Biology subjects

Yasuda, Y.

Publications and source records attributed to Yasuda, Y..

6 recordsLinked to original sources

bioRxiv2026

Dynamic evolution of diaphorin biosynthetic genes against a background of genome conservation in Profftella

The Asian citrus psyllid Diaphorina citri (Insecta: Hemiptera), a major agricultural pest, harbors the unusual obligate bacterial symbiont "Candidatus Profftella armatura" (Profftella), which is intimately integrated with its host yet devotes a remarkably large proportion of its highly reduced genome to the production of diaphorin, a bioactive polyketide. To gain insight into the evolution of this distinctive symbiont, we determined the genomes of Profftella and the co-resident nutritional symbiont "Candidatus Carsonella ruddii" (Carsonella) from Diaphorina lycii and compared them with previously characterized counterparts from D. citri and D. cf. continua. The Profftella and Carsonella chromosomes from D. lycii were 471 and 174 kb, respectively, and Profftella harbored an 8-kb plasmid. Genome organization and gene content were highly conserved across the three lineages. The diaphorin biosynthetic system likewise retained its overall organization, consistent with an important role for diaphorin in the Diaphorina symbiotic system. Remarkably, however, pronounced lineage-specific structural variation was observed in diaphorin synthases DipP and DipT, encoded by two of the 20 diaphorin biosynthetic genes. These synthases contained three extensively amplified tandem arrays of acyl carrier protein (ACP) domains that carry growing polyketide intermediates, with up to seven copies compared with one or two at corresponding positions in the related pederin synthases. All three regions of tandem ACP repeats showed copy-number variation among the Profftella lineages. This variation indicates that ACP amplification remains evolutionarily dynamic despite the otherwise highly constrained symbiotic genome. Together, our findings show that even highly integrated obligate symbionts retain evolutionary flexibility in specialized functional systems.

genomics↗

bioRxiv2026

Diaphorin induces extensive transcriptional remodeling of translation, transport, metabolism, and stress responses in Escherichia coli

Diaphorin is a pederin-family polyketide produced by the obligate bacterial symbiont "Candidatus Profftella armatura" (Betaproteobacteria) of the Asian citrus psyllid Diaphorina citri, in which it accumulates at millimolar concentrations. Diaphorin suppresses the growth of many bacterial species but promotes the growth and metabolic activity of Escherichia coli, highlighting the importance of elucidating the mechanisms underlying these contrasting effects. Although cell-free studies show that diaphorin directly affects bacterial translation, how E. coli transcription changes during growth promotion remains unclear. Here, we analyzed the transcriptome of E. coli after 7 h of exposure to 5 mM diaphorin, when its growth-promoting effect is most pronounced. Diaphorin altered the expression of 1,278 of 4,269 analyzed genes. Functional enrichment analyses revealed strong negative enrichment of translation- and ribosome-related functions, pathway-specific changes in metabolism, and enrichment of transport-, stress-response, and quorum-sensing-related functions. Ribosomal protein genes were broadly downregulated, whereas coordinated upregulation was observed in mdtEF, prpBCDE, the Gad system, and luxS/lsr genes. These findings indicate functionally structured transcriptional remodeling during diaphorin-enhanced growth. Together with previous cell-free evidence, the pronounced translation-related changes observed here highlight bacterial translation as a major process associated with the response of E. coli to diaphorin. One possible interpretation of the apparently contrasting observations is that an earlier stimulation of bacterial translation is followed by compensatory downregulation of translation-related genes as growth proceeds. More broadly, these transcriptional changes provide insight into diaphorin-enhanced growth in E. coli and offer a basis for understanding the strikingly different effects of this symbiont-derived polyketide among bacterial species.

microbiology↗

bioRxiv2026

Multi-color droplet digital PCR assay enables allele-specific quantification of heterogeneous genome editing outcomes

Precise characterization of genome editing outcomes remains a major challenge because edited cell populations contain diverse alleles generated by homology-directed repair, non-homologous end joining (NHEJ), or base editing. While next-generation sequencing enables comprehensive analysis, its routine use is constrained by cost and turnaround time. Here, we developed a multi-color droplet digital PCR (ddPCR) assay that exploits six-color fluorescence detection to quantitatively distinguish multiple edited alleles within a single reaction. Using CRISPR-Cas9 and base editing model systems, we designed sequence-specific probe sets that distinguished recurrent NHEJ alleles generated by CRISPR-Cas9 editing as well as target and bystander alleles generated by base editing. The assay quantitatively resolved individual editing outcomes that could not be distinguished by conventional Sanger sequencing. Together, these results establish multi-color ddPCR as a rapid, scalable, and sequence-specific approach for quantification of genome editing outcomes across multiple editing modalities.

