Weaver 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
Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, South Dakota, USA
Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA.
J Bacteriol. 2017 May 25;199(12). doi: 10.1128/JB.00065-17. Print 2017 Jun 15.
Tools for regulated gene expression in are extremely limited. In this report, we describe the construction of an expression vector for , designated pCIE, utilizing the P 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 family toxin-antitoxin (TA) loci present in , of the pAD1 plasmid and located on the 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 TA system function as well as the regulated expression of other genes in 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 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 and to further elucidate its potential roles in cell physiology.
用于[具体物种]中基因表达调控的工具极其有限。在本报告中，我们描述了一种用于[具体物种]的表达载体的构建，命名为pCIE，它利用了质粒pCF10的P信息素响应启动子。我们证明该启动子受到严格抑制，对纳克量的肽信息素作出响应，并且具有很大的动态范围。为了证明其效用，该启动子被用于控制[具体物种]中存在的两个[具体家族]毒素 - 抗毒素（TA）位点的毒性肽的表达，一个位于pAD1质粒上，另一个位于[具体染色体]上。结果表明这两个相关系统的毒素表达调控模式以及毒素作用存在差异。我们预计该载体将有助于进一步研究[具体物种]的TA系统功能以及其他基因的调控表达。[具体物种]是一种重要的医院病原体，也是用于研究革兰氏阳性球菌遗传学和生理学的模式生物。虽然已经产生了许多用于操纵这种生物体的遗传工具，但用于克隆基因调控表达的载体仍然受到高背景表达以及使用对细胞有不良影响的诱导剂的限制。在这里，我们证明P信息素响应启动子受到足够严格的抑制，从而允许克隆TA系统毒素并在非常低的诱导水平下评估它们的作用。这个工具将使我们能够更全面地研究[具体物种]中的TA系统功能，并进一步阐明其在细胞生理学中的潜在作用。
