|Budget Amount *help
¥26,500,000 (Direct Cost: ¥26,500,000)
Fiscal Year 1991: ¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 1990: ¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 1989: ¥7,500,000 (Direct Cost: ¥7,500,000)
Fiscal Year 1988: ¥7,900,000 (Direct Cost: ¥7,900,000)
The aim of this research is to elucidate the molecular mechanisms of Escherichia coli to sense the concentrations of inorganic phosphate in the medium, and transduce the signal to the regulator protein to regulate the expression of the genes in the phosphate (pho) regulon, which is required for the cell to adapt its physiology to phosphate starvation.
It had been known that the expression of the genes in the pho regulon is dependent on the function of the phoB gene, and the activation of the phoB gene is dependent on the function of phoR or, in the absence of the phoR function, phoM.
(1) To study the functions of phoB and phoR, we purified PhoR and PhoB proteins and demonstrated in vitro that PhoR mediates phosphorylation of PhoB, and the phosphorylated PhoB is the active form that activates the initiation of transcription of the pho genes by binding to the consensus sequences called the pho box, located in the regulatory regions of the pho genes. We also demonstrated that PhoM proten, l
ike PhoR, mediates the phosphorylation of PhoB as well as PhoM-Orf2, providing the mechanism to explain the so-called "cross talk" between the two regulatory systems. These experiments, together with a few similar experiments done simultaneously by other research groups in the world, established the importance of protein phosphorylation for the signal transduction in prokaryotes. Based on the genetic and biochemical studies, we proposed that excess level of phosphate in the medium is sensed by the products of the pstS, C, A, B genes located in the periphery of the membrane, and the signal is transduced to PhoR protein by PhoU protein, which activates the phosphatase activity of PhoR, and PhoR, in turn, inactivates PhoB by dephosphorylation.
(2) We found that the ugpBACQ operon, which is involved in the uptake of sn-glycerol-3-phosphate, is regulated by two promoters, the pho-promoter, induced by phosphate-starvation and the cAMP-CRP regulated promoter, induced by carbon-starvation.
(3) By comparing the DNA sequence of the phn operon of E. coli K-12 and E. coli B strains, we discovered that the inability of E. coli K-12 strain to utilize phosphonate compounds is due to the insertion of an extra 8-bp repeated seqeunce in the phnE gene. Less