Biology subjects

Furuta, K.

Publications and source records attributed to Furuta, K..

13 recordsLinked to original sources

bioRxiv2026

Deletion of the ribosomal protein gene rpmJ activates zntA transcription through a translation-dependent mechanism in Escherichia coli

Bacteria tightly regulate intracellular zinc homeostasis by coordinating zinc uptake and efflux systems. We previously showed that deletion of the ribosomal protein gene rpmJ confers zinc resistance in Escherichia coli in a manner dependent on the zinc efflux transporter zntA. Here, we analyzed the effect of rpmJ deficiency on zntA expression. Under zinc excess conditions, zntA mRNA levels were markedly higher in the rpmJ mutant than in the wild-type strain. Enhanced zntA expression required the native zntA promoter, the native Shine-Dalgarno sequence, and the N-terminal coding region of zntA, indicating that translation initiated from the native Shine-Dalgarno sequence and extending through the N-terminal coding region is required for enhanced transcription initiation from the native zntA promoter. Furthermore, ectopic expression of ykgO, a paralog of rpmJ that is known to replace RpmJ on the ribosome under zinc-limited conditions, abolished the increased zntA expression and zinc resistance conferred by rpmJ deletion. Collectively, these findings suggest that ribosomes lacking RpmJ or YkgO promote transcription initiation from the native zntA promoter through translation of zntA mRNA. IMPORTANCEBacteria must carefully control the amount of zinc inside their cells. Too little zinc prevents essential cellular processes, whereas too much zinc is toxic. We found that removing a small ribosomal protein called RpmJ allows Escherichia coli to survive high zinc levels by increasing production of the zinc exporter zntA. Surprisingly, this increase depends not only on the zntA promoter but also on translation of the beginning of the zntA coding region. Our findings suggest that changes in ribosome composition can stimulate gene transcription through early translation of the same messenger RNA, revealing a previously unrecognized mechanism linking translation and transcription during bacterial adaptation to zinc stress.

molecular biology↗

bioRxiv2025

Overexpression of Escherichia coli yaiX Confers Multidrug Resistance and Enhances Virulence in the Silkworm Infection Model

The emergence of bacteria with both antimicrobial resistance and high virulence has become a global health concern, underscoring the urgent need to elucidate the molecular basis underlying these traits. Here, we employed the silkworm (Bombyx mori) infection model, which is suitable for high-throughput screening, together with an Escherichia coli library containing plasmid clones of all genes from strain W3110, to identify genes whose overexpression enhances virulence. We found that overexpression of the uncharacterized protein YaiX promoted bacterial proliferation in silkworms and increased host lethality. Compared with the empty-vector control, the YaiX-overexpressing strain exhibited resistance to multiple antimicrobial agents with diverse mechanisms of action, including {beta}-lactams, tetracyclines, fluoroquinolones, aminoglycosides, cationic surfactants, and hydrogen peroxide. Sequence analysis revealed that amino acids 18-52 of YaiX contain a transferase hexapeptide domain predicted to form a left-handed parallel {beta}-helix. Overexpression of YaiX mutants lacking regions outside this domain conferred ampicillin resistance, whereas deletion of the hexapeptide domain abolished this phenotype. RNA sequencing and GO enrichment analyses further indicated that YaiX overexpression altered the expression of genes encoding RNA-binding proteins and porins. These findings suggest that YaiX overexpression, through its hexapeptide domain, modulates gene expression and contributes to both multidrug resistance and enhanced virulence in E. coli.

microbiology↗

bioRxiv2025

Autoclaving and long-term storage deplete glutamine sources in complex media and affect bacterial phenotypes

Autoclave sterilization is the most common method for sterilizing reagents and media in biology. However, the effects of heat-induced loss or modification of complex medium components on bacterial growth and phenotypes remain poorly understood. Here, we investigated the impact of autoclaving on glutamine sources in bacterial complex media using an Escherichia coli {Delta}glnA mutant, which requires exogenous glutamine for growth. {Delta}glnA exhibited impaired growth in LB medium after autoclaving compared with non-autoclaved LB, whereas its residual growth indicated the presence of heat-stable glutamine sources. Growth assays and HPLC quantification revealed that free glutamine in LB decreased from 58 {micro}M to 12 {micro}M upon autoclaving, while heat-stable glutamine sources remained at 117 {micro}M. Similar growth defects were observed for {Delta}glnA in autoclaved BHI, TSB, and M17 media compared with their non-autoclaved counterparts. Long-term storage of LB at room temperature for 24 weeks also reduced {Delta}glnA growth regardless of autoclaving, compared with freshly prepared LB. Furthermore, supplementation of glutamine sources into glutamine-deficient MHB medium enhanced biofilm formation by Pseudomonas aeruginosa. Collectively, these results demonstrate that autoclaving and storage reduce glutamine sources in complex media, thereby influencing bacterial growth and phenotypes.

