Transcriptional regulation of the protocatechuate 4,5-cleavage pathway genes in Sphingomonas paucimobilis SYK-6 by LigR, a LysR-type transcriptional regulator

LysR 型转录调节因子 LigR 对少动鞘氨醇单胞菌 SYK-6 中原儿茶酸 4,5-裂解途径基因的转录调节

• 批准号: 16580055
• 负责人: MASAI Eiji
• 金额: $ 2.37万
• 依托单位: Nagaoka University of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16580055/
• 关键词: lignin-derived aromatic compounds; protocatechuate 4,5-cleavage pathway; Sphingomonas; transcriptional regulation; LysR-type transcriptional regulator

The protocatechuate (PCA) 4,5-cleavage pathway is the essential metabolic route for lignin catabolism in Sphingomonas paucimobilis SYK-6. Here, we examined the operon structure of the PCA 4,5-cleavage pathway genes and investigated the transcriptional regulation of the genes. Reverse transcription-PCR analysis indicated that the PCA 4,5-cleavage pathway genes of SYK-6 consist of three transcriptional units, including the ligK-orf1-ligI-lsdA operon, the ligJ-ligA-ligB-ligC operon, and the monocistronic ligR. In the presence of PCA, transcription of the ligK and ligJ operons was induced 10-to 40-fold in SYK-6, however, this induction was no longer observed in the ligR mutant. Inducible transcription of these operons was not influenced by the disruption of ligB, which encodes a large subunit of PCA 4,5-dioxygenase. This result strongly suggested that PCA acts as an inducer of these operons. The promoter regions of these operons were determined by the promoter-lacZ transcriptional fusion analysis. Gel retardation assay exhibited the specific DNA binding of LigR to the promoter regions of ligK and ligJ operons. In conclusion, the PCA 4,5-cleavage pathway genes of SYK-6 consist of three transcriptional units. Transcriptions from the ligK and ligJ promoters are directly up-regulated by LigR, and transcription of these operons requires PCA as an inducer.

原儿茶酸 (PCA) 4,5-裂解途径是少动鞘氨醇单胞菌 SYK-6 中木质素分解代谢的重要代谢途径。在这里，我们检查了 PCA 4,5-裂解途径基因的操纵子结构，并研究了该基因的转录调控。逆转录PCR分析表明，SYK-6的PCA 4,5-裂解途径基因由三个转录单元组成，包括ligK-orf1-ligI-lsdA操纵子、ligJ-ligA-ligB-ligC操纵子和单顺反子ligR。在 PCA 存在的情况下，SYK-6 中 ligK 和 ligJ 操纵子的转录被诱导 10 至 40 倍，然而，在 ligR 突变体中不再观察到这种诱导。这些操纵子的诱导转录不受 ligB 破坏的影响，ligB 编码 PCA 4,5-双加氧酶的大亚基。这一结果强烈表明 PCA 作为这些操纵子的诱导剂。通过启动子-lacZ 转录融合分析确定这些操纵子的启动子区域。凝胶阻滞测定显示 LigR 与 ligK 和 ligJ 操纵子启动子区域的特异性 DNA 结合。总之，SYK-6的PCA 4,5-裂解途径基因由三个转录单位组成。 LigR 直接上调 ligK 和 ligJ 启动子的转录，这些操纵子的转录需要 PCA 作为诱导剂。