Bioscience, Biotechnology, and Biochemistry最新文献

The toxin-antitoxin (TA) genetic module controls various bacterial events. Novel toxins with different functions are still being discovered. This study aimed to determine whether the ECs3274-ECs3275 gene pair encoded by enterohemorrhagic Escherichia coli O157 functions as a TA system. To characterize this putative TA system, we analyzed the growth of E. coli expressing ECs3274, ECs3275, or both; the interaction between ECs3274 and ECs3275 using bacterial adenylate cyclase two-hybrid assays; and the DNA-binding ability of ECs3274 using gel-mobility shift assays. We observed that the ECs3274 antitoxin interacted with the ECs3275 toxin, was destabilized by Lon protease, and repressed its promoter activity via its helix-turn-helix (HTH) motif. These properties are consistent with those of typical type II TA antitoxins. Interestingly, ECs3275 has an HTH motif not observed in other TA toxins and is necessary for ECs3275 toxicity, suggesting that ECs3275 may exert its toxicity by regulating the expression of specific genes.

Recombinant protein production in prokaryotic and eukaryotic cells is a fundamental technology for both research and industry. Achieving efficient protein synthesis is key to accelerating the discovery, characterization, and practical application of proteins. This review focuses on recent advances in recombinant protein production and strategies for more efficient protein production, especially using Escherichia coli and Saccharomyces cerevisiae. Additionally, this review summarizes the development of various functional peptide tags that can be employed for protein production, modification, and purification, including translation-enhancing peptide tags developed by our research group.

Thermophilic actinomycetes significantly contribute to the terrestrial carbon cycle via the rapid degradation of lignocellulosic polysaccharides in composts. In this study, a genome-editing system was constructed for the thermophilic actinomycete Streptomyces thermodiastaticus K5 strain, which was isolated from compost. The genome-editing plasmid (pGEK5) harboring nickase Cas9 was derived from the high-copy plasmid pL99 and used for the K5 strain. It was found that pGEK5 could easily be lost from the transformed clone through cultivation on apramycin-free medium and spore formation, enabling its reuse for subsequent genome-editing cycles. With the aid of this plasmid, mutations were sequentially introduced to 2 uracil-DNA glycosylase genes (Udg1 and Udg2) and 1 β-glucosidase gene (Bgl1). Thus, the genome-editing system using pGEK5 enables us to start the functional modification of this thermophilic actinomycete, especially for improved conversion of lignocellulosic biomass.

Plants, as sessile organisms, must adapt to environmental changes and defend themselves against biotic stress, including pathogen attack. Their immune responses entail recognition of pathogen patterns, activation of defense mechanisms, and accumulation of various antimicrobial compounds. Eugenol, abundant in basil, has antibacterial properties and enhances plant resistance to viruses. However, its priming effects on biotrophic pathogens remain unclear. Thus, we investigated whether eugenol and basil essential oils could prime Arabidopsis thaliana immunity against the hemi-biotroph Pseudomonas syringae pv. maculicola (Psm) MAFF302723. Our study revealed that both eugenol and basil essential oils functioned as priming agents, mitigating disease symptoms upon Psm infection. This priming effect occurred via NPR1-dependent but salicylic acid-independent signaling. Moreover, our gene expression analysis suggested that priming might influence jasmonic acid/ethylene signaling. These findings underscore the potential of employing natural compounds such as basil essential oil to bolster plant immune responses in sustainable agricultural practices.

We previously reported that choline chloride and N-allylglycine stimulate photosynthesis in wheat protoplasts. Treatment of Arabidopsis thaliana and Brassica rapa plants with both compounds promoted growth and photosynthesis. To clarify the relationship between the enhancement of photosynthesis and increased growth, A. thaliana T87 cells, which show photosynthesis-dependent growth, and YG1 cells, which use sugar in the medium for growth, were treated with choline chloride or N-allylglycine. Only the T87 cells showed increased growth, suggesting that choline chloride and N-allylglycine promote growth by increasing photosynthetic activity. Transcriptome analysis using choline chloride- and N-allylglycine-treated plants showed that the most abundant transcripts corresponded to photosynthetic electron transfer-related genes among the genes upregulated by both compounds. Furthermore, the compounds also upregulate genes encoding transcription factors that may control the expression of these photosynthetic genes. These results suggest that choline chloride and N-allylglycine promote photosynthesis through increased expression of photosynthetic electron transfer-related genes.

