UV and Ethyl Methanesulfonate Effects in Hyperthermophilic Archaea and Isolation of Auxotrophic Mutants of Pyrococcus Strains

The lethal and mutagenic effects of ethyl methanesulfonate (EMS) and UV on nine archaeal strains belonging to each of the two described genera of Thermococcales, Pyrococcus and Thermococcus, were investigated. To test the efficiency of the EMS and UV mutagenesis under a variety of experimental conditions, we chose Pyrococcus abyssi strain GE5 as a model strain. We observed a strong induced mutagenicity in both cases, since the spontaneous mutation frequency (expressed as the frequency of resistance to 5-fluoroorotic acid) increased up to 150-fold with EMS and 400-fold with UV, after mutagen exposure. Although a heterogeneous response to the induced effects caused after EMS and UV exposures was detected for all the other sulfothermophilic archaea tested, an efficient mutagenicity of Pyrococcus-like isolates GE27, GE23, and GE9 was observed. Optimal procedures described for UV mutagenesis yielded a number of useful uracil auxotrophic mutant strains of Pyrococcus abyssi. Received: 2 May 1996 / Accepted: 3 July 1996     

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.     

Nuclear and extranuclear mutations in yeast induced by ethyl methanesulfonate

When zero-point mutations were induced in the yeastSaccharomyces cerevisiae using ethyl methanesulfonate (EMS) no differences were found in the frequency of auxotrophic mutants formed by a short and a prolonged treatment of the agent at equal survival level. The expression of a part of the mutations induced by a prolonged EMS treatment was delayed by one or two division cycles. The total frequency of auxotrophs due to both the zero point and delayed mutations, however, is still considerably lower than the frequency of auxotrophs induced by a prolonged treatment of EMS in some bacterial species. Both the prolonged and short EMS treatment induces in yeast also extranuclear respiration-deficient (RD) mutants at a relatively high frequency; in wild strains at equal survival level the prolonged treatment produces a higher number of RD mutants than the short one. In strain which is more susceptible to the lethal EMS effect than wild strain the number of RD mutants produced by the agent is much higher than in the wild strain. The results support the assumption of the different DNA arrangement in yeast nuclei and mitochondria and indicate the possible effect of repair mechanisms during the induction of mutations causing the respiration deficiency.     

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.     

Regulatory mutations affecting the synthesis of cellulase in Bacillus pumilus

A wild strain of Bacillus pumilus was investigated for cellulase production, and putative mutants of this strain were screened for catabolite repression insensitivity after chemical mutagenesis using ethyl methanesulphonate (EMS) as a mutagenic agent. Out of four classes of mutants studied and classified according to their cellulase induction rate and level of cellulase production in the presence of high concentrations of glucose (2.6%[w/v]), classes III and IV exhibited cellulase production up to 6.2 mg cellulase and 11.4 mg cellulase per gram of dry cell mass respectively. These mutants were referred to as catabolite repression-insensitive when compared to the wild strain which exhibited a total repression of cellulase synthesis under the same conditions. How EMS triggered the catabolite repression insensitivity in these mutants was not established. However this mutation brought out new strains of cellulase hyperproducers (mutants 6 and 11) in the presence of glucose when compared to other cellulase producers such as Aspergillus terreus, A. nidulans and Trichoderma reesei, which exhibited catabolite repression of cellulase synthesis. These mutants were selected as the most promising candidates for cellulase synthesis even at high glucose concentration.     

Improving ethanol fermentation performance of Saccharomyces cerevisiae in very high-gravity fermentation through chemical mutagenesis and meiotic recombination

Genome shuffling is an efficient way to improve complex phenotypes under the control of multiple genes. For the improvement of strain’s performance in very high-gravity (VHG) fermentation, we developed a new method of genome shuffling. A diploid ste2/ste2 strain was subjected to EMS (ethyl methanesulfonate) mutagenesis followed by meiotic recombination-mediated genome shuffling. The resulting haploid progenies were intrapopulation sterile and therefore haploid recombinant cells with improved phenotypes were directly selected under selection condition. In VHG fermentation, strain WS1D and WS5D obtained by this approach exhibited remarkably enhanced tolerance to ethanol and osmolarity, increased metabolic rate, and 15.12% and 15.59% increased ethanol yield compared to the starting strain W303D, respectively. These results verified the feasibility of the strain improvement strategy and suggested that it is a powerful and high throughput method for development of Saccharomyces cerevisiae strains with desired phenotypes that is complex and cannot be addressed with rational approaches.     

