Characterization of an anaerobic Escherichia coli K-12 cyclic-di-GMP phosphodiesterase

细菌是地球上适应能力最强的生物。它们适应不同条件的能力的根源是为特定环境选择和表达最佳基因组合的能力。这通常是由被称为转录因子的蛋白质实现的，这些蛋白质感知信号分子，然后作用于打开或关闭特定的基因。有时转录因子并不直接感知信号，而是与充当信使的小分子相互作用。在这些情况下，信号对新陈代谢过程产生影响，从而改变细菌细胞中信使分子的浓度，然后被转录因子感知。最近，在许多细菌中发现了一种称为环-二-GMP的信使分子。环-二-GMP信号与细菌的许多重要性质有关，包括移动和形成生物膜(附着在表面的细菌群落)的能力。然而，尽管循环-二-GMP信号通路广泛存在，但人们对它们的工作方式知之甚少。在这个项目中，我们想要更多地了解一种名为YfgF的蛋白质。我们以前的工作告诉我们，YfgF是由大肠杆菌在缺氧时产生的。我们还发现YfgF具有分解环-二-GMP的能力，因此应该会对该分子控制的过程产生影响，例如运动性和生物膜的形成。由于人们对环-di-GMP信号通路知之甚少，我们想利用YfgF，在已知大肠杆菌作为模式细菌的大量信息的支持下，更多地了解环-di-GMP如何改变基因表达模式。我们的方法将是分离YfgF蛋白，并表征其催化活性以及这些活性在不同条件下的变化。我们还将发现哪些基因受到YfgF的存在或不存在的影响，然后尝试填补所感知的信号、YfgF活性的变化和受调控的基因之间缺失的环节。通过这种方式，我们将更多地了解YfgF是如何工作的，也会了解到一些关于其他与环-双GMP相互作用的蛋白质如何工作的经验教训。这可能具有重要的意义，因为细菌是因为许多感染与缺氧的生物膜有关。

Bacteria are the most adaptable organisms on the planet. At the root of their ability to adapt to different conditions is the ability to select and express optimal combinations of genes for particular environments. This is generally achieved by proteins known as transcription factors which sense signal molecules and then act to turn specific genes on or off. Sometimes the transcription factor does not sense the signal directly but rather interacts with a small molecule that acts as a messenger. In these cases the signal has an effect on a metabolic process that changes the concentration of the messenger molecule in the bacterial cell, which is then sensed by the transcription factor. Recently a messenger molecule called cyclic-di-GMP has been identified in many bacteria. Cyclic-di-GMP signalling is associated with many important properties of bacteria, including the ability to move and the ability to form biofilms (communities of bacteria attached to surfaces). However, despite the widespread occurrence of cyclic-di-GMP signalling pathways little is known about the way they work. In this project we want to learn more about a protein called YfgF. Our previous work has shown us that YfgF is made by the bacterium Escherichia coli when it is starved of oxygen. We have also discovered that YfgF has the ability to breakdown cyclic-di-GMP and so should have an effect on processes controlled by this molecule, e.g. motility and biofilm formation. Because so little is known about cyclic-di-GMP signalling pathways we want to use YfgF, supported by the enormous amount of information that is known about E. coli as a model bacterium, to learn more about how cyclic-di-GMP alters patterns of gene expression. Our approach will be to isolate the YfgF protein and characterise its catalytic activities and how these change under different conditions. We will also discover which genes are influenced by the presence or absence of YfgF and then attempt to fill in the missing links between the signal perceived, the change in YfgF activity and the genes that are regulated. In this way we will learn more about how YfgF works and also learn some general lessons on how other proteins that interact with cyclic-di-GMP work. This could have important implications because bacteria because many infections are associated with oxygen-starved biofilms.