Morphological and genetic effects of benomyl on polyploid brewing yeasts: isolation of auxotrophic mutants.

An enrichment procedure after ethyl methanesulfonate mutagenesis and exposure to the fungicide benomyl yielded mutants auxotrophic for several amino acids from two polyploid Saccharomyces spp. Benomyl treatment was found to have a marked morphological effect on polyploid Saccharomyces cerevisiae, causing cells to adopt a characteristic doublet cell morphology in which buds are nearly as large as the parent cells. Experiments in which nuclear division was monitored in benomyl-induced doublet cells by Giemsa nuclear staining demonstrated an unusual sequence of cytological events which culminated in the formation of binucleate parental and mononucleate bud components. The frequency of formation of doublet and binucleate parent cells was found to depend on the strain employed and the benomyl concentration administered.     

A note on the isolation of auxotrophic mutants of Rhizobium japonicum

Z lotnikov K.M. C hatuev B.M. khmelnitsky , M.I. 1984. A note on the isolation of auxotrophic mutants of Rhizobium japonicum. Journal of Applied Bacteriology 56 , 173–174.
Several different stable auxotrophic mutants of Rhizobium japonicum were isolated following NG mutagenesis. It was found that NG mutagenesis of the rhizobia was most efficient at pH 70.     

The use of vancomycin for the isolation of auxotrophic mutants inMycobacterium smegmatis

A method using vancomycin for the accumulation of auxotrophic mutants ofMycobacterium smegmatis M54/81 induced by N-methyl-N′-nitro-N-nitrosoguanidine was developed. As compared with the simple replication technique the yield of auxotrophic mutants was twenty-fold.     

The potential of genetic engineering for improving brewing, wine-making and baking yeasts

The end of the twentieth century was marked by major advances in life technology, particularly in areas related to genetics and more recently genomics. Considerable progress was made in the development of genetically improved yeast strains for the wine, brewing and baking industries. In the last decade, recombinant DNA technology widened the possibilities for introducing new properties. The most remarkable advances, which are discussed in this Mini-Review, are improved process performance, off-flavor elimination, increased formation of by-products, improved hygienic properties or extension of substrate utilization. Although the introduction of this technology into traditional industries is currently limited by public perception, the number of potential applications of genetically modified industrial yeast is likely to increase in the coming years, as our knowledge derived from genomic analyses increases.     

Use of 5-fluorouracil for the isolation of auxotrophic mutants of Bacillus megaterium

Wachsman, J. T. (University of Illinois, Urbana), and L. Hogg. Use of 5-fluorouracil for the isolation of auxotrophic mutants of Bacillus megaterium. J. Bacteriol. 87:1137-1139. 1964.-The combination of 5-fluorouracil (FU) and uridine was used to selectively kill wild-type cells of Bacillus megaterium KM, thereby providing surviving populations greatly enriched in auxotrophic mutants. Exponentially growing cells were irradiated with ultraviolet light, incubated in a basal medium containing sucrose and, in most experiments, a complete amino acid mixture. Exponentially growing cells were then washed and incubated in the basal medium containing only sucrose, to deplete intracellular reserves. FU and uridine were added, and incubation was continued. After 5 hr, auxotrophs may account for up to 50% of the survivors. Organisms requiring each of the following compounds were identified: alanine, arginine, asparagine, cysteine, histidine, phenylalanine, serine, threonine, tyrosine, adenine, and guanine.     

Isolation and genetic analysis of resistant mutants to the benzimidazole fungicide benomyl inCoprinus cinereus

In total, 404 variants resistant to the antimicrotubule agent benomyl were isolated from UV-irradiated oidia of the basidiomyceteCoprinus cinereus. Part of the variants showed, in addition to benomyl resistance, heat sensitivity or heat dependence. Fifteen variants selected on the basis of different phenotypes were subjected to further analyses. All of the 15 variations were due to single-gene mutations, and the mutations comprised four groups (benA, benB, benC, andbenD) in terms of genetic linkage. Some of the 15 mutations affected nuclear migration in dikaryosis and/or fruiting processes.     

