School of Natural and Computational Sciences, Massey University at Albany, Auckland, New Zealand.
School of Natural and Computational Sciences, Massey University at Albany, Auckland, New Zealand
J Bacteriol. 2020 Jun 9;202(13). doi: 10.1128/JB.00792-19.
HutC is known as a transcriptional repressor specific for histidine utilization () genes in Gram-negative bacteria, including SBW25. However, its precise mode of protein-DNA interactions hasn't been examined with purified HutC proteins. Here, we performed electrophoretic mobility shift assay (EMSA) and DNase I footprinting using His-tagged HutC and biotin-labeled probe of the promoter (P). Results revealed a complex pattern of HutC oligomerization, and the specific protein-DNA interaction is disrupted by urocanate, a histidine derivative, in a concentration-dependent manner. Next, we searched for putative HutC-binding sites in the SBW25 genome. This led to the identification of 143 candidate targets with a value less than 10 HutC interaction with eight selected candidate sites was subsequently confirmed by EMSA analysis, including the type IV pilus assembly protein PilZ, phospholipase C (PlcC) for phosphatidylcholine hydrolyzation, and key regulators of cellular nitrogen metabolism (NtrBC and GlnE). Finally, an isogenic deletion mutant was subjected to transcriptome sequencing (RNA-seq) analysis and phenotypic characterization. When bacteria were grown on succinate and histidine, deletion caused upregulation of 794 genes and downregulation of 525 genes at a value of <0.05 with a fold change cutoff of 2.0. The mutant displayed an enhanced spreading motility and pyoverdine production in laboratory media, in addition to the previously reported growth defect on the surfaces of plants. Together, our data indicate that HutC plays global regulatory roles beyond histidine catabolism through low-affinity binding with operator sites located outside the locus. HutC in is a representative member of the GntR/HutC family of transcriptional regulators, which possess a N-terminal winged helix-turn-helix (wHTH) DNA-binding domain and a C-terminal substrate-binding domain. HutC is generally known to repress expression of histidine utilization () genes through binding to the P promoter with urocanate (the first intermediate of the histidine degradation pathway) as the direct inducer. Here, we first describe the detailed molecular interactions between HutC and its P target site in a plant growth-promoting bacterium, SBW25, and further show that HutC possesses specific DNA-binding activities with many targets in the SBW25 genome. Subsequent RNA-seq analysis and phenotypic assays revealed an unexpected global regulatory role of HutC for successful bacterial colonization .
HutC 是一种在革兰氏阴性菌中特异性转录抑制因子，用于组氨酸利用基因，包括 SBW25。然而，其与纯化的 HutC 蛋白之间的精确蛋白-DNA 相互作用模式尚未被检测到。在这里，我们使用 His 标记的 HutC 和生物素标记的 启动子（P）探针进行电泳迁移率变动分析（EMSA）和 DNase I 足迹分析。结果显示 HutC 寡聚化的复杂模式，并且特定的蛋白-DNA 相互作用被尿刊酸（组氨酸衍生物）以浓度依赖的方式破坏。接下来，我们在 SBW25 基因组中搜索了可能的 HutC 结合位点。这导致鉴定出 143 个候选靶标，其中值小于 10 HutC 与 8 个选定候选位点的相互作用随后通过 EMSA 分析得到证实，包括 IV 型菌毛组装蛋白 PilZ、用于磷脂酰胆碱水解的磷脂酶 C（PlcC）和细胞氮代谢的关键调节剂（NtrBC 和 GlnE）。最后，进行了同源性缺失突变体的转录组测序（RNA-seq）分析和表型特征分析。当细菌在琥珀酸盐和组氨酸上生长时，缺失导致 794 个基因上调和 525 个基因下调，值小于 0.05，倍数变化截止值为 2.0。突变体在实验室培养基中表现出增强的扩散运动和吡咯菌素产生，除了先前报道的在植物表面的生长缺陷。总的来说，我们的数据表明，HutC 通过与位于 基因座外的操纵子位点的低亲和力结合，发挥超出组氨酸分解代谢的全局调控作用。中的 HutC 是 GntR/HutC 家族转录调节剂的代表成员，其具有 N 端翼状螺旋-转角-螺旋（wHTH）DNA 结合结构域和 C 端底物结合结构域。通常认为，HutC 通过与 P 启动子结合并以尿刊酸（组氨酸降解途径的第一个中间产物）作为直接诱导物来抑制组氨酸利用基因的表达。在这里，我们首次描述了植物促生菌 SBW25 中 HutC 与其 P 靶标之间的详细分子相互作用，并进一步表明 HutC 与 SBW25 基因组中的许多靶标具有特异性 DNA 结合活性。随后的 RNA-seq 分析和表型分析揭示了 HutC 对成功细菌定殖的意想不到的全局调控作用。
