title: Construction of upp deletion mutant strains of Lactobacillus casei and Lactococcus lactis based on counterselective system using temperature-sensitive plasmid.
abstract: Integration plasmids are often used in constructing chromosomal mutations, as it enables the alternation of genes at any location by integration or replacement. Food-grade integration vectors can integrate into the host genome without introducing any selectable markers or residual bases, and the recombination often happens in non-coding region. In this study we used the temperature-sensitive pWV01 replicon to construct 2 chloramphenicol-resistant integration plasmids (pGBHC32-upp) containing the uracil phosphoribosyl transferase (upp) gene as a counterselective marker for Lactobacillus casei (L. casei) ATCC393 and Lactococcus lactis (L. lactis) MG1363. We then ligated the designed homologous arms to the pGBHC32-upp plasmids to allow their integration to the bacterial chromosome, and selected upp deletion mutants of L. casei ATCC393 and L. lactis MG1363 in the presence of 5-fluorouracil (5-FU). Analysis of genetic stability, growth curve, carbon utilization and scanning electronic microscopy showed that, except for 5-FU resistance, there were no significant differences between the wild type and mutant lactic acid bacteria. The integration system and the upp deletion strains could be used in the insertion or deletion of genes at any location of the chromosome of both L. casei ATCC 393 and L. lactis MG1363, and the homologous recombination would not introduce any selectable markers or residual bases. These mutant strains can be further investigated for heterologous protein expression and construction of a live mucosal vaccine carrier.
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China.
authors.forename: Li
authors.initials: L
authors.lastname: Song
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Cui
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China.
authors.forename: Lijie
authors.initials: L
authors.lastname: Tang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China.
authors.forename: Xinyuan
authors.initials: X
authors.lastname: Qiao
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China.
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China.
authors.forename: Yanping
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Cui
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China. Electronic address: yijingli@163.com.
authors.forename: Yijing
authors.initials: Y
authors.lastname: Li
mesh_terms: D002702:Chloramphenicol Resistance; D004261:DNA Replication; D055786:Gene Knockout Techniques; D005827:Genetics, Microbial / Q000379:methods*; D007780:Lacticaseibacillus casei / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics*; D010430:Pentosyltransferases / Q000235:genetics*; D010957:Plasmids; D011995:Recombination, Genetic; D012641:Selection, Genetic; D013696:Temperature
chemical_list: D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase
keywords: Gene integration system; Lactobacillus casei; Lactococcus lactis; Temperature-sensitive replicon