on the chromosome was determined by means of induction of back mutations and forward mutations in auxotrophic mutants PAleu and PAmet and in double auxotrophic mutants with methionine as a reference marker.     

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.     

Development of gene transfer systems in Methylobacillus flagellatum KT: Isolation of auxotrophic mutants

A collection of polyauxotrophic mutants of the obligate methylotroph Methylobacillus flagellatum KT was obtained. On the first step two stable auxotrophic mutants with a high requirement for amino acids supplements were isolated by treatment with nitrosoguanidine and selection on complete medium. Spontaneous variants of these mutants with a low requirement for nutrient supplements were the base for obtaining polyauxotrophic strains. It was shown, that the growth of mutants of M. flagellatum KT is inhibited by complete medium. Some amino acids and nucleotides are the inhibitor components of complete media. An approach for selection of auxotrophic mutants of individual genes was worked out on minimal medium. The optimal conditions for nitrosoguanidine mutagenesis of M. flagellatum KT were developed. The possible mechanisms of action of some of the nutrient supplements on the growth of M. flagellatum KT are discussed.     

Direct mating between diploid sake strains of Saccharomyces cerevisiae

Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATα mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.     

Cytogenetic localisation of the purine 1 and guanosine 1 loci of Drosophila melanogaster; the purine 1 locus specifies a vital function.

Summary Three auxotrophic mutants at two loci (pur 1 and gua 1) apparently involved in purine metabolism were mapped cytologically to region 9E1-9E3 on the X-chromosome of Drosophila. Because mutants of pur 1 behaved as semi-lethals against deficiencies even on permissive medium, it appears that the pur 1 locus is vital to the fly and its auxotrophic mutants are hypomorphs.     

Construction of Uracil and Tryptophan Auxotrophic Mutants from Sake Yeasts by Disruption of URA3 and TRP1 Genes

Uracil auxotrophic mutants were constructed from the sake yeasts K-701 and K-901 by successive URA3 gene disruption. First, as sake yeast is diploid, one URA3 gene was disrupted with pURA38 (AURA3 SMR1) and the heterozygous disruptant was isolated on an SM (sulfometuron methyl) plate. The other URA3 gene was disrupted with pURA36 (Δ URA3) and homozygous URA3 disruptants were isolated on FOA (5-fluoro-orotic acid) plate on which only ura3 mutants can grow. Direct URA3 gene disruption with pURA36 (Δ URA3) was also done and the uracil auxotrophic mutant was isolated. Four types of URA3 disruptants were isolated, two of which had no bacterial DNA.

A tryptophan auxotrophic mutant was constructed from one of the URA3 disruptant using pTRP14 (Δ TRP1 URA3) by gene disruption. This TRP1 disruptant was also lacking bacterial DNA.

Laboratory scale sake brewing using the auxotrophic mutants showed that these strains are very useful as recipient strains for molecular breeding of sake yeasts.     

Isolation of auxotrophic mutants and development of conjugation system in KISR methylotrophs

Isolation of auxotrophic mutants and conjugal transfer of plasmids are successfully demonstrated in four obligate methylotrophic strains isolated at Kuwait Institute for Scientific Research (KISR)Out of 11 auxotrophic, mutants, three required methionine and two leucine. Complete transfer of plasmid R68.45 from Pseudomonasto KISR methylotrophs and vice versa have been achieved with a low transfer rate.     

Genetic Recombination in Colletotrichum sublineolum

Genetic recombination without typical parasexuality (parameiosis) was shown in the fungus Colletotrichum sublineolum, causal agent of anthracnose on sorghum. The cross among auxotrophic mutants and mutants resistant to benomyl and cycloheximide confirmed the occurrence of heterokaryosis in this species. Aneuploid and haploid recombinants were obtained from heterokaryons. Some segregants released sectors continually after several replication cycles, and were considered aneuploids in the process of haploidization. Heterokaryons derived from crosses among mutants that presented low pathogenicity produced more virulent recombinants. Parasexuality with parameiosis may represent a natural mechanism for genetic variability amplification explaining the rapid appearance of new C. sublineolum physiological races.     