microbiology↗

bioRxiv2025

Knockout of tusA facilitates flagella formation and cationic antimicrobial resistance by disrupting Fur transcriptional regulation in Escherichia coli

tRNA 2-thiouridine synthesizing protein A (TusA), a sulfur-carrier protein, plays a crucial role in tRNA sulfur modification. Recent studies have reported that tusA deficiency affects iron-sulfur (Fe-S) homeostasis and cluster formation in Escherichia coli; however, its association with this phenotype remains unclear. In this study, we analyzed the phenotype of tusA-deficient E. coli ({Delta}tusA) and its underlying mechanisms using RNA sequencing. We observed that tusA deletion disrupted the expression of genes regulated by the ferric uptake regulator Fur or Fur-regulated transcription factors (flagellar transcriptional regulators D and C [FlhDC] and fumarate and nitrate reduction regulator [Fnr]). Increased expression of flhDC, which is the master regulator of flagellar genes facilitated flagella formation even under conditions in which the wild-type formed few flagella. Additionally, {Delta}tusA was resistant to cationic antibacterial agents, such as cetyltrimethylammonium bromide, cetylpyridinium chloride, and protamine sulfate. This resistance is associated with the increased expression of ompX or ompF, regulated by Fur and Fnr, respectively. Notably, both enhanced flagella formation and resistance to cationic antibacterial agents caused by tusA deletion were abolished in the fur-deficient background. These findings indicate that impaired expression of the fur regulon, possibly because of impaired Fe-S cluster formation, induces multiple phenotypic alterations in {Delta}tusA. IMPORTANCETusA is a sulfur carrier protein involved in tRNA sulfur modification, and its effect on translation has been studied. Recent studies have reported that tusA deficiency affects Fe-S homeostasis and cluster formation in Escherichia coli; however, its association with the phenotype remains unclear. Based on RNA sequencing, we indicated that the altered gene expression in {Delta}tusA resulted from the disruption of fur regulation that is controlled by Fe-S clusters. We further demonstrated that enhanced flagella formation and resistance to cationic drugs were mediated by Fur-dependent gene expression alterations. Our data indicate that the regulation of sulfur allocation for tRNA modification by TusA affects the global gene expression in bacteria.

microbiology↗

bioRxiv2025

Xenopus laevis as an infection model for human pathogenic bacteria

Animal infection models are essential for understanding bacterial pathogenicity and corresponding host immune responses. In this study, we investigated whether juvenile Xenopus laevis could be used as an infection model for human pathogenic bacteria. Xenopus frogs succumbed to intraperitoneal injection containing the human pathogenic bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Listeria monocytogenes. In contrast, non-pathogenic bacteria Bacillus subtilis and Escherichia coli did not induce mortality in Xenopus frogs. The administration of appropriate antibiotics suppressed mortality caused by S. aureus and P. aeruginosa. Strains lacking the agr locus, cvfA (rny) gene, or hemolysin genes in S. aureus, LIPI-1-deleted mutant of L. monocytogenes, which attenuate virulence within mammals, exhibited reduced virulence in Xenopus frogs compared to their respective wild-type counterparts. Bacterial distribution analysis revealed that S. aureus persisted the blood, liver, heart, and muscles of Xenopus frogs until death. These results suggested that intraperitoneal injection of human pathogenic bacteria induces sepsis-like symptoms in Xenopus frogs supporting their use as a valuable animal model for evaluating antimicrobial efficacy and identifying virulence genes in various human pathogenic bacteria.

microbiology↗

bioRxiv2024

Escherichia coli growth under low pH, low temperature, and high osmotic stress conditions requires the wzxE flippase for the enterobacterial common antigen intermediate

