Yasuki Fukuda

Nucleotide sequence and characterization of a Gene conferring resistance to ethionine in yeast Saccharomyces cerevisiae

Abstract The nucleotide sequence of a DNA fragment, when present on a multi-copy plasmid, conferring ethionine resistance to Saccharomyces cerevisiae cells was determined. The fragment contained one long open reading frame and the frame was confirmed to be an ethionine resistant gene caused by frame-shift mutation. Other than ethionine resistance, an increased dosage of the gene directed overaccumulation in cells of S- adenosyl- l -methionine . The protein deduced from the open reading frame consisted of 617 amino acid residues with a calculated molecular weight of 67,977.

Extremely simple, rapid and highly efficient transformation method for the yeast Saccharomyces cerevisiae using glutathione and early log phase cells.

In the presence of polyethylene glycol (PEG), budding cells of Saccharomyces cerevisiae in the early log phase were transformed by exogenous plasmid DNA without additional specific chemical or physical treatments. This capacity of the yeast cells to become competent was strictly dependent on the growth phase, being induced in the early log phase, becoming maximum between the early and mid log phases and then disappearing rapidly in the mid log phase. The transformation was most efficient at pH 6 and the frequency increased with increasing DNA and cell concentrations. PEGs with average molecular sizes between 1000 and 3500 showed almost the same effects and were used most efficiently at 35%. The transformation frequency of S. cerevisiae was markedly enhanced when the oxidized form of glutathione (GSSG), but not the reduced form, was included in the mixture comprising early log phase cells, plasmid DNA, and PEG, and the transformation system with GSSG could be used as a convenient transformation method for the yeast S. cerevisiae.

Direct uptake of alginate molecules through a pit on the bacterial cell surface: A novel mechanism for the uptake of macromolecules

A yellow-pigmented bacterium isolated from a ditch as a potent producer of aglinate lyase was a Gram negative rod with a G + C content of 63 mol%, and was classified in the genus Sphingomonas. Electron microscopy revealed that the bacterial cell surfaces were covered by many large plaits. When grown in a medium containing alginate, a pit of 0.02–0.2 μm in diameter was formed on the cell surface, and a thin section showed the presence of a region where the cell membrane sinks into the cytosol. The pit and its neighborhood on cells grown in the presence of alginate were specifically stained with ruthenium red. On the basis of these results, we propose, for the first time, the existence of a direct uptake mechanism for polysaccharides through a mouth-like pit on the bacterial cell surface. This finding may provide a new insight into the transport of macromolecules in microbial cell systems.

Degradation of rice bran hemicellulose by Paenibacillus sp. strain HC1: gene cloning, characterization and function of β-D-glucosidase as an enzyme involved in degradation

A bacterium (strain HC1) capable of assimilating rice bran hemicellulose was isolated from a soil and identified as belonging to the genus Paenibacillus through taxonomical and 16S rDNA sequence analysis. Strain HC1 cells grown on rice bran hemicellulose as a sole carbon source inducibly produced extracellular xylanase and intracellular glycosidases such as β-d-glucosidase and β-d-arabinosidase. One of them, β-d-glucosidase was further analyzed. A genomic DNA library of the bacterium was constructed in Escherichia coli and gene coding for β-d-glucosidase was cloned by screening for β-d-glucoside-degrading phenotype in E. coli cells. Nucleotide sequence determination indicated that the gene for the enzyme contained an open reading frame consisting of 1,347xa0bp coding for a polypeptide with a molecular mass of 51.4xa0kDa. The polypeptide exhibits significant homology with other bacterial β-d-glucosidases and belongs to glycoside hydrolase family 1. β-d-Glucosidase purified from E. coli cells was a monomeric enzyme with a molecular mass of 50xa0kDa most active at around pH 7.0 and 37°C. Strain HC1 glycosidases responsible for degradation of rice bran hemicellulose are expected to be useful for structurally determining and molecularly modifying rice bran hemicellulose and its derivatives.

