Phosphorylation of MgrA and Its Effect on Expression of the NorA and NorB Efflux Pumps of Staphylococcus aureus

Abstract

MgrA is a global regulator in Staphylococcus aureus that controls the expression of diverse genes encoding virulence factors and multidrug resistance (MDR) efflux transporters. We identified pknB, which encodes the (Ser/Thr) kinase PknB, in the S. aureus genome. PknB was able to autophosphorylate as well as phosphorylate purified MgrA. We demonstrated that rsbU, which encodes a Ser/Thr phosphatase and is involved in the activation of the SigB regulon, was able to dephosphorylate MgrA-P but not PknB-P. Serines 110 and 113 of MgrA were found to be phosphorylated, and Ala substitutions at these positions resulted in reductions in the level of phosphorylation of MgrA. DNA gel shift binding assays using norA and norB promoters showed that MgrA-P was able to bind the norB promoter but not the norA promoter, a pattern which was the reverse of that for unphosphorylated MgrA. The double mutant MgrA S110A-S113A bound to the norA promoter but not the norB promoter. The double mutant led to a 2-fold decrease in norA transcripts and a 2-fold decrease in the MICs of norfloxacin and ciprofloxacin in strain RN6390. Thus, phosphorylation of MgrA results in loss of binding to the norA promoter, but with a gain of the ability to bind the norB promoter. Loss of the ability to phosphorylate MgrA by Ala substitution resulted in increased repression of norA expression and in reductions in susceptibilities to NorA substrates.

Implication of the NorB Efflux Pump in the Adaptation of Staphylococcus aureus to Growth at Acid pH and in Resistance to Moxifloxacin

“…Although acid stress responses do not include increases in major virulence factors or biofilm formation, there were noteworthy changes in several global regulators that control the expression of virulence genes and drug efflux transporters (5), such as the SaeRS and ArlRS, two-component regulators, the Ser/Thr kinase PknB, and its phosphatase Stp1. These global regulators have been shown to be important in modulating virulence factor and drug transporter expression (9,11,22,23,26). Other regulators exhibit increased expression at pH 4.5, including the transcriptional regulator of the iron regulator Fur family (SACOL1541), the histidine kinases PhoP and KdpD, and the VraS protein of the cell wall stimulon response (10,14,16).…”

“…Equal amounts of total cell proteins were separated on 12% SDS-PAGE gel and transferred onto nitrocellulose membranes as previously described (26). All washing steps were performed at room temperature.…”

“…Real-time quantitative reverse transcription-PCR (qRT-PCR) assays were carried out as previously described (23,26). Total S. aureus RNA was prepared by extraction from lysostaphin-treated cells collected at different time points, using an RNeasy midi kit (Qiagen, Valencia, CA).…”

“…The phosphorylation state of MgrA affects its binding to the norA and norB promoters and their gene transcript levels (23,26). Transcriptional profiling data indicated that the level of expression of the Ser/Thr kinase encoded by pknB decreased at least 5-fold at acid pH.…”

“…In contrast, for the mgrA mutant QT1a, there was a lesser loss of viability (0.34-fold) between the two pH conditions (1.37-fold at pH 7.0 versus 4-fold at pH 4.5). Thus, pH-induced differences in cell viability on exposure to moxifloxacin appear to be dependent on intact mgrA, suggesting that the differences in the levels and phosphorylation state of MgrA, which can modulate norB expression (23,26), are also important for the acid-induced increased expression of norB and its consequent reduced susceptibility to moxifloxacin. …”