• Journal of Microbiology and Biotechnology
• /
• v.31 no.12
• /
• pp.1722-1731
• /
• 2021

The genus Streptomyces is intensively studied due to its excellent ability to produce secondary metabolites with diverse bioactivities. In particular, adequate precursors of secondary metabolites as well as sophisticated post modification systems make some high-yield industrial strains of Streptomyces the promising chassis for the heterologous production of natural products. However, lack of efficient genetic tools for the manipulation of industrial strains, especially the episomal vector independent tools suitable for large DNA fragment deletion, makes it difficult to remold the metabolic pathways and streamline the genomes in these strains. In this respect, we developed an efficient deletion system independent of the episomal vector for large DNA fragment deletion. Based on this system, four large segments of DNA, ranging in length from 10 kb to 200 kb, were knocked out successfully from three industrial Streptomyces strains without any marker left. Notably, compared to the classical deletion system used in Streptomyces, this deletion system takes about 25% less time in our cases. This work provides a very effective tool for further genetic engineering of the industrial Streptomyces.

• Journal of Microbiology and Biotechnology
• /
• v.32 no.5
• /
• pp.621-629
• /
• 2022

Bacterial outer membrane vesicles (OMVs) typically contain multiple immunogenic molecules that include antigenic proteins, making them good candidates for vaccine development. In animal models, vaccination with OMVs has been shown to confer protective immune responses against many bacterial diseases. It is possible to genetically introduce heterologous protein antigens to the bacterial host that can then be produced and relocated to reside within the OMVs by means of the host secretion mechanisms. Accordingly, in this study we sought to develop a novel platform for recombinant OMV (rOMV) production in the widely used bacterial expression host species, Escherichia coli. Three different lipoprotein signal peptides including their Lol signals and tether sequences-from Neisseria meningitidis fHbp, Leptospira interrogans LipL32, and Campylobactor jejuni JlpA-were combined upstream to the GFPmut2 model protein, resulting in three recombinant plasmids. Pilot expression studies showed that the fusion between fHbp and GFPmut2 was the only promising construct; therefore, we used this construct for large-scale expression. After inducing recombinant protein expression, the nanovesicles were harvested from cell-free culture media by ultrafiltration and ultracentrifugation. Transmission electron microscopy demonstrated that the obtained rOMVs were closed, circular single-membrane particles, 20-200 nm in size. Western blotting confirmed the presence of GFPmut2 in the isolated vesicles. Collectively, although this is a non-optimized, proof-of-concept study, it demonstrates the feasibility of this platform in directing target proteins into the vesicles for OMV-based vaccine development.

• Journal of the Korean Wood Science and Technology
• /
• v.51 no.4
• /
• pp.270-282
• /
• 2023

Lower termites need symbiotic microbes for cellulose digestion. Elizabethkingia miricola strain BM10 has been proposed as a symbiotic microbe that assists in low-temperature digestion and metabolism of Reticulitermes speratus KMT1, a termite on Bukhan Mountain, Seoul, Korea. In E. miricola strain BM10, β-glucosidase genes expressed at 10℃ were identified, and the psychrophilic enzymatic characteristic was confirmed by heterogeneously expressed proteins. Crude β-glucosidase in the culture broth of E. miricola strain BM10 showed specific enzymatic properties, and its substrate affinity was 4.69 times higher than that of Cellic CTec2. Among the genes proposed as β-glucosidase, two genes, bglB_1 and bglA_2, whose gene expression was more than doubled at 10℃ than at 30℃, were identified. They were heterogeneously expressed in Escherichia coli and identified as psychrophilic enzymes with an optimal reaction temperature of about 20℃-25℃. In this study, E. miricola strain BM10, a symbiotic bacterium of lower termites, produced psychrophilic β-glucosidases that contribute to the spread of the low-temperature habitat of a lower termite, R. speratus KMT1.

• Clinical and Experimental Vaccine Research
• /
• v.12 no.2
• /
• pp.172-175
• /
• 2023

Coronavirus disease 2019 (COVID-19) has become a part of our lives now and we have no more effective way of coping than a vaccine. COVID-19 is a disease that causes severe thrombosis outside the respiratory tract. Vaccines also protect us in this respect, but in some rare cases, thrombosis has been found to develop after vaccination (much less frequently than COVID-19). What was interesting in our case was that it showed how a disaster could happen under three factors that predispose to thrombosis. A 65-year-old female patient with disseminated atherosclerosis was admitted to the intensive care unit with complaints of dyspnea and dysphasia. In the evening of the day, the patient had the vaccination 2 weeks ago, she had active COVID-19. On examination, lower extremity pulses could not be detected. The patient's imaging and blood tests were performed. Multiple complications such as embolic stroke, venous and arterial thrombosis, pulmonary embolism, and pericarditis were observed in the patient. This case may give consideration to anticoagulant therapy studies. We give effective anticoagulant therapy in the presence of COVID-19 in patients at risk of thrombosis. Can anticoagulant therapy be considered after vaccination in patients at risk of thrombosis such as disseminated atherosclerosis?

