| Title: | Examination of Enterococcus faecalis Toxin-Antitoxin System Toxin Fst Function Utilizing a Pheromone-Inducible Expression Vector with Tight Repression and Broad Dynamic Range |
| Author(s): | Weaver KE; Chen Y; Miiller EM; Johnson JN; Dangler AA; Manias DA; Clem AM; Schjodt DJ; Dunny GM; |
| Address: | "Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, South Dakota, USA kweaver@usd.edu. Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA. Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, South Dakota, USA. Oregon Health & Science University, Portland, Oregon, USA" |
| ISSN/ISBN: | 1098-5530 (Electronic) 0021-9193 (Print) 0021-9193 (Linking) |
| Abstract: | "Tools for regulated gene expression in Enterococcus faecalis are extremely limited. In this report, we describe the construction of an expression vector for E. faecalis, designated pCIE, utilizing the P(Q) pheromone-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 two par family toxin-antitoxin (TA) loci present in E. faecalis, par(pAD1) of the pAD1 plasmid and par(EF0409) located on the E. faecalis chromosome. 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 of par TA system function as well as the regulated expression of other genes in E. faecalisIMPORTANCEE. faecalis is 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 P(Q) pheromone-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 in E. faecalis and to further elucidate its potential roles in cell physiology" |
| Keywords: | "Bacterial Toxins/*genetics/*metabolism Enterococcus faecalis/*genetics/*metabolism Gene Expression Regulation/*drug effects *Genetic Vectors Genetics, Microbial/methods Molecular Biology/methods Pheromones/*metabolism Promoter Regions, Genetic Recombinant;" |
| Notes: | "MedlineWeaver, Keith E Chen, Yuqing Miiller, Elly M Johnson, Jake N Dangler, Alex A Manias, Dawn A Clem, Aaron M Schjodt, Daniel J Dunny, Gary M eng R21 AI120601/AI/NIAID NIH HHS/ R35 GM118079/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't 2017/03/30 J Bacteriol. 2017 May 25; 199(12):e00065-17. doi: 10.1128/JB.00065-17. Print 2017 Jun 15" |
