| Summary: | 博士 === 長庚大學 === 生物醫學研究所 === 97 === Bacillus subtilis F29-3 produces an anti-fungal peptidic antibiotic that is synthesized nonribosomally by fengycin synthetases. Our earlier work established that the promoter of the fengycin synthetase operon (fenp) is located 86 nucleotides upstream of the translational initiation codon of fenC. This investigation generated transcriptional fusions with a DNA fragments that contains the region between -105 and +80 in fenp. Analyzing the fusion plasmids revealed that deleting the region between -55 and -42 reduces the promoter activity by 64.5%. Transcriptional fusions generated on the B. subtilis DB2 chromosome also indicate that this region is important to the transcription. In vitro transcription analysis confirms that the transcription is inefficient when the sequence in this region is mutated. The electrophoretic mobility shift and footprinting analyses demonstrate that the C-terminal domain of the RNA polymerase α subunit binds to the region between -55 and -39. These results indicated that the sequence is an UP element. Finally, this UP element is critical to the production of fengycin, since mutating the UP sequence on the chromosome of B. subtilis F29-3 reduces the transcription of the fen operon by 85% and prevents the cells from producing enough fengycin to suppress the germination of Paecilomyces variotii spores on agar plates. Moreover, 8 predicted PerR boxes were found in fenp. In a perR mutant, the transcription activity of fenp was decreases by 86%. However, integrating perR into the chromosome of the perR mutant restores the promoter activity, showing that transcription from fenp is regulated by PerR. Chromatin immunoprecipitation and eletrophoretic mobility shift assay revealed that PerR binds to the region bewteen -20 and +80 in fenp. These results suggested that PerR binds to fenp and positively regulated the transcription activity of fenp. This study demonstrated that fenp has an UP element to activate the transcription activity and PerR may be involved in the transcription regulation of fenp. These results reveal the mechanism by which B. subtilis F29-3 regulates the transcription of fengycin synthetase operon.
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