PDF(Pubmed)
Abstract：
Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes. To overcome this limitation and enable the straightforward investigation of gene functions in S. pyogenes, we have developed a comprehensive genetic toolset. By adapting and combining different tools previously applied in other Gram-positive bacteria, we have created new replicative and integrative plasmids for gene expression and genetic manipulation, constitutive and inducible promoters as well as fluorescence reporters for S. pyogenes. The new replicative plasmids feature low- and high-copy replicons combined with different resistance cassettes and a standardized multiple cloning site for rapid cloning procedures. We designed site-specific integrative plasmids and verified their integration by nanopore sequencing. To minimize the effect of plasmid integration on bacterial physiology, we screened publicly available RNA-sequencing datasets for transcriptionally silent sites. We validated this approach by designing the integrative plasmid pSpy0K6 targeting the transcriptionally silent gene SPy_1078. Analysis of the activity of different constitutive promoters indicated a wide variety of strengths, with the lactococcal promoter P 23 showing the strongest activity and the synthetic promoter P xylS2 showing the weakest activity. Further, we assessed the functionality of three inducible regulatory elements including a zinc- and an IPTG-inducible promoter as well as an erythromycin-inducible riboswitch that showed low-to-no background expression and high inducibility. Additionally, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes. We therefore adapted the chemically defined medium called RPMI4Spy that showed reduced autofluorescence and enabled efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS*, which enabled the scarless deletion of the sagB gene. This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.
摘要：
遗传工具是研究分子机制的基础。尽管最近在细菌基因工程领域取得了许多进展，非模型生物的遗传工具集仍然很少，如强制性的人类病原体化脓性链球菌。为了克服这一限制，并能够直接研究化脓性链球菌的基因功能，我们开发了一套全面的遗传工具。通过适应和组合以前在其他革兰氏阳性细菌中使用的不同工具，我们创造了新的复制和整合质粒，用于基因表达和遗传操作，化脓性链球菌的组成型和诱导型启动子以及荧光报告基因。新的复制质粒具有低拷贝和高拷贝复制子与不同的抗性盒和用于快速克隆程序的标准化多克隆位点的组合。我们设计了位点特异性整合质粒，并通过纳米孔测序验证了它们的整合。为了最小化质粒整合对细菌生理的影响，我们筛选了公开可用的RNA测序数据集的转录沉默位点.我们通过设计靶向转录沉默基因SPy_1078的整合质粒pSpy0K6验证了这种方法。不同组成型启动子的活性分析显示出各种各样的优势，乳球菌启动子P23显示出最强的活性，而合成启动子PxylS2显示出最弱的活性。Further,我们评估了3种诱导型调控元件的功能,包括锌诱导型和IPTG诱导型启动子以及红霉素诱导型核糖开关,它们表现出低至无背景表达和高诱导性.此外,我们证明了两种密码子优化荧光蛋白的适用性，mNeongreen和mKate2，作为酿脓链球菌的记者。因此，我们采用了称为RPMI4Spy的化学成分确定的培养基，该培养基显示出减少的自发荧光，并能够在板读数器测定和荧光显微镜中进行有效的信号检测。最后,我们开发了一种基于质粒的系统，用于化脓性链球菌的基因组工程，其特征是反选择标记pheS*，这使得sagB基因无疤痕缺失。这个新的工具箱简化了以前费力的基因操作程序，并为研究化脓性链球菌基因功能的新方法奠定了基础。从而更好地了解其毒力机制和生理学。
