A-034. Regulation Mechanism of the Staphylococcus aureus NorA Multidrug Efflux Pump by MgrA and NorG Proteins

Q. Truong-Bolduc1,2, Y. Ding1,2, D. C. Hooper1,2;
1Massachusetts General Hosp., Boston, MA, 2Harvard Med. Sch., Boston, MA.

Staphylococcus aureus resists antibiotics by expressing multidrug resistance (MDR) transporters such as NorA, a chromosomally encoded efflux pump. We previously found that at least three regulators MgrA, NorG, and ArlRS were involved in the regulation of norA expression. MgrA and NorG were discovered by their affinities for the norA promoter. In this study, we attempted to determine the possible roles of interactions and post-tranlational modification of MgrA and NorG in regulation of norA expression. Histidine-tagged MgrA heterologously expressed in Escherichia coli and purified by Ni affinity chromatography was incubated with crude extracts from S. aureus RN6390 (rsbU-) or SH1000 (rsbU+), and subsequently re-purified. Mass spectroscopy revealed putative phosphorylation at two residues, threonine 109 and serine 161 of MgrA after incubation with the RN6390 extract. Western blots with phosphoserine and phosphothreonine antibodies were carried out to confirm the phosphorylation at these two amino acids. After separation on phospho-binding columns, phosphorylated (MgrA-P) and unphosphorylated (MgrA) were used in gel-mobility shift assays. MgrA and NorG generated the expected gel shift pattern with norA promoter DNA, and the pattern was similar when NorG and MgrA were together. In contrast MgrA-P did not bind to the norA promoter but modified the pattern of NorG binding. RT-PCR was used to measure the level of norA transcripts from strain SH1000 and its mutants, SH1 (mgrA) and SH11 (norG), and strain RN6390 and its mutants, RN1(mgrA) and RN11(norG) grown to late-log phase. We observed a 3-fold increase and 2-fold decrease in the norA transcripts for SH1 and RN1, relative to their respective parent strains. No significant change was found for SH11 and RN11. Our data indicate that MgrA-P binds norA promoter DNA poorly but affects binding of NorG, suggesting that levels of phosphorylation of MgrA may affect its function. In addition, MgrA-mediated regulation of norA expression differed in the presence and absence of rsbU. The link among RsbU, MgrA phosphorylation, and norA regulation is under study.