Fig 1.
Transposon-sequencing identifies sweD and sweC as synthetic lethal with lytE.
(A) Transposon insertion profiles from wild-type (WT) and ΔlytE libraries. Three regions of the genome are shown. The height of each line reflects the number of sequencing reads at this genomic position. Transposon insertions in cwlO, ftsE, ftsX, sweD (yqzD) and sweC (yqzC) are underrepresented in the ΔlytE library compared to WT. No insertions were mapped to the essential gene trxB in either library. (B) sweD and sweC mutants are synthetically lethal with ΔlytE. Spot dilutions of the indicated strains in the presence and absence of inducer. All strains were grown in the presence of IPTG (500 μM) to OD600 ~2.0. The cultures were washed twice without inducer, resuspended at an OD600 of 1.5, and 10-fold serially diluted. Five microliters of each dilution was spotted onto LB agar plates with and without IPTG. Representative plates from one of three biological replicates are shown. (C) Schematic representations of SweD and SweC. Transmembrane segments (TM); putative coiled-coil (CC); helix-turn-helix motif (HTH); and LysM homology domain are indicated.
Fig 2.
ΔsweDC mutants have morphological defects that resemble ΔcwlO and ΔftsEX.
(A) Representative images of wild-type (WT) (BDR2649), ΔsweDC (BYB370), ΔcwlO (BYB371), ΔsweDC ΔcwlO (BYB374), ΔftsEX (BYB372), and ΔlytE (BYB373) are shown. Exponentially growing cells in CH medium were stained with the membrane dye TMA-DPH and examined by fluorescence microscopy. (B) Representative images of a ΔlytE strain during depletion of SweDC. The indicated strain (BYB362) harboring cytoplasmic mCherry was grown to exponential phase in CH medium in the presence of 500 μM IPTG, washed twice with medium lacking inducer, and used to inoculate CH medium at an OD600 of 0.05. The cells were examined by fluorescence microscopy at the indicated times. Membranes were visualized with TMA-DPH (top) and cytoplasmic mCherry and phase contrast (bottom). (C) Comparison of the terminal phenotypes of strains lacking LytE and depleted of SweDC (BYB362), CwlO (BYB279), or FtsEX (BYB439) The indicated strains were grown to exponential phase in the presence of IPTG (500 μM), washed twice with medium lacking inducer, back-diluted to an OD600 of 0.05 (BYB362, BYB439) or 0.1 (BYB279) in CH medium, and grown to mid-exponential phase in the absence of inducer. Cells were examined by fluorescence microscopy. Images of SweDC, CwlO, and FtsEX depletions are from 230, 60, and 150 min after removal of IPTG, respectively. The different times required to reach the terminal phenotype likely reflect the abundance and half-lives of the individual proteins. All strains contained cytoplasmic mCherry, and an overlay of mCherry and phase contrast is shown adjacent to TMA-DPH-stained membranes. The representative images in this figure are from one of three independent experiments. Curved cell morphologies are highlighted (yellow carets). Scale bar indicates 2 μm.
Fig 3.
SweD and SweC are Type II membrane proteins.
(A) Fractionation of SweD and SweC analyzed by immunoblot. Lysates from exponentially growing wild-type cells were subject to centrifugation to separate soluble (S100) and membrane-associated (P100) proteins. The membrane fraction was incubated with buffer or buffer supplemented with TritonX-100 and the solubilized proteins (S100) were separated from insoluble material (P100) by a second round of centrifugation. Equivalent amounts of each fraction including the input protoplasts (proto) were separated by SDS-PAGE and analyzed by immunoblot. The membrane protein EzrA and cytoplasmic protein ScpB served as membrane and cytoplasmic controls. (B) Protease accessibility analysis of SweD and SweC analyzed by immunoblot. Protoplasts of strain BDR776 were treated with buffer or buffer supplemented with Proteinase K in the absence or presence of sodium-lauroyl-sarcosinate (Sarcosyl). Reactions were resolved by SDS-PAGE and analyzed by immunoblot. SweD and SweC were inaccessible to cleavage by Proteinase K. Degradation of the extracellular domain of SpoIVFA (FA) served as a protease accessible control. The membrane protein EzrA and the cytoplasmic protein FtsE served as protease inaccessible controls. The immunoblots shown are from one of three independent experiments. Molecular weight markers (in kDa) are indicated.
