• Journal of Microbiology and Biotechnology
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• v.26 no.3
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• pp.627-636
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• 2016

The causative agent of pandemic cholera, Vibrio cholerae, infects the anaerobic environment of the human intestine. Production of cholera toxin (CT), a major virulence factor of V. cholerae, is highly induced during anaerobic respiration with trimethylamine N-oxide (TMAO) as an alternative electron acceptor. However, the molecular mechanism of TMAO-stimulated CT production is not fully understood. Herein, we reveal that CT production during anaerobic TMAO respiration is affected by glucose fermentation. When the seventh pandemic V. cholerae O1 strain N16961 was grown with TMAO and additional glucose, CT production was markedly reduced. Furthermore, an N16961 Δcrp mutant, devoid of cyclic AMP receptor protein (CRP), was defective in CT production during growth by anaerobic TMAO respiration, further suggesting a role of glucose metabolism in regulating TMAO-mediated CT production. TMAO reductase activity was noticeably decreased when grown together with glucose or by mutation of the crp gene. A CRP binding region was identified in the promoter region of the torD gene, which encodes a structural subunit of the TMAO reductase. Gel shift assays further confirmed the binding of purified CRP to the torD promoter sequence. Together, our results suggest that the bacterial ability to respire using TMAO is controlled by CRP, whose activity is dependent on glucose availability. Our results reveal a novel mechanism for the regulation of major virulence factor production by V. cholerae under anaerobic growth conditions.

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2002.10a
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• pp.144-145
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• 2002

어패류를 날 것으로 즐겨먹는 우리나라와 일본에서는 매년 여름철이면 병원성 비브리오균에 의한 식중독 사고가 많이 발생한다. 현재까지 알려져 있는 40 여종의 비브리오균 중 12종이 사람에게 질병을 일으키는 것으로 알려져 있으며, 우리나라에서 주로 문제를 일으키는 것은 Vibrio parahaemolytius, V. vulnificus 등 이다. 그러나 최근에는 V. cholerae non-Ol의 병원성에 관한 연구도 많으며, 특히 2001년에는 우리나라에서 발생율이 희박하던 V. cholerae Ol에 의한 환자가 140명을 상회하는 대형사고가 발생하기도 하였다. (중략)

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.4
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• pp.556-561
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• 1997

Vibrio cholerae non-O1 FM-3 was isolated from sea water, and it showed the same bacteriological characteristics as Vibrio cholerae non-O1 ATCC 25872. V. cholerae non-O1 FM-3 presented the highest hemolytic activity at stationary phase of its growth. The hemolytic activity was decreased in accordance with increasing of pretense activity of its cultural supernatant. The characteristics of the hemolysin produced by V. cholerae non-O1 FM-3 were investigated after partial purification with a Sephadex G-100 chromatography. The hemolytic activity of purified protein was stable at $4^{\circ}C$ while it was completely lost by heating at $60^{\circ}C$ for 30 min. The activity of hemolysin was increased by addition of divalent cations such as $Ca^{2+},\;Mg^{2+},\;and\;Mn^{2+}$ while it was inhibited by additions of $Zn^{2+}$. When the hemolysin was incubated with suspensions of erythrocytes at $4^{\circ}C$ and $37^{\circ}C$, respectively, hemolysis was not observed at $4^{\circ}C$ but at $37^{\circ}C$. It means that hemolysis by purified hemolysin was temperature-dependent while its binding step of hemolysin to cell membrane was temperature-independent.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.41 no.4
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• pp.243-247
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• 2008

The hemolysin of Vibrio cholerae non-O1 CT isolated from sea water was purified and characterized. The purified hemolysin displayed an optimum at $37^{\circ}C$ and exhibited more than 70% of residual hemolytic activity after incubation at $45^{\circ}C$ for 120 min. However, the activity dropped dramatically at temperature above $55^{\circ}C$. The purified protein showed the highest hemolytic activity at pH 7.0, while the activity was completely lost outside of the pH ranges of 5.0 and 10.0. The activity of hemolysin was inactivated by addition of divalent cations, such as $Cu^{2+},\;Fe^{2+},\;Hg^{2+},\;Mn^{2+},\;and\;Zn^{2+}$, however, the activity was not completely inhibited by additions of $Ca^{2+},\;Mg^{2+},\;K^+,\;Na^+,\;and\;Li^+$.

