ArticleApplied and environmental microbiology2009
USER friendly cloning coupled with chitin-based natural transformation enables rapid mutagenesis of Vibrio vulnificus.
Article in Applied and environmental microbiology, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 46 papers.
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Who cites it
46 citing papers in PubMed, 80 citations in OpenAlex.
- Molecular mechanisms and applications of natural transformation in bacteria.Frontiers in microbiology · 2025Review
- Regulators of natural competence in Vibrio parahaemolyticus.Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] · 2022Article
- Chemotactic invasion in deep soft tissue byVirulence · 2020Article
- CryoEM structure of the type IVa pilus secretin required for natural competence in Vibrio cholerae.Nature communications · 2020Article
- Comparison of chitin-induced natural transformation in pandemic Vibrio cholerae O1 El Tor strains.Environmental microbiology · 2020Article
- Regulation of Chitin-Dependent Growth and Natural Competence inMicroorganisms · 2020Article
- Article
- Identification ofFrontiers in microbiology · 2019Article
- Natural Transformation in Vibrio parahaemolyticus: a Rapid Method To Create Genetic Deletions.Journal of bacteriology · 2018Article
- Multiplex Genome Editing by Natural Transformation (MuGENT) for Synthetic Biology in Vibrio natriegens.ACS synthetic biology · 2017Article
- Lrp, a global regulator, regulates the virulence of Vibrio vulnificus.Journal of biomedical science · 2017Article
- The Opportunistic PathogenFrontiers in microbiology · 2017Article
- Influence of Niche-Specific Nutrients on Secondary Metabolism in Vibrionaceae.Applied and environmental microbiology · 2016Article
- MARTX toxins as effector delivery platforms.Pathogens and disease · 2015Review
- Clinical and environmental genotypes of Vibrio vulnificus display distinct, quorum-sensing-mediated, chitin detachment dynamics.Pathogens and disease · 2015Article
- Direct regulation of the natural competence regulator gene tfoX by cyclic AMP (cAMP) and cAMP receptor protein (CRP) in Vibrios.Scientific reports · 2015Article
- TfoX-based genetic mapping identifies Vibrio fischeri strain-level differences and reveals a common lineage of laboratory strains.Journal of bacteriology · 2015Article
- Application of chitosan microparticles for reduction of vibrio species in seawater and live oysters (Crassostrea virginica).Applied and environmental microbiology · 2015Article
- Article
- Multiplex genome editing by natural transformation.Proceedings of the National Academy of Sciences of the United States of America · 2014Article
Corrections and comments
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
Abstract
Vibrio vulnificus is a bacterial contaminant of shellfish and causes highly lethal sepsis and destructive wound infections. A definitive identification of virulence factors using the molecular version of Koch's postulates has been hindered because of difficulties in performing molecular genetic analysis of this opportunistic pathogen. For example, conjugation is required to introduce plasmid DNA, and allelic exchange suicide vectors that rely on sucrose sensitivity for counterselection are not efficient. We therefore incorporated USER friendly cloning techniques into pCVD442-based allelic exchange suicide vectors and other expression vectors to enable the rapid and efficient capture of PCR amplicons. Upstream and downstream DNA sequences flanking genes targeted for deletion were cloned together in a single step. Based on results from Vibrio cholerae, we determined that V. vulnificus becomes naturally transformable with linear DNA during growth on chitin in the form of crab shells. By combining USER friendly cloning and chitin-based transformation, we rapidly and efficiently produced targeted deletions in V. vulnificus, bypassing the need for two-step, suicide vector-mediated allelic exchange. These methods were used to examine the roles of two flagellin loci (flaCDE and flaFBA), the motAB genes, and the cheY-3 gene in motility and to create deletions of rtxC, rtxA1, and fadR. Additionally, chitin-based transformation was useful in moving antibiotic resistance-labeled mutations between V. vulnificus strains by simply coculturing the strains on crab shells. The methods and genetic tools that we developed should be of general use to those performing molecular genetic analysis and manipulation of other gram-negative bacteria.
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