Fig 4.
SweD and SweC depend on each other for stability.
(A) Immunoblot analysis of SweD and SweC in different mutant backgrounds. The indicated strains were grown in LB medium to mid-exponential phase and SweD, SweC, CwlO, FtsE, FtsX and SigA were assessed by immunoblot analysis. SweC levels were almost undetectable in cells lacking SweD (ΔD) but were largely restored when SweD was produced under IPTG control [P(IPTG)-D] from an ectopic locus. Similar results were obtained for SweD in cells lacking SweC (ΔC). By contrast, the levels of FtsE, FtsX, and CwlO were unaffected by the absence of SweD and/or SweC. CwlO levels were partially reduced in the absence of FtsEX. The SigA immunoblot serves to control for loading. The immunoblots shown are from one of three independent experiments. (B) Cell-association of CwlO partially depends on FtsEX but not on SweDC. Immunoblot analysis of wild-type (PY79) and cells lacking cwlO (BJM54), ftsEX (BJM272) and sweDC (BYB339). Strains were grown in LB medium to mid-exponential phase and equivalent amounts of whole cell lysate (C) and culture medium (M) concentrated by trichloroacetic acid precipitation were separated by SDS-PAGE. CwlO and the cytoplasmic protein SMC were assessed by immunoblot analysis. The SMC immunoblot serves to control for loading. See Methods for a detailed protocol describing how CwlO was recovered from the medium and the plastic microfuge tube where it bound non-specifically during culture collection. The immunoblots are from one of three independent experiments. Molecular weight markers (in kDa) are indicated.
Fig 5.
The cytoplasmic domains of SweD and SweC are important for function.
(A) Spot dilutions of the indicated strains on LB and CH media in the presence of either xylose or IPTG. All strains except wild-type (WT) are ΔsweDC ΔlytE double mutants and contain a xylose-regulated lytE allele. These strains also harbor IPTG-regulated alleles of the sweDC operon with the indicated mutations. The strains were grown in LB in the presence of xylose (10 mM) to an optical density of ∼2.0. The cultures were washed twice without inducer, resuspended at an OD600 of 1.5, and 10-fold serially diluted. Five microliters of each dilution was spotted onto LB agar plates supplemented with xylose (10 mM) or IPTG (500 μM) and CH agar plates supplemented with IPTG (500 μM). Representative plates from one of three biological replicates are shown. (B) Schematic representations of SweD and SweC domain-deletions and point-mutations. HTH* represents amino acid substitutions D104A and V105A in the second helix of the helix-turn-helix motif. (C) Immunoblot analysis of the SweDC domain deletion and point mutants. The indicated strains were grown in LB medium to mid-exponential phase. SweD(ΔCC) partially stabilizes SweC. SweD(ΔHTH) is virtually undetectable but fully stabilizes SweC suggesting it is produced but poorly recognized by the anti-SweD antibody. Similarly SweC(ΔLysM) is undetectable but stabilizes SweD. We have not been able to establish the nature of the higher molecular weight band observed in the SweD immunoblot that appears to be an anomalously migrating SweD species but is only present in a subset of lysates and at different levels. The SigA immunoblot serves to control for loading. Molecular weight markers (in kDa) are indicated. Representative immunoblots from one of three independent experiments is shown.
Fig 6.
Point mutations in ftsE and ftsX suppress the ΔlytE ΔsweDC synthetic lethality.
