Functional analysis of feedback inhibition-insensitive aspartate kinase identified in a threonine-accumulating mutant of Saccharomyces cerevisiae.

Humans and mammals need to ingest essential amino acids (EAAs) for protein synthesis. In addition to their importance as nutrients, EAAs are involved in brain homeostasis. However, elderly people are unable to efficiently consume EAAs from their daily diet due to reduced appetite and variations in the contents of EAAs in foods. On the other hand, strains of the yeast Saccharomyces cerevisiae that accumulate EAAs would enable elderly people to intakegest adequate amounts of EAAs and thus might slow down the neurodegenerative process, contributing to the extension of their healthy lifespan. In this study, we isolated a mutant (strain HNV-5) that accumulates threonine, an EAA, derived from a diploid laboratory yeast by conventional mutagenesis. Strain HNV-5 carries a novel mutation in the HOM3 gene encoding the Ala462Thr variant of aspartate kinase (AK). Enzymatic analysis revealed that the Ala462Thr substitution significantly decreased the sensitivity of AK activity to threonine feedback inhibition even in the presence of 50 mM threonine. Interestingly, Ala462Thr substitution did not affect the catalytic ability of Hom3, in contrast to previously reported amino acid substitutions that resulted in reduced sensitivity to threonine feedback inhibition. Furthermore, yeast cells expressing the Ala462Thr variant showed an approximately threefold increase in intracellular threonine content compared to that of the wild-type Hom3. These findings will be useful for the development of threonine-accumulating yeast strains that may improve the quality of life in elderly people.IMPORTANCEFor humans and mammals, essential amino acids (EAAs) play an important role in maintaining brain function. Therefore, increasing the intake of EAAs by using strains of the yeast Saccharomyces cerevisiae that accumulate EAAs may inhibit neurodegeneration in elderly people and thus contribute to extending healthy lifespan and improving their quality of life. Threonine, an EAA, is synthesized from aspartate. Aspartate kinase (AK) catalyzes the first step in threonine biosynthesis and is subject to allosteric regulation by threonine. Here, we isolated a threonine-accumulating mutant of S. cerevisiae by conventional mutagenesis and identified a mutant gene encoding a novel variant of AK. In contrast to previously isolated variants, the Hom3 variant exhibited AK activity that was insensitive to feedback inhibition by threonine but retained its catalytic ability. This resulted in increased production of threonine in yeast. These findings open up the possibility for the rational design of AK to increase threonine productivity in yeast.

Morphological Changes in Spores during Germination in Bacillus cereus and Bacillus subtilis.

Processes from spore germination to outgrowth were observed in detail using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for Bacillus cereus and Bacillus subtilis. At 15 and 30 min after germination induction, SEM observation and SEM-EDX analysis of Bacillus spores prepared by freeze substitution showed that spherical structures including compounds having the same elemental ratio as that of the spore were observed on the surface of the spores. The results suggested the leakages of the cellular materials from the spores. At 360 min, B. cereus spores in outgrowth phase elongated with hemispherical structures at the end of the long side of the cells. The discoid structures with a hole (20-30 nm diameter) in the center was observed at 360 min. Confocal laser scanning microscopy after staining with fluorescence-labeled anti-spore antibodies showed that the hemispherical and discoid structures originated from the spore coat. These structures broke down after detached from the cells in outgrowth phase.

Method for rapid detection and identification of chaetomium and evaluation of resistance to peracetic acid.

In the beverage industry, peracetic acid has been increasingly used as a disinfectant for the filling machinery and environment due to merits of leaving no residue, it is safe for humans, and its antiseptic effect against fungi and endospores of bacteria. Recently, Chaetomium globosum and Chaetomium funicola were reported resistant to peracetic acid; however, little is known concerning the detail of peracetic acid resistance. Therefore, we assessed the peracetic acid resistance of the species of Chaetomium and related genera under identical conditions and made a thorough observation of the microstructure of their ascospores by transmission electron microscopy. The results of analyses revealed that C. globosum and C. funicola showed the high resistance to peracetic acid (a 1-D antiseptic effect after 900 s and 3-D antiseptic effect after 900 s) and had thick cell walls of ascospores that can impede the action mechanism of peracetic acid. We also developed specific primers to detect the C. globosum clade and identify C. funicola by using PCR to amplify the ß-tubulin gene. PCR with the primer sets designed for C. globosum (Chae 4F/4R) and C. funicola (Cfu 2F/2R) amplified PCR products specific for the C. globosum clade and C. funicola, respectively. PCR with these two primer sets did not detect other fungi involved in food spoilage and environmental contamination. This detection and identification method is rapid and simple, with extremely high specificity.

Difference of EGCg adhesion on cell surface between Staphylococcus aureus and Escherichia coli visualized by electron microscopy after novel indirect staining with cerium chloride.

We developed a novel method using indirect staining with cerium chloride for visualization of the catechin derivative epigallocatechin gallate (EGCg) on the surface of particles, i.e., polystyrene beads and bacterial cells, by electron microscopy. The staining method is based on the fact that in an alkaline environment, EGCg produces hydrogen peroxide, and then hydrogen peroxide reacts with cerium, resulting in a cerium hydroperoxide precipitate. This precipitate subsequently reacts with EGCg to produce larger deposits. The amount of precipitate is proportional to the amount of EGCg. Highly EGCg-sensitive Staphylococcus aureus and EGCg-resistant Escherichia coli were treated with EGCg under various pH conditions. Transmission electron microscopy observation showed that the amount of deposits on S. aureus increased with an increase in EGCg concentration. After treating bacterial cells with 0.5mg/mL EGCg (pH 6.0), attachment of EGCg was significantly lower to E. coli than to S. aureus. This is the first report that shows differences in affinity of EGCg to the cell surfaces of Gram-positive and -negative bacteria by electron microscopy.