Biozentrum, University of Basel, Basel, Switzerland.
Appl Environ Microbiol. 2023 Jun 28;89(6):e0021123. doi: 10.1128/aem.00211-23. Epub 2023 May 18.
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 P, 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 / system with powerful fluorescence-activated cell sorting (FACS) to select functionally optimized variants. Using flow cytometry and live-cell fluorescence microscopy, we demonstrate that P responds rapidly and homogenously to the inducer cumate in a graded manner at the single-cell level. P and cumate are orthogonal to the frequently used isopropyl β-d-thiogalactopyranoside (IPTG)-regulated -P 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. 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 P, 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 and . 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.
诱导型基因表达系统是研究细菌生理学的强大遗传工具，可用于探测必需和毒性基因功能、基因剂量效应以及过表达表型。对于机会性病原体铜绿假单胞菌来说，专门的诱导型基因表达系统却很少。在本研究中，我们开发了一种最小的合成 4-异丙基苯甲酸（伞形酸）诱导启动子 P，其可调范围为几个数量级。这是通过组合半随机化的管家启动子文库和来自恶臭假单胞菌 F1 /系统的控制元件，并利用强大的荧光激活细胞分选（FACS）来选择功能优化的变体来实现的。我们使用流式细胞术和活细胞荧光显微镜证明，P 在单细胞水平上以分级的方式对诱导剂伞形酸快速且均匀地响应。P 和伞形酸与常用的异丙基 β-D-硫代半乳糖苷（IPTG）调控的 -P 表达系统正交。这里提出的基于 cumate 诱导表达盒的模块化设计和 FACS 富集策略便于携带，因此可作为针对广泛细菌定制基因表达系统的蓝图。
反向遗传学是通过依赖于成熟的遗传工具（如诱导型启动子）来研究细菌生理学和行为的强大方法。对于人类病原体铜绿假单胞菌，特征明确的诱导型启动子却很少。在本工作中，我们使用基于合成生物学的方法为铜绿假单胞菌开发了一种伞形酸诱导启动子，称为 P，它在单细胞水平上具有出色的诱导特性。该遗传工具为定性和定量基因功能研究提供了手段，可用于描述铜绿假单胞菌的生理学和毒力。由于构建物种特异性诱导型启动子的这种合成方法具有可移植性，因此可以作为类似定制基因表达系统在细菌中的蓝图，而这些细菌在很大程度上缺乏此类工具，包括例如人类微生物群的代表。