(A) Spot dilutions of the indicated strains on permissive and restrictive media. All strains were grown in LB in the presence of IPTG (500 μM) to an optical density of ∼2.0. The cultures were washed twice without inducer, resuspended at an OD600 of 1.5, and 10-fold serially diluted. Five microliters of each dilution was spotted onto the indicated agar plates without inducer. The permissive condition contains CH medium supplemented with 0.25 M sucrose and 20 mM MgCl2. The ΔsweDC (ΔDC) ΔlytE double mutant, like the parental strain used to select for suppressors, can only grow under permissive conditions. A walH deletion or point mutations in ftsE or ftsX support growth on CH medium lacking IPTG but not LB. Combining ΔwalH with either point mutation supports growth on LB. Representative plates from one of three biological replicates are shown. (B) Cytological analysis of the ftsEX suppressors strains under permissive and restrictive conditions. The indicated strains were grown to exponential phase in CH medium in presence of IPTG (500 μM). The cultures were washed twice in CH medium lacking inducer and back-diluted to an OD of 0.01 in permissive and restrictive culture media lacking IPTG. Cells were analyzed using differential interference contrast (DIC) microscopy after 4 generations. Mutations in ftsE or ftsX restore rod-shape morphology to the ΔsweDC ΔlytE double mutant when grown under permissive conditions and restored viability and partial rod-shape morphology when grown under restrictive conditions. Scale bar indicates 2 μm. Representative images from one of three independent experiments are shown.
Fig 7.
SweD and SweC reside in a complex with FtsX.
(A) Immunoblot analysis of co-immunoprecipitation assays. Digitonin-solubilized membrane fractions derived from exponentially growing wild-type (WT) (PY79) and ΔftsX (BJM72) mutant cells were incubated with anti-FtsX antisera and precipitated with Protein A Sepharose. The detergent-solubilized fraction prior to immunoprecipitation (load; L), the supernatants after immunoprecipitation (flow through; FT), and the immunoprecipitates (IP) were subjected to immunoblot analysis probing for FtsX, SweD, SweC and a control membrane protein WalI. Molecular weight markers (in kDa) are indicated. Immunoblots are representative of one of two independent experiments. Since the anti-FtsX polyclonal antibodies were not covalently coupled to the Protein A Sepharose the heavy and light chains are present in the IP fractions. The bands and smears detected in the FtsX and WalI immunoblots result from the secondary antibodies recognizing light chains that migrate heterogeneously at ~25 kDa. (B) The bacterial adenylate cyclase two-hybrid (BACTH) assay detects an interaction between SweD and FtsX. The BTH101 E. coli reporter strain containing plasmids with the indicated proteins fused to the T18 or T25 domains of the Bordetella adenylate cyclase were spotted on LB(X-gal) indicator plates. Interactions can be detected between FtsE and FtsX, SweD and FtsX, and SweD and itself. T18 and T25 fusions to E. coli TolB and Pal were used as positive and negative controls. (C) Analysis of SweD truncation variants. Interactions can be detected between FtsX and SweD lacking its putative coiled-coil domain (SweDΔCC) and lacking its entire intracellular domain [SweD(TM)]. No interaction was detected between full-length SweD and either of these SweD variants. The BACTH assays were performed in triplicate and photographs of representative agar plates are shown.
Fig 8.
FtsEX complexes employ distinct co-factors to regulate PG hydrolysis during cell division and cell wall elongation.
Schematic models comparing the E. coli FtsEX-EnvC-Amidase complex that functions during division (left) with the B. subtilis SweDC-FtsEX-CwlO elongation hydrolase complex (right). In E. coli, FtsE (E)—FtsX (X) complex promotes divisome assembly [32, 33] and regulates septal cell wall cleavage by the amidases AmiA and AmiB (Ami) via the coiled-coil domain-containing regulator EnvC [27, 31]. FtsEX has been found to interact with FtsZ (Z) [81] and FtsA (A) [32] and FtsEX ATPase activity is required for both PG synthesis [32, 34] and amidase activity [27]. SweD and SweC reside in a membrane complex with FtsEX and function as co-factors in regulating the D,L-endopeptidase activity of CwlO. Based on ribosome profiling and our two-hybrid analysis, the stoichiometry of the SweD-SweC-FtsE-FtsX complex is proposed to be 4:2:2:2. Our analysis suggests that SweDC regulate the FtsEX ATPase activity. Genetic evidence suggests that the hydrolase complex functions in the same pathway as the peptidoglycan elongation machinery (not depicted) [22]. Whether this is direct or indirect is currently unknown.