Effect of substrate exposure and other growth condition manipulations on norA expression

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Abstract
Objectives: Multidrug efflux is a resistance mechanism that simultaneously affects susceptibility to many structurally unrelated compounds. The regulation of norA expression, which encodes the Staphylococcus aureus NorA multidrug efflux pump, is not well understood but the MgrA global regulator and the arlRS locus are involved. The expression of genes encoding proteins related to NorA, such as QacA of S. aureus and Bmr of Bacillus subtilis, is affected by pump substrates. In these instances, substrate interacts with regulatory proteins such that pump gene transcription is increased. The goal of this study was to identify if a similar substrate-level effect exists, or an effect of other growth condition manipulations, on the expression of norA. Methods: A transcriptional fusion between norA and lacZ was created in single copy on the chromosome of S. aureus SH1000. β-Galactosidase activity was quantified following exposure of the fusion strain to various NorA substrates, salicylate, a high salt concentration, putative soluble factors elaborated during growth, and different incubation temperatures. Results and conclusions: Exposure to several substrates significantly increased norA expression whereas salicylate and osmotic stress had no effect and no stable soluble factor affecting norA expression was detectable. An inverse relationship between norA expression and incubation temperature was observed and this effect was related, at least in part, to changes in norA mRNA half-life. However, concomitant changes in translational efficiency at different temperatures could not be ruled out. We conclude that there is a substrate-level effect on norA expression and propose that this may be mediated through substrate interaction with a regulatory protein.

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