molecular biology↗

bioRxiv2026

Epithelial Glutamate Retention Protects against Colitis

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that encompasses ulcerative colitis and Crohns disease. Here we identify the cystine/glutamate antiporter xCT as being markedly upregulated in the inflamed intestinal epithelium of patients with IBD. To clarify its functional contribution to disease pathogenesis, we performed genetic loss-of-function study and found that inhibition of xCT confers robust protection against dextran sulfate sodium (DSS)-induced colitis in mice. Intestinal epithelial cell (IEC)-specific deletion of xCT markedly attenuated colitis severity, demonstrating that epithelial xCT upregulation acts as a disease-exacerbating factor in IBD. Mechanistically, xCT deficiency preserved intracellular glutamate levels and protein polyglutamylation, thereby maintaining epithelial barrier integrity and protecting IECs from inflammatory injury. Consistently, pharmacological inhibition of glutamine synthetase, which increases intracellular glutamate, exerted a potent anti-inflammatory effect on the DSS-induced colitis. These findings identify intracellular glutamate retention in IECs as a previously unrecognized mechanism of epithelial protection and highlight both inhibition of xCT-dependent glutamate efflux and suppression of glutamine synthetase as potential therapeutic strategies for IBD.

molecular biology↗

bioRxiv2024

Highly reduced complementary genomes of dual bacterial symbionts in the mulberry psyllid Anomoneura mori

The genomes of obligately host-restricted bacteria suffer from accumulating mildly deleterious mutations, resulting in a drastic size reduction. Psyllids (Hemiptera) are phloem sap-sucking insects with a specialized organ called the bacteriome, which typically harbors two vertically transmitted bacterial symbionts: the primary symbiont "Candidatus Carsonella ruddii" (Gammaproteobacteria) and a secondary symbiont phylogenetically diverse among psyllid lineages. Genomes of several Carsonella lineages were revealed to be drastically reduced (158-174 kb), AT-rich (14.0-17.9% GC), and structurally conserved with similar gene inventories devoted for synthesizing essential amino acids scarce in the phloem sap. However, genomic information for secondary symbionts was limited. Hence, this study analyzed genomes of the bacteriome-associated dual symbionts, Secondary_AM (Gammaproteobacteria) and Carsonella_AM, in the mulberry psyllid Anomoneura mori (Psyllidae). This revealed that the Secondary_AM genome is as small and AT-rich (229,822 bp, 17.3% GC) as those of Carsonella, including Carsonella_AM (169,120 bp, 16.2% GC), implying that Secondary_AM is an evolutionarily ancient obligate mutualist like Carsonella. Phylogenomic analyses demonstrated that Secondary_AM is sister to "Candidatus Psyllophila symbiotica" of Cacopsylla spp. (Psyllidae), whose genomes (221-237 kb, 17.3-18.6% GC) were recently reported. The Secondary_AM and Psyllophila genomes showed highly conserved synteny, sharing all genes for complementing the incomplete tryptophan biosynthetic pathway of Carsonella and genes for synthesizing B vitamins. However, sulfur assimilation and carotenoid synthesizing genes were retained only in Secondary_AM and Psyllophila, respectively, indicating ongoing gene silencing. Average nucleotide identity, gene ortholog similarity, genome-wide synteny, and substitution rates suggested that the Secondary_AM/Psyllophila genomes are more labile than the Carsonella genomes.

genomics↗

bioRxiv2023

Functional consequences of shifting transcript boundaries in glucose starvation

Glucose is a major source of carbon and essential for the survival of many organisms, ranging from yeast to human. A sudden 60-fold reduction of glucose in exponentially growing fission yeast induces transcriptome-wide changes in gene expression. This regulation is multilayered, and the boundaries of transcripts are known to vary, with functional consequences at the protein level. By combining direct RNA sequencing with 5-CAGE and short-read sequencing, we accurately defined the 5- and 3-ends of transcripts that are both poly(A) tailed and 5-capped in glucose starvation, followed by proteome analysis. Our results confirm previously experimentally validated loci with alternative isoforms and reveal several transcriptome-wide patterns. First, we show that sense-antisense gene pairs are more strongly anti-correlated when a time lag is taken into account. Secondly, we show that the glucose starvation response initially elicits a shortening of 3-UTRs and poly(A) tails, followed by a shortening of the 5-UTRs at later time points. These result in domain gains and losses in proteins involved in the stress response. Finally, the relatively poor overlap both between differentially expressed genes (DEGs), differential transcript usage events (DTUs), and differentially detected proteins (DDPs) highlight the need for further study on post-transcriptional regulation mechanisms in glucose starvation.

molecular biology↗