Biology subjects

Yagi, N.

Publications and source records attributed to Yagi, N..

5 recordsLinked to original sources

bioRxiv2025

Species identification and genotyping of Citrobacter spp. using genes with high nucleotide diversity.

Citrobacter spp. are facultative anaerobic Gram-negative bacillus found in a wide range of habitats. Recently, there have been reports of an increasing number of cases of Citrobacter spp. being the cause of nosocomial infections, and of increasing highly antimicrobial-resistant (AMR) Citrobacter spp. In clinical laboratory testing, Citrobacter spp., such as C. freundii and C. braakii, are handled collectively as the C. freundii complex. This can be obstacle to collect information regarding species identification and genetic lineage which are necessary for estimating distribution mechanism and evaluating AMR degree for each Citrobacter species. This study investigated gene combinations that allow for Citrobacter spp. identification and genetic lineage estimation with as few genes as possible. Citrobacter genomes of 18 species were collected as many as possible from GenBank. After the gene annotation, nucleotide diversity of 1,113 genes contained in all 453 genomes was calculated. Genotypes of top seven highest nucleotide diversity (HND) genes were confirmed and investigated their distributions among Citrobacter genomes. Except for C. werkmanii and C. cronae, it was suggested that the genotype of the top HND gene, groups_3152, and combinations of the genotypes of the top two HND genes were a species- and genetic lineage-specific distribution, respectively. Considering that the genomes of C. werkmanii and C. cronae were highly similar and indistinguishable each other by conventional genotyping methods, it was suggested that the combination of the top two HND genes could be used to Citrobacter spp. identification and genetic lineage classification. Importance StatementMolecular epidemiology of individual Citrobacter species has not been widely performed, partly because Citrobacter spp. are handled as the C. freundii complex in the clinical testing and the multi-locus sequencing typing (MLST) has been performed targeting the entire genus Citrobacter. This study proposed a combination of two genes for identification of Citrobacter spp. and classify their genetic lineages. Currently, phylogenetic tree analysis has become possible using core-genome MLST and core-genome single nucleotide polymorphisms (SNPs) after whole genome sequencing (WGS) of bacterial isolates. But these phylogenetic analyses require a considerable amount of cost. In this regard, using two genes for Citrobacter species identification and genetic lineage classification reduces the required cost compared to WGS, encouraging its introduction into clinical testing and further performing molecular epidemiology of individual Citrobacter species.

genomics↗

bioRxiv2024

A rapid method to determine the genetic lineage of Escherichia coli using open reading frame composition in the shallow sequencing

Determining the genetic background of bacterial isolates and evaluating the genetic relatedness among these isolates in a short time period are important to identify the spreading route(s) in cases of healthcare-associated infections and outbreaks caused by antimicrobial-resistant bacteria. Previously, we proposed a shallow sequencing (Shall-seq) procedure to determine the genetic backgrounds of clinical isolates using the minimum amount of sequence data. However, it took a longer time, such as longer than 10 h, to determine the genetic background of one clinical isolate. In this study, we developed a search procedure using open reading frame (ORF) composition to select the reference genome sequence with the highest matching ratio (>90%), the indicator genome sequence (IGS), for the examined bacterial isolate. Consequently, IGSs were selected for 28 (96.6%) of the examined 29 isolates and selection was performed within 30 min for each bacterial isolate. More importantly, the comparison of IGSs indicated that the IGSs, determined by ORF composition, of the examined bacterial isolates were closely related to the genome sequences determined using the Shall-seq procedure. Taken together, these results suggest that our newly developed search procedure can quickly determine the genetic background of bacterial isolates.

microbiology↗

bioRxiv2024

Shallow sequencing (Shall-seq) procedure: A method for determining the genetic lineages and detecting antimicrobial resistance genes of antimicrobial-resistant bacteria using minimum Nanopore sequencing data

Antimicrobial-resistant bacteria could cause nosocomial infections and outbreaks in healthcare facilities. Phylogenetic analyses based on whole-genome sequencing (WGS) could become the gold-standard method for understanding the route of antimicrobial-resistant bacterial spreading. However, generally, the WGS needs to analyze much amount of data. Therefore, sufficient resources such as budget and data analysis system are needed and it is a burden for introduction of the WGS in the routine clinical examination of pathogenic bacterial isolates. In this study, we used Escherichia coli as a model and evaluated whether determination of the genetic background and detection of antimicrobial-resistance genes of 29 E. coli clinical isolates were achieved by searching databases using sequence reads output by the Nanopore sequencer as the search keys. Consequently, only 66.2 MB data was sufficient to search for a genome sequence with [≥]90% range of coverage rate. Importantly, AMR genes and plasmid replicon types were also detected with minimum data, and the detected AMR genes and phenotypes of the E. coli isolates did not present any discrepancy. Taken together, this shallow sequencing (Shall-seq) procedure consists of "shallow of coverage" sequencing using the Nanopore sequencer and data search using minimum data could be used to analyze bacterial isolates cost-effectively.

microbiology↗

bioRxiv2023

Near-infrared imaging of phytochrome-derived autofluorescence in plant nuclei

Capturing images of the nuclear dynamics within live cells is an essential technique for comprehending the intricate biological processes inherent to plant cell nuclei. While various methods exist for imaging nuclei, including combining fluorescent proteins and dyes with microscopy, there is a dearth of commercially available dyes for live-cell imaging. In Arabidopsis thaliana, we discovered that nuclei emit autofluorescence in the near-infrared (NIR) range of the spectrum and devised a non-invasive technique for the visualization of live cell nuclei using this inherent NIR autofluorescence. Our studies demonstrated the capability of the NIR imaging technique to visualize the dynamic behavior of nuclei within primary roots, root hairs, and pollen tubes, which are tissues that harbor a limited number of other organelles displaying autofluorescence. We further demonstrated the applicability of NIR autofluorescence imaging in various other tissues by incorporating fluorescence lifetime imaging techniques. Nuclear autofluorescence was also detected across a wide range of plant species, enabling analyses without the need for transformation. The nuclear autofluorescence in the NIR wavelength range was not observed in animal or yeast cells. Genetic analysis revealed that this autofluorescence was caused by the phytochrome protein. Our studies demonstrated that nuclear autofluorescence imaging can be effectively employed not only in model plants but also for studying nuclei in non-model plant species.

plant biology↗

bioRxiv2020

Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings

AO_SCPLOWBSTRACTC_SCPLOWSynthetic chemical fluorescent dyes are promising tools for many applications in biology. SNAP tagging provides a unique opportunity for labeling of specific proteins in vivo with synthetic dyes for studying for example endocytosis, or super-resolution microscopy. However, despite the potential, chemical dye tagging has not been used effectively in plants. A major drawback was the limited knowledge regarding cell wall and membrane permeability of synthetic dyes. Twenty-six out of 31 synthetic dyes were taken up into BY-2 cells, eight were not taken up and can thus serve for measuring endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine (TMR) and silicon-rhodamine (SiR) 647 were used to SNAP tag -tubulin. Successful tagging was verified by live cell imaging and visualization of microtubules arrays in interphase and during mitosis. Fluorescence activation-coupled protein labeling (FAPL) with DRBG-488 was used to observe PIN2 endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the newly forming cell plate during mitosis. Together the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety to cell biological processes.

cell biology↗