Essential amino acids (EAAs) are important for the maintenance of brain functions. Therefore, the yeast Saccharomyces cerevisiae that accumulates EAAs would help elderly people ingest appropriate levels of EAAs, which in turn could slow neurodegeneration, extend the healthy lifespan, and improve quality of life. Here, we isolated 2 mutant strains, ETH-80 and ETH-129, that accumulate the EAA methionine. Both strains were derived from a diploid laboratory yeast by conventional mutagenesis and carry a novel mutation in the MET13 gene, which encodes the Ser443Phe variant of methylenetetrahydrofolate reductase. Enzymatic analysis revealed that the Ser443Phe substitution abolished the sensitivity to S-adenosyl methionine (SAM)-mediated inhibition even in the presence of 2 m m SAM, while increasing the activity for NADPH-dependent reduction. Furthermore, yeast cells expressing the Ser443Phe variant showed a 4-fold increase in intracellular methionine content compared to the wild-type Met13. These findings will be useful for the future development of methionine-accumulating yeast strains.

Cold atmospheric plasma (CAP) irradiation exhibits a sterilizing effect without causing thermal denaturation or leaving behind residual toxicants. CAP also has potential applications in various fields, including agriculture, leading to research efforts in recent years. This study investigated the effects of CAP on the seed germination rate of spinach (Spinacia oleracea), which typically has a low seed germination rate. Our results confirmed that irradiation with N2-CAP and Air-CAP significantly enhanced the germination rate of spinach seeds. Notably, we discovered that CAP irradiation promoted germination even in spinach seeds coated with a fungicide (thiuram) and a disinfectant (Captan), which are commonly used. Additionally, we examined whether the interval between CAP irradiation and the subsequent germination-induction treatment influenced the germination efficiency. We found that the germination-promoting effect of CAP on spinach seeds persisted for at least 30 days, demonstrating the high utility and practicality of CAP in the agricultural sector.

Foam cells are primarily formed through scavenger receptors that mediate the uptake of various modified low-density lipoproteins (LDL) into cells. In addition to the receptor-dependent pathway, macropinocytosis is an essential nonreceptor endocytic pathway for vascular smooth muscle cells (VSMCs) to take up lipids. However, the molecular mechanisms underlying this process remain unclear. Primary cultured VSMCs were stimulated with 200 ng/mL lipopolysaccharide (LPS) and 200 µg/mL native LDL (nLDL). We observed a significant increase in Toll-like receptor 4 (TLR4) protein expression and a significant activation of macropinocytosis, which correlated with the highest uptake of nLDL and intracellular lipid deposition in WT VSMCs. However, macropinocytosis was inhibited and lipid accumulation decreased after treatment with macropinocytosis inhibitors and Syk inhibitors in WT VSMCs. Consistently, TLR4 knockout significantly suppressed macropinocytosis and lipid droplets accumulation in VSMCs. Taken together, our findings suggest a critical role of TLR4/Syk signaling in promoting receptor-independent macropinocytosis leading to VSMC-derived foam cells formation.

Glycoside hydrolase family 65 (GH65) includes glycoside hydrolases active on various α-glucosides. We previously demonstrated that the GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) is a kojibiose hydrolase and determined its 3-dimensional structure. In this study, the effects of glucosidase inhibitors on FjGH65A and their complex structures were analyzed to elucidate their inhibition mechanism. FjGH65A was competitively inhibited by 1-deoxynojirimycin (DNJ) and noncompetitively inhibited by castanospermine (CSP) with Ki values of 2.95 and 3.69 µm, respectively. The crystal structures of FjGH65A complexed with the inhibitors indicated that DNJ was bound to subsite -1 of FjGH65A, while CSP was bound to subsites -1 and +1 of FjGH65A. Compared with the glucose complex structure, the conformation of Tyr337 was changed in the CSP complex structure. These results provide new structural insights into the mechanism of inhibition against GH65 α-glucoside hydrolases.

ABCF proteins (ABCFs) are key components of prokaryotic translation systems, resolving ribosomal stalling. These ATPases contain two ATPase domains and interdomain linker, the length and composition of which are key determinants of their function. Antibiotic resistance ABCF (ARE-ABCFs) proteins, counteract ribosome-targeting antibiotics by binding to the E site of the 70S ribosome, promoting drug dissociation. In contrast, housekeeping ABCF proteins, such as YfmR and YkpA in Bacillus subtilis, resolve intrinsic translation challenges without conferring antibiotic resistance. YfmR addresses stalling at proline-rich motifs, while YkpA resolves stalling caused by charged motifs. This review draws on the work of Chadani, Boël, Fega, and our own studies to compare the structural and functional diversity of ABCF proteins across bacterial species. It highlights the key roles of ARD/PtlM domains in defining their specific functions and explores future research directions to further our understanding of ABCF proteins in translation control.