Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Microbiol Spectr. 2024 Nov 5;12(11):e0130624. doi: 10.1128/spectrum.01306-24. Epub 2024 Sep 20.
Gene expression systems that transcend species barriers are needed for cross-species analysis of gene function. In particular, expression systems that can be utilized in both model and pathogenic bacteria underpin comparative functional approaches that inform conserved and variable features of bacterial physiology. In this study, we develop replicative and integrative vectors alongside a novel, IPTG-inducible promoter that can be used in the model bacterium K-12 as well as strains of the antibiotic-resistant pathogen, . We generate modular vectors that transfer by conjugation at high efficiency and either replicate or integrate into the genome, depending on design. Embedded in these vectors, we also developed a synthetic, IPTG-inducible promoter, P, that induces to a high level but is less leaky than the commonly used promoter. We show that P is titratable at both the population and single-cell levels, regardless of species, highlighting the utility of our expression systems for cross-species functional studies. Finally, as a proof of principle, we use our integrating vector to develop a reporter for the envelope stress σ factor, RpoE, and deploy the reporter in and , finding that does not recognize RpoE-dependent promoters unless RpoE is heterologously expressed. We envision that these vector and promoter tools will be valuable for the community of researchers who study the fundamental biology of and .IMPORTANCE is a multidrug-resistant, hospital-acquired pathogen with the ability to cause severe infections. Understanding the unique biology of this non-model bacterium may lead to the discovery of new weaknesses that can be targeted to treat antibiotic-resistant infections. In this study, we provide expression tools that can be used to study the gene function in , including in drug-resistant clinical isolates. These tools are also compatible with the model bacterium, , enabling cross-species comparisons of gene function. We anticipate that the use of these tools by the scientific community will accelerate our understanding of biology.
需要能够跨越物种障碍的基因表达系统来进行基因功能的跨物种分析。特别是，能够在模型和致病性细菌中使用的表达系统为比较功能方法提供了基础，这些方法可以告知细菌生理学的保守和可变特征。在这项研究中，我们开发了复制和整合载体，以及一种新型的 IPTG 诱导启动子，该启动子可用于模型细菌 K-12 以及抗生素抗性病原体的菌株。我们生成了模块化载体，这些载体通过高效的接合转移，根据设计，要么复制，要么整合到基因组中。在这些载体中，我们还开发了一种合成的、IPTG 诱导的启动子 P，它可以高水平诱导，但比常用的启动子泄漏性低。我们表明，无论物种如何，P 都可以在群体和单细胞水平上进行滴定，突出了我们的表达系统在跨物种功能研究中的实用性。最后，作为原理验证，我们使用我们的整合载体开发了一个用于 包膜应激 σ 因子 RpoE 的报告基因，并在 和 中部署了该报告基因，发现除非异源表达 RpoE，否则 不会识别依赖于 RpoE 的启动子。我们设想，这些载体和启动子工具将对研究 和 基础生物学的研究人员具有重要价值。
