Selective enrichment of aromatic amino acid auxotrophs in Hansenula polymorpha.

Aromatic amino acid auxotrophs of the methanol-utilizing yeast Hansenula polymorpha were effectively selected by the use of nystatin and a medium that inhibits the growth of tyrosine auxotrophs. The procedure resulted in a frequency of aromatic auxotrophs of 2% of survivors and an enrichment of 20-fold. The new procedure also takes less time than traditional procedures. Of the auxotrophic mutants isolated, two-thirds required tyrosine and the remainder were tyrosine-phenylalanine double auxotrophs.     

Selective enrichment of aromatic amino acid auxotrophs in Hansenula polymorpha.

Aromatic amino acid auxotrophs of the methanol-utilizing yeast Hansenula polymorpha were effectively selected by the use of nystatin and a medium that inhibits the growth of tyrosine auxotrophs. The procedure resulted in a frequency of aromatic auxotrophs of 2% of survivors and an enrichment of 20-fold. The new procedure also takes less time than traditional procedures. Of the auxotrophic mutants isolated, two-thirds required tyrosine and the remainder were tyrosine-phenylalanine double auxotrophs.     

Isolation of Candida tropicalis auxotrophic mutants

An enrichment scheme using nystatin was designed for the isolation of auxotrophic mutants from the diploid-alkane-utilizing yeast Candida tropicalis. A collection of 194 auxotrophs representing 7 phenotypes was isolated. One class of mutants was identified as having a defect in histidinol dehydrogenase activity and a second class of mutants was identified as having a defect in orotidine monophosphate decarboxylase activity. These strains are good candidates to be carrying mutations corresponding to the HIS4 and URA3 genes of Saccharomyces cerevisiae.     

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.     

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.     

Optimization on the selection of auxotrophic mutants in Candida utilis by snail enzyme treatment

The determination of the best conditions for the application of the snaill enzyme digestion method in the enrichment of auxotrophic mutants in Candida utilis was carried out following Box and Wilson's mathematical method. The selection procedure proposed was tested in the enrichment of auxotrophic mutants from a mutagenized culture of a wild-type strain. Mutant frequency was increased 46-fold by treatment with snail enzyme. The method also proved useful in the selection of additional auxotrophic mutations from single auxotrophs.     

A new mutation protocol for obtaining auxotrophic mutants of the yeastPhaffia rhodozyma

Summary A new mutation strategy, which involves -irradiation of cells followed by a selective enzymatic enrichment step, was worked out to obtain auxotrophic mutants from the astaxanthin-producing yeast P. rhodozyma. Under the optimized conditions described, different mutants suitable for strain improvement were isolated.     

Development of an integrative DNA transformation system for the yeast Candida tropicalis.

We developed the alkane and fatty-acid utilizing yeast Candida tropicalis as a host for DNA transformations. The system is based on an auxotrophic mutant host of C. tropicalis which is defective in orotidine monophosphate decarboxylase (ura3). The ura3 host was isolated by mutagenesis and a double-selection procedure that combined nystatin enrichment selection and 5-fluoro-orotic acid resistance selection. As a selectable marker, we isolated and characterized the C. tropicalis URA3 gene. Plasmid vectors that contained the C. tropicalis URA3 gene transformed the C. tropicalis mutant host at a frequency of 10(3) to 10(4) transformants per micrograms of plasmid DNA. Vectors that contained the Saccharomyces cerevisiae URA3 gene could not transform C. tropicalis. DNA transfer was accomplished by modified versions of either spheroplast generation (CaCl2-polyethylene glycol)-fusion or cation (LiCl) procedures developed for S. cerevisiae. Plasmid vectors that had been cut within the C. tropicalis URA3 fragment integrated by homologous recombination at the URA3 locus.     

