FtsA acts through FtsW to promote cell wall synthesis during cell division in Escherichia coli

Abstract

In Escherichia coli, FtsQLB is required to recruit the essential septal peptidoglycan (sPG) synthase FtsWI to FtsA, which tethers FtsZ filaments to the membrane. The arrival of FtsN switches FtsQLB in the periplasm and FtsA in the cytoplasm from a recruitment role to active forms that synergize to activate FtsWI. Genetic evidence indicates that the active form of FtsQLB has an altered conformation with an exposed domain of FtsL that acts on FtsI to activate FtsW. However, how FtsA contributes to the activation of FtsW is not clear, as it could promote the conformational change in FtsQLB or act directly on FtsW. Here, we show that the overexpression of an activated FtsA (FtsA*) bypasses FtsQ, indicating it can compensate for FtsQ's recruitment function. Consistent with this, FtsA* also rescued FtsL and FtsB mutants deficient in FtsW recruitment. FtsA* also rescued an FtsL mutant unable to deliver the periplasmic signal from FtsN, consistent with FtsA* acting on FtsW. In support of this, an FtsW mutant was isolated that was rescued by an activated FtsQLB but not by FtsA*, indicating it was specifically defective in activation by FtsA. Our results suggest that in response to FtsN, the active form of FtsA acts on FtsW in the cytoplasm and synergizes with the active form of FtsQLB acting on FtsI in the periplasm to activate FtsWI to carry out sPG synthesis.

Keywords: FtsA; FtsQ; FtsW; cytokinesis; septal PG.

Fig. 1.

Recruitment and activation of FtsWI in E. coli. (A) The hierarchical divisome assembly pathway and activation mechanism. The FtsQLB complex lies between FtsK and FtsWI in the pathway. FtsWI is recruited to the Z-ring in an FtsQ-dependent manner with the cytoplasmic domain of FtsL being required to recruit FtsWI. The arrival of FtsN (E domain in yellow and cyto domain in red) switches FtsA in the cytoplasm and FtsQLB in the periplasm to the “on” state, which synergize to activate FtsWI to carry out sPG synthesis. Part of this activation is due to a conformational change in FtsQLB, which exposes a domain of FtsL (AWI) that interacts with FtsI that affects FtsW. The work presented in this study indicates that FtsA* acts directly on FtsW. Note that some cell division proteins are not depicted, including ZipA and FtsEX. Also, the deletion of FtsQ, loss of FtsL^cyto or disruption of the FtsB–FtsQ interaction prevents the recruitment of FtsWI. (B) Bypassing ftsQ with the FtsW–FtsK^cyto fusion and FtsA*. The FtsW–FtsK^cyto fusion was used to bypass the requirement for FtsQ for the recruitment to the Z-ring. This fusion bypasses ftsQ, provided it carries an ftsW* mutation and ftsA* is present. It is likely that FtsL and FtsB are back recruited by the FtsWI complex since they cannot be deleted. (C) Bypass of ftsQ by overexpression of ftsA* in the presence of ftsW*. The results presented herein indicate that, in the absence of FtsQ, FtsA* rescues the recruitment of FtsW by interacting with it in the cytoplasm. FtsA* can also activate FtsWI in the absence of the signal from FtsL^AWI.

Fig. 2.

Deletion of ftsQ in the presence of ftsW*–ftsK^cyto and ftsA*. P1 grown on JOE417 (ftsQ14::kan/pBAD33-ftsQ) was used to transduce W3110 to kanamycin resistance. The strain carried plasmids that constitutively expressed ftsQ (pSEB468) or ftsW*–ftsK^cyto (pND16*) and either ftsA* inducible with IPTG (pSEB306*[P₂₀₆::ftsA*]) or a vector. Transductants were selected at 30 °C on plates containing 200 µM IPTG. Colonies from the Top and Bottom were purified and then grown in liquid medium of the same composition. The strain containing plasmids expressing ftsW*–ftsK^cyto and ftsA* was centrifuged, washed, and resuspended in LB with or without IPTG. Samples were taken 2 h later for photography. Colonies also arose on plates expressing just ftsW*–ftsK^cyto but grew slower and were of variable size. Colonies also arose on plates expressing just ftsA*, but cells displayed elongated and chaining phenotypes (SI Appendix, Fig. S3). Upon restreaking, growth of these colonies was poor, and when they were cultured in LB broth, cells were filamentous and not studied further. Note that sporadic colonies appeared on plates of the strain with just the vector plasmids, suggesting they arose because of suppressor mutations.