• Korean Journal of Microbiology
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• v.35 no.3
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• pp.248-252
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• 1999

The PI-oduction of virulence factors such as cholera toxin, heinolysin and hemagglutinin in V cliolerae non-01 and non-0139 were examined. Among 65 strains isolated from environmental and clinical blood sources, 29 (14.6%) strains produced hemolysin only, 35(53.9%) sh.ains produced both hemolysin and hemagglutinin. From one 037 slrain isolated from environmenl, cholera toxin, ctx gene, hemolysin, and hemagglutinin were detected. All of the strains isolated from clinical and environmental sources showed hemolytic activity against human 0 group e~ythrocytes. In inhibition patterns of heinagglotination, 5 of 18 clinical strains (27.8%) were inhibited by less than 1% mannose and galactose, while, among the 47 environmental isolates. hose paltems by less than 1% mannose and galactose 55.4% wel-e inhibited. Thel-ehre, exohamagglutinin positive rate was high in clinical blood isolates but in environnlental sources, the rate was almost similar lo ihe rate or endohemagglutinin positive. These results indicaled that V cholerae non-01 and non-0139 produced various virnlence factors such as cholera toxin, hemolysin, and hemagglutinin but not a single factor. Further studies are need for epidemiological or bacteriological shtdies of V cholerae 037 isolated from environment.

• Microbiology and Biotechnology Letters
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• v.49 no.4
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• pp.510-518
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• 2021

Bacteriophages are considered excellent sensing elements for platforms detecting bacteria. However, their lytic cycle has restricted their efficacy. Here, we used the minor coat protein pIII domain (N1N2) of phage CTXφ to construct a novel surface plasmon resonance (SPR) biosensor that could detect Vibrio cholerae. N1N2 harboring the domains required for phage adsorption and entry was obtained from Escherichia coli using recombinant protein expression and purification. SDS-PAGE revealed an approximate size of 30 kDa for N1N2. Dot blot and transmission electron microscopy analyses revealed that the protein bound to the host V. cholerae but not to non-host E. coli K-12 cells. Next, we used amine-coupling to develop a novel recombinant N1N2 (rN1N2)-functionalized SPR biosensor by immobilizing rN1N2 proteins on gold substrates and using SPR to monitor the binding kinetics of the proteins with target bacteria. We observed rapid detection of V. cholerae in the range of approximately 10³ to 10⁹ CFU/ml but not of E. coli at any tested concentration, thereby confirming that the biosensor exhibited differential recognition and binding. The results indicate that the novel biosensor can rapidly monitor a target pathogenic microorganism in the environment and is very useful for monitoring food safety and facilitating early disease prevention.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2199-2205
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• 2016

Vibrio cholerae O1 serogroup Wave 3 El Tor strains are presently prevalent worldwide. The Wave 3 El Tor strains contain a TLC:RS1:CTX array on chromosome 1, and no element is integrated on chromosome 2. A multiplex PCR optimized to identify the TLC:RS1:CTX array of Wave 3 strains has been developed in this study. By using eight primers, the multiplex PCR can identify the characteristic CTX and RS1 array of Wave 3 strains from various arrays of strains belonging to other Waves. The four amplified DNA fragments of Wave 3 strains have been cloned in a vector, which could be used as a positive control for the multiplex PCR. This multiplex PCR and the positive control set could be useful tools for rapid recognition of Wave 3 El Tor strains.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.10a
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• pp.8-8
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• 2019