A reusable method for construction of non-marker large fragment deletion yeast auxotroph strains: A practice in Torulopsis glabrata

A reusable method for construction of yeast auxotrophic mutants with large deletions that do not contain markers was developed and successfully applied to a pyruvate overproducing yeast strain, Torulopsis glabrata CCTCC M202019. A URA3 knockout fragment with a large deletion of the open reading frame and long homologous arms was constructed by fusion PCR and transformed into the parent strain by high-efficiency electroporation. A high concentration of transformed yeast was achieved by subsequently culturing in rich medium for 24 h and in nitrogen-free minimal medium for 4 h. Potential Deltaura3 auxotrophic mutants were enriched by treatment with nystatin and selection on uracil-limited medium. Mutants were confirmed by streaking cultures and colony PCR. Deltaarg8 and Deltaura3Deltaarg8 double auxotroph mutants were also successfully constructed using this method.     

Biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces noursei

The polyene macrolide antibiotic nystatin, produced commercially by the bacterium Streptomyces noursei, is an important antifungal agent used in human therapy for treatment of certain types of mycoses. Early studies on nystatin biosynthesis in S. noursei provided important information regarding the precursors utilised in nystatin biosynthesis and factors affecting antibiotic yield. New insights into the enzymology of nystatin synthesis became available after the gene cluster governing nystatin biosynthesis in S. noursei was cloned and analysed. Six large polyketide synthase proteins were implicated in the formation of the nystatin macrolactone ring, while other enzymes, such as P450 monooxygenases and glycosyltransferase, were assumed responsible for ring decoration. The latter data, supported by analysis of the polyene mixture synthesised by the nystatin producer, helped elucidate the complete nystatin biosynthetic pathway. This information has proved useful for engineered biosynthesis of novel nystatin analogues, suggesting a plausible route for the generation of potentially safer and more efficient antifungal drugs.     

Isolation of Clostridium thermocellum Auxotrophs

Spontaneous and UV irradiation-induced auxotrophic mutants of Clostridium thermocellum, an anaerobic cellulolytic thermophile, were isolated after penicillin enrichment in a chemically defined medium.     

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.     

Genetic, biochemical, and developmental studies of nystatin resistant mutants in Dictyostelium discoideum

Summary Conditions are given for the isolation of nystatin resistant mutants of the cellular slime mold Dictyostelium discoideum. These mutants fall into three phenotypic groups; corresponding to three genes: nysA, nysB, and nysC. Mutants in nysB and nysC affect sterol metabolism since they have altered sterol compositions. Each group contains several unique, but as yet unidentified, sterols in place of the wild type sterol. The nysC strains are most nystatin resistant, display altered sensitivity to some drugs, and grow on nystatin from amoebae or spores. All other mutants are nystatin resistant only as amoebae. Although nysC mutants grow normally, they make small fruiting bodies which appear to result from the formation of smaller aggregates.Supported by N.I.H. grant GM 18476     

Comparison of therapeutic effect of aqueous extract of garlic and nystatin mouthwash in denture stomatitis

doi: 10.1111/j.1741‐2358.2011.00544.x Comparison of therapeutic effect of aqueous extract of garlic and nystatin mouthwash in denture stomatitis Introduction: Denture stomatitis (DS) is the most common form of chronic oral candidiasis. The standard treatment for DS is nystatin, which is accompanied with complications such as a bitter taste. The aim of this study was to compare the effect of garlic with nystatin in DS. Material and Methods: This randomised clinical trial study was performed on 40 patients with DS. After obtaining written consent, patients were divided into two groups while members of each group were given either nystatin or garlic extract for 4 weeks. The length and width of erythema area was measured at the end of the first, second, third, and the fourth weeks using a calliper. Data were analysed by SPSS and statistical tests including variance analysis with anova repeated measures, chi‐square, and least square differences. Results: The changes in the length and width of erythema at different times according to the type of treatment were found to be significant while an accelerated recovery was demonstrated for nystatin (p < 0.001). Both regimens resulted in significant recovery (p < 0.0001). Greater satisfaction with the use of garlic rather than nystatin was mentioned (p < 0.0001). Conclusion: Considering the efficacy of garlic and lack of side effects for this compound and also regarding the nystatin‐associated complications, garlic extract can be introduced as a substitution for standard treatment in DS.     

