Transcriptional regulation of proH, lutA, lutP,

• Main Authors: Chen-Jyun Lin, 林辰駿
• Other Authors: Gwo-Chyuan Shaw
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/92617251195287402491

碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 102 === The salt-inducible proHJ operon of Bacillus subtilis 168 encodes enzymes involved in the synthesis of the osmoprotectant proline for osmotic stress resistance. In this study we have found that glucose also can induce expression of the proH-lacZ fusion in a dose-dependent manner. Glucose induction of proH-lacZ expression is only partially CcpA-dependent. No typical catabolite-responsive element (CRE) sequence is present in the regulatory region of proH, implying that CcpA may positively control proH expression indirectly. Inactivation of the ptsH or hprK gene, which is involved in glucose uptake into cells, totally abolished the inductive effect of glucose on proH-lacZ expression, indicating their importance in this glucose induction. Mutation and complementation analysis revealed that ccpA, ptsH, and hprK can also contribute to proH expression in the absence of exogenous glucose. Furthermore, deletion and mutation analysis showed that a direct repeat, 5’-CGAACAAA-N4-CGAACAAA-3’, which is located downstream of the σA promoter of proH and conserved in some other Bacillus species, can negatively regulate proH transcription. Together, these results indicate that proH expression in B. subtilis is subject to both positive and negative controls. The lutABC operon of B. subtilis RO-NN-1 encodes three iron-sulfur-containing proteins required for L-lactate utilization. The transcriptional regulator LutR of the GntR family negatively regulates lutABC expression. The lutP gene, which is situated immediately upstream of lutR and encodes an L-lactate permease, is also negatively regulated by LutR. In this study we show that lutA expression is not subject to catabolite repression by glucose whereas lutP expression is subject to partial catabolite repression. Mutation analysis revealed that a CRE sequence located downstream of the σA promoter of lutP is responsible for this catabolite repression. We also showed that lutR expression is not autoregulated but lutP expression is still partially induced by L-lactate in the lutP mutant, consistent with the previous proposal that LutP is a major permease for L-lactate import and other permease(s) may also have a partial contribution to the uptake of L-lactate. Moreover, deletion analysis revealed that a DNA segment upstream of the lutP promoter is important for lutP expression. In contrast to the truncated LutR of the laboratory strain PY79, the full-length LutR of the undomesticated strain RO-NN-1 cannot interact with the regulatory regions of ispA and aprE as revealed by electrophoretic mobility shift assays. Likewise, there is no interaction between the truncated LutR and the regulatory region of lutA or lutP. The absence or presence of the N-terminal 21 amino acids of the full-length LutR, which encompass a small part of the predicted winged helix-turn-helix DNA-binding motif, may probably alter the DNA-binding specificity or affinity of LutR.