Breaking the walls to wake-up bacterial cells

细菌可以通过减缓它们的主要生命过程来在恶劣的环境条件下生存下来。它们看起来像正常细胞，但在被称为复苏促进因子(RPFS)的蛋白质刺激之前，行为与死亡细胞相似。RPFS不断被正常生长的细菌分离，在细胞膜的形成中起着重要的作用。然而，睡眠或休眠的细菌不会产生rPFS。这个项目的主要目标是找出rPFS是如何唤醒睡眠细胞的。RPFS是一种消化细菌细胞壁的酶(如溶菌酶)，但与后者不同的是，它们不会杀死细胞，也不会以某种方式改变它们的包膜。RPFS可以简单地打破沉睡细胞的僵硬细胞壁，使它们自由生长和扩张。在更复杂的模型中，rPFS在消化过程中释放的细胞壁片段具有非常特殊的信号分子功能。换句话说，它们是休眠细胞的唤醒消息。这些“信号”与细菌表面的其他分子结合，启动了一个称为蛋白激酶级联的复杂系统，这是一种在多细胞生物体中很好地描述的细胞调节机制。为了解决这两种假设，研究人员将分离细菌包膜的主要部分，称为murein，并用不同的rPFS消化它。所产生的产品将用于休眠细胞的复苏，并将对其组成进行分析和表征。其他实验将被设计来确定rPFS在murein中切割的是什么键，以及这种切割如何影响细菌细胞包膜的结构。在该项目的不同部分，研究人员将试图了解细菌细胞是如何控制酶的，这些酶能够通过‘吃掉’细菌包膜来摧毁它们。RPFS蛋白将被用作这种细胞壁降解酶的一个例子。该项目的成果不仅满足了科学家对细菌细胞功能的好奇心，而且为控制细菌的生理状态，进一步提高其在生物技术、医学中的应用，特别是对抗由休眠细菌引起的持续感染提供了新的方法。

Bacteria can survive harsh environmental conditions by slowing down their major living processes. They look like normal cells but behave like the dead ones until stimulated by proteins called the Resuscitation-promoting factors (Rpfs). Rpfs are constantly sectereted by normal growing bacteria and play an important role in making of cell envelope. However, sleeping or dormant bacteria do not produce the Rpfs. The main goal of this project is to find out how Rpfs wake-up sleeping cells. Rpfs are enzymes which digest bacterial cell wall (like lyzosyme) but unlike the latter they don't kill cells and somehow modify their envelope. Rpfs may simply break quite inflexible cell wall of sleeping cells and make them 'free' to grow and expand. In more sophisticated model fragments of cell wall, released by Rpfs during digestion, have a very special function as signalling molecules. In other words they are waking-up messages for dormant cells. These 'signals' bind to other molecules on the bacterial surface and initiate a complex system called a protein kinase cascade, a well described mechanisms of cell regulation in multicellular organisms. To address both hypotheses the investigator will isolate the major part of bacterial envelope, called murein and digest it with different Rpfs. The resulting products will be used for resuscitation of dormant cells and their composition will be analysed and characterised. Other experiments will be designed to establish what bonds the Rpfs cut in murein and how this cleavage influences the structure of bacterial cell envelope. In separate part of the project the investigators will attempt to understand how bacterial cells control the enzymes which able to destroy them by 'eating' bacterial envelope. Rpfs proteins will be applied as an example of such cell-wall degrading enzymes. The results of the project will not only satisfy scientific curiosity on bacterial cell function but also provide novel methods for manipulating of physiological condition of bacteria and further improving their application in biotechnology, medicine and specifically for combating of persisting infections caused by dormant bacteria.