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Abstract：
Inducible gene expression systems are powerful genetic tools to study bacterial physiology, probing essential and toxic gene functions, gene dosage effects, and overexpression phenotypes. For the opportunistic human pathogen Pseudomonas aeruginosa, dedicated inducible gene expression systems are scarce. In the current study, we developed a minimal synthetic 4-isopropylbenzoic acid (cumate)-inducible promoter, called PQJ, that is tunable over several orders of magnitude. This was achieved by combining semirandomized housekeeping promoter libraries and control elements from the Pseudomonas putida strain F1 cym/cmt system with powerful fluorescence-activated cell sorting (FACS) to select functionally optimized variants. Using flow cytometry and live-cell fluorescence microscopy, we demonstrate that PQJ responds rapidly and homogenously to the inducer cumate in a graded manner at the single-cell level. PQJ and cumate are orthogonal to the frequently used isopropyl β-d-thiogalactopyranoside (IPTG)-regulated lacIq-Ptac expression system. The modular design of the cumate-inducible expression cassette together with the FACS-based enrichment strategy presented here facilitates portability, thus serving as a blueprint for the development of tailored gene expression systems for a wide range of bacteria. IMPORTANCE Reverse genetics is a powerful approach to study bacterial physiology and behavior by relying on well-developed genetic tools, such as inducible promoters. For the human pathogen Pseudomonas aeruginosa, well-characterized inducible promoters are scarce. In the current work, we used a synthetic biology-based approach to develop a cumate-inducible promoter for P. aeruginosa, termed PQJ, that shows excellent induction properties at the single-cell level. This genetic tool provides the means for qualitative and quantitative gene function studies describing P. aeruginosa\'s physiology and virulence in vitro and in vivo. Because this synthetic approach to constructing species-specific inducible promoters is portable, it can serve as a blueprint for similar tailored gene expression systems in bacteria largely lacking such tools, including, for example, representatives of the human microbiota.
摘要：
诱导型基因表达系统是研究细菌生理学的强大遗传工具，探测必需和毒性基因功能，基因剂量效应，和过表达表型。对于机会性人类病原体铜绿假单胞菌，专用的诱导型基因表达系统是稀缺的。在目前的研究中,我们开发了一个最小的合成4-异丙基苯甲酸（cumate）诱导型启动子，叫做PQJ，在几个数量级上是可调的。这是通过将来自恶臭假单胞菌菌株F1cym/cmt系统的半随机看家启动子文库和控制元件与强大的荧光激活细胞分选（FACS）相结合来选择功能优化的变体来实现的。使用流式细胞术和活细胞荧光显微镜，我们证明了PQJ在单细胞水平上以分级的方式对诱导剂cumate快速且均匀地响应。PQJ和cumate与常用的异丙基β-d-硫代吡喃半乳糖苷（IPTG）调节的lacIq-Ptac表达系统正交。cumate诱导型表达盒的模块化设计与本文提出的基于FACS的富集策略一起促进了便携性。从而成为为各种细菌开发定制基因表达系统的蓝图。重要性反向遗传学是一种强大的方法来研究细菌的生理和行为，依靠完善的遗传工具，如诱导型启动子。对于人类病原体铜绿假单胞菌，良好表征的诱导型启动子是稀缺的。在目前的工作中,我们使用基于合成生物学的方法来开发铜绿假单胞菌的cumate诱导型启动子，称为PQJ，在单细胞水平显示出优异的诱导特性。这种遗传工具提供了定性和定量基因功能研究的手段，描述了铜绿假单胞菌的生理和毒力的体外和体内。因为这种构建物种特异性诱导型启动子的合成方法是便携式的，它可以作为类似的定制基因表达系统的蓝图，在细菌中缺乏这样的工具，包括,例如,人类微生物群的代表。
