title: A counterselection method for Lactococcus lactis genome editing based on class IIa bacteriocin sensitivity.
abstract: In this paper, we present a new counterselection method for deleting fragments from Lactococcus lactis chromosome. The method uses a non-replicating plasmid vector, which integrates into the chromosome and makes the cell sensitive to bacteriocins. The integration vector carries pUC ori functional in Escherichia coli but not in L. lactis, an erythromycin resistance gene for selecting single crossover integrants, and two fragments from L. lactis chromosome for homologous recombinations. In addition, the integration vector is equipped with the Listeria monocytogenes gene mptC encoding the mannose-phosphotransferase system component IIC, the receptor for class IIa bacteriocins. Expression of mptC from the integration vector renders the naturally resistant L. lactis sensitive to class IIa bacteriocins. This sensitivity is then used to select the double crossover colonies on bacteriocin agar. Only the cells which have regained the endogenous bacteriocin resistance through the loss of the mptC plasmid will survive. The colonies carrying the desired deletion can then be distinguished from the wild-type revertants by PCR. By using the class IIa bacteriocins leucocin A, leucocin C or pediocin AcH as the counterselective agents, we deleted 22- and 33-kb chromosomal fragments from the wild-type nisin producing L. lactis strain N8. In conclusion, this counterselection method presented here is a convenient, efficient and inexpensive technique to generate successive deletions in L. lactis chromosome.
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 56, Helsinki, Finland.
authors.forename: Xing
authors.initials: X
authors.lastname: Wan
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 56, Helsinki, Finland.|Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, P.O. Box 16100, Espoo, Finland.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Usvalampi
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 56, Helsinki, Finland.
authors.forename: Per E J
authors.initials: PE
authors.lastname: Saris
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 56, Helsinki, Finland. timo.takala@helsinki.fi.
authors.forename: Timo M
authors.initials: TM
authors.lastname: Takala
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001430:Bacteriocins / Q000378:metabolism*; D000072669:Gene Editing / Q000379:methods*; D005822:Genetic Vectors; D059765:Homologous Recombination; D013294:Lactococcus lactis / Q000187:drug effects* / Q000235:genetics*; D050296:Microbial Viability / Q000187:drug effects*; D010957:Plasmids; D012641:Selection, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D001430:Bacteriocins
keywords: Class IIa bacteriocin; Counterselection; Genome editing; Lactic acid bacteria