1992 Fiscal Year Final Research Report Summary
STUDIES ON THE MOLECULAR GENETICS OF REGULATORY MECHANISM OF THETA TOXIN PRODUCTION IN C. perfringens
| Project/Area Number |
03670213
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
細菌学
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| Research Institution | OSAKA UNIVERSITY |
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Principal Investigator |
HIGASHI Yasushi COLLEGE OF BIO-MEDICAL TECHNOLOGY, OSAKA UNIV., PROFESSOR, 医療技術短期大学部, 教授 (60028371)
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| Co-Investigator(Kenkyū-buntansha) |
IMAGAWA Tadashi OSAKA UNIV., MEDICAL SCHOOL, DEPT. OF BACTERIOLOGY, ASSISTANT PROFESSOR, 医学部・細菌学, 助手 (10036478)
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| Project Period (FY) |
1991 – 1992
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| Keywords | Clostridium perfrinegens / Theta toxin deficient mutants / "Substance A" / Regulon-like regulatory mechanism |
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| Research Abstract |
Theta toxin-deficient mutants of Clostridium perfringens could be devided into two groups, a and b. Group a mutants are deficient in theta toxin but release a dialysable substance ("substance A"), which restores theta toxin activity to group b mutants, into culture supernatant at the exponentially-growing phase ; group b mutants are defective in "substance A" release. The phenomenon occurring between them has been demonstrated not only in theta toxin-deficient mutants but also mutants deficient in kappa- and lambda-toxins and HA of virulence factors. These results suggest that there exists "regulon"-like regulatory mechanism, in which "substance A" plays a key role, on the expression of genes for certain virulence factors of C1. perfringens. Two important themes of this research project were firstly to examine conditions for the stabilization of "substance A" itself and secondly to analyze "substane A"-associated regulatory mechanism on the expression of virulence-factor genes by means
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of techniques of genetic engineering. (1) Studies on the characterization and stabilization of "substance A" clarified that "substance A" was produced mostly in exponentially-growing phase and was also preserved at least one month in the cold with no activity loss if the pII of culture filtrate is lowered to 5.0 or less before lyophilized. "Substance A" was purified about 20 folds by means of silica-resin column chromatography of a preserved lyophilized-material. It was inactivated promptly at 37ﾟC and alkaline pH but the peculiarity was found that the activity increased by two to three hundred % for the first several hours of incubation at pH 5.0 and 0ﾟC. (2) Attempts were made at the gene manipulation of cloned theta-toxin gene in order to demonstrate that there exist the homologous consensus-sequence in the upper stream of each gene for four virulence factors to be associated with "substance A". Analysis of possible indirect-interaction (through a possible DNA-binding protein) between "substance A" and the consensus signal sequence has never been developped yet although attempts were made at electroporation-mediated transformation of group a mutant strain("substance A" producer) under various electrical and cultural conditions with deletants in the upper stream of theta toxin gene. Less
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