ArticleApplied and environmental microbiology2012
Counterselection system for Geobacillus kaustophilus HTA426 through disruption of pyrF and pyrR.
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 24 papers.
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Who cites it
24 citing papers in PubMed.
- Atypical catabolite repression ofMicrobiology (Reading, England) · 2026Article
- Development of a markerless tool for targeted chromosome modification in the thermophilic and methylotrophic bacterium Bacillus methanolicus.Microbial cell factories · 2025Article
- Efficient pathway-drivenApplied and environmental microbiology · 2024Article
- Development of a thermophilic host-vector system for the production of recombinant proteins at elevated temperatures.Applied microbiology and biotechnology · 2023Article
- Loss in the Antibacterial Ability of a PyrR Gene Regulating Pyrimidine Biosynthesis after Using CRISPR/Cas9-Mediated Knockout for Metabolic Engineering inMicroorganisms · 2023Article
- Genome-Wide Pathway Exploration of theMicroorganisms · 2023Article
- New Platform for Screening Genetic Libraries at Elevated Temperatures: Biological and Genomic Information and Genetic Tools of Geobacillus thermodenitrificans K1041.Applied and environmental microbiology · 2022Article
- A novel method for transforming Geobacillus kaustophilus with a chromosomal segment of Bacillus subtilis transferred via pLS20-dependent conjugation.Microbial cell factories · 2022Article
- Development of aJournal of Zhejiang University. Science. B · 2021Article
- Extremophiles, a Nifty Tool to Face Environmental Pollution: From Exploitation of Metabolism to Genome Engineering.International journal of environmental research and public health · 2021Review
- Frequent Transposition of Multiple Insertion Sequences inFrontiers in microbiology · 2021Article
- A plasmid vector that directs hyperproduction of recombinant proteins in the thermophiles Geobacillus species.Extremophiles : life under extreme conditions · 2020Article
- Genome-wide systematic identification of methyltransferase recognition and modification patterns.Nature communications · 2019Article
- A novel method for transforming the thermophilic bacterium Geobacillus kaustophilus.Microbial cell factories · 2018Article
- Genetic Tools and Techniques for Recombinant Expression in Thermophilic Bacillaceae.Microorganisms · 2018Review
- Article
- Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp.Microbial cell factories · 2017Article
- Development and implementation of rapid metabolic engineering tools for chemical and fuel production inBiotechnology for biofuels · 2017Article
- Unique plasmids generated via pUC replicon mutagenesis in an error-prone thermophile derived from Geobacillus kaustophilus HTA426.Applied and environmental microbiology · 2015Article
- Engineering the genome of Thermus thermophilus using a counterselectable marker.Journal of bacteriology · 2015Article
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3 authors.
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Abstract
Counterselection systems facilitate marker-free genetic modifications in microbes by enabling positive selections for both the introduction of a marker gene into the microbe and elimination of the marker from the microbe. Here we report a counterselection system for Geobacillus kaustophilus HTA426, established through simultaneous disruption of the pyrF and pyrR genes. The pyrF gene, essential for pyrimidine biosynthesis and metabolization of 5-fluoroorotic acid (5-FOA) to toxic metabolites, was disrupted by homologous recombination. The resultant MK54 strain (ΔpyrF) was auxotrophic for uracil and resistant to 5-FOA. MK54 complemented with pyrF was prototrophic for uracil but insensitive to 5-FOA in the presence of uracil. To confer 5-FOA sensitivity, the pyrR gene encoding an attenuator to repress pyrimidine biosynthesis by sensing uracil derivatives was disrupted. The resultant MK72 strain (ΔpyrF ΔpyrR) was auxotrophic for uracil and resistant to 5-FOA. MK72 complemented with pyrF was prototrophic for uracil and 5-FOA sensitive even in the presence of uracil. The results suggested that pyrF could serve as a counterselection marker in MK72, which was demonstrated by efficient marker-free integrations of heterologous β-galactosidase and α-amylase genes. The integrated genes were functionally expressed in G. kaustophilus and conferred new functions on the thermophile. This report describes the first establishment of a pyrF-based counterselection system in a Bacillus-related bacterium, along with the first demonstration of homologous recombination and heterologous gene expression in G. kaustophilus. Our results also suggest a new strategy for establishment of counterselection systems.
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