A note on auxotrophic mutants of cowpea rhizobia

We have examined a variety of common mutagens in producing auxotrophic mutants in cowpea rhizobia strains JRC23 and IRC256. While NTG (N-methyl-N-nitro-N-nitrosoguanidine), EMS (ethylmethane sulfonate), NA (nitrous acid), and UV (ultraviolet) irradiation were mutagenic with the strain JRC23, these mutagenic agents did not mutate strain IRC256. On the contrary, transposon mutagenesis with Tn5 yielded auxotrophs in strain IRC256 but not in strain JRC23, while only methionine (Met^–) auxotrophs from strain JRC23, histidine (His^–), and adenine plus thiamine (Ade^–+Thi^–) auxotrophs from strain IRC256 were isolated.     

Amber mutants of bacteriophage T4D: their isolation and genetic characterization

We have isolated a large number of mutants of bacteriophage T4D that are unable to form plaques on strain B of Escherichia coli, but are able to grow (nearly) normally on some other strains of E. coli, in particular strain CR63. These mutants, designated amber (am), have been characterized by complementation tests, by genetic crosses, and by their response to chemical mutagens. It is concluded that a particular subclass of base substitution mutations may give rise to amber mutants and that such mutants occur in many genes, which are widely distributed over the T4 genome.     

Morphological,genetic and molecular characteristics of barley root hair mutants

Root hairs are tubular outgrowths of specialized epidermal cells called trichoblasts. They affect anchoring plants in soil, the uptake of water and nutrients and are the sites of the interaction between plants and microorganisms. Nineteen root hair mutants of barley representing different stages of root hair development were subjected to detailed morphological and genetic analyses. Each mutant was monogenic and recessive. An allelism test revealed that nine loci were responsible for the mutated root hair phenotypes in the collection and 1–4 mutated allelic forms were identified at each locus. Genetic relationships between the genes responsible for different stages of root hair formation were established. The linkage groups of four loci rhl1, rhp1, rhi1 and rhs1, which had previously been mapped on chromosomes 7H, 1H, 6H and 5H, respectively, were enriched with new markers that flank the genes at a distance of 0.16 cM to 4.6 cM. The chromosomal position of three new genes – two that are responsible for the development of short root hairs (rhs2 and rhs3) and the gene that controls an irregular root hair pattern (rhi2) – were mapped on chromosomes 6H, 2H and 1H, respectively. A comparative analysis of the agrobotanical parameters between some mutants and their respective parental lines showed that mutations in genes responsible for root hair development had no effect on the agrobotanical performance of plants that were grown under controlled conditions. The presented mutant collection is a valuable tool for further identification of genes controlling root hair development in barley.     

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.     

Adenine auxotrophic mutants of Aspergillus oryzae: development of a novel transformation system with triple auxotrophic hosts

adeA and adeB genes homologous to Saccharomyces cerevisiae ADE1 and ADE2, respectively, were cloned from Aspergillus oryzae. AdeA and AdeB share 62.8% and 52.5% identities with S. cerevisiae Ade1 and Ade2, respectively. In order to obtain triple auxotrophic mutants from A. oryzae, 12 red-colored mutant colonies were isolated by UV mutagenesis of a double auxotrophic host, NS4 (niaD(-), sC(-)), as a parent strain. All the mutants exhibited adenine auxotrophy and showed fluorescence in the vacuoles due to accumulation of a purine biosynthetic pathway precursor. Adenine auxotrophy of all the mutants was restored by introduction of either A. oryzae adeA or adeB genes. Sequence analysis demonstrated that substitutions or deletions of a single base pair occurred, inducing substitutions or frame shifts of amino acid sequences in both ade genes complementing the mutants. This study provides a novel host-vector system with triple auxotrophy in A. oryzae.     

Selective antimicrobial activity of chitosan on beer spoilage bacteria and brewing yeasts

Chitosan (0.1 g l(-1)), assayed in a simple medium, reduced the viability of four lactic acid bacteria isolated during the beer production process by 5 logarithmic cycles, whereas activity against seven commercial brewing yeasts required up to 1 g chitosan l(-1). Antimicrobial activity was inversely affected by the pH of the assay medium. In brewery wort, chitosan (0.1 g l(-1)) selectively inhibited bacterial growth without altering yeast viability or fermenting performance.