title: Examination of Enterococcus faecalis Toxin-Antitoxin System Toxin Fst Function Utilizing a Pheromone-Inducible Expression Vector with Tight Repression and Broad Dynamic Range.
abstract: Tools for regulated gene expression inEnterococcus faecalisare extremely limited. In this report, we describe the construction of an expression vector forE. faecalis, designated pCIE, utilizing the PQpheromone-responsive promoter of plasmid pCF10. We demonstrate that this promoter is tightly repressed, responds to nanogram quantities of the peptide pheromone, and has a large dynamic range. To demonstrate its utility, the promoter was used to control expression of the toxic peptides of twoparfamily toxin-antitoxin (TA) loci present inE. faecalis,parpAD1of the pAD1 plasmid andparEF0409located on theE. faecalischromosome. The results demonstrated differences in the modes of regulation of toxin expression and in the effects of toxins of these two related systems. We anticipate that this vector will be useful for further investigation ofparTA system function as well as the regulated expression of other genes inE. faecalisIMPORTANCEE. faecalisis an important nosocomial pathogen and a model organism for examination of the genetics and physiology of Gram-positive cocci. While numerous genetic tools have been generated for the manipulation of this organism, vectors for the regulated expression of cloned genes remain limited by high background expression and the use of inducers with undesirable effects on the cell. Here we demonstrate that the PQpheromone-responsive promoter is repressed tightly enough to allow cloning of TA system toxins and evaluate their effects at very low induction levels. This tool will allow us to more fully examine TA system function inE. faecalisand to further elucidate its potential roles in cell physiology.
authors.affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, South Dakota, USA kweaver@usd.edu.
authors.forename: Keith E
authors.initials: KE
authors.lastname: Weaver
authors.affiliation: Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Yuqing
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, South Dakota, USA.
authors.forename: Elly M
authors.initials: EM
authors.lastname: Miiller
authors.affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, South Dakota, USA.
authors.forename: Jake N
authors.initials: JN
authors.lastname: Johnson
authors.affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, South Dakota, USA.
authors.forename: Alex A
authors.initials: AA
authors.lastname: Dangler
authors.affiliation: Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Dawn A
authors.initials: DA
authors.lastname: Manias
authors.affiliation: Oregon Health & Science University, Portland, Oregon, USA.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Clem
authors.affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, South Dakota, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Schjodt
authors.affiliation: Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Gary M
authors.initials: GM
authors.lastname: Dunny
mesh_terms: D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism*; D013293:Enterococcus faecalis / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods; D008967:Molecular Biology / Q000379:methods; D010675:Pheromones / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D010675:Pheromones; D011994:Recombinant Proteins; C473319:fst toxin, Enterococcus faecalis
keywords: Enterococcus; expression vector; pheromone; toxin-antitoxin systems