Biology subjects

Saito, R.

Publications and source records attributed to Saito, R..

23 records · Page 2Linked to original sources

bioRxiv2023

Functional and structural analysis of a cyclization domain in a cyclic beta-1,2-glucan synthase

Cyclic {beta}-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic {beta}-1,2-glucans (C{beta}Gs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that {beta}-glucosidase-resistant compounds are produced by TiCGSCy with linear {beta}-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are C{beta}Gs. Next, action pattern analyses using {beta}-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive transglycosylation without hydrolysis and a hexasaccharide as the minimum length of the substrate. Nevertheless, longer substrate {beta}-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce C{beta}Gs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of {beta}-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear difference in the reaction pathways between these enzymes. Therefore, we propose a novel reaction mechanism of TiCGSCy. Thus, we propose that the present group of CGSs should be defined as a new glycoside hydrolase family, GHxxx. Statements and DeclarationsCompeting Interests: The authors declare no competing interests. Key PointsIt was clearly evidenced that cyclization domain alone produces cyclic {beta}-1,2-glucans. The domain exclusively catalyzes transglycosylation without hydrolysis. The present catalytic domain should be defined as a new glycoside hydrolase family.

biochemistry↗

bioRxiv2021

Mitochondrial fission dysfunction alleviates heterokaryon incompatibility-triggered cell death in the industrial filamentous fungus Aspergillus oryzae

In filamentous fungi, cell-to-cell recognition is a fundamental requirement for the formation, development, and maintenance of complex hyphal networks. Basically, self/compatible individuals within the fungal species are capable of fusing together, a step important for crossbreeding, which results in the formation of viable vegetative heterokaryons. Conversely, the fusion of incompatible individuals does not result in the formation of viable hyphal networks, but it often leads to growth inhibition or cell death. Even though a number of studies have been conducted to investigate such incompatibility, the understanding of the associated molecular mechanism is still limited, and this restricts the possibility of crossbreeding incompatible individuals. Therefore, in this study, the characteristics of compatibility/incompatibility in the industrial filamentous fungus, Aspergillus oryzae, were comprehensively investigated. Protoplast fusion and co-culture assays indicated the existence of a correlation between strain phylogeny and compatibility/incompatibility features. Time-course fluorescence observations were employed to investigate the types of incompatible responses that are induced at different cellular levels upon incompatible cell fusion, which eventually lead to cell death. Propidium iodide-indicated cell death, ROS accumulation, and mitochondrial fragmentation were identified as the major responses, with mitochondrial fragmentation showing the most significant subcellular change immediately after incompatible cell fusion. Furthermore, the deletions of mitochondrial fission-related genes Aofis1 and Aodnm1 in incompatible pairing alleviated cell death, indicating that mitochondrial fission is an important mechanism by which incompatibility-triggered cell death occurs. Therefore, this study provides new insights about heterokaryon incompatibility. IMPORTANCEFor a long time, it was believed that as an asexual fungus, A. oryzae does not exhibit any sexual cycle. However, the fungus has two mating types, indicating the potential for sexual reproduction besides a known parasexual cycle. Therefore, given that viable heterokaryon formation following cell fusion is an important step required for genetic crossing, we explored the mechanism of incompatibility, which restricts the possibility of cell fusion in A. oryzae. Protoplast fusion and co-culture assays led to the identification of various vegetative compatible groups. Mitochondrial fragmentation was found to be the most significant incompatible cellular response that occurred in organelles during incompatible pairing, while the deletion of mitochondrial fission-related genes was identified as a strategy used to alleviate incompatibility-triggered cell death. Thus, this study revealed a novel mechanism by which mitochondrial fission regulates incompatible responses.

microbiology↗

bioRxiv2021

Small Vessel Diseases: 3D Characteristics of the Vasculature and White Matter

Cerebral small vessel disease (SVD) is associated with white matter hyperintensities (WMHs), thereby contributing to vascular dementia and movement disorder. However, the pathomechanisms responsible for WMH-related small vessel degeneration remain poorly understood due to the technical limitations of current methods. The aim of this study was to clarify the 2- and 3-dimensional (2D and 3D) pathological features of small vessels and white matter (WM) in the brains of patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), HTRA1-autosomal dominant disease (HTRA1-AD) and sporadic SVD (sSVD). From a cohort of 86 consecutive autopsied patients with SVDs, we retrieved those with genetically confirmed CADASIL and HTRA1-AD (three and three, respectively), and four with sSVD. We quantitatively evaluated WM and vascular changes in the frontal portion of the centrum semiovale and temporal lobe using conventional 2D and chemically cleared 3D analytical methods with light-sheet fluorescence microscopy. Quantitatively, the WM pathology, including the density of myelin, axons and gliosis, was most severe in CADASIL, but unexpectedly sSVD was second in order of severity, followed by HTRA1-AD. The density of clasmatodendrocytes, known to be irreversibly injured astrocytes, was considerably highest in HTRA1-AD. The vascular pathology, including arteriole and capillary sclerosis and the extent of the perivascular space, was most severe in CADASIL, whereas the density of smooth muscle actin (SMA) positivity was most decreased in HTRA1-AD. 3D immunohistochemistry for SMA demonstrated two distinct patterns of SMA loss within the vessels: (1) CADASIL and sSVD: diffuse loss, being prominent in small branches, (2) HTRA1-AD: selective loss in main branches. Overall, the extent of WM and vascular degeneration is most severe in CADASIL, whereas SMA loss is most evident in HTRA1-AD. These differences in the size and distribution of affected vessels may be related to the heterogeneous WM pathology and underlying pathomechanisms of SVD.

