Control of bacterial peptidoglycan biosynthesis through protein phosphorylation and regulated proteolysis

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Peptidoglycan is the main component of the bacterial cell wall. It surrounds the cell as a three-dimensional network that withstands the internal turgor pressure and is important for cell shape maintenance. Biosynthesis, reorganization and degradation of peptidoglycan must be tightly coordinated with the size and shape changes that occur during cell growth and division in order to maintain the integrity of the bacterium. We have discovered a novel pathway for regulation of peptidoglycan biosynthesis in the pathogenic bacterium Listeria monocytogenes and this pathway is also conserved in other Gram-positive bacteria. According to our results, MurA, the first committed step enzyme of peptidoglycan biosynthesis, acts as a key control point for the regulation of the entire pathway. We have demonstrated that L. monoctyogenes MurA is degraded by the ClpCP protease and that this ClpCP-dependent degradation of MurA depends on the phosphorylation state of the small cytosolic protein ReoM. We also have shown that ReoM phosphorylation is PrkA-dependent. PrkA is a transmembrane serine/threonine protein kinase with extracellular PASTA domains and senses peptidoglycan fragments that accumulate during reorganization and degradation of peptidoglycan. Using this route, activation of PrkA and phosphorylation of ReoM influence the proteolytic stability of MurA and finally control peptidoglycan biosynthesis to adjust it to the current growth conditions and repair needs.Fundamental questions regarding the PrkA→ReoM→ClpCP→MurA signaling cascade should be answered in this project. Substrates of the L. monocytogenes ClpCP protease should be identified using proteomic approaches. The question to what degree ReoM and its two auxiliary factors ReoY and MurZ control degradation of ClpCP substrates should then be answered in subsequent analyses. Furthermore, a possible function of ReoM, ReoY and MurZ as potential ClpC adaptors should be tested in genetic and biochemical experiments. Different screening approaches should be used to identify novel determinants that are important for the proteolytic degradation of MurA. Furthermore, growth conditions leading to stimulation of ReoM phosphorylation in vivo should be identified. Finally, epistasis experiments should be used to investigate the role of the PrkA→ReoM→ClpCP→MurA signaling pathway in the intrinsic resistance of L. monocytogenes against cephalosporins. This project should lead to a deeper understanding of the regulation of peptidoglycan biosynthesis in Gram positive bacteria and their response to cell wall damaging conditions and antibiotics. The project should contribute to the development of new antimicrobial compounds through the identification of novel target structures.