Toolbox of Characterized Genetic Parts for <i>Staphylococcus aureus</i>

Staphylococcus aureus is an important clinical bacterium prevalent in human-associated microbiomes and the cause of many diseases. However, S. aureus has been intractable to synthetic biology approaches due to limited characterized genetic parts for this nonmodel Gram-positive bacterium. Moreover, genetic manipulation of S. aureus has relied on cumbersome and inefficient cloning strategies. Here, we report the first standardized genetic parts toolbox for S. aureus, which includes characterized promoters, ribosome binding sites, terminators, and plasmid replicons from a variety of bacteria for precise control of gene expression. We established a standard relative expression unit (REU) for S. aureus using a plasmid reference and characterized genetic parts in standardized REUs using S. aureus ATCC 12600. We constructed promoter and terminator part plasmids that are compatible with an efficient Type IIS DNA assembly strategy to effectively build multipart DNA constructs. A library of 24 constitutive promoters was built and characterized in S. aureus, which showed a 380-fold activity range. This promoter library was also assayed in Bacillus subtilis (122-fold activity range) to demonstrate the transferability of the constitutive promoters between these Gram-positive bacteria. By applying an iterative design-build-test-learn cycle, we demonstrated the use of our toolbox for the rational design and engineering of a tetracycline sensor in S. aureus using the PXyl‑TetO aTc-inducible promoter that achieved 25.8-fold induction. This toolbox greatly expands the growing number of genetic parts for Gram-positive bacteria and will allow researchers to leverage synthetic biology approaches to study and engineer cellular processes in S. aureus.金黄色葡萄球菌（Staphylococcus aureus）是一类广泛定殖于人类相关微生物组的重要临床病原菌，可引发多种疾病。然而，作为非模式革兰氏阳性菌（Gram-positive bacterium），由于已表征的遗传元件有限，金黄色葡萄球菌的合成生物学研究一直难以开展。此外，针对金黄色葡萄球菌的遗传操作长期依赖繁琐且低效的克隆策略。本研究报道了首个针对金黄色葡萄球菌的标准化遗传元件工具箱，该工具箱包含来自多种细菌的已表征启动子、核糖体结合位点、终止子与质粒复制子，可实现基因表达的精准调控。研究人员以质粒为参照，建立了金黄色葡萄球菌的标准相对表达单位（Relative Expression Unit, REU），并在金黄色葡萄球菌ATCC 12600中对标准化REU体系下的遗传元件进行了表征。本研究还构建了适配高效Type IIS型DNA组装策略的启动子和终止子元件质粒，可高效构建多片段DNA构建体。研究人员构建并表征了包含24个组成型启动子的文库，该文库在金黄色葡萄球菌中的活性范围达380倍。同时，该启动子文库还在枯草芽孢杆菌（Bacillus subtilis）中进行了活性检测，其活性范围达122倍，证明了组成型启动子在这类革兰氏阳性菌间的可转移性。通过应用迭代设计-构建-测试-学习（design-build-test-learn）循环，本研究展示了利用该工具箱合理设计并工程化改造金黄色葡萄球菌中的四环素传感器：采用PXyl-TetO aTc诱导型启动子，实现了25.8倍的诱导倍数。该工具箱极大扩充了革兰氏阳性菌已有的遗传元件库，将助力研究人员利用合成生物学手段研究并工程化改造金黄色葡萄球菌的细胞过程。