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bioRxiv · 10.1101/2020.07.30.230052

Midcell localization of PBP4 of Escherichia coli is essential for the timing of divisome assembly

Verheul, J.·Lodge, A.·Yau, H.·Liu, X.·Solovyova, A. S.·Typas, A.·Banzhaf, M.·Vollmer, W.·den Blaauwen, T.
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Abstract

Insertion of new material into the Escherichia coli peptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and hydrolytic activities to maintain cell shape and avoid lysis. The hydrolytic enzymes outnumber the enzymes that insert new PG by far and very little is known about their specific function. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Microscale thermophoresis with isolated proteins shows that domain 3 is needed for the interaction with NlpI, but not PBP1A or LpoA. In vivo crosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that PBP4 functions together with the amidases AmiA and B to create denuded glycan strands to attract the initiator of septal PG synthesis, FtsN. Consistent with this model, we found that the divisome assembly at midcell was significantly affected in cells lacking PBP4. IMPORTANCEPeptidoglycan biosynthesis is a major target for antibacterials. The covalently closed peptidoglycan mesh, called sacculus, protects the bacterium from lysis due to its turgor. Sacculus growth is facilitated by the balanced activities of synthases and hydrolases, and disturbing this balance leads to cell lysis and bacterial death. Because of the large number and possible redundant functions of peptidoglycan hydrolases, it has been difficult to decipher their individual functions. In this paper we show that the DD-endopeptidase PBP4 localizes at midcell during septal peptidoglycan synthesis in Escherichia coli and is important for the timing of the assembly of the division machinery. This shows that inhibition of certain hydrolases could weaken the cells and might enhance antibiotic action.

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Verheul, J., Lodge, A., Yau, H., Liu, X., Solovyova, A. S., Typas, A., Banzhaf, M., Vollmer, W., den Blaauwen, T.. 2020-07-31. Midcell localization of PBP4 of Escherichia coli is essential for the timing of divisome assembly. https://doi.org/10.1101/2020.07.30.230052

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