Patricia Rothe (Wernigerode / DE), Sabrina Wamp (Wernigerode / DE), Lisa Rosemeyer (Wernigerode / DE), Jeanine Rismondo (Göttingen / DE; London / GB), Jörg Döllinger (Berlin / DE), Sven Halbedel (Wernigerode / DE; Magdeburg / DE)
Listeria monocytogenes, a Gr+ bacterium, is characterised by a thick cell wall consisting mainly of peptidoglycan (PG). The cell wall counteracts the high internal turgor pressure and ensures viability. To fully understand PG biosynthesis and identify potential pharmaceutical targets, we are studying a regulatory pathway that controls PG production in L. monocytogenes and related Gr+ bacteria1,2. This pathway controls the proteolytic degradation of MurA, the enzyme that catalyses the first committed step of PG biosynthesis. MurA is degraded by the protease ClpCP in the presence of unphosphorylated ReoM. However, when ReoM is phosphorylated by the PASTA-domain containing protein serine/threonine kinase PrkA, the interaction of ReoM with MurA is inhibited and MurA degradation is prevented. The phosphorylation can be reversed by the phosphatase PrpC. In this way, PrkA and PrpC are involved in the decision whether PG biosynthesis is switched on or off.
We here have studied the phosphorylation profile of ReoM in living L. monocytogenes cells to clarify under which growth conditions ReoM is phosphorylated and thus PG biosynthesis activated, respectively.
Our results show that ReoM is fully phosphorylated under most tested laboratory growth conditions. However, we also found factors that prevented the phosphorylation of ReoM as, for example, ReoM phosphorylation was reduced in a mutant lacking the divisome gene gpsB. Artificial overexpression of MurA exerted a negative feedback on ReoM phosphorylation and this required a direct contact between ReoM and MurA, as well as MurA enzymatic activity and substrate binding.
These findings indicate that MurA and PrkA compete for ReoM as their shared interaction partner and suggest that the involved proteins constitute a safety valve mechanism ensuring controlled degradation of excess MurA to align the MurA amounts and the rate of PG biosynthesis with nutrient supply and growth.
1Wamp et al. (2020) Elife, 9, e56048
2Wamp et al. (2022) PLoS Pathogens, 18(3), e1010406