A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis

Background  

Arabidopsis thaliana is the main model species for plant molecular genetics studies and world-wide efforts are devoted to identify the function of all its genes. To this end, reverse genetics by TILLING (Targeting Induced Local Lesions IN Genomes) in a permanent collection of chemically induced mutants is providing a unique resource in Columbia genetic background. In this work, we aim to extend TILLING resources available in A. thaliana by developing a new population of ethyl methanesulphonate (EMS) induced mutants in the second commonest reference strain. In addition, we pursue to saturate the number of EMS induced mutations that can be tolerated by viable and fertile plants.     

Physical and chemical mutagens improved Sporotrichum thermophile,strain ST20 for enhanced Phytase activity

ObjectivePhosphorous is an essential micronutrient of plants and involved in critical biological functions. In nature, phosphorous is mostly present in immobilized inorganic mineral and in the fixed organic form including phytic acid and phosphoesteric compounds. However, the bioavailability of bound phosphorous could be enhanced by the use of phosphate solubilizing microorganisms such as bacteria and fungi. The phytases are widespread in an environment and have been isolated from different sources comprising bacteria and fungi.MethodologyIn current studies, we show the successful use of gamma rays and EMS (Ethyl Methane Sulphonate) mutagenesis for enhanced activity of phytases in a fungal strain Sporotrichum thermophile.ResultsWe report an improved strain ST2 that could produce a clear halo zone around the colony, up to 24 mm. The maximum enzymatic activity was found of 382 U/mL on pH 5.5. However, the phytase activity was improved to 387 U/ml at 45 °C. We also report that the mutants produced through EMS showed the greater potential for phytase production.ConclusionThe current study highlights the potential of EMS mutagenesis for strain improvement over physical mutagens.     

Amplification of the tryptophan operon gene in Escherichia coli chromosome to increase l-tryptophan biosynthesis

Classical mutagenesis could desensitize the feedback inhibition of l-tryptophan (l-Trp) biosynthesis. Among the mutants, a5-fluorotryptophan-resistant strain, Escherichia coli EMS4-C25 produced 3 g/l of l-Trp within 18 h. The feedback-resistant l-Trp operon gene (trp) prepared from E. coli EMS4-C25 was inserted into pUC19 and pHSG576 to generate pTC701 and pTC576, respectively. When pHSG576 and pTC701 were introduced into E. coli EMS4-C25, chromosomal integration occured through homologous recombination. By using Souther hybridization, we demostrated that the integrated plasmids existed as multicopies. The strains with integrated foreign trp operon gene had higher activities of anthranilate synthase and Trp synthase than those found for the host strain and produced 9.2 g/l of l-Trp with 13% conversion yield from d-glucose. The integration and implification of the trp-operon-beraing plasmid avoided the plasmid instability and increased l-TRp production. Correspondence to: E.-C. Chan     

Improvement of lincomycin production by mutant selection and metabolic regulation

Lincomycin is a lincosamide antibiotic produced by Streptomyces lincolnensis. Through mutagenesis by ethylmethansulfonate (EMS) and ultraviolet (UV) irradiation repeatedly, M2 was picked out in plate with glutamine and propylproline orderly. In 50-L stirred bioreactor, the production of lincomycin, fermented by M2, was increased to 8136?u/ml under the optimal condition as compared to original strain S. lincolnensis 07–5 (6634?u/ml). Two-dimensional gel electrophoresis (2-D GE) and mass spectrometry (MS)-shown LmbG, LmbI, and acetohydroxy acid isomeroreductase were remarkably synthesized in M2. The gene lmbG and lmbI are responsible for methylation in the lincomycin biosynthetic cluster, while acetohydroxy acid isomeroreductase contributes to stronger metabolic capability. Finally, we obtained a better strain for industrial production.     

