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B.S., Nanjing University-2004
Thesis Advisor: Ann Stock, Ph.D.
Graduate Program in Biochemistry & Molecular Biology
CABM Room 010
Thursday, July 26, 2012
Two-component systems (TCS) are commonly utilized in bacteria in response to different environmental stimuli. Understanding of the regulation mechanism of TCS is important for general bacterial research such as studies of virulence and antibiotic discovery. PhoR/PhoB is one of the well-studied TCS in E. coli. In response to inorganic phosphate (Pi) starvation, response regulator PhoB is activated by PhoR, the histidine kinase, through phosphorylation. Phosphorylated PhoB activates the expression of a set of phosphate starvation-inducible (psi) genes including phoBR operon and other genes involved in phosphorus assimilation. This thesis is mainly focused on the transcription regulation of PhoR/PhoB at both spatial and time scales.
In the spatial scale, to explore the function of PhoB as a transcription regulator, a genomic search of genes controlled by PhoB was done with chromatin immunoprecipitation on microarrays (ChIP-chip). Two antibodies were used in ChIP to eliminate non-specific binding caused by the quality of antibodies. We verified direct control of phoBR, pstSCABphoU, phoA, phoE, ugpBAECQ by PhoB under a Pi limiting condition. No biding of DNA was detected for PhoB under a Pi excess condition, suggesting that unphosphorylated PhoB cannot serve as a transcription factor.
In the time sale, we examined the kinetics of regulation of gene expression and protein levels under Pi limitation. The activation of transcription was transient, as shown by mRNA profile, even though the phosphorylation level of PhoB was stable for several hours. Stationary phase sigma factor RpoS repressed the expression of the pho regulon under Pi limitation, possible by competition with sigma factor 70, which was utilized for transcription initiation of pho regulon genes.
The kinetics of the regulation when Pi was reintroduced to E. coli under Pi starvation was explored, too. The amount of components of the pho system were gradually diluted by growth. This was the underlying mechanism for a ‘learning behavior’ of PhoR/PhoB when Pi limitation was recounted. The PhoB was de-phosphorylated quickly by PhoR after addition of Pi. The phosphorylation level of PhoB was controlled by the equilibrium between phosphatase and histidine kinase function of PhoR. The quick regulation of the phosphorylation level of PhoB indicated the sensitivity of PhoR in response to environmental Pi alternation.
The results of these experiments will help illuminate the transcription regulation of PhoB and provide a general understanding of regulation mechanisms of TCS. The comprehensive analysis of the pho regulon together with the kinetic characterization of activation under Pi high to low switch and low to high switch provide a foundation for further investigations on the activation mechanism of TCS.