Fig 1.
Structure of a SEDS-bPBP protein complex and a model for FtsN-induced activation of FtsWI.
(A) Structure of T. thermophilus RodA-PBP2 (PDB ID#: 6P5L) and the location of the corresponding residues in FtsW and FtsI analyzed in this study. TtRodA is colored green and TtPBP2 is colored cyan, the unstructured region in ECL4 of TtRodA is colored orange. The residues are numbered according to E. coli FtsW and the catalytic residues of TtRodA and TtPBP2 are colored red. Residues altered by mutations studied here are indicated and colored orange. (B) The signal transduction pathway leading to the activation of FtsWI. FtsN switches FtsA and FtsQLB to the ON conformations via its cytoplasmic domain (Cyto) and its E domain (E), respectively. In the cytoplasm, the activation signal in FtsA is transmitted to FtsW and this is regulated by the ATPase activity of FtsEX via an interaction between FtsX and FtsA. In the periplasm, the action of the E domain of FtsN likely causes a conformational change of the FtsQLB complex at the CCD domain of FtsB and FtsL, resulting in the AWI domain (Activation of FtsWI) of FtsL becoming available to interact with FtsI. These signals synergize to activate FtsW.
Fig 2.
Overexpression of FtsWM269I bypasses FtsN.
(A) IPTG-dependent growth of ftsN::kan transductants from S2 Fig. A single transductant of each strain was restreaked on LB plates with ampicillin, kanamycin, 1 mM sodium citrate and with or without 60 μM IPTG. Plates were incubated at 37°C overnight and photographed. (B) Morphology of WT and FtsWM269I cells with or without FtsN. Cells from an overnight culture of SD534 [W3110, ftsN::kan ftsWM269I/pSEB429-M269I (pDSW208, P204::ftsWM2690I)] were collected by centrifugation and washed twice with LB to remove IPTG and resuspended in the same volume of LB. The culture was diluted 1:100 and split in half, one with IPTG and one without IPTG and grown at 37°C. The control cultures (W3110 and SD488 [W3110, leu::Tn10 ftsWM269I]) were diluted 1:100 in fresh LB medium with antibiotics and grown at 37°C. Samples were taken for photography 2.5 hours after removal of IPTG. The scale bar is 5 μm.
Fig 3.
FtsWM269I is a unique substitution that suppresses an FtsN depletion strain.
(A) Complementation test of FtsW mutants. Plasmid pDSW208, pSEB429 or its derivatives carrying ftsW mutations were transformed into strain SD237 [W3110, leu::Tn10, ftsW::kan/pDSW406 (pBAD33, PBAD::ftsW)] on LB plates with ampicillin, kanamycin, chloramphenicol and 0.2% arabinose. The next day, a single transformant of each resulting strain was resuspended in 1 ml of LB medium, serially diluted. 3 μl of each dilution was spot on LB plates with antibiotics, with or without 0.2% arabinose and increasing concentrations of IPTG. Plates were incubated at 37°C overnight and photographed. (B) Spot test of the ability of FtsW mutants to rescue the growth of an FtsN depletion strain. Colonies of strain SD264 [W3110, ftsN::kan/pBL154 (pSC101ts, Psyn::ftsN, spc)] harboring pDSW208 or pSEB429 expressing FtsW variants were resuspended in 1 ml of LB medium and serially diluted. 3 μl of each dilution was spotted on LB plates with antibiotics with or without IPTG. Plates were incubated at 30°C for 24 hours or at 42°C overnight and photographed.
Fig 4.
(A) Complementation test of FtsW mutants. Plasmid pDSW208, pSEB429 or its derivatives carrying ftsW mutations were transformed into strain SD237 [W3110, leu::Tn10, ftsW::kan/pDSW406 (pBAD33, PBAD::ftsW) on LB plates with ampicillin, kanamycin, chloramphenicol and 0.2% arabinose. The spot test was performed as Fig 3A. (B) Western blot to test the stability of FtsW mutants. Details about the blot is described in Materials and Methods.
Fig 5.
FtsWM269K and FtsWA270T localize to the division site and recruit FtsI.
(A) Localization of FtsW mutants. Localization of the FtsW mutants was assessed in the FtsW depletion strain SD237 [W3110, leu::Tn10 ftsW::kan/pDSW406 (PBAD::ftsW)] using plasmid pSD347 (P206::ftsW-l60-gfp) and derivatives carrying ftsW mutations. After removal of arabinose, IPTG was added to the culture to induce the fusion protein and 4 hours post induction cells were immobilized on 2% agarose pad for photographing. (B) Localization of FtsI. Overnight cultures of SD288 [W3110, ftsW::kan gfp-ftsI/pSD257 (pSC101ts, ftsW)] carrying plasmid pBAD33 or pDSW406 (pBAD33, PBAD::ftsW) or its derivatives grown at 30°C were diluted 1:100 in fresh LB medium with antibiotics and grown at 30°C for 2 h. The cultures were then diluted 1:10 in fresh LB medium with antibiotics, 0.2% arabinose and 10 μM IPTG and grown at 37°C for 4 hours. Cells were immobilized on 2% agarose pads for photography. The scale bar is 5 μm.
Fig 6.
FtsIK211I and FtsWE289G rescue inactive FtsL mutants.
