N-methyl-N′-nitro-N-nitrosoguanidine and UV induced mutants of the dinoflagellate Crypthecodinium cohnii*

SYNOPSIS. Mutant strains were chemically induced by treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and UV irradiation. UV and NTG mutation rates were obtained that were both consistent with the organism being haploid. Three types of mutants were produced: (a) strains deficient in both β- and γ-carotene, the only carotenoids found in the wild type; phenotypes include albinos (translucent, dull white, “snow white”) and cream-colored on agar as compared to the yellow-orange color of wild type colonies; (b) strains requiring adenine, guanine or cytosine in addition to the minimal medium for growth; (c) mutants that grow at a rate less than 40% of the wild type in minimal medium.     

基因组改组（genome－shuffling）提高

首先采用紫外线与亚硝基胍两种传统微生物诱变方法对干酪乳杆菌进行诱变,经低pH平板、碳酸钙平板和摇瓶试验获得了5株耐酸性提高的突变菌株。以获得的突变菌株为出发菌株,应用灭活双亲原生质体融合后致死损伤得到互补获得活性融合子的方法,对其进行基因组改组,经过低pH平板、碳酸钙平板和摇瓶筛选,获得4株可以在pH3.8平板上旺盛生长且产酸量较高的改组菌株。将改组菌株与原始菌株分别于pH 3.8和3.4的YE液体培养基中培养,改组菌株能够在原始菌株无法生存的pH条件（pH 3.4）下生长。在pH 3.8的条件下,对改组菌株与原始菌株的发酵特征进行比较,37℃发酵48小时后,改组菌株产酸量为原始菌株的2.4倍,表明基因组改组技术能有效提高多基因调控表型的进化。     

Isolation of paraquat-resistant mutants of Escherichia coli: lack of correlation between resistance and the activity of superoxide dismutase

Abstract Paraquat-resistant Escherichia coli mutants were isolated. The mutants were 10- to 50-fold more resistant to paraquat than the wild type. The wild type was more responsive to the presence of paraquat by inducing higher levels of the manganese-containing superoxide dismutase (MnSOD). Thus, in minimal medium, 0.1 mM paraquat caused a 5-fold increase in MnSOD in the wild type while it had no effect on the level of MnSOD in the mutants. Yet, 50 mM paraquat exerted a dramatic induction of SOD in the mutant strains when grown in trypticase soy yeast extract (TSY) medium. In TSY medium, catalase was not significantly affected by paraquat in all the strains tested. Resistance to paraquat in these mutant strains is, therefore, unrelated to their capacity to detoxify superoxide or hydrogen peroxide.     

Isolation of a sulphate reductase-less mutant of the blue-green alga Nostoc muscorum.

The wild type Nostoc muscorum (UW strain) has yielded various physiological mutants altered in utilization of sulphate, following mutagenic treatments with N-methyl, N'-nitro N-nitrosoguanidine (NTG). One of the mutant strains designated as Sat-20 failed to grow in a medium containing sulphate (MgSO4.7 H2O). However, the mutant strain could grow when supplemented with thiosulphate (Na2S2O3.5 H2O), while methionine could fulfil the sulphur requirement only partially. On comparative reasons, the wild type as well as the mutant showed preference for thiosulphate over other sulphur sources employed.     

亚硝基胍消除链霉菌FR-008的线性质粒

【目的】利用亚硝基胍(NTG)消除链霉菌FR-008线性质粒以简化其基因组,获得背景清晰的菌株,作为抗生素异源生物合成的底盘细胞。【方法】NTG溶液处理链霉菌FR-008孢子悬液,从存活的诱变株中筛选砷敏感的突变株,再通过脉冲场凝胶电泳(PFGE)检测线性质粒是否被消除;用生测实验定性检测各个线性质粒消除突变株杀念菌素合成的能力,最后通过HPLC定量比较突变株和野生型产生杀念菌素的差异。【结果】从103个诱变株中筛选到3株砷敏感的突变株(10#、59#、115#)。PFGE检测发现它们均丢失了大线性质粒p SSFR1,此外,42#突变株的小线性质粒p SSFR2被消除,在此基础上,第二轮NTG诱变获得了双质粒消除的突变株。大线性质粒p SSFR1消除率约为3%,小线性质粒p SSFR2消除率约为1%。发酵结果显示:10#、115#突变株杀念菌素有效组分III产量分别提高了40%和30%。【结论】首次发现NTG是一种有效消除链霉菌线性质粒的诱变剂,2株大线性质粒消除的突变株杀念菌素的产量得到提高。此方法可以用来消除特定链霉菌菌株中的巨型线性质粒以高效简化其基因组,因而是一种有效的抗生素遗传育种的方法。     

