| Summary: | 碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 98 === The Bacillus subtilis ycgMNO operon, which encodes proline dehydrogenase, ??-pyrroline-5-carboxylate dehydrogenase, and proline permease, is known to be responsible for proline uptake and subsequent conversion of proline to glutamate. However, regulation of expression of the ycgMNO operon has not been extensively studied. A search in the genome of B. subtilis revealed that the ycgP gene, whose function is unknown, is located immediately downstream of the ycgO gene. The ycgP-encoded putative protein shows no significant overall amino acid sequence homology to other proteins with known functions in the database. Nevertheless, YcgP contains a putative DNA-binding motif at its C-terminal region. In this study, we have demonstrated that the ycgP gene is not only essential for ycgM expression in Luria-Bertani medium, but is also required for proline induction of ycgM expression in M9 minimal medium. Electrophoretic mobility shift assays showed that purified His-tagged YcgP protein could specifically interact with an inverted repeat sequence located upstream of the ycgM promoter. Deletion analysis and site-directed mutagenesis confirmed the importance of the inverted repeat sequence in ycgM expression. Cell growth assays have also established that both ycgP and ycgM are essential for metabolism of proline as a sole carbon or nitrogen source in B. subtilis. Taken together, these results support that the novel transcriptional activator YcgP can activate transcription of ycgMNO in vivo through binding to the inverted repeat sequence. In addition, we found that the ycgP gene was constitutively expressed at a low level in M9 minimal medium and that ycgP expression was not subject to negative autoregulation and proline induction. Disruption of the yusM gene, which encodes an YcgM homolog, did not affect ycgM expression. The expression level of yusM was low both in wild-type cells and in the ycgM mutant. These results are consistent with the idea that YcgM is the only functional proline dehydrogenase in B. subtilis. Moreover, sporulation efficiency analysis showed that ycgM but not ycgP is required for normal sporulation and that the sporulation defect of the ycgM mutant could not be suppressed by the addition of glutamate. Finally, we found that casamino acid could repress ycgM expression in a CodY-independent manner. The precise mechanism underlying casamino acid repression remains to be clarified.
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