Bacterial cell enlargement and microinjection into the cell
Bacterial protoplasts/spheroplasts are generated by lysing the cell wall using lysozyme. When cell wall synthesis is inhibited, bacterial protoplasts/spheroplasts can enlarge without dividing. We used the Marine Broth including penicillin to enlarge bacterial cells. DNA replication occurs during cell enlargement. For microinjection, cells must be 15 μm or larger in diameter. Vacuoles are formed in these cells. Nishida (2020) summarized the factors that influence bacterial cell enlargement. Takahashi et al. (2020) succeeded in generating cells of E. faecalis and L. amnigena suitable for microinjection. In addition, Takahashi and Nishida (2022) microinjected heterogeneous genomic DNAs into the cytoplasm of of E. faecalis.
YouTube: microinjection of calligraphy ink into a bacterial cell
Evolution of fungi
Not only fungi but also bacteria synthesize lysine via aminoadipic acid. Thermus thermophilus was the first bacterium in which lysine synthesis via aminoadipic acid was discovered. Nishida et al. (1999) showed that the Thermus pathway is not identical to fungal one, which is distributed to the archaeon Pyrococcus. This lysine biosynthesis led a light on the evolution of amino acid biosynthesis.
Ascomycota and basidiomycota are evolutionarily sister groups. Mixia osmundae had belonged to the ascomycota until 1995. Nishida et al. (1995) showed that M. osmundae is a member of the basidiomycota. In addition, Nishida et al. (2012) analyzed the genome of M. osmundae. M. osmundae was described as a basidiomycetous yeast in The Yeasts 5th edition, a taxonomic study (Kurtzman, Fell, and Boekhout 2011).
Nishida and Sugiyama (1994) proposed archiascomycetes as the earliest ascomycetous lineage. Introductory Mycology 4th edition (Alexopoulos, Mims, and Blackwell 1996) included a chapter on archiascomycetes. Ascomycota consist of three lineages; archiascomycetes, ascomycetous yeasts, and filamentous ascomycetes.
Although animals and plants lack intron in their replication-dependent histone genes, some fungi have introns. Basidiomycota and filamentous ascomycetes have introns in their histone genes. Yun and Nishida (2011) suggested that during the fungal evolution, archiascomycetes and ascomycetous yeasts had lost the introns on histone genes; basidiomycetes and filamentous ascomycetes had acquired introns independently.
Genome comparison
When clarifying the phylogenetic relationships of evolution in the distant past, such as the origin of life, accurate phylogenetic relationshiops cannot be obtained by comparing only a single gene or protein. Nishida et al. (2011) showed the phylogenetic position of Dictyoglomus, inferred form whole-genome comparison. There are three major strategies of whole-genome comparison; comparison of orthologous sequences, that of gene content, and that of genome signature. Evolutionary and ecological factors strongly affect the base mutations and gene contents, respectively. It is uncertain how evolutionary and/or ecological factors affect the genome signature.
Histones/DNA complex
Genomic base composition (guanine-cytosine content, GC content) is maintained by GC content-dependent DNA-binding proteins. Histone is a GC content-dependent DNA-binding protein. Eukaryotic genomic DNA is packaged with histone proteins to form chromatin. The most fundamental repeating unit of chromatin is the nucleosome. Nucleosomes consist of an octamer of histones, around which genomic DNA is wrapped. GC and AT rich sequences, respectively, favor and disfavor core nucleosomes, although some variations of preferred sequences exist between species.
Nishida et al. (2006) showed that nucleosome depletion occurs in the vicinity of the transcription start site in human cells. Genome-wide nucleosome mapping revealed that nucleosome-free regions are pervasive in gene promoters. Nucleosome positioning plays an important role in gene expression as well as genomic DNA maintenance. Nucleosomes downstream from the nucleosome-depleted region are well positioned, with positioning decaying with increasing distance into protein coding region.
Bacteria do not have histones, but archaea do. A subpopulation of archaea possesses histones, which are similar to eukaryotic histones H3 and H4. However, archaeal histones are smaller than H3 and H4, and are not post-translationally modified. Archaeal histones are more diverse in amino acid sequences than eukaryotic histones. Nishida and Oshima (2017) showed that distribution of archaeal histones is associated with genomic GC content. Thus, archaeal histones have evolved concomitantly with their genomic GC content.
Sake production and kuratsuki bacteria
Sake production maintains a critical position in traditional Japanese cultures. In the sake production process, the two eukaryotic microorganisms, the koji mold Aspergillus oryzae and the sake yeast Saccharomyces cerevisiae are used. A. oryzae converts starch of rice to sugar, but S. cerevisiae cannot digest the starch. The sake yeast converts the sugar to ethanol, but the koji mold cannot produce ethanol. The sake yeast produces not only ethanol but also other chemical compounds that affect the flavor and taste of sake.
Sake yeast interacts with other microorganisms during the sake production process. As is known as far, kuratsuki yeasts and kuratsuki lactic acid bacteria have existed in sake breweries. The Japanese words "kura" and "tsuki" correspond to "sake brewery" and "inhabiting", respectively. Terasaki et al. (2021) reported kuratsuki actinobacteria, which were isolated from Narimasa Sake Brewery. Kanamoto et al. (2021) reported kuratsuki firmicutes, which were isolated from Shiraki-Tsunesuke Sake Brewery.
The kuratsuki bacteria we are studying do not produce compounds directly related to the flavor and taste of sake, and have been overlooked until now. Nishida (2024) summarized the effects of the kuratsuki bacteria on the flavor and taste of sake through interaction between the sake yeast and the kuratsuki bacteria.
TOYOWebStyle [in Japanese]: our research concept for kuratsuki bacteria in sake making