Colanic acid and enterobacterial common antigen (ECA) are cell surface polysaccharides that are produced by many E. coli isolates. Colanic acid is induced under low pH, low temperature, and hyperosmotic conditions and is important in E. coli resistance to these stresses; however, the role of the ECA is unclear. In this study, we observed that knockout of the flippase wzxE, which converts ECA intermediates from the cytoplasmic side of the inner membrane to the periplasmic side, resulted in low pH sensitivity in E. coli. The wzxE-knockout mutant showed reduced growth potential and viable counts in the extracts of several vegetables (cherry tomatoes, carrots, celery, lettuce, and spinach), which are known to be low pH environments. A double knockout strain of wzxE and wecF, which encodes an enzyme that synthesizes an ECA intermediate, did not show sensitivity to low pH, nor did a double knockout mutant of wzxE and wcaJ, which encodes a colanic acid synthase. The wzxE-knockout mutant was sensitive to low temperature or hyperosmotic conditions, which induced colanic acid synthesis, and these sensitivities were abolished by the additional knockout of wcaJ. These results suggest that ECA intermediates cause E. coli susceptibility to low pH, low temperature, and high osmotic pressure in a colanic acid-dependent manner, and that wzxE suppresses this negative effect. ImportancePolysaccharides covering bacterial cell surfaces, such as colanic acid, confer resistance to various stresses, such as low pH. However, the role of enterobacterial common antigens, carbohydrate antigens that are conserved throughout enterobacteria, in stress resistance is unclear. Our results suggest that lipid III enterobacterial common antigen, a substrate of flippase, causes sensitivity of Escherichia coli to low pH, low temperature, and high osmolarity in dependence on colanic acid synthesis, while wzxE inhibits this negative effect. The wzxE-knockout mutant was sensitive to crude vegetable extracts, suggesting that the creation of WzxE inhibitors could lead to new food poisoning prevention agents.

microbiology↗

bioRxiv2024

Overexpression of Ribosomal Proteins Leads to Zn Resistance in Escherichia coli

Knockout of ribosomal protein bL36 (RpmJ) leads to Zn resistance in Escherichia coli, and the expression of ribosomal protein genes other than RpmJ increase in the rpmJ-knockout strain. In this study, we examined whether the overexpression of ribosomal proteins causes Zn resistance using an E. coli overexpression gene library (ASKA clone library). The overexpression of 48 of the 54 ribosomal proteins led to Zn resistance. However, the overexpression of proteins other than ribosomal proteins did not lead to Zn resistance, suggesting that Zn resistance is a phenomenon specific to the overexpression of ribosomal proteins. In addition, the overexpression of ribosomal proteins did not lead to resistance to metal ions other than Zn (Cu2+, Ni2+, Mn2+, and Ag+), suggesting a Zn-specific resistance mechanism. Deletion of ZntA, a Zn efflux pump, resulted in the loss of Zn resistance in a ribosomal protein-overexpressing strain. Deletion of Lon protease, which is responsible for degrading misfolded proteins, in a ribosomal protein-overexpressing strain resulted in the accumulation of overexpressed ribosomal proteins and loss of Zn resistance. These results suggest that the overexpression of ribosomal proteins leads to Zn resistance in E. coli via ZntA and Lon protease. ImportanceThe ribosome is a complex comprising ribosomal RNAs and more than 50 types of ribosomal proteins. Ribosomal proteins play an important role in ribosomal function responsible for protein translation; however, their involvement in other cellular processes is not fully understood. Based on the finding that ribosomal protein expression increases in a Zn-resistant E. coli mutant, we analyzed 54 ribosomal proteins and found that the overexpression of 48 ribosomal proteins led to Zn resistance. This finding suggests a role for ribosomal proteins in resistance to zinc stress.

microbiology↗

bioRxiv2024

Active fluctuations of cytoplasmic actomyosin networks facilitate dynein-driven intracellular transport along microtubules

Inside cells, molecular motors transport cargoes within the actively fluctuating environment known as the cytoplasm. How fluctuations in the cytoplasm affect motor-driven transport is not fully understood. In this study, we investigated the role of fluctuations for transport along microtubules using C. elegans early embryos, focusing on transport driven by cytoplasmic dynein. An artificial motor-cargo complex showed faster transport in vivo than in vitro, suggesting an in vivo acceleration mechanism. We also found that endogenous early endosome transport by dynein is significantly enhanced by the fluctuations in the cytoplasm, which is attributed to the activity of actomyosin networks. An in vitro force measurement of dynein suggests that the asymmetric force response would play a key role in the acceleration. This study provides insights into a regulatory mechanism of molecular motors within actively fluctuating cytoplasm, potentially utilizing random force originating from fluctuating dynamics in the cytoplasm to increase transport efficiency.

