ArticleJournal of bacteriology1993
High-efficiency gene inactivation and replacement system for gram-positive bacteria.
Article in Journal of bacteriology, 1993. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 212 papers.
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Who cites it
212 citing papers in PubMed, 444 citations in OpenAlex.
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- Chromosomal and Plasmid-Based CRISPRi Platforms for Conditional Gene Silencing inInternational journal of molecular sciences · 2025Article
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- Development of the First Shuttle Vector System and Optimization of Transformation inJournal of microbiology and biotechnology · 2025Article
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- Application of the SpCas9 inhibitor BRD0539 for CRISPR/Cas9-based genetic tools inBioscience of microbiota, food and health · 2025Article
- An improved temperature-sensitive shuttle vector system for scarless gene deletion in human-gut-associatediScience · 2024Article
- Constitutive activation of two-component systems reveals regulatory network interactions in Streptococcus agalactiae.Nature communications · 2024Article
- Review
- Assessing immunogenicity of CRISPR-NCas9 engineered strain against porcine epidemic diarrhea virus.Applied microbiology and biotechnology · 2024Article
- Streptolysin S is required for Streptococcus pyogenes nasopharyngeal and skin infection in HLA-transgenic mice.PLoS pathogens · 2024Article
- Acyl-AcpB, a FabT corepressor inJournal of bacteriology · 2023Article
- Negative chemotaxis of Ligilactobacillus agilis BKN88 against gut-derived substances.Scientific reports · 2023Article
- Characterization of TelE, a T7SS LXG Effector Exhibiting a Conserved C-Terminal Glycine Zipper Motif Required for Toxicity.Microbiology spectrum · 2023Article
- pBFK, a new thermosensitive shuttle vector forHeliyon · 2023Article
- Wild-typeFrontiers in microbiology · 2023Article
- The Role of Regulator Catabolite Control Protein A (CcpA) in Streptococcus agalactiae Physiology and Stress Response.Microbiology spectrum · 2022Article
- The Streptococcus pyogenes hyaluronic acid capsule promotes experimental nasal and skin infection by preventing neutrophil-mediated clearance.PLoS pathogens · 2022Article
152 more citing papers are in PubMed but not listed here.
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
4 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
A system for high-efficiency single- and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37 degrees C. A nested set of L. lactis chromosomal fragments cloned onto pG+host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37 degrees C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28 degrees C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40% of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.