ArticleApplied and environmental microbiology2012
Novel system for efficient isolation of Clostridium double-crossover allelic exchange mutants enabling markerless chromosomal gene deletions and DNA integration.
Article in Applied and environmental microbiology, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 54 papers.
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54 citing papers in PubMed, 121 citations in OpenAlex.
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- A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792.Applied and environmental microbiology · 2020Article
- Consolidated bioprocessing for butanol production of cellulolytic Clostridia: development and optimization.Microbial biotechnology · 2020Review
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- Clostridial Genetics: Genetic Manipulation of the Pathogenic Clostridia.Microbiology spectrum · 2019Review
- Development of a novel selection/counter-selection system for chromosomal gene integrations and deletions in lactic acid bacteria.BMC molecular biology · 2019Article
- An efficient method for markerless mutant generation by allelic exchange inBiotechnology for biofuels · 2019Article
Corrections and comments
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Authors and funding
3 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Isolation of Clostridium mutants based on gene replacement via allelic exchange remains a major limitation for this important genus. Use of a heterologous counterselection marker can facilitate the identification of the generally rare allelic exchange events. We report on the development of an inducible counterselection marker and describe its utility and broad potential in quickly and efficiently generating markerless DNA deletions and integrations at any genomic locus without the need for auxotrophic mutants or the use of the mobile group II introns. This system is based on a codon-optimized mazF toxin gene from Escherichia coli under the control of a lactose-inducible promoter from Clostridium perfringens. This system is potentially applicable to almost all members of the genus Clostridium due to their similarly low genomic GC content and comparable codon usage. We isolated all allelic-exchange-based gene deletions (ca_p0167, sigF, and sigK) or disruptions (ca_p0157 and sigF) we attempted and integrated a 3.6-kb heterologous DNA sequence (made up of a Clostridium ljungdahlii 2.1-kb formate dehydrogenase [fdh] gene plus a FLP recombination target [FRT]-flanked thiamphenicol resistance marker) into the Clostridium acetobutylicum chromosome. Furthermore, we report on the development of a plasmid system with inducible segregational instability, thus enabling efficient deployment of the FLP-FRT system to generate markerless deletion or integration mutants. This enabled expeditious deletion of the thiamphenicol resistance marker from the fdh integrant strain as well as the sigK deletion strain. More generally, our system can potentially be applied to other organisms with underdeveloped genetic tools.
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