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

类杆菌是健康的人和养殖动物肠道微生物区系的关键成员。它们能够从宿主、饮食和微生物来源降解大量复杂的多糖，这是它们无处不在地在人类肠道中定居的关键。这些细菌面临的挑战是感知并快速应对肠道竞争环境中持续不断的营养波动。它们通过被称为多糖利用基因座(Puls)的基因定位于不同的糖链。通常，每个PUL针对不同的糖链，编码细胞表面的酶、结合蛋白和转运蛋白。当部分降解的寡糖输入周质时，特定的寡糖线索被一种称为混合双组分系统(HTCS)的调节蛋白结合并识别。HTCs包括典型细菌双组分系统的所有结构域(感受器结构域、组氨酸激酶结构域、受体结构域和DNA结合域)，但存在于单一的跨膜蛋白中。通过结合寡糖激活HTCs，导致PUL表达迅速上调2-3个数量级。这种反应使细菌能够输入并迅速降解可利用的多糖。先前的数据显示，每个调节子感知不同的低聚糖，使细菌能够区分目标多糖的连接和单糖含量。有趣的是，DNA结合域在激活时不会切割到胞浆中，这表明它们的DNA靶标必须非常接近细胞膜。这个项目将结合生物化学、细菌遗传学和全球转录分析(ChIP-Seq和RNA-Seq)来研究这些HTC如何能够直接调控膜上的基因表达。这个项目将使我们能够了解这些不寻常的调节器能够指导碳水化合物活性酶快速表达的机制，以响应肠道微生物群中的营养波动。这为合理设计这些菌株以释放特定分子或酶以响应饮食干预铺平了道路。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/饲料和微生物群之间的机械相互作用。拟杆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某些碳水化合物并对其做出特定反应，对于了解它们在肠道中的成功定植至关重要。这些生物的遗传易操纵性意味着，一旦我们在分子水平上了解它们如何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，酶活性或分泌受特定膳食纤维/补充剂调控。

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.