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The alarmone (p)ppGpp mediates physiological-responsive control at the sigma 54-dependent Po promoter
1Department of Cell and Molecular Biology, Umeå University, Sweden.
Abstract:
Transcription from the Pseudomonas-derived sigma 54-dependent Po promoter of the dmp operon is mediated by the aromatic-responsive regulator DmpR. However, physiological control is superimposed on this regulatory system causing silencing of the DmpR-mediated transcriptional response in rich media until the transition between exponential and stationary phase is reached. Here, the positive role of the nutritional alarmone (p)ppGpp in DmpR regulation of the Po promoter has been identified and investigated in vivo. Overproduction of (p)ppGpp in a Pseudomonas reporter system was found to allow an immediate transcriptional response under normally non-permissive conditions. Conversely (p)ppGpp-deficient Escherichia coli strains were found to be severely defective in DmpR-mediated transcription, demonstrating the requirement for this metabolic signal. A subset of mutations in the beta, beta' and sigma 70 subunits of RNA polymerase, which confer prototrophy on ppGpp0 E. coli, was also found to restore specific DmpR-mediated transcription from Po, suggesting that the metabolic signal is mediated directly through the sigma 54-RNA polymerase. These data provide a direct mechanistic link between the physiological status of the cell and expression from sigma 54 promoters.
Insights
The study reveals that the alarmone (p)ppGpp is crucial for activating DmpR-mediated transcription from the sigma 54-dependent Po promoter in Pseudomonas. This metabolic signal links cellular physiological status to gene expression from sigma 54 promoters.
Area of Science:
- Microbiology
- Molecular Biology
- Gene Regulation
Background:
- DmpR regulates transcription from the sigma 54-dependent Po promoter in Pseudomonas.
- DmpR-mediated transcription is typically silenced in rich media until the cell transitions to stationary phase.
- The physiological control mechanisms overriding DmpR regulation are not fully understood.
Purpose of the Study:
- To investigate the role of the nutritional alarmone (p)ppGpp in DmpR regulation of the Po promoter.
- To elucidate the mechanism by which (p)ppGpp influences DmpR-mediated transcription.
- To establish a link between cellular physiological status and sigma 54 promoter activity.
Main Methods:
- In vivo studies using Pseudomonas reporter systems.
- Investigation of (p)ppGpp-deficient Escherichia coli strains.
- Analysis of RNA polymerase subunit mutations affecting DmpR-mediated transcription.
Main Results:
- Overproduction of (p)ppGpp enabled immediate DmpR-mediated transcription under non-permissive conditions.
- (p)ppGpp-deficient E. coli strains exhibited severe defects in DmpR-mediated transcription.
- Specific mutations in RNA polymerase subunits restored DmpR-mediated transcription in ppGpp0 E. coli, implicating the sigma 54-RNA polymerase complex.
Conclusions:
- The nutritional alarmone (p)ppGpp plays a positive regulatory role in DmpR-mediated transcription from the Po promoter.
- The metabolic signal (p)ppGpp is required for efficient DmpR-mediated transcription.
- The sigma 54-RNA polymerase complex is a likely mediator of the (p)ppGpp signal, linking cellular physiology to gene expression.