Selective activation of sar promoters with the use of green fluorescent protein transcriptional fusions as the detection system in the rabbit endocarditis model

The global regulatory locus sar is composed of three overlapping transcript s initiated from a triple promoter system (designated P1, P3, and P2). To e xplore if the individual sar promoters are differentially expressed in vitr o and in vivo, we constructed a shuttle plasmid (pALC1434) containing a pro moterless gfp(UV) gene (a gfp derivative [Clontech]) preceded by a polylink er region. Recombinant shuttle vectors containing individual sar promoters upstream of the gfp(UV) reporter gene were then introduced into Staphylococ cus aureus RN6390. Northern and immunoblot analysis revealed that P1 is str onger than the P2 and P3 promoters in vitro. Additionally, the levels of th e gfp, transcript driven by individual snr promoters also correlated with t he growth cycle dependency of these promoters in liquid cultures, thus sugg esting the utility of pALC1434 as a vehicle for reporter fusion. Using the rabbit endocarditis model, me examined the expression of these three GFP(UV ) fusions in vivo by fluorescence microscopy of infected cardiac vegetation s 24 h after initial intravenous challenge. similar to the in vitro finding s, P1 was activated both in the center and on the surface of the vegetation s. In contrast, the P3 promoter was silent both in vivo and in vitro as det ermined by fluorescence microscopy. Remarkably, P2 was silent in vitro but became highly activated in vivo. In particular, the sar P2 promoter was act ivated on the surface of the vegetation but not in the center of the lesion . These data imply that in vivo promoter activation of sar differed from th at observed in vitro. Moreover, the individual sar promoters may be differe ntially expressed in different areas within the same anatomic niche, presum ably reflecting the microbial physiological response to distinct host micro environments, As the sar locus controls the synthesis of both extracellular and cell mall virulence determinants, these promoter-gfp(UV) constructs sh ould be useful to characterize many aspects of S. aureus gene regulation in vivo.