Fig. 3.

FtsA* and FtsW* synergize to bypass ftsQ. P1 grown on JOE417 (ftsQ14::kan/pBAD33-ftsQ) was used to transduce SD247 (ftsW*) to kanamycin resistance. The strain also contained plasmids (pSEB306 [P₂₀₆::ftsA] or pSEB306* [P₂₀₆::ftsA*]) containing ftsA alleles inducible with IPTG. Kanamycin-resistant transductants were selected at 30 °C in the presence of 200 µM IPTG (to induce ftsA or ftsA*). Transductants obtained from the strain containing ftsA* were restreaked under the same conditions. Colonies were grown in LB in the presence of 200 µM IPTG, centrifuged, and grown with or without IPTG. Samples were taken for photography, as described in the legend to Fig. 2. At least 25 µM IPTG was required to have a nonfilamentous morphology.

Fig. 4.

Overexpression of ftsA* or ftsW* but not ftsN rescues an ftsL allele deficient in activating ftsW. SD399 (ftsL::kan/pSC101^ts-ftsL) containing pSD296-2 (P_ara::ftsL^L86F/E87K) and pSEB417 (P₂₀₄::ftsN), pSEB306* (P₂₀₆::ftsA*), pSEB429 (P₂₀₄::ftsW), pSEB429-1 (P₂₀₄::ftsW*), or pKTP100 (P_tac::ftsL) was spotted at 37 °C to deplete WT ftsL. Arabinose was added to induce ftsL^L86F/E87K, and IPTG was added to induce ftsL, ftsN, ftsA, ftsA*, ftsW, or ftsW*. The control plate lacked arabinose, whereas the test plates contained 0.2% arabinose to induce ftsL^L86F/E87K and increasing concentrations of IPTG to induce the other genes. Note that the basal expression of ftsL from pKTP100 is sufficient for complementation in the absence of IPTG.

Fig. 5.

FtsA* and FtsW* synergize to bypass ftsN and ftsEX. (A) Bypass of ftsN. To examine conditions for bypassing ftsN, P1 prepared on CH34 (ftsN::kan/pCH201 [P_lac:: gfp-ftsN]) was used to transduce SD247 (ftsW*) containing plasmids pSEB306 (P₂₀₆::ftsA), pSEB306* (P₂₀₆::ftsA*), or pSEB417 (P₂₀₄::ftsN) to kanamycin resistance. Transductants were selected at 30 °C on LB agar in the presence of 200 µM IPTG. A transductant growing exponentially in LB with 200 μM IPTG was centrifuged and resuspended in LB with or without 200 µM IPTG, and samples were taken for photography, as described in the legend to Fig. 2. (B) Bypass of ftsN and ftsEX. To see if ftsN and ftsEX could be bypassed simultaneously, a kanamycin-resistant transductant from part A, PK7 (ftsW* ftsN::kan/p306*[P₂₀₆::ftsA*]), was transduced to chloramphenicol resistance with P1 grown on SD205 (ftsEX::cat). A transductant was grown for photography, as described in the legend to Fig. 2.

Fig. 6.

Overexpression of ftsA* rescues an ftsB allele that disrupts the hierarchical recruitment pathway. To see if FtsA* could rescue a strain in which the hierarchical recruitment pathway was disrupted (because of the lack of FtsQ–FtsB interaction), P1 phage prepared on SD368 (ftsB::kan/pSD255 [pSC101^ts/P_syn135::ftsB]) was used to transduce SD247 (ftsW*) containing plasmids expressing ftsB^1–54 (pSD295-54/P_ara::ftsB^1–54) and ftsA* (pSEB306*/P₂₀₆::ftsA*) to kanamycin resistance. FtsB^1–54 is a C-terminal–truncated FtsB mutant that interacts with FtsL but not FtsQ and cannot support the hierarchical pathway of divisome assembly. Transductants were selected on kanamycin plates with 0.2% arabinose (to induce ftsB^1–54) and 200 µM IPTG (to induce ftsA*). No transductants were obtained with SD247 (ftsW*) expressing ftsB^1–54 or ftsB^1–54 and pSEB306 (P₂₀₆::ftsA).