Beta-fructofuranosidase production by 2-deoxyglucose resistant mutants of aspergillus niger in submerged and solid-state fermentation

Aspergillus niger produces extracellular beta-fructofuranosidase under submerged (SmF) and solid state fermentation (SSF) conditions. After UV mutagenesis of conidiospores of A. niger, 2-deoxyglucose (10 g/l) resistant mutants were isolated on Czapek's minimal medium containing glycerol as a carbon source and the mutants were examined for improved production of beta-fructofuranosidase in SmF and SSF conditions. One of such mutant DGRA-1 overproduced beta-fructofuranosidase in both SmF and SSF conditions. In SmF, the mutant DGRA-1 showed higher beta-fructofuranosidase productivity (110.8 U/l/hr) than the wild type (48.3 U/l/hr). While in SSF the same strain produced 322 U/l/hr of beta-fructofuranosidase, 2 times higher than that of wild type (154.2 U/l/hr). In SmF, both wild type and mutants produced relatively low level of beta-fructofuranosidase in medium containing sucrose with glucose than from the sucrose medium. However in SSF, the DGRA-1 mutant grown in sucrose and sucrose+ glucose did not show any difference with respect to beta-fructofuranosidase production. These results indicate that the catabolite repression of beta-fructofuranosidase synthesis is observed in SmF whereas in SSF such regulation was not prominent.     

High-level production of extracellular lipase by Yarrowia lipolytica mutants from methyl oleate

The yeast Yarrowia lipolytica degrades efficiently low-cost hydrophobic substrates for the production of various added-value products such as lipases. To obtain yeast strains producing high levels of extracellular lipase, Y. lipolytica DSM3286 was subjected to mutation using ethyl methanesulfonate (EMS) and ultraviolet (UV) light. Twenty mutants were selected out of 1600 mutants of Y. lipolytica treated with EMS and UV based on lipase production ability on selective medium. A new industrial medium containing methyl oleate was optimized for lipase production. In the 20 L bioreactor containing new industrial medium, one UV mutant (U6) produced 356 U/mL of lipase after 24h, which is about 10.5-fold higher than that produced by the wild type strain. The properties of the mutant lipase were the same as those of the wild type: molecular weight 38 kDa, optimum temperature 37°C and optimum pH 7. Furthermore, the nucleotide sequences of extracellular lipase gene (LIP2) in wild type and mutant strains were determined. Only two silent substitutions at 362 and 385 positions were observed in the ORF region of LIP2. Two single substitutions and two duplications of the T nucleotide were also detected in the promoter region. LIP2 sequence comparison of the Y. lipolytica DSM3286 and U6 strains shows good targets to effective DNA recombinant for extracellular lipase of Y. lipolytica.     

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.     

The properties of UV sensitive mutants of Escherichia coli K12 which are also refractory to colicin E2

Summary Among mutants refractory to colicin E2 at low temperatures but sensitive at high temperatures (designated Ref-II), three strains are described which are also UV sensitive. Although colicin refractivity is temperature dependent UV sensitivity is expressed at all temperatures. Although the UV sensitive lesion appears to be similar in its effects to that in Rec (recombinationless) strains, mutants specifically isolated as Rec strains are in fact more sensitive to E2 than are wild type strains. It is suggested that E2 refractivity and UV sensitivity in the mutants probably reflects the phenotypic expression of distinct although linked genes. It is also suggested that the degradation of DNA stimulated by adsorption of E2 to wild type bacteria may be caused by the same enzyme(s) which causes enhanced breakdown of DNA in some rec ^– mutants after UV irradiation.     

