title: An Effective Counterselection System for Listeria monocytogenes and Its Use To Characterize the Monocin Genomic Region of Strain 10403S.
abstract: Construction ofListeria monocytogenesmutants by allelic exchange has been laborious and time-consuming due to lack of proficient selection markers for the final recombination event, that is, a marker conveying substance sensitivity to the bacteria bearing it, enabling the exclusion of merodiploids and selection for plasmid loss. In order to address this issue, we engineered a counterselection marker based on a mutated phenylalanyl-tRNA synthetase gene (pheS*). This mutation renders the phenylalanine-binding site of the enzyme more promiscuous and allows the binding of the toxicp-chloro-phenylalanine analog (p-Cl-phe) as a substrate. WhenpheS*is introduced intoL. monocytogenesand highly expressed under control of a constitutively active promoter, the bacteria become sensitive top-Cl-phe supplemented in the medium. This enabled us to utilizepheS*as a negative selection marker and generate a novel, efficient suicide vector for allelic exchange inL. monocytogenesWe used this vector to investigate the monocin genomic region inL. monocytogenesstrain 10403S by constructing deletion mutants of the region. We have found this region to be active and to cause bacterial lysis upon mitomycin C treatment. The future applications of such an effective counterselection system, which does not require any background genomic alterations, are vast, as it can be modularly used in various selection systems (e.g., genetic screens). We expect this counterselection marker to be a valuable genetic tool in research onL. monocytogenesIMPORTANCEL. monocytogenesis an opportunistic intracellular pathogen and a widely studied model organism. An efficient counterselection marker is a long-standing need inListeriaresearch for improving the ability to design and perform various genetic manipulations and screening systems for different purposes. We report the construction and utilization of an efficient suicide vector for allelic exchange which can be conjugated, leaves no marker in the bacterial chromosome, and does not require the use of sometimes leaky inducible promoters. This highly efficient genome editing tool forL. monocytogeneswill allow for rapid sequential mutagenesis, introduction of point mutations, and design of screening systems. We anticipate that it will be extensively used by the research community and yield novel insights into the diverse fields studied using this model organism.
authors.affiliation: Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.
authors.forename: Tal
authors.initials: T
authors.lastname: Argov
authors.affiliation: Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.
authors.forename: Lev
authors.initials: L
authors.lastname: Rabinovich
authors.affiliation: Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.
authors.forename: Nadejda
authors.initials: N
authors.lastname: Sigal
authors.affiliation: Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel anathe@post.tau.ac.il.
authors.forename: Anat A
authors.initials: AA
authors.lastname: Herskovits
mesh_terms: D001430:Bacteriocins / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D005819:Genetic Markers / Q000235:genetics; D008089:Listeria monocytogenes / Q000235:genetics* / Q000254:growth & development; D016685:Mitomycin / Q000494:pharmacology*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D010652:Phenylalanine-tRNA Ligase / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012641:Selection, Genetic / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics
chemical_list: D001430:Bacteriocins; D005819:Genetic Markers; D010649:Phenylalanine; D016685:Mitomycin; D010652:Phenylalanine-tRNA Ligase
keywords: Listeria monocytogenes; allelic exchange; bacteriocins; counterselection; monocin; mutagenesis; pheS