Effect of ethylmethanesulfonate on Chlamydomonas reinhardi

Summary Ethylmethane sulfonate (EMS) is known to cause a considerably high mutation rate in higher plants. In our experiments with Chlamydomonas reinhardi however, the mutagenic effect was unexpectedly low, whereas the toxic effect was quite remarkable. It is supposed that the reason for the low rate of mutants is the high toxicity, since non-toxic EMS concentrations induce no mutants. The toxic effect on Chlamydomonas cells is caused not only by the products of hydrolysis of the EMS, but also by the EMS itself. The damaged cells begin to bleach, furthermore they are not able to deliver their daughter cells. To a certain degree both effects are reversible. Finally it was found that the sensivity to EMS was higher in cells of the mating type — than it was in those of the+mating type.     

Genome assembly of the Chinese maize elite inbred line RP125 and its EMS mutant collection provide new resources for maize genetics research and crop improvement

Maize is an important crop worldwide, as well as a valuable model with vast genetic diversity. Accurate genome and annotation information for a wide range of inbred lines would provide valuable resources for crop improvement and pan-genome characterization. In this study, we generated a high-quality de novo genome assembly (contig N50 of 15.43 Mb) of the Chinese elite inbred line RP125 using Nanopore long-read sequencing and Hi-C scaffolding, which yield highly contiguous, chromosome-length scaffolds. Global comparison of the RP125 genome with those of B73, W22, and Mo17 revealed a large number of structural variations. To create new germplasm for maize research and crop improvement, we carried out an EMS mutagenesis screen on RP125. In total, we obtained 5818 independent M2 families, with 946 mutants showing heritable phenotypes. Taking advantage of the high-quality RP125 genome, we successfully cloned 10 mutants from the EMS library, including the novel kernel mutant qk1 (quekou: “missing a small part” in Chinese), which exhibited partial loss of endosperm and a starch accumulation defect. QK1 encodes a predicted metal tolerance protein, which is specifically required for Fe transport. Increased accumulation of Fe and reactive oxygen species as well as ferroptosis-like cell death were detected in qk1 endosperm. Our study provides the community with a high-quality genome sequence and a large collection of mutant germplasm.     

A new class of arginine-requiring mutants in Chlamydomonas reinhardi

Summary Forward mutations to auxotrophy have been induced in Chlamydomonas reinhardi after treatment of wild-type cells with ethyl methanesulfonate (EMS) and plating on a selective medium supplemented with yeast extract but deprived of ammonium chloride.Among the 41 stable mutants isolated, 6 were arginine-requiring. Reconstruction experiments show that the growth of all these arginine auxotrophs is inhibited on media containing high concentrations of NH ₄ ⁺ ions. It is concluded that the isolation of this new class of mutants was made possible because of the low ammonium concentration of the plating medium.It appears therefore that in Chlamydomonas, as in other microorganisms, the specificity of mutations may be imposed by the selective medium.Chargé de Recherches du Fonds National de la Recherche Scientifique.     

Nouvelles données sur l'écologie et le comportement entomopathogène expérimental de l'entomophthoraleConidiobolus coronatus (Zygomycètes)

Résumé Quelle que soit l'origine de la souche (sol, arthropodes, lésions humaines), les conidies de l'EntomophthoraleConidiobolus coronatus sont capables d'infecter le puceronAcyrthosiphon pisum (Hémiptères, Aphididae) et les chenilles deGalleria mellonella (Lépidoptères, Pyralidae). De plus, les filtrats, de culture en milieu liquide agité des huit souches éprouvées sont toxiques par inoculation à l'égard de ces derniers insectes; cette toxicité se traduit d'abord par une hémorragie au trou de pip?re, qui peut durer plusiers heures, puis par un noircissement du corps de l'insecte et enfin par la mort de celui-ci. L'effet anticoagulant de substances produites par une Entomophthorale est ici signalé pour la première fois. Les deux souches les plus virulentes à l'égard deAcyrthosiphon pisum, parmi lesquelles se trouve celle isolée de lésions humaines, sont également les plus toxinogènes. L'étude de la vitesse de croissancein vitro du champignon à des températures comprises entre 4 et 37°C révèle une adaptation des souches aux conditions thermiques en relation avec la région d'origine de l'isolat. Notamment, les souches d'origine tropicale se développent plus, rapidement à 26–29°C que les souches originaires de France. La vitesse maximale de croissance est constatée à 29°C pour les différentes souches, sauf pour celle isolée de lésions humaines qui se développe le plus rapidement à 37°C. Cette température est létale pour les autres souches sauf pour une souche isolée d'un myriapode de l'?le de la Réunion. C'est la première fois que l'aptitude à cro?tre à cette température est reconnue chez un isolat deConidiobolus coronatus provenant d'un arthropode.

