Development of a regulatable expression system for the functional study of Vibrio vulnificus essential genes.

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.