Department of Biochemistry, Microbiology, and Immunology, Wayne State Universitygrid.254444.7 School of Medicine, Detroit, Michigan, USA.
J Bacteriol. 2021 Aug 20;203(18):e0018721. doi: 10.1128/JB.00187-21.
Vibrio cholerae infects human hosts following ingestion of contaminated food or water, resulting in the severe diarrheal disease cholera. The watery diarrhea that is characteristic of the disease is directly caused by the production of cholera toxin (CT). A complex regulatory cascade controls the production of CT and other virulence factors. However, ultimately, a single protein, ToxT, directly binds to virulence gene promoters and activates their transcription. Previously, we identified two ToxT binding sites, or toxboxes, within the cholera toxin promoter (P). The toxboxes overlap the two promoter-proximal GATTTTT heptad repeats found within P in classical biotype V. cholerae strain O395. These heptad repeats were previously found to be located within a large DNA region bound by H-NS, a global transcriptional repressor present in Gram-negative bacteria. The current model for the control of P transcription proposes complete H-NS displacement from the DNA by ToxT, followed by direct activation by ToxT-RNA polymerase (RNAP) contacts. The goal of this study was to determine more precisely where H-NS binds to P and test the hypothesis that ToxT completely displaces H-NS from the P promoter before activating transcription. The results suggest that H-NS binds only to the region of P encompassing the heptad repeats and that ToxT displaces H-NS only from its most promoter-proximal binding sites, calling for a revision of the current model involving H-NS and ToxT at P. H-NS is a global negative regulator of transcription in Gram-negative bacteria, particularly in horizontally acquired genetic islands. Previous work in Vibrio cholerae suggested that H-NS represses the transcription of cholera toxin genes by binding to a large region upstream of its promoter and that the virulence activator ToxT derepresses transcription by removing H-NS from the promoter. Here, new data support a revised model in which ToxT displaces only H-NS bound to the most promoter-proximal DNA sites that overlap the ToxT binding sites, leaving the upstream sites occupied by H-NS. This introduces a higher-resolution mechanism for the antirepression of H-NS in the control of cholera toxin production.
霍乱弧菌经食用受污染的食物或水感染人类宿主，导致严重的腹泻病霍乱。疾病的特征性水样腹泻直接由霍乱毒素 (CT) 的产生引起。复杂的调控级联控制 CT 和其他毒力因子的产生。然而，最终，单个蛋白 ToxT 直接结合毒力基因启动子并激活其转录。此前，我们在霍乱毒素启动子 (P) 内鉴定了两个 ToxT 结合位点或 toxboxes。toxboxes 重叠了在经典生物型 V. cholerae 菌株 O395 中 P 内发现的两个启动子近端 GATTTTT 七肽重复。这些七肽重复先前被发现位于 H-NS 结合的大 DNA 区域内，H-NS 是革兰氏阴性细菌中存在的一种全局转录抑制剂。目前关于 P 转录的控制模型提出，ToxT 通过完全取代 H-NS 从 DNA 上位移，然后通过 ToxT-RNA 聚合酶 (RNAP) 接触直接激活。本研究的目的是更精确地确定 H-NS 与 P 的结合位置，并检验 ToxT 在激活转录之前是否完全将 H-NS 从 P 启动子上置换的假说。结果表明，H-NS 仅结合 P 中包含七肽重复的区域，而 ToxT 仅从其最靠近启动子的结合位点置换 H-NS，这要求对涉及 P 上 H-NS 和 ToxT 的当前模型进行修订。H-NS 是革兰氏阴性细菌中转录的全局负调控因子，特别是在水平获得的遗传岛中。霍乱弧菌的先前工作表明，H-NS 通过结合其启动子上游的大片段 DNA 来抑制霍乱毒素基因的转录，而毒力激活因子 ToxT 通过将 H-NS 从启动子上移除来解除转录抑制。在这里，新的数据支持一个修订后的模型，其中 ToxT 仅置换与 ToxT 结合位点重叠的最靠近启动子的 DNA 位点上结合的 H-NS，而上游位点仍被 H-NS 占据。这为控制霍乱毒素产生中 H-NS 的反阻遏作用引入了一种更高分辨率的机制。
