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The McCleary Laboratory


Our lab is currently engaged in research to understand the molecular mechanisms by which the simple bacterium, Escherichia coli senses and responds to changes in environmental phosphate. We are studying this question because it is an extremely useful model to understand complex regulatory schemes in all living organisms. At the heart of this question is a two-component signal transduction system that senses environmental phosphate and controls the expression of many genes for the high affinity acquisition of phosphate and the utilization of alternate sources of phosphorous.

Our current model for the regulation of this response is that the PstSCAB transporter (a high affinity ABC transporter of phosphate) senses phosphate levels and communicates through the PhoU protein to the bifunctional histidine kinase, PhoR, which interacts with and controls the steady state phosphorylation level of the response regulator, PhoB. Phospho-PhoB binds to specific DNA sequences upstream of Pho regulon genes, interacts with the sigma70 subunit of RNA polymerase and stimulates transcription. Phosphate sufficiency generates a signal that shuts off the Pho regulon by activating the phospho-PhoB phosphatase activity of PhoR. However, when phosphate is limiting or when mutations eliminate any component of the PstSCAB transporter or PhoU, the Pho regulon is turned on. This low-phosphate signal stimulates the kinase activity of PhoR allowing it to serve as an efficient phospho-donor to PhoB. Phosphate starvation also triggers the general stress response in which cells become increasingly resistant to many environmental stresses. The master regulator of this response is sigmaS (RpoS), an alternate sigma factor that competes with sigma70 to direct the transcription of genes under its control. The regulation of sigmaS is very complex and has numerous inputs. Its cellular levels are controlled at the levels of transcription, translation, protein turnover and activity.

We are currently using genetic, molecular genetic, biochemical and biophysical techniques to determine the molecular mechanisms of signal transduction in the Pho regulon. Students who work and study in our lab gain experience in mastering multiple techniques and in learning to address difficult problems through a multifaceted approach.