Plasmids pDSW208, pSEB429 (pDSW208, P204::ftsW), pLY91 (pDSW208, P204::ftsI) or their derivatives carrying different ftsW or ftsI alleles were transformed into strain SD399 (W3110, ftsL::kan /pSD256) harboring plasmid pSD296-L86F (pBAD33, PBAD::ftsLL86F) or pSD296-E87K (pBAD33, PBAD::ftsLE87K) and transformants selected on LB plates with antibiotics and glucose. The next day, a single colony of each resulting strain was subject to spot tests. Plates were incubated at 30°C for 24 hours or at 42°C overnight and photographed.
Fig 7.
FtsIK211I and FtsWE289G bypass FtsN.
(A) Morphology of WT and FtsIK211I cells with or without FtsN. Overnight cultures of W3110, LYA8 (W3110, leu::Tn10 ftsIK211I) and LYA9/pLY105 [W3110, leu::Tn10 ftsN::kan ftsIK211I/pLY105 (P204::ftsIK211I)] were diluted 1:100 in fresh LB medium with antibiotics and grown at 42°C. 60 μM IPTG was added to the culture of LYA9/pLY105. 2 hours later, samples were taken for photography. The scale bar is 5 μm. (B) Morphology of WT and FtsWE289G cells with or without FtsN. Overnight cultures of W3110, SD488, SD264 (W3110, leu::Tn10 ftsN::kan/pBL154 (pSC101ts, Psyn::ftsN)] and SD530 (W3110, leu::Tn10 ftsN::kan ftsWE289G) were diluted 1:100 in fresh LB medium with antibiotics and grown at 37°C. Samples were taken for photography 2 hours later. The scale bar is 5 μm.
Fig 8.
Overexpression of FtsWE289G suppresses FtsI mutations defective in activation of septal PG synthesis.
Plasmid pDSW208, pSEB429 (pDSW208, P204::ftsW) or its derivatives carrying different ftsW alleles were co-transformed with plasmid pBAD33 carrying different ftsI alleles into strain MCI23 ΔrecA (MC4100, ftsI23 recA::spec). Transformants were selected on LB plates with antibiotics and glucose at 30°C. The next day, a single colony of each resulting strain was subjected to a spot test.
Fig 9.
FtsBE56A and FtsIK211I suppress FtsWA270T but not FtsWM269K.
(A) Spot test to assess the ability of FtsBE56A to suppress FtsWA270T and FtsWM269K. Plasmid pDSW208, pSEB429 or its derivatives carrying different ftsW alleles were transformed into strain SD367 [TB28, ftsW::kan/pSD257(pSC101ts, ftsW)] or SD368 [TB28, ftsBE56A, ftsW::kan/pSD257(pSC101ts, ftsW)] on LB plates with ampicillin, kanamycin and spectinomycin at 30°C. The spot test was performed as in Fig 5. (B) Spot test of the ability of FtsIK211I to suppress FtsWA270T and FtsWM269K. The test was done in strain SD292 [W3110, ftsW::kan recA56 slrD::Tn10 /pSD257 (pSC101ts, ftsW)] containing plasmid pLY91 (P204::ftsI) or pLY105 (P204::ftsIK211I). SD292 was transformed with plasmid pBAD33 carrying different ftsW alleles and transformants selected at 30°C. Test was done as (A) on LB plates with or without arabinose and IPTG at 30°C and 42°C.
Fig 10.
ftsIK211I and ftsWE289G are intragenic suppressors of inactive FtsI and FtsW mutants respectively.
(A) ftsIK211I suppresses inactive FtsI mutations. To test the ability of various FtsI mutants to be rescued by K211I, plasmid pDSW208, pLY91 (pDSW208, P204::ftsI) or its derivatives carrying different alleles of ftsI were transformed into strain MCI23ΔrecA (W3110, recA::spec, ftsI23) at 30°C. Complementation test was done at 42°C (to inactivae FtsI23) and in the presence of IPTG to induce the ftsI alleles contained on the plasmids. (B) ftsWE289G suppresses inactive FtsW mutants. To test the ability of E289G to rescue deficient FtsW alleles, plasmid pDSW208, pSEB429 or its derivatives carrying different alleles of ftsW were transformed into strain SD237 (W3110, leu::Tn10, ftsW::kan/pDSW406 [pBAD33, PBAD::ftsW]) on LB plates with ampicillin, kanamycin, chloramphenicol and 0.2% arabinose at 37°C. The complementation test was done by removing arabinose (to deplete WT FtsW) and adding IPTG to induce the ftsW alleles on the plasmids.
Fig 11.
Location of residues on the TtRodA-PBP2 structure that correspond to mutations that affect the activity of SEDS-bPBP protein complexes.
TtRodA is colored green, whereas TtPBP2 is colored cyan (only a part of the pedestal domain is shown). Mutations that increased the activity of FtsWI or RodA-PBP2 are colored orange, whereas mutations that inhibit the activity of these SEDS-bPBP protein complex are colored magenta. The FtsW putative catalytic residue is colored red. Most of the active/inactive mutations are located at the interaction interface between ECL4 of TtRodA and the tip of the lower part of the pedestal domain of TtPBP2.
Table 1.
Active (orange) and inactive (magenta) FtsW/RodA and FtsI/PBP2 mutations.