Zhang Yan-Jiao, Chen Guozhong, Lin Huiyuan, Wang Pan, Kuang Baozhi, Liu Jing, Chen Shiyong
Shandong Province Key Laboratory of Applied Mycology, School of Life Sciences, Qingdao Agricultural University, Qingdao, 266109, Shandong, People's Republic of China.
Central Laboratory, Qingdao Agriculture University, Qingdao, 266109, Shandong, People's Republic of China.
Antonie Van Leeuwenhoek. 2017 Apr;110(4):607-614. doi: 10.1007/s10482-016-0827-x. Epub 2017 Jan 2.
We developed a regulatable gene expression system for Vibrio vulnificus, which contains a lacIq-pTrc cassette. Monomeric red fluorescence protein (mRFP) was used as a reporter to test this system. The results showed that this system tightly controlled the expression of mRFP without leaky expression and was suitable for the controlled expression of genes encoding recombinant proteins in V. vulnificus. To demonstrate the utility of this system, a dominant negative form of V. vulnificus VVMO6_RS04990, a homolog of Escherichia coli LolD that is essential in lipoprotein transport and membrane biogenesis, was inducibly expressed. Expression of the dominant negative LolD homolog, which has a mutation in the ATPase domain, resulted in a growth defect in V. vulnificus cells and impaired cell envelope stability. This result suggests that the V. vulnificus LolD homolog plays a role in cell envelope biogenesis. This tight and titratable expression system will therefore be a valuable tool for the study of essential genes in V. vulnificus.
我们开发了一种用于创伤弧菌的可调控基因表达系统，该系统包含一个lacIq-pTrc盒。使用单体红色荧光蛋白（mRFP）作为报告基因来测试该系统。结果表明，该系统紧密控制mRFP的表达，无渗漏表达，适用于创伤弧菌中重组蛋白编码基因的可控表达。为证明该系统的实用性，诱导表达了创伤弧菌VVMO6_RS04990的显性负性形式，它是大肠杆菌LolD的同源物，在脂蛋白转运和膜生物合成中至关重要。具有ATP酶结构域突变的显性负性LolD同源物的表达导致创伤弧菌细胞生长缺陷并损害细胞包膜稳定性。这一结果表明创伤弧菌LolD同源物在细胞包膜生物合成中发挥作用。因此，这种紧密且可滴定的表达系统将成为研究创伤弧菌必需基因的有价值工具。