• Journal of Plant Biotechnology
• /
• v.50
• /
• pp.225-231
• /
• 2023

Cold stress is one of the most vulnerable environmental stresses that affect plant growth and crop yields. With the recent advancements in genetic approaches using Arabidopsis and other model systems, genes involved in cold-stress response have been identified and the key cold signaling factors have been characterized. Exposure to low-temperature stress triggers the activation of a set of genes known as cold regulatory (COR) genes. This activation process plays a crucial role in enhancing the resistance of plants to cold and freezing stress. The inducer of the C-repeatbinding factor (CBF) expression 1-CBF module (ICE1-CBF module) is a key cold signaling pathway regulator that enhances the expression of downstream COR genes; however, this signaling module in Panax ginseng remains elusive. Here, we identified cold-signaling-related genes, PgCBF1, PgCBF3, and PgICE1 and conducted functional genomic analysis with a heterologous system. We confirmed that the overexpression of cold- PgCBF3 in the cbf1/2/3 triple Arabidopsis mutant compensated for the cold stress-induced deficiency of COR15A and salt-stress tolerance. In addition, nuclearlocalized PgICE1 has evolutionarily conserved phosphorylation sites that are modulated by brassinsteroid insensitive 2 (PgBIN2) and sucrose non-fermenting 1 (SNF1)-related protein kinase 3 (PgSnRK3), with which it physically interacted in a yeast two-hybrid assay. Overall, our data reveal that the regulators identified in our study, PgICE1 and PgCBFs, are evolutionarily conserved in the P. ginseng genome and are functionally involved in cold and abiotic stress responses.

• Molecules and Cells
• /
• v.46 no.11
• /
• pp.710-724
• /
• 2023

The plant defense responses to microbial infection are tightly regulated and integrated with the developmental program for optimal resources allocation. Notably, the defense-associated hormone salicylic acid (SA) acts as a promoter of flowering while several plant pathogens actively target the flowering signaling pathway to promote their virulence or dissemination. Ralstonia pseudosolanacearum inject tens of effectors in the host cells that collectively promote bacterial proliferation in plant tissues. Here, we characterized the function of the broadly conserved R. pseudosolanacearum effector RipL, through heterologous expression in Arabidopsis thaliana. RipL-expressing transgenic lines presented a delayed flowering, which correlated with a low expression of flowering regulator genes. Delayed flowering was also observed in Nicotiana benthamiana plants transiently expressing RipL. In parallel, RipL promoted plant susceptibility to virulent strains of Pseudomonas syringae in the effector-expressing lines or when delivered by the type III secretion system. Unexpectedly, SA accumulation and SA-dependent immune signaling were not significantly affected by RipL expression. Rather, the RNA-seq analysis of infected RipL-expressing lines revealed that the overall amplitude of the transcriptional response was dampened, suggesting that RipL could promote plant susceptibility in an SA-independent manner. Further elucidation of the molecular mechanisms underpinning RipL effect on flowering and immunity may reveal novel effector functions in host cells.

• International Journal of Oral Biology
• /
• v.33 no.1
• /
• pp.1-5
• /
• 2008

Adenosine 5'-triphosphate (ATP) is an important extracellular signaling molecule which is involved in a variety of physiological responses in many different tissues and cell types, by acting at P2 receptors, either ionotropic (P2X) or G protein-coupled metabotropic receptors (P2Y). P2X receptors have seven isoforms designated as $P2X_{1^-}P2X_7$. In this study, we investigated the electrophysiological and pharmacological properties of rat $P2X_{1^-}P2X_4$ currents by using whole-cell patch clamp technique in a heterologous expression system. When ATP-induced currents were analyzed in human embryonic kidney (HEK293) cells following transient transfection of rat $P2X_{1^-}P2X_4$, the currents showed different pharmacological and electrophysiological properties. ATP evoked inward currents with fast activation and fast desensitization in $P2X_{^1-}$ or $P2X_{3^-}$ expressing HEK293 cells, but in $P2X_{2^-}$ or $P2X_{4^-}$ expressing HEK293 cells, ATP evoked inward currents with slow activation and slow desensitization. While PPADS and suramin inhibited $P2X_2$ or $P2X_3$ receptor-mediated currents, they had little effects on $P2X_4$ receptor-mediated currents. Ivermectin potentiated and prolonged $P2X_4$ receptor-mediated currents, but did not affect $P2X_2$ or $P2X_3$ receptor-mediated currents. We suggest that distinct pharmacological and electrophysiological properties among P2X receptor subtypes would be a useful tool to determine expression patterns of P2X receptors in the nervous system including trigeminal sensory neurons and microglia.