Fig. 7.

Overexpression of ftsA* rescues an ftsL allele deficient in recruitment FtsW. To investigate whether FtsA* recruits FtsW, we tested whether the overexpression of ftsA* rescues ftsL^Δcyto, which cannot recruit FtsW. To do this, P1 prepared on SD439 (ftsL::kan/pSD296 [P_ara::ftsL]) was used to transduce W3110 containing plasmid pKTP107 (P_ara::ftsL^Δcyto]) or pSEB306* (P₂₀₆::ftsA*) inducible with IPTG to kanamycin resistance. For a control, W3110 contained pKTP100 (P_tac::ftsL), which expresses ftsL at a high level to overcome the dominant-negative effect caused by ftsL^Δcyto. Kanamycin-resistant transductants were selected at 30 °C on plates containing 0.2% arabinose, 200 µM IPTG, chloramphenicol, and ampicillin. Transductants were grown in LB with 200 µM IPTG and photographed, as in Fig. 2.

Fig. 8.

The effect of hyperactive periplasmic and cytoplasmic signals on the rescue of FtsW cytoplasmic mutants. (A) Diagram indicating FtsW residues targeted for site-directed mutagenesis. Conserved residues in cytoplasmic loops 2 and 4 of FtsW were altered by site-directed mutagenesis and tested for loss of function and dominant-negative phenotypes (SI Appendix, Fig. S11). (B) Four FtsW loss-of-function mutants that are also dominant negative were tested to see if they could be rescued by the expression of ftsL** (hyperactive periplasmic signal, Top) or ftsA* (hyperactive cytoplasmic signal, Bottom). To do this, SD295 (ftsW::kan/pSD257 [repA^TS ftsW]) was transformed with plasmids carrying various ftsW alleles under the arabinose promoter control (pDSW406/P_ara::ftsW) along with plasmids expressing ftsA* (pSEB306*[P₂₀₆::ftsA*]) or ftsL^E88K/G92D (pKTP100* [P_tac::ftsL^E88K/G92D]) under IPTG control. Plates were incubated at 37 °C to deplete WT ftsW, and 0.2% arabinose (to induce ftsW alleles) and IPTG (to induced ftsA* or ftsL**) were added.

Fig. 9.

An ftsW activation mutation (ftsW*) is an intragenic suppressor of ftsW^R172D. To test if ftsW* was an intragenic suppressor of ftsW^R172D, we used P1 transduction. P1 grown on EC912 (ftsW::kan/pDSW406 [P_ara::ftsW]) was used to transduce W3110 to kanamycin resistance. W3110 contained derivatives of pSEB439 (P₂₀₄::ftsW) carrying either ftsW* or ftsW^*/R172D. Colonies from the plates were grown to the exponential phase in liquid culture and photographed. The panel to the extreme Right shows the cells of W3110, containing a vector for comparison. Cell lengths were measured and plotted.

Fig. 10.

ftsW^R172D reduces the interaction between FtsA and FtsW. (A) Plasmids carrying various alleles of FtsA and FtsW were transformed into BTH101. Three colonies from each transformation were picked into LB and spotted onto indicator plates that were incubated for 24 to 36 h at 30 °C. (B) The β-galactosidase activity was quantitated in liquid cultures from the four strains containing T18–FtsA*^/R300E (the SE is indicated). The controls contained FtsL and FtsW or an empty vector (Vec) and FtsW.

Fig. 11.

Updated model for the activation of FtsW for sPG synthesis. In the activation model, the arrival of FtsN switches FtsA (cytoplasm) and FtsQLB (periplasm) to the “on” state, which activates FtsWI. Our earlier study indicated that the FtsN^E domain alters the FtsQLB conformation so that a domain of FtsL is exposed and contacts FtsI. In this study, we find that FtsN^cyto, which induces the FtsA*-like state, causes FtsA* to act on FtsW.