Physical and biochemical properties of freeze-tolerant mutants of a yeast Saccharomyces cerevisiae

Freeze-tolerant mutants of a yeast Saccharomyces cerevisiae were obtained through repeated mutations. The freeze-tolerance of the yeast cells was thought to be partially induced by the increasing rigidity of the cell surface, although the tolerance altered the susceptibility of the yeast cells to some toxic chemicals.

Adaptation mechanism of yeast to extreme environments: Construction of salt-tolerance mutants of the yeast Saccharomyces cerevisiae

Abstract A salt-tolerant mutant was derived from a haploid yeast, Saccharomyces cerevisiae DKD-5D-H, through repeated mutations with ethyl methanesulfonate. The mutant showed decreased cell size and could grow in the presence of NaCl up to 10%. A similar mutation method was also used to induce salt-tolerance in a diploid yeast, S. cerevisiae Kyokai no. 7. The mutant of the strain showed drastically decreased cell size, almost comparable with that of bacteria, and was resistant to NaCl up to 18%. These two mutation studies on haploid and diploid yeast strains indicated that the salt-tolerance was acquired through the decrease in yeast cell size.

Enzymes and germination of spores of the yeast Saccharomyces cerevisiae

Abstract Enzymes in spores of the yeast Saccharomyces cerevisiae were determined and compared with those in vegetative cells. The activities of phosphatase, oxidase and enzymes in the glycolytic bypass were high (about 3–10 fold) in spores, whereas those of dipeptidase, protease, DNase, RNase and TCA cycle enzymes were low in spores, being only 50% of the activities in vegetative cells. Enzymes in the glycolysis, glutathione and acetate metabolic pathways remained unchanged before and after sporulation. Germination of the yeast spores was repressed in the presence of d -aspartate, d -glutamate, d,l -methionine, d,l -cysteine, l -isoleucine, l -histidine and l -threonine, or bivalent metal ions such as Ni 2+ , Co 2+ and Zn 2+ .

Cloning of a gene for S-adenosylmethionine synthesis in Saccharomyces cerevisiae

SummaryThe gene for ethionine resistance was isolated, and its phenotypic characteristics were investigated. The cells transformed with this gene showed strong resistance to DL-ethionine, and S-adenosylmethionine (SAM) was remarkably accumulated within the cells. Judging from the restriction map of this gene, it suggests that the gene is not the gene SAM1 but SAM2.

Growth repression of yeast and fungus by bacterial DNAs: A possible physiological function of DNA other than as a carriage of genetic information

Deoxyribonucleic acid (DNA) of bacteria (Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis) repressed the growth of yeasts (Saccharomyces cerevisiae, Hansenula anomala, and Schizosaccharomyces pombe) and a fungus (Aspergillus niger), whereas the DNA of S. cerevisiae did not significantly repress the growth of bacteria. Chemically synthesized single-stranded oligonucleotides with the CpG dinucleotide motif also repressed the growth of S. cerevisiae. The effect of E. coli DNA was partially abolished after complete depolymerization or methylation of the DNA, and that of the oligonucleotides with the CpG motif decreased when the cytosine in the motif was methylated. The observed repression of eukaryotic microbial growth by bacterial DNA is thought to be due to the presence of the CpG motif. The results indicate that DNAs have some physiological function in addition to being carriers of genetic information.

Cloning of genes for spermine resistance in Saccharomyces cerevisiae and their effects on S-adenosyl-l-methionine accumulation

Abstract Among the polyamines tested, spermine strongly inhibited the growth of Saccharomyces cerevisiae DKD-5D-H. Two kinds of DNA fragments that confer strong and weak resistances to spermine were cloned onto a vector plasmid, YEp13. The restriction map of the DNA fragment conferring strong resistance was the same as that of a DNA fragment responsible for ethionine resistance in the same yeast cells. (Shiomi et al., Appl. Microbiol. Biotechnol., 29, 302–304, 1988) The yeast cells with the DNA fragment conferring strong resistance to spermine were resistant to ethionine and accumulated S- adenosyl- l -methionine intracellularly.