Abstract：
The ability to genetically manipulate bacteria is a staple of modern molecular microbiology. Since the 2000s, marker-less mutants of Streptococcus pneumoniae (Spn) have been made by allelic exchange predominantly using the kanR-rpsL cassette known as \"Janus.\" The conventional Janus protocol involves two transformation steps using multiple PCR-assembled products containing the Janus cassette and the target gene\'s flanking DNA. We present an innovative strategy to achieve marker-less allelic replacement through a single transformation step. Our strategy involves integrating an additional copy of the target\'s downstream region before the Janus cassette, leading to a modified genetic arrangement. This single modification reduced the number of required PCR fragments from five to four, lowered the number of assembly reactions from two to one, and simplified the transformation process to a single step. To validate the efficacy of our approach, we implemented this strategy to delete in Spn serotype 4 strain TIGR4 the virulence gene pspA, the entire capsular polysaccharide synthesis locus cps4, and to introduce a single-nucleotide replacement into the chromosome. Notably, beyond streamlining the procedure, our method markedly reduced false positives typically encountered during negative selection with streptomycin when employing the traditional Janus protocol. Furthermore, and as consequence of reducing the amount of exogenous DNA required for construct synthesis, we show that our new method is amendable to the use of commercially available synthetic DNA for construct creation, further reducing the work needed to obtain a mutant. Our streamlined strategy, termed easyJanus, substantially expedites the genetic manipulation of Spn facilitating future research endeavors.
OBJECTIVE: We introduce a new strategy aimed at streamlining the process for marker-less allelic replacement in Streptococcus pneumoniae, a Gram-positive bacterium and leading cause of pneumonia, meningitis, and ear infections. Our approach involves a modified genetic arrangement of the Janus cassette to facilitate self-excision during the segregation step. Since this new method reduces the amount of exogenous DNA required, it is highly amendable to the use of synthetic DNA for construction of the mutagenic construct. Our streamlined strategy, called easyJanus, offers significant time and cost savings while concurrently enhancing the efficiency of obtaining marker-less allelic replacement in S. pneumoniae.
OBJECTIVE: We introduce a new strategy aimed at streamlining the process for marker-less allelic replacement in Streptococcus pneumoniae, a Gram-positive bacterium and leading cause of pneumonia, meningitis, and ear infections. Our approach involves a modified genetic arrangement of the Janus cassette to facilitate self-excision during the segregation step. Since this new method reduces the amount of exogenous DNA required, it is highly amendable to the use of synthetic DNA for construction of the mutagenic construct. Our streamlined strategy, called easyJanus, offers significant time and cost savings while concurrently enhancing the efficiency of obtaining marker-less allelic replacement in S. pneumoniae.
摘要：
基因操纵细菌的能力是现代分子微生物学的主要内容。自2000年以来，肺炎链球菌（Spn）的无标记突变体已通过等位基因交换制备，主要使用kanR-rpsL盒称为“Janus”。“传统的Janus方案涉及两个转化步骤，使用包含Janus盒和靶基因侧翼DNA的多个PCR组装产物。我们提出了一种创新策略，通过单个转化步骤实现无标记等位基因置换。我们的策略包括在Janus盒式磁带之前整合目标下游区域的额外副本，导致基因排列的改变。这种单一的修饰将所需的PCR片段的数量从五个减少到四个，将组装反应的数量从两个减少到一个，并将转换过程简化为一个步骤。为了验证我们方法的有效性，我们实施此策略以在Spn血清型4菌株TIGR4中删除毒力基因pspA，整个荚膜多糖合成基因座cps4，并将单核苷酸置换引入染色体。值得注意的是,除了精简程序，我们的方法显著减少了采用传统Janus方案时链霉素阴性选择过程中通常遇到的假阳性.此外，并且由于减少了构建体合成所需的外源DNA的量，我们表明，我们的新方法是可修正的使用市售合成DNA的构建物的创建，进一步减少获得突变体所需的工作。我们精简的战略，叫做easyJanus，大大加快了Spn的遗传操作，促进了未来的研究工作。
目的：我们引入了一种新的策略，旨在简化肺炎链球菌的无标记等位基因替代过程，一种革兰氏阳性细菌和肺炎的主要原因，脑膜炎,耳部感染。我们的方法涉及Janus盒的改良遗传排列，以促进分离步骤中的自我切除。由于这种新方法减少了所需的外源DNA的数量，使用合成DNA构建诱变构建体是高度可修正的。我们精简的战略，叫做easyJanus，提供显著的时间和成本节约,同时提高在肺炎链球菌中获得无标记等位基因替代的效率。
目的：我们引入了一种新的策略，旨在简化肺炎链球菌的无标记等位基因替代过程，一种革兰氏阳性细菌和肺炎的主要原因，脑膜炎,耳部感染。我们的方法涉及Janus盒的改良遗传排列，以促进分离步骤中的自我切除。由于这种新方法减少了所需的外源DNA的数量，使用合成DNA构建诱变构建体是高度可修正的。我们精简的战略，叫做easyJanus，提供显著的时间和成本节约,同时提高在肺炎链球菌中获得无标记等位基因替代的效率。