• Korean Journal of Microbiology
• /
• v.51 no.3
• /
• pp.187-193
• /
• 2015

Candida (also known as Pseudozyma) antarctica lipase B (CAL-B) has been intensely studied in academic and industrial fields. However, the research related to its homologous enzymes has been rarely reported. In the current investigation, protein sequence similarity search of CAL-B has been conducted and six homologous protein sequences were identified. After the syntheses of their codon-optimized genes, the synthetic genes have been cloned into a periplasmic expression vector to express in Escherichia coli. Among six homologous sequences, four sequences were successfully expressed in E. coli. The hydrolytic activities of the expressed proteins towards 4-nitrophenyl acetate and 4-nitrophenyl butyrate were measured and compared with those of CAL-B to identify whether the expressed proteins work as a hydrolase. It has been revealed that the expressed proteins can hydrolyze the substrates and the specific activities were determined as $(1.3-30){\times}10^{-2}{\mu}mol/min/mg$, which are lower than those of CAL-B. Among these homologous enzymes, Pseudozyma hubeiensis SY62 exhibits the comparable enantioselectivity to that of CAL-B towards the hydrolysis of (${\pm}$)-1-phenylethyl acetate.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.1
• /
• pp.122-135
• /
• 2018

Listeriolysin O (LLO), one of the most immunogenic proteins of Listeria monocytogenes and its main virulence factor, mediates bacterial escape from the phagosome of the infected cell. Thus, its expression in a nonpathogenic bacterial host may enable effective delivery of heterologous antigens to the host cell cytosol and lead to their processing predominantly through the cytosolic MHC class I presentation pathway. The aim of this project was to characterize the delivery of a model antigen, chicken egg ovalbumin (OVA), to the cytosol of dendritic cells by recombinant Bacillus subtilis vegetative cells expressing LLO. Our work indicated that LLO produced by non-sporulating vegetative bacteria was able to support OVA epitope presentation by MHC I molecules on the surface of antigen presenting cells and consequently influence OVA-specific cytotoxic T cell activation. Additionally, it was proven that the genetic context of the epitope sequence is of great importance, as only the native full-sequence OVA fused to the N-terminal fragment of LLO was sufficient for effective epitope delivery and activation of $CD8^+$ lymphocytes. These results demonstrate the necessity for further verification of the fusion antigen potency of enhancing the MHC I presentation, and they prove that LLO-producing B. subtilis may represent a novel and attractive candidate for a vaccine vector.

• Biomolecules & Therapeutics
• /
• v.1 no.2
• /
• pp.236-243
• /
• 1993

Immunologic potential of DA-125, a new anthracycline antitumor antibiotic, was investigated using guinea pigs and mice. In antigenicity experiments, guinea pigs were sensitized subcutaneously with DA-125 or DA-125 incorporated in complete Freund's adjuvant (CFA) once a week for three weeks. No systemic anaphylaxis was induced by intravenous injection of DA-125 or DA-125 incubated with guinea pig serum after 3 weeks from the last sensitization. None of sera of these animals showed any passive cutaneous anaphylactic reaction (PCA) when DA-125 or DA-125 incubated with guinea pig serum was used as a challenging antigen in homologous PCA experiment. On the other hand the treatment of guinea pigs with ovalbumin Incorporated in CFA induced systemic anaphylactic reaction when challenged by intravenous injection of 5 mg/body of ovalbumin. Immunodiffusion test revealed no precipitating antibodies as detected in guinea pigs sensitized with DA-125. In 24-hour heterologous PCA reaction with sera of C57BL/6 mice immunized with DA-125 or DA-125 mixed with aluminum hydroxide gel (Alum), None of sera showed positive reaction when DA-125 or DA-125 incubated with rat serum was used as a challenging antigen. Sera of animals immunized with a mixture of ovalbumin and alum showed positive PCA reaction when 5 mg/body of ovalbumin was injected as a challenging antigen. In lymphocyte proliferation tests, spleen lymphocyte proliferation to PHA and LPS was similarly impaired by 12 mg/kg of DXR or 36 mg/kg of DA-125, and the immunodepressive activity of DA-125 showed a dose-dependent manner. From these results, it could be concluded that immunosupression of DA-125 would be comparable to that of DXR and that DA-125 would not induce systemic allergic reaction in its clinical use.