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

Abstract

SEDS family peptidoglycan (PG) glycosyltransferases RodA and FtsW require their cognate transpeptidases PBP2 and FtsI (class B penicillin binding proteins) to synthesize PG along the cell cylinder and at the septum, respectively. The activities of these SEDS-bPBPs complexes are tightly regulated to ensure proper cell elongation and division. In Escherichia coli FtsN switches FtsA and FtsQLB to the active forms that synergize to stimulate FtsWI, but the exact mechanism is not well understood. Previously, we isolated an activation mutation in ftsW (M269I) that allows cell division with reduced FtsN function. To try and understand the basis for activation we isolated additional substitutions at this position and found that only the original substitution produced an active mutant whereas drastic changes resulted in an inactive mutant. In another approach we isolated suppressors of an inactive FtsL mutant and obtained FtsWE289G and FtsIK211I and found they bypassed FtsN. Epistatic analysis of these mutations and others confirmed that the FtsN-triggered activation signal goes from FtsQLB to FtsI to FtsW. Mapping these mutations and others affecting the activities of FtsWI on the RodA-PBP2 structure revealed they are located at the interaction interface between the extracellular loop 4 (ECL4) of FtsW and the pedestal domain of FtsI (PBP3). This supports a model in which the interaction between the ECL4 of SEDS proteins and the pedestal domain of their cognate bPBPs plays a critical role in the activation mechanism.Author summaryBacterial cell division requires the synthesis of septal peptidoglycan by the widely conserved SEDS-bPBP protein complex FtsWI, but how the complex is activated during cell division is still poorly understood. Previous studies suggest that FtsN initiates a signaling cascade in the periplasm to activate FtsW. Here we isolated and characterized activated FtsW and FtsI mutants and confirmed that the signaling cascade for FtsW activation goes from FtsN to FtsQLB to FtsI and then to FtsW. The residues corresponding to mutations affecting FtsWI activation are clustered to a small region of the interaction interface between the pedestal domain of FtsI and the extracellular loop 4 of FtsW, suggesting that this interaction mediates activation of FtsW. This is strikingly similar to the proposed activation mechanism for the RodA-PBP2 complex, another SEDS-bPBP complex required for cell elongation. Thus, the two homologous SEDS-bPBP complexes are activated similarly by completely unrelated activators that modulate the interaction interface between the SEDS proteins and the bPBPs.

Integrative structural biology of the penicillin-binding protein-1 from Staphylococcus aureus, an essential component of the divisome machinery

“…The equilibrium between the conformations was suggested to be regulated by MreC (or other proteins) of the elongasome complex [62] . This same regulation was proposed recently for the bPBP:SEDS complex of the divisome, despite the very different regulatory proteins of the divisome compared to the elongasome [77] . We explored this hypothesis by modeling the sa PBP1: sa FtsW complex of S. aureus , using the crystal structure of the T. thermophilus PBP2:RodA (PDB code 6PL6) as the template, in a lipid bilayer membrane ( Fig.…”

FtsN activates septal cell wall synthesis by forming a processive complex with the septum-specific peptidoglycan synthase in E. coli

“…Under these two conditions, nearly all FtsW molecules are engaged in sPG synthesis and hence exhibit only one slow-moving, active population on the sPG-track, identical to that of FtsN. Moreover, tracking of various mutant derivatives of FtsN revealed that the only domain required for the processive complex formation on the sPG-track is the Essential (E) domain, which is proposed to interact with the sPG synthesis machinery FtsWI, likely via the FtsQLB complex 15,16,18,19 . Most importantly, such a complex is crucial for activating and sustaining sPG synthesis in a processive manner, as a double point mutation that inactivates the E domain (WYAA) prevents formation of the processive complex and causes failure of cell division.…”

“…Other noteworthy divisome proteins include FtsA, which links FtsZ polymers to the membrane 12 , the core septal PG (sPG) synthase complex composed of the polymerase FtsW and transpeptidase FtsI 13,14 , and the FtsQLB complex, which regulates FtsWI activity 15,16 (also see reviews [5][6][7] ). According to current models, FtsN acts through FtsA in the cytoplasm and the FtsQLB complex in the periplasm to activate synthesis of sPG by the FtsWI synthase complex [15][16][17][18][19][20][21] .…”

The coiled-coil domain ofE. coliFtsLB is a structurally detuned element critical for modulating its activation in bacterial cell division

“…In current models, FtsN-mediated activation of FtsWI occurs through the FtsLB complex 28,29,31 . FtsLB is a heterotetramer consisting of two FtsL and two FtsB subunits, both of which are single-pass membrane proteins.…”