Roles of ATP Hydrolysis by FtsEX and Interaction with FtsA in Regulation of Septal Peptidoglycan Synthesis and Hydrolysis

Abstract

<jats:title>ABSTRACT</jats:title> <jats:p>In <jats:named-content content-type="genus-species">Escherichia coli</jats:named-content>, FtsEX coordinates peptidoglycan (PG) synthesis and hydrolysis at the septum. It acts on FtsA in the cytoplasm to promote recruitment of septal PG synthetases and recruits EnvC, an activator of septal PG hydrolases, in the periplasm. Following recruitment, ATP hydrolysis by FtsEX is thought to regulate both PG synthesis and hydrolysis, but how it does this is not well understood. Here, we show that an ATPase mutant of FtsEX blocks septal PG synthesis similarly to cephalexin, suggesting that ATP hydrolysis by FtsEX is required throughout septation. Using mutants that uncouple the roles of FtsEX in septal PG synthesis and hydrolysis, we find that recruitment of EnvC to the septum by FtsEX, but not ATP hydrolysis, is required to promote cell separation when the NlpD-mediated cell separation system is present. However, ATP hydrolysis by FtsEX becomes necessary for efficient cell separation when the NlpD system is inactivated, suggesting that the ATPase activity of FtsEX is required for optimal activity of EnvC. Importantly, under conditions that suppress the role of FtsEX in cell division, disruption of the FtsEX-FtsA interaction delays cell separation, highlighting the importance of this interaction in coupling the cell separation system with the septal PG synthetic complex.</jats:p> <jats:p><jats:bold>IMPORTANCE</jats:bold> Cytokinesis in Gram-negative bacteria requires coordinated invagination of the three layers of the cell envelope; otherwise, cells become sensitive to hydrophobic antibiotics and can even undergo cell lysis. <jats:italic>In</jats:italic> <jats:named-content content-type="genus-species">E. coli</jats:named-content>, the ABC transporter FtsEX couples the synthesis and hydrolysis of the stress-bearing peptidoglycan layer at the septum by interacting with FtsA and EnvC, respectively. ATP hydrolysis by FtsEX is critical for its function, but the reason why is not clear. Here, we find that in the absence of ATP hydrolysis, FtsEX blocks septal PG synthesis similarly to cephalexin. However, an FtsEX ATPase mutant, under conditions where it cannot block division, rescues <jats:italic>ftsEX</jats:italic> phenotypes as long as a partially redundant cell separation system is present. Furthermore, we find that the FtsEX-FtsA interaction is important for efficient cell separation.</jats:p>

Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes

“…In support of this, mutations in the ECL4 of RodA and in the pedestal domain of PBP2 activate the complex (14,16). Similarly, here we found that mutations in the ECL4 of FtsW can lock the protein in an inactive state (M269K) that no longer 22 responds to the activation of FtsN or in an active state (E289G and to a less extent M269I) that no longer requires FtsN for division. Mutations in the pedestal domain of FtsI can also activate (K211I) or inactivate (G57D, S61F, L62P and R210C) the FtsWI complex in vivo.…”

“…To prove that FtsW E289G and FtsI K211I were active division mutants, we tested if they suppressed other mutations that blocked septal PG synthesis. First, we tested the ATPase defective mutant of FtsEX, FtsE D162N X, which was previously shown to block septal PG synthesis and to be suppressed by FtsW M269I (19,22,32). As shown in Fig.…”

Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes

“…Although FtsI is sufficient for the PG glycosyltranferase activity of FtsW in vitro [3], this appears to be a basal activity and another layer of regulation exists in vivo. This layer includes FtsA, FtsEX, FtsQLB, and FtsN (Fig 1B ) with FtsN arriving last and triggering FtsWI activity [18][19][20][21][22][23][24][25]. FtsN is a bitopic membrane protein with a short N-terminal cytoplasmic domain (FtsN cyto ), a transmembrane segment, followed by an essential domain (FtsN E ) and a SPOR domain in the periplasm [26].…”

Coregulated assembly of actin-like FtsA polymers with FtsZ during Z-ring formation and division inEscherichia coli

“…Several proteins have been suggested to interact directly with FtsA to potentially modify the polymerization state, including ZipA, FtsN, and FtsEX [14,16,17,45,63]; all have been suggested to destabilize or bypass FtsA-FtsA interactions. Interestingly, FtsA is highly conserved indicating that the ATP hydrolysis and polymerization functions of FtsA have been evolutionarily preserved and are therefore critical for function [46].…”

“…We predict that other cell division proteins, such as ZipA or FtsEX may contribute to regulating the FtsA polymerization state [17,[43][44][45]. Once recruited by FtsA, FtsN would then activate FtsQBL and FtsWI for cell wall synthesis [18,45].…”