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Summary Whatever the substrate of origin of the strain (soil, arthropods, human phycomycosis), conidia ofConidiobolus coronatus (Zygomycotina, Entomophthorales) are able to infect the pea aphid,Acyrthosiphon pisum (Hemiptera, Aphididae) and the greater wax moth,Galleria mellonella (Lepidoptera, Pyralidae). Moreover, intrahemocoelic injections of filtrates from shake cultures of all the 8 strains tested proved to be toxic for the later insect; the symptoms are successively haemorrhage, which could last for a few hours, blackening of the blood and at last death. This is the first mention of an anti-coagulant effect of metabolic products from any Entomophthorale. The strain isolated from an human phycomycosis is both the most virulent against the pea aphid and the most effective with regard to toxicity. Comparison of fungal growthin vitro at different temperatures, ranging from 4 to 37°C, revealed a certain relation between the temperature for the optimal growth and the locality of origin of a strain. In particular, strains from tropical areas were growing at 26–29°C faster than those from temperate regions. The higher growth rate was noticed at 29°C for all the 8 tested strains, except for the strain from human phycomycosis, for which the optimum temperature was 37°C. This temperature was lethal for the other strains, but for a strain isolated from a myriapod from the Reunion Island. It is the first time that the ability of a strain ofC. coronatus isolated from an arthropod to growth at 37°C is ascertained.

     

Regulation of the Pyrimidine Biosynthetic Pathway in <Emphasis Type="Italic">Pseudomonas mucidolens</Emphasis>

Control of pyrimidine biosynthesis was examined in Pseudomonas mucidolens ATCC 4685 and the five de novo pyrimidine biosynthetic enzyme activities unique to this pathway were influenced by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. When uracil was supplemented to glucose-grown ATCC 4685 cells, activities of four de novo enzymes were depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the glucose-grown mutant strain cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase activities to increase by more than 3-fold while OMP decarboxylase activity increased by 2.7-fold. The syntheses of the de novo enzymes appeared to be regulated by pyrimidines. At the level of enzyme activity, aspartate transcarbamoylase activity in P. mucidolens ATCC 4685 was subject to inhibition at saturating substrate concentrations. Transcarbamoylase activity was strongly inhibited by UTP, ADP, ATP, GTP and pyrophosphate.     

Regulation of methanol metabolism in the yeast Hansenula polymorpha

A study of enzyme profiles in Hansenula polymorpha grown on various carbon substrates revealed that the synthesis of the methanol dissimilatory and assimilatory enzymes is regulated in the same way, namely by catabolite repression and induction by methanol. Mutants of H. polymorpha blocked in dihydroxyacetone (DHA) synthase (strain 70 M) or DHA kinase (strain 17 B) were unable to grow on methanol which confirmed the important role attributed to these enzymes in the biosynthetic xylulose monophosphate (XuMP) cycle. Both mutant strains were still able to metabolize methanol. In the DNA kinase-negative strain 17 B this resulted in accumulation of DHA. Although DHA kinase is thought to be involved in DHA and glycerol metabolism in methylotrophic yeasts, strain 17 B was still able to grow on glycerol at a rate similar to that of the wild type. DHA on the other hand only supported slow growth of this mutant when relatively high concentrations of this compound were provided in the medium. This slow but definite growth of strain 17 B on DHA was not based on the reversible DHA synthase reaction but on conversion of DHA into glycerol, a reaction catalyzed by DNA reductase. The subsequent metabolism of glycerol in strain 17 B and in wild type H. polymorpha, however, remains to be elucidated.Abbreviations XuMP xylulose monophosphate - DHA dihydroxyacetone - EMS ethyl methanesulphonate     

