2000 Fiscal Year Final Research Report Summary
Charactorization of expression mechanisms for a novel glucan-binding protein C in S.mutans
| Project/Area Number |
09671869
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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 |
Morphological basic dentistry
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| Research Institution | TOKYO DENTAL COLLEGE |
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Principal Investigator |
SATO Yutaka Tokyo Dental College, Department of Dentistry, Associate professor, 歯学部, 助教授 (70085827)
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| Project Period (FY) |
1997 – 2000
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| Keywords | S.mutans / gbpC / glucan / aggregation / Two-component system / gcrR / xylitol / wall protein |
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| Research Abstract |
I have identified the gbpC gene encoding the glucan-binding protein C which is involved in dextran (glucan)-dependent aggregation (ddag) of Streptococcus mutans by random mutagenesis. It was generally thought that S.mutans does not exhibit this ddag phenotype, since this phenotype was appeared only under certain stress conditions. I have introduced random mutation into S.mutans with one of the integration vectors pVA891 and have screened the mutants for the ddag- negative phenotype to isolate genes involved in this property. Insertion of pVA891 containing a Sau3AI-digested host DNA fragment into the chromosome occurs following homologous recombination via a Campbell-like mechanism. However, most of mutants that we obtained appeared to have resulted from chromosomal rearrangements as well as pVA891 insertion. I found that several dozen mutants exhibiting the non-aggregation phenotype harbored the intact gbpC gene and that these mutants possessed a large and characteristic duplication of
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a region of the chromosome which was responsible for the phenotype. Based upon characterization of these duplications, I developed a strategy to introduce a duplication into any specific region of the chromosome of these organisms. The 690 bp gene responsible for the ddag- phenotype was identified within a 60 kb region by observing ddag (positive or negative) phenotypes of successively constructed specific duplication mutants. The gene was one of the response-regulator gene of the Two-component regulatory systems, and was designated as the gene gcrR.
I also found a new phenomenon that cells repeatedly cultured in the presence of xylitol evolved into those exhibiting the elevated dextran-dependent aggregation phenotype. This phenotype was confirmed to result from expression of the gbpC gene by constructing of a S.mutans isogenic mutant carrying the gbpC : : lacZ fusion gene. I found that gbpC expression of the such cells was elevated 20-fold. It was of interest that these cells also exhibited decreased adhesion to glass surfaces when grown with shaking in the presence of sucrose. This may be one of the ways by which some populations of S.mutans are removed from dental plaques. Less
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