Xanthomonas campestris, a novel stress tolerant, phosphate-solubilizing bacterial strain from saline–alkali soils

A total of 198 bacterial strains were isolated from various niches of saline–alkali soils, out of which 85 strains were able to solubilize phosphate on plates at 30 °C. The strain RMLU-26, identified as Xanthomonas campestris, was the most efficient with its ability to solubilize P, subjected to N-methyl-N′-nitro-N-nitrosoguanidine (NTG) for development of mutants. The P solubilizing ability of X. campestris is reported for the first time. The wild type and mutant strains of X. campestris revealed a differential response to various stress factors (high pH, temperature, and salt concentration). The mutant strain revealed maximum P solubilization (67.1%) at 30 °C and pH 8.0 while the wild type strain showed maximum solubilization (41.9%) at 35 °C and pH 7.0. Percent P₂O₅ solubilization by both strains revealed a steep decline in tricalcium phosphate solubilization with an increase in NaCl concentration from 0.5 to 10% along with a concomitant drop in pH of the medium from 8.0 to 4.5 in wild type and 4.0 in mutant strain. However, a 1.5- to 2-fold increase in ‘P’ solubilization was observed in the mutant strain when compared to the wild type strain in the presence of NaCl. The overall improved tolerance of the strains to alkalinity and salinity could be due to accumulation and/or secretion of specific solute (xanthan).     

Combining induced mutation and protoplasting for strain improvement of Aspergillus oryzae for kojic acid production

By combining induced mutation, using NTG and UV irradiation, and protoplasting of a wild type strain of Aspergillus oryzae ATCC 22788, a hyper-producing strain was obtained that accumulated 41 g kojic acid l(-1) in shake-flasks, which was 100-fold higher than that in the wild type strains. Similar production of kojic acid was obtained in 5 l stirred-tank fermentations.     

Mutant Analysis and Enzyme Subunit Complementation in Bacterial Bioluminescence in Photobacterium fischeri

Chemical mutagens were used to obtain mutants deficient in bioluminescence in the marine bacterium Photobacterium fischeri strain MAV. Acridine dyes were effective in the production of dark mutants but not in the production of auxotrophs. These dark mutants were all of one type and appeared to contain lesions blocking the synthesis of luciferase. ICR-191 was especially effective in the production of aldehyde mutants, i.e., dark strains that luminesce when a long-chain aldehyde such as n-decanal is added to them. However, other mutant types were isolated after treatment with ICR-191. N-methyl-N'-nitro-N-nitrosoguanidine induced many bioluminescence-deficient types with respect to both the site of the lesion and the quantitative effect on the luminescent system. We characterized the dark and dim mutants with respect to their response to exogenous decanal, levels of in vivo and in vitro luminescence, and their rates of reversion to wild type. In addition, the luciferases of the mutant strains were examined by subunit complementation. On the basis of these analyses, we identified mutants which synthesize altered luciferase, strains which are deficient in synthesis of luciferase, and aldehyde mutants. The results of analysis of luciferase from the aldehyde mutants and the complementation studies indicate that the lesions in these strains are in the luciferase itself. Results obtained with wild-type cells grown in minimal medium, and aldehyde mutant cells grown either in complete or minimal medium, indicate that a "natural aldehyde factor" is involved in in vivo light emission. These same studies showed that the long-chain aldehyde(s) could only partially substitute for the natural "aldehyde factor." The possibility that the in vivo aldehyde factor is not a long-chain aldehyde is discussed.     

Cellular inactivation and mitotic recombination induced by ultraviolet radiation in aneuploid and euploid strains of Candida albicans

Prototrophic aneuploid and euploid derivatives of wild type Candida albicans strain 207 were produced by fusing protoplasts of complementing auxotrophs obtained from strain 207. Comparisons of cell survivals and incidences of mitotic recombinants ocurring after ultraviolet irradiation (UV) of these strains indicate that (i) aneuploids are categorically less efficient than euploids for repair of pyrimidine dimers induced in DNA by UV and that (ii) such repair is enhanced by growing irradiated cells at 25° C, on minimal medium or in the presence of ergosterol rather than at 37° C, on amino acid enriched medium or medium unsupplemented with ergosterol. In addition, the comparisons establish than one cannot discriminate between strains of C. albicans which differ in cellular DNA contents or genomic constitutions on the basis of their UV survival curves.     

NH(4)-Excreting Azospirillum brasilense Mutants Enhance the Nitrogen Supply of a Wheat Host