The role of umuC gene product in mutagenesis by simple alkylating agents

Summary This paper describes studies to determine the role of the umuC gene product in the process of alkylation induced mutagenesis. An active umuC gene is necessary for most MMS induced mutagenesis but it is not essential for EMS nor for MNNG induced mutagenesis in either normal or adapted cultures. In this respect the umuC mutation differs from lexA mutations which have a striking effect on MNNG induced mutagenesis (Schendel, et al., 1978). These findings have prompted a re-evaluation of these previously published data and the advancement of an hypothesis which explains the lexA effect without evoking a role for error-prone repair in the process of alkylation induced mutagenesis.It was also observed that exposure to MNNG is capable of generating a small amount of W-reactivation and W-mutagenesis capacity in a umuC strain which is totally blocked for UV induced reactivation. In light of this result a possible function for the umuC gene product is discussed.     

A survey of natural and ethyl methane sulfonate-induced variations of eIF4E using high-resolution melting analysis in Capsicum

Allele mining is a method used to find undiscovered natural variations or induced mutations in a plant, and has become increasingly important as more genomic information is available in plants. A high-throughput method is required to facilitate the identification of novel alleles in a large number of samples. In this paper we describe the application of a high-resolution melting (HRM) method to detect natural variations and ethyl methane sulfonate (EMS)-induced mutations in Capsicum. We have scanned single polymorphic mutations in the first exon of the eIF4E gene, wherein the mutations confer resistance to potyviruses. Sixteen allelic variations out of 248 germplasm collections were identified using HRM analysis, and one accession carrying an allelic variation (pvrHRM1 ¹³ ) was confirmed to be resistant to the TEV-HAT strain. In addition, five single polymorphic mutations in the eIF4E gene were identified in an EMS-induced mutant population. These results demonstrate that HRM allows for the rapid identification of new allelic variants in both natural and artificial mutant populations.     

Morphometric and Biochemical Changes in <i>Agave americana</i> L. Plantlets Induced By Ethyl Methanesulfonate

A. americana L. is a crop with very little genetic variability. In order to evaluate the effect of ethyl methanesulfonate (EMS) to induce variability in in vitro plantlets of A. americana, different explants (meristems, leaves and roots) were evaluated for the production of callus. MS medium supplemented with ANA (2.68 μM) and BAP (2.68 μM) was used. Callus obtained from apical meristem were treated with 15 mM EMS for two hours after which shoot formation was induced using 2,4-D (0.11 μM) and BAP (44 μM). The EMS induced variations in the morphometric and morphological parameters of the plantlets obtained, with 60% of the plantlets presenting differences such as dwarfism and different leaf forms, without the presence of spines, as well as an increase in fructan content of 30% with respect to the control plantlets. PAL was increased and this activity is related with higher anthocyanins concentration in A. americana L. plantlets.     

高皂苷轮叶党参EMS诱变突变体研究

以轮叶党参为材料,采用甲基磺酸乙酯(EMS)处理离体叶片和愈伤组织对轮叶党参进行诱变,选择最佳诱变组合,并对诱变再生群体进行遗传分析。结果表明:(1)轮叶党参叶片和愈伤组织经EMS处理的存活率和分化率均低于对照,并且随EMS浓度的升高和处理时间的延长而下降。(2)叶片和愈伤组织的致死处理组合分别是0.4%EMS处理4h和0.3%EMS处理4h,半致死组合分别为0.3%EMS处理2h和0.2%EMS处理2h,愈伤组织是EMS诱变轮叶党参的的最佳材料。(3)筛选出的6号变异株皂苷含量为5.061mg/g,较对照平均值提高了5.48%。(4)对诱变再生苗进行遗传分析,8个特异引物对10个供试材料共扩增出59条带,具有多态性的谱带数为44条,占74.6%。材料间的相似系数变化范围0.453～0.912,其中3号、7号株与其他8株达到了品种间遗传差异。研究认为,EMS处理可应用于轮叶党参无性变异系的诱变,3号、6号、7号植株为诱变产生的具有较高皂苷含量的初选植株。