biophysics↗

bioRxiv2024

Overexpression of diglucosyldiacylglycerol synthase leads to daptomycin resistance in Bacillus subtilis

The lipopeptide antibiotic daptomycin exhibits bactericidal activity against gram-positive bacteria by forming a complex with phosphatidylglycerol (PG) and lipid II in the cell membrane, causing membrane perforation. With the emergence of daptomycin-resistant bacteria, understanding the mechanism of bacterial resistance to daptomycin has gained great importance. In the present study, we found that overexpression of ugtP, which encodes a diglucosyldiacylglycerol synthase, resulted in daptomycin resistance in Bacillus subtilis. The amount of diglucosyldiacylglycerol (Glc2DAG) increased in the ugtP-overexpressed strain, whereas the amounts of the acidic phospholipids cardiolipin (CL) and PG and the basic phospholipid lysylphosphatidylglycerol (Lys-PG) decreased. Moreover, ugtP overexpression did not alter the cell surface charge or the susceptibility to the cationic antimicrobial peptide nisin or the cationic surfactant hexadecyltrimethylammonium bromide (CTAB). Furthermore, under daptomycin exposure, we obtained daptomycin-resistant mutants that carried ugtP mutations and showed increased amounts of Glc2DAG and more than 4-fold increase in the MIC of daptomycin. These results suggest that an increase in the amount of Glc2DAG changes the phospholipid composition and leads to daptomycin resistance without altering the bacterial cell surface charge in B. subtilis. ImportanceDaptomycin is one of the last-resort drugs for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections, and the emergence of daptomycin-resistant bacteria has become a problem. Understanding the mechanism of daptomycin resistance is important for establishing clinical countermeasures against daptomycin-resistant bacteria. In the present study, we found that overexpression of ugtP, which encodes diglucosyldiacylglycerol synthase, causes daptomycin resistance in B. subtilis, a model organism for gram-positive bacteria. Overexpression of ugtP increased diglucosyldiacylglycerol levels, resulting in altered phospholipid composition and daptomycin resistance. These findings are important for establishing clinical strategies against daptomycin-resistant bacteria, including their detection.

microbiology↗

bioRxiv2022

Knockout of ribosomal protein RpmJ leads to zinc resistance in Escherichia coli

Zinc is an essential metal for cells, but excess amounts are toxic. Other than by regulating the intracellular zinc concentration by zinc uptake or efflux, the mechanisms underlying bacterial resistance to excess zinc are unknown. In the present study, we searched for zinc-resistant mutant strains from the Keio collection, a gene knockout library of Escherichia coli, a model gram-negative bacteria. We found that knockout mutant of RpmJ, a 50S ribosomal protein, exhibited zinc resistance. The rpmJ mutant was sensitive to protein synthesis inhibitors and had altered translation fidelity, indicating ribosomal dysfunction. In the rpmJ mutant, the intracellular zinc concentration was decreased under excess zinc conditions. RNA sequence analysis revealed that rpmJ knockout decreased the expression of synthetic genes for iron-sulfur cluster proteins, which are toxic targets for zinc. These findings suggest that knocking out RpmJ causes zinc resistance by decreasing zinc targets and lowering the intracellular zinc concentration. Knockouts of other ribosomal proteins, including RplA, RpmE, RpmI, and RpsT, also led to a zinc-resistant phenotype, suggesting that deletion of ribosomal proteins is closely related to zinc resistance.

microbiology↗

bioRxiv2022

Knockout of ykcB, a putative glycosyltransferase, leads to vancomycin resistance in Bacillus subtilis

