Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural Universitygrid.27871.3b, Nanjing, China.
Appl Environ Microbiol. 2022 Mar 22;88(6):e0239021. doi: 10.1128/aem.02390-21. Epub 2022 Feb 9.
5-Hydroxypicolinic acid (5HPA), an important natural pyridine derivative, is microbially degraded in the environment. Previously, a gene cluster, , responsible for 5HPA degradation, was identified in Alcaligenes faecalis JQ135. However, the transcription regulation mechanism of the cluster is still unknown. In this study, the transcription start site and promoter of the operon was identified. Quantitative reverse transcription-PCR and promoter activity analysis indicated that the transcription of the operon was negatively regulated by a TetR family regulator, HpaR, whereas the transcription of itself was not regulated by HpaR. Electrophoretic mobility shift assay and DNase I footprinting revealed that HpaR bound to two DNA sequences, covering the -35 region and -10 region, respectively, in the promoter region of the operon. Interestingly, the two binding sequences are partially palindromic, with 3 to 4 mismatches and are complementary to each other. 5HPA acted as a ligand of HpaR, preventing HpaR from binding to promoter region and derepressing the transcription of the operon. The study revealed that HpaR binds to two unique complementary sequences of the promoter of the operon to negatively regulate the catabolism of 5HPA. This study revealed that the transcription of the operon was negatively regulated by a TetR family regulator, HpaR. The binding of HpaR to the promoter of the operon has the following unique features: (i) HpaR has two independent binding sites in the promoter of the operon, covering -35 region and -10 region, respectively; (ii) the palindrome sequences of the two binding sites are complementary to each other; and (iii) both of the binding sites include a 10-nucleotide partial palindrome sequence with 3 to 4 mismatches. This study provides new insights into the binding features of the TetR family regulator with DNA sequences.
5-羟吡啶酸（5HPA）是一种重要的天然吡啶衍生物，可在环境中被微生物降解。先前，在 Alcaligenes faecalis JQ135 中鉴定了一个负责 5HPA 降解的基因簇。然而，该簇的转录调控机制尚不清楚。在本研究中，鉴定了 操纵子的转录起始位点和启动子。定量反转录-PCR 和启动子活性分析表明， 操纵子的转录受 TetR 家族调节剂 HpaR 的负调控，而 HpaR 本身不调节 的转录。电泳迁移率变动分析和 DNase I 足迹实验表明，HpaR 结合到两个 DNA 序列上，分别覆盖启动子区域的 -35 区域和 -10 区域。有趣的是，这两个结合序列部分是回文序列，有 3 到 4 个错配，彼此互补。5HPA 作为 HpaR 的配体，阻止 HpaR 结合到启动子区域并解除对 操纵子转录的抑制。该研究表明，HpaR 结合到 操纵子启动子的两个独特互补序列上，负调控 5HPA 的分解代谢。本研究表明， 操纵子的转录受 TetR 家族调节剂 HpaR 的负调控。HpaR 与 操纵子启动子的结合具有以下独特特征：（i）HpaR 在 操纵子启动子上具有两个独立的结合位点，分别覆盖 -35 区域和 -10 区域；（ii）两个结合位点的回文序列彼此互补；（iii）两个结合位点均包含一个 10 个核苷酸的部分回文序列，有 3 到 4 个错配。本研究为 TetR 家族调节剂与 DNA 序列的结合特征提供了新的见解。
