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Updated: Jun 30, 2026
A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues
Published on: February 19, 2019
Posttranslational modification influences the effects of MgrA on norA expression in Staphylococcus aureus
Que Chi Truong-Bolduc1, Yanpeng Ding, David C Hooper
1Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114-2696, USA.
Abstract:
MgrA is a global regulator in Staphylococcus aureus. Differences in the effects of MgrA on norA expression have been reported for different strains, which varied in rsbU, a gene that affects the expression of sigB, which encodes an alternative sigma factor involved in stress responses. We hypothesized that MgrA was modified by sigB-dependent factors that affected its ability to control the expression of the norA efflux pump. Heterologously expressed MgrA purified from Escherichia coli was incubated with crude extracts (CE) from strains RN6390 (rsbU) and SH1000 (rsbU(+)) and tested for binding to the norA promoter. Purified MgrA exhibited greater binding to norA promoter DNA after being incubated with SH1000 CE than MgrA incubated with the RN6390 CE. Phosphorylation of MgrA occurring in cell extracts caused it to lose the ability to bind norA promoter DNA. Overexpression of pknB, encoding a candidate serine/threonine kinase, produced increased phospho-MgrA and led to a fivefold increase in the transcript level of norA for both RN6390 and SH1000, as well as a fourfold increase in the MICs of norfloxacin and ciprofloxacin for these two strains. The levels of expression of pknB in RN6390 and SH1000, however, indicated that additional factors related to rsbU or sigB contribute to the differential regulatory effects of MgrA on norA expression.
Insights
Staphylococcus aureus MgrA regulation of norA efflux pump expression is influenced by phosphorylation. Strain variations in rsbU and sigB affect MgrA binding to the norA promoter, impacting antibiotic resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- MgrA is a global regulator in Staphylococcus aureus.
- Differential regulation of the norA efflux pump by MgrA has been observed across strains, linked to variations in rsbU and sigB.
- sigB encodes an alternative sigma factor crucial for stress responses.
Purpose of the Study:
- To investigate the hypothesis that sigB-dependent factors modify MgrA's ability to regulate norA expression.
- To elucidate the role of phosphorylation in MgrA's regulatory function.
- To understand strain-specific differences in MgrA-mediated norA regulation.
Main Methods:
- Heterologous expression and purification of MgrA from Escherichia coli.
- Incubation of purified MgrA with crude extracts from S. aureus strains RN6390 (rsbU) and SH1000 (rsbU(+)).
- Assessment of MgrA binding to the norA promoter DNA using electrophoretic mobility shift assays.
- Analysis of MgrA phosphorylation status and its effect on norA expression and antibiotic susceptibility.
Main Results:
- MgrA bound more effectively to the norA promoter after incubation with SH1000 crude extract compared to RN6390 crude extract.
- Phosphorylation of MgrA in cell extracts led to a loss of its DNA-binding ability to the norA promoter.
- Overexpression of pknB resulted in increased phospho-MgrA, a fivefold increase in norA transcript levels, and a fourfold increase in MICs for norfloxacin and ciprofloxacin.
Conclusions:
- Phosphorylation is a key mechanism by which MgrA's regulatory activity on norA expression is modulated.
- Strain-specific differences in rsbU and sigB likely contribute to the varied regulatory effects of MgrA on norA.
- pknB plays a significant role in MgrA phosphorylation and subsequent regulation of norA expression and antibiotic resistance in S. aureus.
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