Vancomycin resistance of gram-positive bacteria poses a serious health concern around the world. In this study, we searched for vancomycin-resistant mutants from a gene deletion library of a model gram-positive bacterium, Bacillus subtilis, to elucidate the mechanism of vancomycin resistance. We found that knockout of ykcB, a glycosyltransferase that is expected to utilize C55-P-glucose to glycosylate cell surface components, caused vancomycin resistance in B. subtilis. Knockout of ykcB altered the susceptibility to multiple antibiotics, including sensitization to {beta}-lactams, and increased the pathogenicity to silkworms. Furthermore, the ykcB-knockout mutant had: i) an increased content of diglucosyl diacylglycerol, a glycolipid that shares a precursor with C55-P-glucose, ii) a decreased amount of lipoteichoic acid, and iii) decreased biofilm formation ability. These phenotypes and vancomycin resistance were abolished by knockout of ykcC, a ykcB-operon partner involved in C55-P-glucose synthesis. Overexpression of ykcC enhanced vancomycin resistance in both wild-type B. subtilis and the ykcB-knockout mutant. These findings suggest that ykcB deficiency induces structural changes of cell surface molecules depending on the ykcC function, leading to resistance to vancomycin, decreased biofilm formation ability, and increased pathogenicity to silkworms. IMPORTANCEAlthough vancomycin is effective against gram-positive bacteria, vancomycin-resistant bacteria is a major public health concern. While the vancomycin resistance mechanisms of clinically important bacteria such as Staphylococcus aureus, Enterococcus faecium, and Streptococcus pneumoniae are well-studied, they remain unclear in other gram-positive bacteria. In the present study, we searched for vancomycin-resistant mutants from a gene deletion library of a model gram-positive bacterium, Bacillus subtilis, and found that knockout of a putative glycosyltransferase, ykcB, caused vancomycin resistance in B. subtilis. Notably, unlike the previously reported vancomycin-resistant bacterial strains, ykcB-deficient B. subtilis exhibited increased virulence while maintaining its growth rate. Our results broaden the fundamental understanding of vancomycin-resistance mechanisms in gram-positive bacteria.

microbiology↗

bioRxiv2018

Kinesin-6 Klp9 plays motor-dependent and -independent roles in collaboration with Kinesin-5 Cut7 and the microtubule crosslinker Ase1 in fission yeast

Bipolar mitotic spindles play a critical part in accurate chromosome segregation. During late mitosis, spindle microtubules undergo drastic elongation towards the cell cortex in a process called anaphase B. Two kinesin motors, Kinesin-5 and Kinesin-6, are thought to generate outward forces to drive spindle elongation, and the microtubule crosslinker Ase1/PRC1 maintains structural integrity of antiparallel microtubules. However, how these three proteins orchestrate this process remains unknown. Here we explore the functional interplay among fission yeast Kinesin-5/Cut7, Kinesin-6/Klp9 and Ase1. Using total internal reflection fluorescence microscopy, we show that Klp9 is a processive plus end-directed motor. klp9{Delta}ase1{Delta} is synthetically lethal. Surprisingly, this lethality is not ascribable to the defective motor activity of Klp9; instead, it is dependent upon an NLS and coiled coil domains within the non-motor region. We isolated a cut7 mutant (cut7-122) that displays temperature sensitivity only in the absence of Klp9. Interestingly, cut7-122 is impaired specifically in late mitotic stages. cut7-122klp9{Delta} double mutant cells exhibit additive defects in spindle elongation. Together, we propose that Klp9 plays dual roles during anaphase B; one is motor-dependent that collaborates with Cut7 in force generation, while the other is motor-independent and ensures structural integrity of spindle microtubules together with Ase1.

cell biology↗

bioRxiv2017

Small teams of myosin Vc coordinate their stepping for efficient cargo transport on actin bundles

Myosin Vc (myoVc) is unique among vertebrate class V myosin isoforms in that it requires teams of motors to transport cargo. Single molecules of myoVc cannot take multiple steps on single actin filaments, in stark contrast to the well-studied myosin Va (myoVa) isoform. Consistent with in vivo studies (1), only teams of myoVc motors can move continuously on actin bundles at physiologic ionic strength (2), raising the question of how motor-motor interactions cause this preference. Here, using DNA nanostructures as synthetic cargos for linking defined numbers of myoVa or myoVc molecules, we compared the stepping behavior of myoVa versus myoVc teams, and myoVc stepping patterns on single actin filaments versus actin bundles. Run lengths of both myoVa and myoVc teams increased with motor number, but the run lengths of myoVc teams were longer on actin bundles than on filaments. By resolving the stepping behavior of individual myoVc motors with a Qdot bound to the motor domain, we found that coupling of two myoVc molecules significantly decreases futile back/side steps, which were frequently observed for single myoVc motors. Data showing how changes in the inter-motor distance of two coupled myoVc motors affected stepping dynamics suggested that mechanical tension coordinates the stepping behavior of two molecules for efficient directional motion. Our study thus provides a molecular basis to explain how teams of myoVc motors are suited to transport cargos such as zymogen granules on actin bundles.

biophysics↗