Biology subjects

Ogata, M.

Publications and source records attributed to Ogata, M..

4 recordsLinked to original sources

bioRxiv2025

MoChia1 is a family 18 glycoside hydrolase from the rice blast fungus, Magnaporthe oryzae, that releases reducing-end GlcNAc from chitin oligosaccharides

MoChia1, a family 18 glycoside hydrolase from the rice blast fungus Magnaporthe oryzae, was identified as an enzyme that binds chitin oligosaccharide elicitor to suppress the chitin-induced rice immune response. It hydrolyzed chitin oligosaccharides with a degree of polymerization of at least 3 [(GlcNAc)n (n[≥]3)] in an exo-splitting manner with anomeric retention. HPLC analysis of (GlcNAc)n cleavage by MoChia1 revealed that it specifically recognizes the {beta}-anomer of the substrate at the + 1 subsite and cleaves the terminal glycosidic linkage at the reducing end. It also hydrolyzed the partially deacetylated chitotrisaccharide GlcN-(GlcNAc)2 to GlcN-GlcNAc and GlcNAc, but not (GlcN)2-GlcNAc, indicating the requirement of the N-acetyl group at the -1 subsite for the hydrolytic reaction. MoChia1 released p-nitrophenol from pNP-(GlcNAc)2, p-nitrophenyl di-N-acetyl-{beta}-chitotrioside. Furthermore, it hydrolyzed the chitotriose derivatives with a modified GlcNAc residue on the reducing end, (GlcNAc)2-G, 4-O-{beta}-di-N-acetylchitobiosyl-2-acetamido-2-deoxy-2,3-anhydro-glucopyranose and (GlcNAc)2-L, 4-O-{beta}-di-N-acetylchitobiosyl-2-acetamido-2-deoxy-2,3-anhydro-glucono-{delta}-lactone, to (GlcNAc)2 and the modified GlcNAc, respectively. However, it did not hydrolyze 4-O-{beta}-di-N-acetylchitobiosyl moranoline [(GlcNAc)2-M], reduced chitotriose (chitotriitol), and -(GlcNAc)2 fluoride [(-(GlcNAc)2-F]. MoChia1 did not bind to chitin and barely hydrolyzed the polymeric substrate, glycol chitin. Taken together, we concluded that MoChia1 is a GH18 reducing end GlcNAc-releasing exo-oligochitinase with the {beta}-anomer selectivity. Allosamidin, a potent inhibitor of GH18 chitinases, was found to bind to MoChia1 and inhibit its hydrolytic activity with an IC50 of 54.4 {+/-} 6.91 M, indicating that allosamidin may be a potential candidate for a pesticide to prevent rice blast infection by inhibiting the chitinase activity of MoChia1.

biochemistry↗

bioRxiv2022

Cleavage of α-1,4-Glycosidic Linkages by the Glycosylphosphatidylinositol-Anchored α-Amylase AgtA Decreases the Molecular Weight of Cell Wall α-1,3-Glucan in Aspergillus oryzae

Aspergillus fungi contain -1,3-glucan with a low proportion of -1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored -amylases are conserved in Aspergillus fungi. The GPI-anchored -amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall -1,3-glucan but not to degrade it in vivo. However, the detailed mechanism of cell wall -1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by the Aspergillus oryzae agtA gene, an ortholog of the A. nidulans amyD gene. Similar to findings in A. nidulans, agtA overexpression in A. oryzae grown in submerged culture decreased the amount of cell wall -1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wild-type strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced in Pichia pastoris and found that it degraded soluble starch, but not linear bacterial -1,3-glucan. Furthermore, rAoAgtA cleaved 3--maltotetraosylglucose with a structure similar to the predicted boundary structure between the -1,3-glucan main chain and a short spacer composed of -1,4-linked glucose residues in cell wall -1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the -1,4-glycosidic bonds of 3--maltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall -1,3-glucan. Consistent with this hypothesis, heterologous overexpression of agtA in A. nidulans decreased the molecular weight (MW) of cell wall -1,3-glucan. These in vitro and in vivo properties of AoAgtA suggest that GPI-anchored -amylases can degrade the spacer -1,4-glycosidic linkages in cell wall -1,3-glucan before its insolubilization, and this spacer cleavage decreases the MW of cell wall -1,3-glucan in vivo.

microbiology↗

bioRxiv2018

Potential Application of Bacteriophages in Enrichment Culture for Improved Prenatal Streptococcus agalactiae Screening

Vertical transmission of Streptococcus agalactiae can cause neonatal infections. A culture test in the late stage of pregnancy is used to screen for the presence of maternal S. agalactiae for intrapartum antibiotic prophylaxis. For the test, vaginal-rectal swab sampling is immediately followed by enrichment culture and bacterial identification. In some cases, Enterococcus faecalis competes with and overgrowths S. agalactiae in the enrichment culture. Consequently, the identification test occasionally yields false-negative results. Bacterial viruses, bacteriophages (phages), infect and kill specific host bacteria. In the current study, we explored the feasibility of using phages to minimize the undesirable E. faecalis outgrowth and facilitate S. agalactiae detection in an experimental setting. Phage mixture was prepared using three phages that specifically infect E. faecalis: phiEF24C, phiEF17H, and phiM1EF22. The mixture inhibited the growth of 86.7% (26/30) of E. faecalis strains tested in the enrichment broth. When single strains of E. faecalis and S. agalactiae were inoculated in the enrichment broth containing the phage mixture, bacterial growth was inhibited or facilitated, respectively. Further, several sets of S. agalactiae and E. faecalis strains were co-cultured, and bacteria were detected on chromogenic agar after the enrichment culture. S. agalactiae was dominant after plating a phage mixture-treated co-culture, while it was barely detected after plating the untreated co-culture. Considering these observations, the phage mixture can be employed in the S. agalactiae culture test to increase test accuracy.

microbiology↗

bioRxiv2017

Genome-wide stability of the DNA replication program in single mammalian cells

Here, we report the establishment of a single-cell DNA replication sequencing method, scRepli-seq, which is a simple genome-wide methodology that measures copy number differences between replicated and unreplicated DNA. Using scRepli-seq, we demonstrate that replication domain organization is conserved among individual mouse embryonic stem cells (mESCs). Differentiated mESCs exhibited distinct replication profiles, which were conserved from cell to cell. Haplotype-resolved scRepli-seq revealed similar replication timing profiles of homologous autosomes, while the inactive X chromosome was clearly replicated later than its active counterpart. However, a small degree of cell-to-cell replication timing heterogeneity was present, and we discovered that developmentally regulated domains are a source of such variability, suggesting a link between cell-to-cell heterogeneity and developmental plasticity. Together, our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into 3D genome organization.

genomics↗