| Project/Area Number |
12670247
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bacteriology (including Mycology)
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| Research Institution | Gunma University |
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Principal Investigator |
FUJIMOTO Shuhei Denartment of Microbiology, School of Medicine, Gunma University, lecturer, 医学部, 講師 (90241869)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Enterococcus faecalis / plasmid / conjugation / pheromone / receptor / TraA / peptide binding / DNA binding |
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| Research Abstract |
We investigated relationship between structure and function of Enterococcus faecalis pheromone receptor TraA family proteins employing chimera protein formation between different members of the protein family. Fusion of pPD1 TraA and pAD1 TraA genes as tagged protein with strep-tag was performed with a conventional restriction enzyme cleavage and ligation method and Overlap Extension (OLE) PCR method. We developed the peptide associated protein-tag chromatography (PPAC) method to observe peptide pheromone binding of the tagged proteins using unmodified non-radioactive peptide. We observed the DNA binding, the pheromone binding, and the pheromone-to-DNA-binding signaling of the chimera proteins independently by DPAC (DNA associated protein-tag chromatography) and PPAC. A pPD1 TraA sustained its DNA binding properties when 149 aa from its N-terminal were kept on the chimera protein. It sustained its pheromone binding properties when more than 146 aa from its N-terminal were kept on chimera proteins. The DNA binding domain of pPD1 TraA was supposed to reside on N-terminus (Ca. 150 aa) and the pheromone binding domain was supposed to reside on C-terminus (Ca. 170 aa). We purified pAD1 TraA protein, pPD1 TraA protein, and PrgX of pCF10 as tagged proteins and observed DNA and pheromone binding specificities of these proteins by DPAC and PPAC. These proteins bound exclusively to their own cognate DNA fragments and pheromones. We investigated the pheromone response of clinical isolates of vancomycin resistant enterococci (VRE). A strain showed clumping response against two different synthetic pheromones cAD1 and cCF10. We constructed new E. faecalis- E. coli shuttle vectors and showed that pAD1 TraA protein works in-trans in vivo.
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