Due to its potential for use in bacterial engineering applications, we previously cloned the SPI-1 type 3 secretion system (T3SS) genes from the genome of Salmonella enterica serovar Typhimurium strain LT2. We have documented that this clone, while functionally expressed in S. Typhimurium strains, displays a severe expression defect in other Gram negative backgrounds including Escherichia coli. To address this issue, we compared SPI-1 DNA sequence across different backgrounds, fully sequenced the original SPI-1 clone, and cloned SPI-1 from other S. Typhimurium strains. In this process, we were able to successfully obtain SPI-1 clones that are functionally expressed in E. coli indicating the first such result for a full-length SP-1 T3SS clone. We discovered that the original cloning technique using a DNA homology-based capture method was the root of the expression defect and that the FRT-Capture technique is preferable over the homology-based method. This result paves the way for future studies and applications using cloned SPI-1 and other T3SS in non-Salmonella bacterial backgrounds.

中文翻译：

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克隆的SPI-1 3型分泌系统可以在沙门氏菌背景之外进行功能表达。

由于其在细菌工程应用中的潜力，我们先前从肠道沙门氏菌血清鼠伤寒沙门氏菌菌株LT2的基因组中克隆了SPI-1 3型分泌系统（T3SS）基因。我们已经证明，该克隆虽然在鼠伤寒沙门氏菌菌株中功能表达，但在包括大肠杆菌在内的其他革兰氏阴性背景中显示出严重的表达缺陷。为了解决此问题，我们比较了不同背景下的SPI-1 DNA序列，对原始SPI-1克隆进行了完全测序，并从其他鼠伤寒沙门氏菌菌株中克隆了SPI-1。在此过程中，我们能够成功获得在大肠杆菌中功能性表达的SPI-1克隆，这表明全长SP-1 T3SS克隆首次获得此类结果。我们发现使用基于DNA同源性的捕获方法的原始克隆技术是表达缺陷的根源，而FRT-Capture技术优于基于同源性的方法。该结果为在非沙门氏菌属细菌背景下使用克隆的SPI-1和其他T3SS进行进一步的研究和应用铺平了道路。