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, as well as others affecting the activity 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.

Identification of the potential active site of the septal peptidoglycan polymerase FtsW

“…For FtsW to synthesize septal PG, it must bind its substrate lipid II and polymerize the disaccharide pentapeptide into glycan strands. In addition, previous studies showed that FtsW must bind FtsI as it is activated by a pathway that operates through its cognate bPBP (PBP3/FtsI) [30][31][32]. Thus, there are at least three possibilities for the observed defect in septal PG synthesis: inability to bind lipid II, a defect in catalysis or a defect in the activation step.…”

“…Accumulated evidence suggests that the activity of the SEDS-bPBP complexes in vivo is regulated by an activation pathway that goes through bPBP to stimulate the PG polymerase activity of its cognate SEDS protein [10,11,[30][31][32]. In the elongasome, the activity of RodA-PBP2 is believed to be regulated by MreC and MreD [11,33], two critical components of the elongasome.…”

“…Consistent with this model, purified FtsQLB activates the polymerase activity of the FtsW-FtsI complex in vitro with FtsL playing a key role [ 31 ], likely through an interaction with FtsI [ 30 ]. Moreover, activating mutations in FtsW or FtsI can bypass the entire activation pathway initiated by FtsN [ 32 ]. Strikingly, most of the activating mutations occur in the pedestal domain of FtsI or the ELC4 of FtsW [ 32 , 40 , 41 ].…”

“…Moreover, activating mutations in FtsW or FtsI can bypass the entire activation pathway initiated by FtsN [ 32 ]. Strikingly, most of the activating mutations occur in the pedestal domain of FtsI or the ELC4 of FtsW [ 32 , 40 , 41 ]. On the other hand, other mutations in these regions block the activity of FtsW-FtsI, presumably by disrupting the interaction [ 30 , 32 ].…”

The coiled-coil domain of Escherichia coli FtsLB is a structurally detuned element critical for modulating its activation in bacterial cell division

“…In current models, activation begins with the midcell localization of FtsN ( 18 , 19 , 20 ) ( Fig. 1 C ), which communicates with FtsWI through a cytoplasmic route involving FtsA ( 21 , 22 , 23 ) and through a periplasmic route involving the FtsLB complex ( 21 , 24 , 25 , 26 , 27 ). In this work, we focus on the latter periplasmic activation route.…”