Development of a heterodimer plasmid system for the introduction of heterologous genes into streptococci.
Development of a heterodimer plasmid system for the introduction of heterologous genes into streptococci.
复制标题
开发异二聚体质粒系统，用于将异源基因引入链球菌。
作者：
T. Shiroza;H. Kuramitsu
我们以前已经构建了一个模型分泌系统的口腔链球菌使用的分泌结构域的变形链球菌GS-5 gtfB基因。作为开发用于分泌含有来自链球菌的葡聚糖结合多肽的蛋白融合物的系统的初始步骤，将对应于来自菌株GS-5的葡糖基转移酶-S酶的葡聚糖结合结构域（GBD）的DNA片段融合到gtfB分泌结构域。然而，利用大肠杆菌将该杂合基因克隆到大肠杆菌-链球菌穿梭质粒中是不可能的。coli作为宿主细胞。利用一种新的异源二聚体质粒系统，将杂合GBD基因直接整合到戈登链球菌染色体中。异源二聚体是通过将两个质粒连接在一起而构建的，每个质粒含有与S. gordonii染色体侧翼的杂合GBD基因的双交换重组事件。然而，这种单拷贝整合体分泌有限量的GBD蛋白。为了实现该蛋白的高效表达，将杂合GBD基因整合到S.在构建中间异源二聚体质粒后，这些质粒含有GBD基因，其侧翼是与驻留质粒区域同源的序列。来自携带多拷贝质粒的转化体的培养液中GBD蛋白的存在证明了功能性GBD蛋白的分泌。成功开发的在链球菌中分泌GBD的策略应该适用于其他可用基因转移系统的生物体。此外，当构建体不能稳定地保持在中间宿主如大肠杆菌内的穿梭载体中时，这些系统将允许直接引入遗传构建体。杆菌
We have previously constructed a model secretion system for oral streptococci using the secretory domain of the Streptococcus mutans GS-5 gtfB gene. As an initial step in developing systems for secreting protein fusions containing a glucan-binding polypeptide from streptococci, a DNA fragment corresponding to the glucan-binding domain (GBD) of the glucosyltransferase-S enzyme from strain GS-5 was fused to the gtfB secretory domain. However, it was not possible to clone the hybrid gene into Escherichia coli-streptococcal shuttle plasmids using E. coli as host cells. Integration of the hybrid GBD gene into the Streptococcus gordonii chromosome was directly accomplished utilizing a strategy involving a novel heterodimeric plasmid system. The heterodimer was constructed by ligating together two plasmids each containing DNA fragments homologous with a corresponding region of the S. gordonii chromosome flanking the hybrid GBD gene following a double crossover recombination event. However, this single-copy integrant secreted limited quantities of the GBD protein. In order to achieve high-level expression of the protein, the hybrid GBD gene was integrated into resident plasmids in S. gordonii following construction of intermediate heterodimeric plasmids. These plasmids contained the GBD gene flanked by sequences homologous to regions of the resident plasmids. The presence of the GBD protein in culture fluids from transformants harboring the multicopy plasmids demonstrated the secretion of the functional GBD protein. The strategy successfully developed for secreting the GBD in the streptococci should be adaptable for other organisms for which gene transfer systems are available. In addition, these systems will allow the direct introduction of genetic constructs when the constructs cannot be stably maintained in shuttle vectors within intermediate hosts such as E. coli.