Development of shuttle vectors for evaluation of essential regulator regulated gene expression in Staphylococcus aureus.

We describe the construction of a series of shuttle vectors for Staphylococcus aureus. In order to determine transcriptional regulation by essential regulators, we constructed promoterless luxABCDE reporter system using a TetR-regulated antisense RNA expression vector, pJYJ909, which is composed of S. aureus plasmid pE194, the Gram(-) plasmid pUC18, a TetR regulatory cassette, and Pxyl/teto-driven yhcS antisense expression construct. The reformed shuttle vector was utilized to construct an opuCA promoter-luxABCDE fusion and simultaneously examine transcriptional regulation by measuring bioluminescence intensity during down-regulating yhcSR. In addition, we utilized the same plasmid, pJYJ909, and constructed a Pspac-driven constant expression system, which allows us to determine the complementary effect of overexpression of opuCA operon modulated by yhcSR. These plasmids provide important tools for elucidating regulatory mechanisms for genes that are essential for bacterial growth in S. aureus.