Improved technique for the isolation of stable mutants ofZymomonas mobilis

Addition of caffeine to the recovering medium after mutagenesis ofZymomonas mobilis by N-methyl-N-nitrosoguanidine increased 4-fold the number of auxotrophic mutants obtained. Moreover, while the mutants isolated without caffeine survived only a few repeated serial transfers on minimal medium supplemented with the required growth factor, 40 % of those obtained in the presence of caffeine were stable.     

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.     

Enrichment of auxotrophic mutants inHansenula polymorpha

Summary An enrichment scheme was designed for the isolation of auxotrophic mutants of the thermotolerant and methanol-utilizing yeast,Hansenula polymorpha, by the use of the polyene antibiotic nystatin. This treatment resulted in auxotrophic isolates at a frequency of 75%. With or without nystatin enrichment, the vast majority of obtained auxotrophs required nucleic acid bases.     

Selection of spontaneous mutants ofYarrowia lipolytica by inositol-less death

Summary A procedure was developed for the selection of spontaneous mutants of the yeastYarrowia lipolytica. An inositol-requiring mutant of a wild-typeY. lipolytica, YB 3-122, was derived by mutagenesis and screening. The mutant had a reversion frequency of less than 6×10^–9. A mutant selection procedure based on inositolless death was then developed using this mutant strain. The selection procedure killed growingY. lipolytica cells and enriched for mutants yielding cultures that consisted of 60–98% spontaneous mutants after two rounds of inositol-less death. The procedure enriched for four classes of mutants, strains that were auxotrophic, metabolite analog sensitive, temperature sensitive, or unable to grow on citric acid as the sole carbon source. Since strain YB 3-122 is now available to yeast researchers, inositol-less death will be useful for the routine isolation of spontaneous mutants ofY. lipolytica.     

Mutation of the cyanobacterium Anacystis nidulans (Synechococcus PCC 6301): Improved conditions for the isolation of auxotrophs

Previous attempts to isolate auxotrophic mutants of Anacystis nidulans produced only a limited range of phenotypes. The frequency of recovery of auxotrophic mutants has been quantified following different mutagenic and selective treatments, and their yield has been improved by using (1) a complete medium, (2) additional mutagens, (3) multiple cycles of penicillin enrichment and (4) altered pre-enrichment starvation conditions. These modified induction and selection conditions permitted the isolation of mutants defective in nitrate reductase, nitrite reductase or malate dehydrogenase, unable to reduce sulphate, or deficient in the synthesis of biotin, thiamine, paminobenzoate, serine, glutamate, adenine or uracil.     

Enrichment for Auxotrophic Mutants of Tetrahymena Using Density Gradient Centrifugation*

SYNOPSIS. A protocol based on density differences between starved and fed cells and employing density gradient centrifugation has been devised to facilitate the isolation of auxotrophic mutants of cell lines derived from Tetrahymena thermophila strain B1868. First, a mass phenotype screening procedure was established whereby true auxotrophic mutants and slow-growing wild-type cells such as strain C* could readily be distinguished. Second, simulation experiments were performed in which wild-type cells starved first in non-nutritive buffer, then suspended in a defined medium lacking a single essential amino acid became significantly denser than the same cells when starved, then suspended in a complete defined medium. Finally, using the same protocol, a reconstruction experiment was carried out which resulted in effective separation of wild-type cells from cells of a tyrosine auxotroph. The overall procedure resulted in a 9-fold increase in the relative frequency of auxotrophic cells, while the density gradient centrifugation alone provided a 400-fold enrichment.     

Mutagenic effect of N-Methyl-N-nitrosourea onMycobacterium phlei

N-Methyl-N-nitrosourea was used to induce auxotrophic, scotochromogenic and isonicotinic acid hydrazide resistant mutants inMycobacterium phlei and its effect was compared with that of nitrosoguanidine. Seventeen auxotrophic mutants requiring arnino acids or vitamins and 52 sootochromosgenic mutants with orange colonies were induced. The frequency of isonicotinic acid hydrazide-resistant mutants increased by two orders of magnitude.