| Summary: | 碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 96 === Bacillus subtilis cells can produce a 35-kDa cell wall hydrolase, CwlF (LytE), during vegetative growth. LytE is involved in cell separation and cell wall turnover, and thus regulation of lytE expression is important to B. subtilis. Previously, a search in the B. subtilis genome database for possible SigI target genes had identified a putative σI promoter in the regulatory region of lytE. Reporter gene analysis showed that overexpression of sigI could stimulate expression of the lytE regulatory region-bgaB fusion, but the putative σI promoter itself exhibited no promoter activity in normal conditions and under heat stress or sigI overexpression. This is quite different from those observed for other known SigI target genes. In this study, Western blotting and zymography analysis were used to confirm that sigI overexpression could remarkably increase the expression of lytE. Deletion analysis and site-directed mutagenesis were also conducted to examine the role of the putative σI promoter in lytE expression. The results revealed that the presence of both σI promoter-like sequence and σA promoter sequence are required for the stimulatory effect of sigI overexpression on lytE expression. Meanwhile, previous results showed that heat stress could induce expressions of sigI and lytE. When the chromosomal sigI gene was inactivated, heat stress could still induce the expression of the lytE regulatory region-bgaB fusion. In this study we explored how heat stress could induce lytE expression and the physiologic function of LytE in heat stress. The results suggest that YycF may also contribute to heat induction of lytE, and lytE expression is required for cell viability at high temperature.
Carbon catabolite control protein A (CcpA), a member of the LacI / GalR family of regulatory proteins, constitutes the master transcription regulator in catabolite control regulation in B. subtilis. It functions by interacting with various catabolite responsive elements (CREs), thereby either activating or repressing catabolic genes. It is known that approximately 10% genes in B. subtilis genome are under the control of CcpA, underscoring its vital metabolic role. Besides sensing carbon metabolite, CcpA is also known to be able to regulate gene expression in a glucose-independent manner. In this study, we investigated whether CcpA could also autoregulate its own expression. The results suggest that the expression of ccpA is subject to negative autoregulation regardless of whether glucose was added to the culture medium.
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