pathology↗

bioRxiv2020

Marker-assisted selection of trees with MALE STERALITY 1 in Cryptomeria japonica D. Don

Practical use of marker-assisted selection (MAS) is limited in conifers because of the difficulty with developing markers due to a rapid decrease in linkage disequilibrium, the limited genomic information available, and the diverse genetic backgrounds among breeding material collections. First, in this study, two families were produced by artificial crossing between two male-sterile trees, Shindai11 and Shindai12, and a plus tree, Suzu-2 (Ms1/ms1) (S11-S and S12-S families, respectively). The segregation ratio between male-sterile and male-fertile trees did not deviate significantly from the expected 1:1 ratio in either family. These results clearly suggested that the male-sterile gene of Shindai11 and Shindai12 is MALE STERALITY 1 (MS1). Because some markers reported previously have not been linkage mapped, we constructed a partial linkage map of the region encompassing MS1 using the S11-S and S12-S families. For the S11-S and S12-S families, 19 and 18 markers were mapped onto the partial linkage maps of MS1 region, respectively. There was collinearity (conserved gene order) between the two partial linkage maps. Two markers (CJt020762_ms1-1 and reCj19250_2335) were mapped to the same position as the MS1 locus on both maps. Of these markers, we used CJt020762 for MAS in this study. According to the MAS results for 650 trees from six prefectures of Japan (603 trees from breeding materials and 47 trees from the Ishinomaki natural population), five trees in Niigata Prefecture and one tree in Yamagata Prefecture had heterozygous ms1-1, and three trees in Miyagi Prefecture had heterozygous ms1-2. The results obtained in this study suggested that there may be geographical hotspots for the ms1-1 and ms1-2 alleles. Because MAS can be used effectively to reduce the labor and time required for selection of trees with a male-sterile gene, the number of breeding materials should increase in the future.

genetics↗

bioRxiv2018

Gene composition as a potential barrier to large recombinations in the bacterial pathogen Klebsiella pneumoniae

Klebsiella pneumoniae (Kp) is one of the most important nosocomial pathogens world-wide, being responsible for frequent hospital outbreaks and causing sepsis and multi-organ infections with a high mortality rate and frequent hospital outbreaks. The most prevalent and widely disseminated lineage of K. pneumoniae is clonal group 258 (CG258), which includes the highly resistant \"high-risk\" genotypes ST258 and ST11. Recent studies revealed that very large recombination events have occurred during the recent emergence of Kp lineages. A striking example is provided by ST258, which has undergone a recombination event that replaced over 1 Mb of the genome with DNA from an unrelated Kp donor. Although several examples of this phenomenon have been documented in Kp and other bacterial species, the significance of these very large recombination events for the emergence of either hyper-virulent or resistant clones remains unclear. Here we present an analysis of 834 Kp genomes that provides data on the frequency of these very large recombination events (defined as those involving >100Kb), their distribution within the genome, and the dynamics of gene flow within the Kp population. We note that very large recombination events occur frequently, and in multiple lineages, and that the majority of recombinational exchanges are clustered within two overlapping genomic regions, which result to be involved by recombination events with different frequencies. Our results also indicate that certain non-CG258 lineages are more likely to act as donors to CG258 recipients than others. Furthermore, comparison of gene content in CG258 and non-CG258 strains agrees with this pattern, suggesting that the success of a large recombination depends on gene composition in the exchanged genomic portion.\n\nAuthor SummaryKlebsiella pneumoniae (Kp) is an opportunistic bacterial pathogen, a major cause of deadly infections and outbreaks in hospitals worldwide. This bacterium is able to exchange large genomic portions (up to a fourth of the entire genome) within a single recombination event. Indeed, the most epidemiologically important Kp clone, is actually a hybrid which emerged after a > 1Mb recombination event. In this work, we investigated how recombinations affected the evolution of the most studied Kp Clonal Group, CG258. We found that large recombinations occurred frequently during Kp evolution, and occurred preferentially in a well-delimited genomic region. Furthermore, we found that four epidemiologically important clones emerged after large recombinations. We identified the donors of several large recombinations: despite many Kp lineages acted as donors during CG258 evolution, two of them have been involved more frequently. We hypothesize that the observed pattern of donors-recipients in recombinations, and the presence of a large recombinogenic region in Kp genome, could be related to gene composition. Indeed, genomic analyses showed a pattern compatible with this hypothesis, suggesting that gene content can represent a main factor in the success of a large recombination.

evolutionary biology↗