A Simple and Rapid Method for Transformation of Vibrio Species by Electroporation

eThe type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells. Citrobacter freundii strain CF74 has a complete T6SS genomic island (GI) that contains clpV, hcp-2, and vgr T6SS genes. We constructed clpV, hcp-2, vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74⌬T6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased in clpV, hcp-2, vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74⌬T6SS, CF74⌬clpV, and CF74⌬hcp-2 mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role in C. freundii.

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“…For transformation of plasmids into CF74, electrocompetent cells were prepared as described previously (29).…”

Section: Methodsmentioning

confidence: 99%

The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

et al. 2015

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“…Transformation of V. parahaemolyticus was carried out by the method of Hamashima et al (1995). V. parahaemolyticus cells were cultured in LB medium until mid-exponential phase was reached.…”

Section: Methodsmentioning

confidence: 99%

“…Occurrence of a second recombination should yield cells resistant to sucrose but susceptible to chloramphenicol, and several such candidates were identified. After checking deletion of the vmeAB region by PCR, we picked a single isolate for further use and designated it TM3.Transformation of V. parahaemolyticus was carried out by the method of Hamashima et al (1995). V. parahaemolyticus cells were cultured in LB medium until mid-exponential phase was reached.…”

mentioning

confidence: 99%

VmeAB, an RND-type multidrug efflux transporter in Vibrio parahaemolyticus

et al. 2007

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Genes vmeA and vmeB, encoding a multidrug efflux transporter in the halophilic bacterium Vibrio parahaemolyticus, have been cloned using a drug-hypersusceptible Escherichia coli strain as the host. Cells of E. coli KAM33 (DacrAB DydhE) carrying the vmeAB region from V. parahaemolyticus conferred much higher MICs for a variety of antimicrobial agents than did control cells. Cells possessing VmeAB under energized conditions maintained very low intracellular concentrations of ethidium. This was as expected for an energy-dependent efflux system, and supports the notion -based on sequence homology -that VmeAB belongs to the resistance nodulation cell division (RND) family of multidrug efflux transporters. It is likely that VmeAB forms functional complexes with the outer-membrane protein TolC in E. coli, because introduction of vmeAB into cells of E. coli KAM43, which lacks the tolC gene, failed to elevate the MICs for any of the antimicrobial agents tested. Therefore, a V. parahaemolyticus homologue of tolC was also cloned, designated vpoC, and was introduced together with vmeAB into cells of E. coli KAM43. The MICs of all agents tested were raised and were comparable to the values observed in E. coli KAM33 harbouring a plasmid carrying vmeAB. Finally, a vmeAB-deficient mutant of V. parahaemolyticus was constructed (designated TM3). TM3 showed slightly higher susceptibility than the parental V. parahaemolyticus to some antimicrobial agents. Survival rate of the TM3 when exposed to deoxycholate decreased compared with that of the parent. INTRODUCTIONVibrio parahaemolyticus, a slightly halophilic marine bacterium, is a major cause of food poisoning in Japan (Obata et al., 2001; WHO, 1999;Yamazaki et al., 2003) and many other countries (Cabanillas-Beltran et al., 2006; CDC, 2006;Lozano-Leon et al., 2003;McLaughlin et al., 2005; Sen et al., 2007;Su et al., 2005a). The organism has some peculiar physiological characteristics, such as a very fast growth rate, a Na + requirement for growth (Baumann & Schubert, 1984) and an ability to live in diverse environments including brackish water (Kumazawa & Kato, 1985), on estuarine algae (Kumazawa et al., 1991), and in mammalian hosts, including humans (Yamamoto & Yokota, 1989).In achieving human infection, V. parahaemolyticus survives in the intestine and especially in the duodenum, where bile acids and their conjugates are abundant. Bile acids are anionic detergents and support the digestion of fats in the intestine. In addition, bile has bactericidal effects due to its membrane-solvent property (Provenzano et al., 2000). Thus, bile resistance of enteropathogenic bacteria is thought to be important for their survival in the intestine. Some enteropathogenic bacteria have been shown to possess systems to protect from the actions of bile. In the case of Vibrio cholerae, the outer-membrane component TolC Vc , which had already been identified as a component of some multidrug efflux transporters (sometimes called multidrug efflux pumps), was found to contribute to resistance to bile,...

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“…Plasmid transformation was done by electroporation as described by Hamashima et al (18). After electroporation, the cells were grown for 1 h with agitation at 37°C and then streaked on plates with the appropriate antibiotic.…”

Section: Methodsmentioning

confidence: 99%

Adhesion of Vibrio cholerae to Granular Starches

Gancz¹,

Niderman-Meyer

Broza

et al. 2005

Appl Environ Microbiol

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Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. Cholera can become epidemic and deadly without adequate medical care. Appropriate rehydration therapy can reduce the mortality rate from as much as 50% of the affected individuals to <1%. Thus, oral rehydration therapy (ORT) is an important measure in the treatment of this disease. To further reduce the symptoms associated with cholera, improvements in oral rehydration solution (ORS) by starch incorporation were suggested. Here, we report that V. cholerae adheres to starch granules incorporated in ORS. Adhesion of 98% of the cells was observed within 2 min when cornstarch granules were used. Other starches showed varied adhesion rates, indicating that starch source and composition play an important role in the interaction of V. cholerae and starch granules. Sugars metabolized by V. cholerae showed a repressive effect on the adhesion process. The possible mechanisms involved are discussed. Comparing V. cholerae adhesion with the adhesion of other pathogens suggests the involvement of starch degradation capabilities. This adhesion to granular starch can be used to improve ORT.

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A Simple and Rapid Method for Transformation of Vibrio Species by Electroporation

Cited by 18 publications

References 2 publications

The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

VmeAB, an RND-type multidrug efflux transporter in Vibrio parahaemolyticus

Adhesion of Vibrio cholerae to Granular Starches

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