利用原生质体融合技术提高黑曲霉产酶活力的研究

以黑曲霉Ｎ３４３和ＵＶ—１１为出发株,分别经亚硝酸诱变处理,再以制霉菌素浓缩处理,从中筛得３株维生素缺陷型和１株氨基酸缺陷型的突变株。选取来自Ｎ３４３的突变株ＮＢ_３（Ｂ_１^－,ＰＰ^－,ＦＡ^－）和来自ＵＶ—１１的突变株ＵＢＩ（Ｐｒｏ^－）为融合亲本,在研究其原生质体释放和再牛条件的基础上,以ＰＥＧ诱导进行了原生质体融合。用直接选择法检出９８个融合子,经过筛选得到两株稳定的融合子：Ｆ     

The response ofDatura innoxia wild-type and auxotrophic cells to several enrichment agents

A number of growth-lethal agents were tested for their ability to discriminate between growing and non-growing cells of wild-type and several auxotrophic cell lines ofDatura innoxia P. Mill. Of these agents, 1--4-arabinofuranosylcytosine (ara-C), 5-FU and FUdR were shown to decrease the growth of an adenine-requiring auxotroph only slightly at concentrations at which wild-type cells were killed. Only nystatin and arsenate had a degree of discrimination with the adenine-requirer sufficient for counter-selection. However, neither of these agents could be used to differentiate between growing and non-growing cells of two otherDatura auxotrophs, an isoleucine-valine-and a pantothenate-requirer.The efficacy of general enrichment methods for plant cell auxotrophs is discussed.Abbreviations Ara-C 1--4-arabinofuranosylcytosine - FPGA filter-paper-growth-assay - 5-FU 5-fluorouridine - FUdR 5-fluoro-2-deoxyuridine - GDW glass-distilled water     

Use of the beta-lactamase inhibitor clavulanic acid in the isolation of auxotrophic mutants of Bacillus licheniformis.

The isolation of auxotrophic mutants of Bacillus licheniformis, a microbe containing constitutive beta-lactamase activity, was found to be facilitated by the addition of clavulanic acid and cefotaxime during enrichment.     

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.     

Effect of glucose limitation and specific mutations in the module 5 enoyl reductase domains in the nystatin and amphotericin polyketide synthases on polyene macrolide biosynthesis

Enoyl reductase (ER) domains in module 5 of nystatin and amphotericin polyketide synthase (PKS) are responsible for reduction of the C28–C29 unsaturated bond on the nascent polyketide chain during biosynthesis of both macrolides, resulting in production of tetraenes nystatin A₁ and amphotericin A, respectively. Data obtained in fermentations under glucose limitation conditions demonstrated that the efficiency of the ER5 domain can be influenced by carbon source availability in the amphotericin producer Streptomyces nodosus, but not in the nystatin producer Streptomyces noursei. Two S. noursei ER5 domain mutants were constructed, GG5073SP and S5016N, both producing the heptaene nystatin analogue S44HP with unsaturated C28–C29 bond. While the GG5073SP mutant, with altered ER5 NADPH binding site, produced S44HP exclusively, the S5016N mutant synthesized a mixture of nystatin and S44HP. Comparative studies on the S5016N S. noursei mutant and S. nodosus, both producing mixtures of tetraenes and heptaenes, revealed that the ratio between these two types of metabolites was significantly more affected by glucose limitation in S. nodosus. These data suggest that mutation S5016N in NysC “locks” the ER5 domain in a state of intermediate activity which, in contrast to the ER5 domain in the amphotericin PKS, is not significantly influenced by physiological conditions.