Development of An Antibiotic Marker-Free Gene Delivery System in Streptococcus gordonii

Abstract

Streptococcus gordonii, a commensal oral bacterium, is considered a good candidate to function as a live oral vaccine vector. The introduction of vaccine antigen genes into S. gordonii relies on the use of antibiotic resistance genes as selectable markers, which is undesirable. In this study, we used auxotrophic complementation (deletion of an essential gene from the chromosome and insertion into a plasmid) as a means to create an antibiotic marker-free gene delivery system in S. gordonii. S. gordonii ?thyA was created and complemented by an antibiotic marker-free expression plasmid containing the intact thyA gene, pDL276/thyAdelkan. Transformation of pDL276/thyAdelkan into the mutant gave an unexpected 100-fold increase in transformation efficiency as compared to pDL276. The transformants arose from both single and double crossing over. The increase in transformation efficiency suggests that a highly efficient antibiotic marker-free system to deliver genes to the chromosome has been created using thyA complementation.