Spontaneous ethylenediamine-resistant mutants of Azospirillum brasilense were selected on the basis of their excretion of NH(4). Two mutants exhibited no repression of their nitrogenase enzyme systems in the presence of high (20 mM) concentrations of NH(4). The nitrogenase activities of these mutants on nitrogen-free minimal medium were two to three times higher than the nitrogenase activity of the wild type. The mutants excreted substantial amounts of ammonia when they were grown either under oxygen-limiting conditions (1 kPa of O(2)) or aerobically on nitrate or glutamate. The mutants grew well on glutamate as a sole nitrogen source but only poorly on NH(4)Cl. Both mutants failed to incorporate [C]methylamine. We demonstrated that nitrite ammonification occurs in the mutants. Wild-type A. brasilense, as well as the mutants, became established in the rhizospheres of axenically grown wheat plants at levels of > 10 cells per g of root. The rhizosphere acetylene reduction activity was highest in the preparations containing the mutants. When plants were grown on a nitrogen-free nutritional medium, both mutants were responsible for significant increases in root and shoot dry matter compared with wild-type-treated plants or with noninoculated controls. Total plant nitrogen accumulation increased as well. When they were exposed to a N(2)-enriched atmosphere, both A. brasilense mutants incorporated significantly higher amounts of N inside root and shoot material than the wild type did. The results of our nitrogen balance and N enrichment studies indicated that NH(4)-excreting A. brasilense strains potentially support the nitrogen supply of the host plants.     

Isolation of endopolygalacturonase hyperproducing mutants ofAspergillus sp. CH-Y-1043

Summary With the aim of obtaining hyperproducing strains of pectinases,Aspergillus sp. CH-Y-1043 was mutated with NTG and mutants resistant to glycerol catabolic repression were selected. Among the mutants obtained, CH-SS/ M63 produced an endo-PG activity 400% higher than the wild type, using lemon peel as the sole carbon source.     

The role of replication initiation control in promoting survival of replication fork damage

Dam methylase mutants were recovered in a screen for mutants sensitive to UV irradiation or mild inhibition of replication elongation. Dam's role in tolerance of DNA damage is to provide binding sites for SeqA, because seqA mutants showed similar sensitivity that was genetically epistatic to dam. The sensitivity of seqA mutants to UV irradiation and to the replication inhibitors hydroxyurea (HU) and azidothymidine (AZT) was suppressed by alleles of dnaA that reduce the efficiency of replication initiation. These results suggest that for survival of replication fork damage, SeqA's repression of replication initiation is more important than its effects on nucleoid organization. Convergence of forks upon DNA damage is a likely explanation for seqA mutant sensitivity, because its poor survival of UV was suppressed by reducing secondary initiation through minimal medium growth. Surprisingly, growth in minimal medium reduced the ability of seqA+ strains to form colonies in the presence of low levels of AZT. Double dnaA seqA mutants exhibited plating efficiencies much superior to wild-type strains during chronic low-level AZT exposure in minimal medium. This suggests that mild inhibition of replication fork progression may actively restrain initiation such that seqA+ strains fail to recover initiation capacity after sustained conditions of replication arrest.     

Repair of Ultraviolet Radiation Damage in Sensitive Mutants of Micrococcus radiodurans

Various aspects of the repair of ultraviolet (UV) radiation-induced damage were compared in wild-type Micrococcus radiodurans and two UV-sensitive mutants. Unlike the wild type, the mutants are more sensitive to radiation at 265 nm than at 280 nm. The delay in deoxyribonucleic acid (DNA) synthesis following exposure to UV is about seven times as long in the mutants as in the wild type. All three strains excise UV-induced pyrimidine dimers from their DNA, although the rate at which cytosine-thymine dimers are excised is slower in the mutants. The three strains also mend the single-strand breaks that appear in the irradiated DNA as a result of dimer excision, although the process is less efficient in the mutants. It is suggested that the increased sensitivity of the mutants to UV radiation may be caused by a partial defect in the second step of dimer excision.     

Improvement of rifamycin B production using mutant strains of Amycolatopsis mediterranei

Two mutant strains of Amycolatopsis mediterranei VA17 and VA18 were isolated using physical (UV) and chemical (NTG) mutagens gave high rifamycin B than the parent type when grown in the same fermentation medium with a pH of 7.2, temperature 32v°C for a period of 12 days. The cultural conditions of both mutant strains are similar to the parent strain except temperature which was higher by 4v°C. By this mutation and selection study, rifamycin B production was improved from 1400 mg/l to 2450 mg/l.     

Mutants of luminous bacteria with an altered control of luciferase synthesis.

Arginine is known to increase the luminescence in vivo and in vitro of the marine bacterium Beneckea harveyi growing in minimal medium. Mutants in which this arginine effect is either diminished, or absent were isolated as bright clones on a minimal medium after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. On a minimal medium both with and without added arginine and also on complex medium, these "minimal bright" mutants produce higher levels of luminescence than the wild type both in vivo and in vitro. This is attributed to the production of an increased amount of luciferase, which itself is wild type in terms of its specific activity.