The stringent response (SR) is characterized as a bacterial defense mechanism in response to various growth-inhibiting stresses. It is activated by accumulation of a small nucleotide regulator, (p)ppGpp, and induces global changes in bacterial transcription and translation. Recent work from our group has shown that (p)ppGpp plays a critical role in virulence and survival in Vibrio cholerae. The genes, relA and relV, are involved in the production of (p)ppGpp, while the spoT gene encodes an enzyme that hydrolyzes it in V. cholerae. A mutant strain defective in (p)ppGpp production (i.e. ${\Delta}relA{\Delta}relV{\Delta}spoT$ mutant) lost the ability to produce cholera toxin (CT) and lost their viability due to uncontrolled production of organic acids, when grown with extra glucose. In contrast, the ${\Delta}relA{\Delta}spoT$ mutant, a (p)ppGpp overproducer strain, produced enhanced level of CT and exhibited better growth in glucose supplemented media via glucose metabolic switch from organic fermentation to acetoin, a neutral fermentation end product, fermentation. These findings indicates that (p)ppGpp, in addition to its well-known role as a SR mediator, positively regulates CT production and maintenance of growth fitness in V. cholerae. This implicates SR as a promising drug target, inhibition of which may possibly downregulate V. cholerae virulence and survival fitness. Therefore, we screened a chemical library and identified a compound that induces medium acidification (termed iMAC) and thereby loss of wild type V. cholerae viability under glucose-rich conditions. Further, we present a potential mechanism by which the compound inhibits (p)ppGpp accumulation. Together, these results indicate that iMAC treatment causes V. cholerae cells to produce significantly less (p)ppGpp, an important regulator of the bacterial virulence and survival response, and further suggesting that it has a therapeutic potential to be developed as a novel antibacterial agent against cholera.

• Journal of Microbiology and Biotechnology
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• v.22 no.8
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• pp.1107-1112
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• 2012

Outbreaks of foodborne diseases associated with Vibrio species such as V. parahaemolyticus, V. vulnificus, and V. cholerae frequently occur in countries having a dietary habit of raw seafood consumption. For rapid identification of different Vibrio species involved in foodborne diseases, whole-cell protein pattern analysis for 13 type strains of 12 Vibrio species was performed using SDS-PAGE analysis. Pathogenic Vibrio species such as V. parahaemolyticus, V. vulnificus, V. cholerae, V. alginolyticus, V. fluvialis, and V. mimicus were included in the 12 Vibrio species used in this study. Each of the 12 Vibrio species showed clearly specific band patterns of its own. Two different strains of V. parahaemolyticus showed two different SDS-PAGE whole-cell protein patterns, giving the possibility of categorizing isolated strains in the same V. parahaemolyticus species into two subgroups. The 36 Vibrio isolates collected from sushi restaurants in Busan were all identified as V. parahaemolyticus by comparing their protein patterns with those of Vibrio type strains. The identified isolates were categorized into two different subgroups of V. parahaemolyticus. The whole-cell protein pattern analysis by SDS-PAGE can be used as a specific, rapid, and simple identification method for Vibrio spp. involved in foodborne diseases at the subspecies level.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.430-434
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• 2009

Bacterial 16S rRNA gene sequences have been widely used for the studies on molecular phylogeny, evolutional history, and molecular detections. Bacterial genomes have multiple rRNA operons, of which gene sequences sometimes are variable. In the present study, heterogeneity of the Vibrio 16S rRNA gene sequences were investigated. Vibrio 16S rRNA sequences were obtained from GenBank databases, considering the completion of gene annotation of Vibrio genome sequences. These included V. cholerae, V. harveyi, V. parahaemolyticus, V. splendidus, and V. vulnificus. Chromosome 1 of the studied Vibrio had 7~10 copies of the 16S rRNA gene, and their intragenomic variations were less than 0.9% dissimilarity (more than 99.1% DNA similarity). Chromosome 2 had none or single 16S rRNA gene. Intragenomic 16S rRNA genotypes were detected at least 5 types (V. vulnificus #CMCP6) to 8 types (V. parahaemolyticus #RIMD 2210633, V. harveyi #ATCC BAA-1116). These suggest that Vibrio has high heterogeneity of the 16S rRNA gene sequences.