Midcell localization of PBP4 ofEscherichia colimodulates the timing of divisome assembly

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. Domain 3 is also needed for the interaction of PBP4 with NlpI, but not for its interactions with PBP1A or LpoA. 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 arrival of FtsN and other cell division proteins at midcell was significantly delayed in cells lacking PBP4.

PBP4 Is Likely Involved in Cell Division of the Longitudinally Dividing Bacterium Candidatus Thiosymbion Oneisti

“…To investigate the PBP4 TO localization, we fixed Ca . T. oneisti cells and immunolabeled them with a PBP4 EC antibody [ 19 ]. The antibody specificity and recognition of PBP4 TO were verified by Western blot in Ca .…”

“…Some PBPs, from all classes, have been found to form dimers both in vivo and vitro [ 21 , 42 , 43 ]. The PBP4 EC forms dimers as isolated protein and its domain 3 is not essential for this dimerization [ 19 ]. PBP4 TO and PBP4 TO S69A also appears to form dimers and higher homo-oligomeric complexes in native PAGE gel ( Figure 5 d).…”

“…The PBP4 EC localizes in between PG layer and outer membrane. Domain III of PBP4 EC is essential for its midcell localization during cell division and for the interaction with the outer membrane lipoprotein NlpI [ 19 , 61 ]. PBP4 TO has a very small domain III, which likely does not interact with the partners of PBP4 EC .…”

“…Analysis of a set of mutants with multiple class C PBPs deleted demonstrates that the loss of PBP4 in a PBP5/PBP6 double mutant background induces severe shape deficiencies [ 18 ]. PBP4 localizes specifically at midcell during septation to participate in septal PG synthesis and its absence (Δ dacB strain) affects the timing of the divisome assembly [ 19 ]. The overexpression of PBP4 is toxic and leads to reduced numbers of PG crosslinks and pentapeptides side chains in vitro and in vivo [ 20 ].…”

Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes inEscherichia coli

“…This is not supported by the analyses of PG recycling in conditions of selective inhibition of PBP3 by aztreonam (Uehara and Park, 2008). The endopeptidases involved in septum formation have not been identified, except for a contribution of PBP4, which has an effect on the timing of septation (Verheul et al, 2020). MepK is a candidate for this function in 3→3 cross-linked PG although this is currently not supported by any experimental evidence and it remains to be seen if endopeptidases are needed for septum PG synthesis.…”