Breaking the walls to wake-up bacterial cells

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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.