Abstract
ABSTRACTCell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The synthesis or activity of several key enzymes along the PG biosynthetic pathway is controlled by the Hanks-type serine/threonine kinases (STKs). In Gram-positive bacteria, inactivation of genes encoding STKs is associated with a range of phenotypes, including cell division defects and changes in cell wall metabolism, but only a few kinase substrates and associated mechanisms have been identified. We previously demonstrated that STK-PrkC plays an important role in cell division, cell wall metabolism, and resistance to antimicrobial compounds in the human enteropathogen Clostridioides difficile. In this work, we characterized a PG hydrolase, CwlA, which belongs to the NlpC/P60 family of endopeptidases and hydrolyses cross-linked PG between daughter cells to allow cell separation. We identified CwlA as the first PrkC substrate in C. difficile. We demonstrated that PrkC-dependent phosphorylation inhibits CwlA export, thereby controlling hydrolytic activity in the cell wall. High levels of CwlA at the cell surface led to cell elongation, whereas low levels caused cell separation defects. Thus, we provided evidence that the STK signaling pathway regulates PGH homeostasis to precisely control PG hydrolysis during cell division.
Highlights
Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG)
Typical regulation occurs through a signal, such as iron concentration, that regulates the transcription of the autolysin-encoding gene isdP in Staphylococcus lugdunensis [7] or through twocomponent systems, such as WalK-WalR, that monitor the expression of the lytE and cwlO genes encoding endopeptidases in Bacillus subtilis by sensing and responding to their cleavage products [8]
We found that CwlA is phosphorylated by PrkC and demonstrated that PrkC-dependent phosphorylation controls the export of CwlA required for cytokinesis
Summary
Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). In Gram-positive bacteria, inactivation of genes encoding STKs is associated with a range of phenotypes, including cell division defects and changes in cell wall metabolism, but only a few kinase substrates and associated mechanisms have been identified. We previously demonstrated that STK-PrkC plays an important role in cell division, cell wall metabolism, and resistance to antimicrobial compounds in the human enteropathogen Clostridioides difficile. We demonstrated that PrkC-dependent phosphorylation inhibits CwlA export, thereby controlling hydrolytic activity in the cell wall. PrkC-dependent phosphorylation controls the export of CwlA, modulating its levels and, its activity in the cell wall. We found that CwlA is phosphorylated by PrkC and demonstrated that PrkC-dependent phosphorylation controls the export of CwlA required for cytokinesis This represents a novel and original mechanism for CW hydrolysis regulation by STK phosphorylation
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