How do hybrid two-component systems in gut bacteria regulate global gene expression?

拟杆菌是健康人类和养殖动物肠道微生物群的关键成员。它们降解来自宿主、饮食和微生物来源的各种复杂多糖的能力是它们在人类肠道中无处不在的定植的关键。这些细菌面临的挑战是感知并快速响应肠道竞争环境中持续的营养波动。它们通过称为多糖利用位点（普尔斯）的位点靶向不同的聚糖。通常，每个PUL靶向不同的聚糖，并编码细胞表面酶、结合蛋白和转运蛋白。在将部分降解的寡糖导入周质后，特定的寡糖提示物被称为杂交双组分系统（HTCS）的调节蛋白结合并识别。HTCS包含典型细菌双组分系统的所有结构域（传感器结构域、组氨酸激酶、受体结构域和DNA结合结构域），但在单个跨膜蛋白中。通过结合寡糖激活HTCS然后导致PUL表达快速上调2 - 3个数量级。这种反应使细菌能够迅速输入和降解可用的聚糖。先前的数据表明，每个调节器都能感知不同的寡糖，从而使细菌能够区分目标聚糖的连接和单糖含量。有趣的是，DNA结合结构域在激活时不会裂解到胞质溶胶中，这表明它们的DNA靶必须非常接近膜。该项目将研究这些HTCS如何能够直接调节膜上的基因表达，使用生物化学，细菌遗传学和全局转录分析（ChIP-Seq和RNA-Seq）的组合。该项目将使我们能够了解这些不寻常的调节剂能够指导碳水化合物活性酶快速表达的机制，以响应肠道微生物组中的营养波动。这为合理改造这些菌株以释放特定分子或酶来响应饮食干预铺平了道路，增强了它们的益生菌特性。该项目符合BBSRC的农业和食品安全战略目标，特别是食品安全和营养重点，其中包括食品/饲料和微生物组之间的机械相互作用。拟杆菌属细菌是人类和动物肠道中复杂碳水化合物的主要降解者，了解它们如何能够识别并特异性地响应某些碳水化合物对于了解它们在肠道中的成功至关重要。这些生物体的遗传易处理性意味着，一旦我们了解它们如何在分子水平上调节它们对碳水化合物的反应，我们就可以开始将它们作为潜在的益生菌菌株来操纵，例如，通过特定的膳食纤维/补充剂调节酶活性或分泌。

The Bacteroides are key members of the healthy human and farmed-animal gut microbiota. Their ability to degrade a huge variety of complex polysaccharides from host, dietary and microbial sources is key to their ubiquitous colonisation of the human gut. The challenge facing these bacteria is sensing and rapidly responding to the constant nutrient fluctuations in the competitive, environment of the gut. They target different glycans via loci known as polysaccharide utilisation loci (PULs). Typically, each PUL is targeted towards a different glycan, and encodes cell surface enzymes, binding proteins and transporters. Upon import of partially degraded oligosaccharides into the periplasm, a specific oligosaccharide cue is bound and recognised by a regulator protein known as a hybrid two-component system (HTCS). HTCS comprise all the domains of a typical bacterial two-component system (sensor domain, histidine kinase, acceptor domain and DNA binding domain), but in a single transmembrane protein. The activation of an HTCS by binding oligosaccharide then leads to rapid upregulation of PUL expression by 2-3 orders of magnitude. This response enables the bacterium to import and degrade the available glycan rapidly. Previous data shows that each regulator senses a different oligosaccharide, allowing the bacterium to distinguish between linkage and monosaccharide content of a target glycan. Intriguingly, the DNA binding domains are not cleaved into the cytosol upon activation, suggesting their DNA targets must be in close proximity to the membrane. This project will investigate how these HTCS are able to directly regulate gene expression at the membrane, using a combination of biochemistry, bacterial genetics, and global transcriptional analysis (ChIP-Seq and RNA-Seq). This project will enable us to understand the mechanism by which these unusual regulators are able to direct the rapid expression of carbohydrate active enzymes, in response to nutrient fluctuations in gut microbiomes. This paves the way for rational engineering of these strains to release specific molecules or enzymes in response to dietary interventions, enhancing their probiotic properties.This project fits within the agriculture and food security strategic aim of the BBSRC, specifically the food safety and nutrition focus, which includes mechanistic interactions between food/feed and the microbiome. Bacteroidetes bacteria are the dominant degraders of complex carbohydrates in human and animals guts, and understanding how they are able to recognise and specifically respond to certain carbohydrates is critical to understanding their success in colonising the gut. The genetic tractability of these organisms means that once we understand how they regulate their response to carbohydrates at the molecular level, we can begin to manipulate them as potential probiotic strains with e.g. enzymatic activity or secretion regulated by specific dietary fibre/supplements.