SPOR Proteins Are Required for Functionality of Class A Penicillin-Binding Proteins in Escherichia coli

Strains, plasmids, and growth conditions.E. coli strains and plasmids used are listed in Table S2. Primers and methods used to construct the strains and plasmids are detailed in Text S1.

Unless stated otherwise, E. coli cells were grown in Miller Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, 1% NaCl) for protein production. When appropriate, antibiotics were added to the medium (20 μg ml⁻¹ chloramphenicol, 50 μg ml⁻¹ kanamycin, 100 μg ml⁻¹ ampicillin).

Protein purification.The following proteins were purified following published protocols: PBP1A (20), PBP1B (19), and FtsN-His (45).

(i) RlpA. LOBSTR (low-background strain) E. coli cells containing plasmid pPZW25 were grown in 2 liters of LB medium supplemented with kanamycin at 37°C to an optical density at 578 nm (OD₅₇₈) of 0.4 to 0.5. Protein overproduction was induced by addition of 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside) to the cell culture, which was further incubated for 4 h at 37°C. Cells were harvested by centrifugation (6,200 × g, 15 min, 4°C), and the pellet was resuspended in buffer I (25 mM Tris-HCl, 1 M NaCl [pH 7.5]). After addition of 200 μM phenylmethylsulfonylfluoride (PMSF), a 1-in-1,000 dilution of protease inhibitor cocktail (Sigma-Aldrich) and DNase, the cells were disrupted by sonication (Branson Digital). The cell lysate was centrifuged (130,000 × g, 60 min, 4°C), and the supernatant was incubated overnight with 4 ml of nickel-nitrilotriacetic acid (Ni-NTA) Superflow (Qiagen), which had been pre-equilibrated in buffer I containing 20 mM imidazole, at 4°C with gentle stirring. The resin was poured into a gravity column and washed with 25 volumes of wash buffer (25 mM Tris-HCl, 1 M NaCl, 10 mM MgCl₂, 20 mM imidazole [pH 7.5]). Bound protein was eluted with elution buffer (25 mM Tris-HCl, 1 M NaCl, 10 mM MgCl₂, 400 mM imidazole [pH 7.5]). The Ni-NTA-eluted protein was dialyzed against 1 liter of dialysis buffer I (25 mM Tris-HCl, 500 mM NaCl, 10 mM MgCl₂ [pH 7.5]) for 30 min; 500 ml of dialysis buffer I was replaced with 500 ml dialysis buffer II (25 mM Tris-HCl, 300 mM NaCl, 10 mM MgCl₂ [pH 7.5]) and further dialyzed for 30 min. Restriction-grade thrombin (4 U ml⁻¹; Merck Millipore, Darmstadt, Germany) was added to remove the oligohistidine tag during overnight dialysis against 1 liter of dialysis buffer II at 4°C. The sample was diluted 1:1 with buffer no salt (25 mM Tris-HCl, 10 mM MgCl₂ [pH 7.5]) and applied in AKTA A buffer (25 mM Tris-HCl, 10 mM MgCl₂, 150 mM NaCl [pH 7.5]) to a 5-ml HiTrap Q HP column using an ÄKTA Prime system (GE Healthcare Bio-Sciences) for anion-exchange chromatography (flow rate, 1 ml min⁻¹). Although a gradient from 150 mM to 1 M NaCl was applied, the protein was present in the flowthrough and wash. The protein was dialyzed against 3 liters of storage buffer (25 mM HEPES-NaOH, 500 mM NaCl, 10 mM MgCl₂, 10% glycerol [pH 7.5]) and stored at −80°C.

(ii) His-DamX. LOBSTR cells containing plasmid pPZW23 were grown in 2 liters of LB medium supplemented with kanamycin at 37°C to an OD₅₇₈ of 0.4 to 0.5. Protein overproduction was induced by addition of 0.5 mM IPTG to the cell culture, which was further incubated for 4 h at 37°C. Cells were harvested by centrifugation (6,200 × g, 15 min, 4°C), and the pellet was resuspended in buffer I (25 mM Tris-HCl, 1 M NaCl [pH 7.5]). After addition of 200 μM PMSF, a 1-in-1,000 dilution of protease inhibitor cocktail (Sigma-Aldrich) and DNase, the cells were disrupted by sonication (Branson Digital), and the cell lysate was centrifuged (130,000 × g, 60 min, 4°C). The supernatant was discarded, and the membrane pellet was resuspended in extraction buffer (25 mM Tris-HCl, 1 M NaCl, 2% Triton X-100 reduced, 10 mM MgCl₂, 10% glycerol [pH 7.5]) and incubated overnight with mixing at 4°C. Resuspended sample was centrifuged (130,000 × g, 60 min, 4°C), and the supernatant was incubated with Ni-NTA Superflow (Qiagen) as described for RlpA. The protein did not bind to the resin and was collected from the flowthrough. The flowthrough containing the protein was dialyzed against 3 liters of dialysis buffer I (25 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl₂ [pH 7.5]) overnight. The sample was applied in AKTA A buffer (25 mM Tris-HCl, 10 mM MgCl₂, 150 mM NaCl, 0.2% Triton X-100 reduced [pH 7.5]) to a 5-ml HiTrap Q HP column using an ÄKTA Prime system (GE Healthcare Bio-Sciences), and the protein was collected in the flowthrough. The protein was dialyzed against 3 liters of dialysis buffer II (10 mM sodium acetate, 150 mM NaCl, 10 mM MgCl₂ [pH 4.8]) overnight. The sample was applied in dialysis buffer II containing 0.2% Triton X-100 reduced to a 5-ml HiTrap SP HP column using an ÄKTA Prime system (GE Healthcare Bio-Sciences) for cation-exchange chromatography (flow rate, 1 ml min⁻¹). The protein was eluted using a gradient from 150 mM to 2 M NaCl. Protein-containing fractions were dialyzed against 3 liters of storage buffer (25 mM HEPES NaOH, 10 mM MgCl₂, 150 mM NaCl, 10% glycerol [pH 7.5]) and stored at −80°C.

(iii) DamX-His and sDamX-His. LOBSTR cells containing plasmid pPZW26 and pPZW27 were grown in 2 liters of LB medium supplemented with kanamycin at 37°C to an OD₅₇₈ of 0.4 to 0.5. Protein overproduction was induced by addition of 0.5 mM IPTG to the cell culture which was further incubated for 4 h at 37°C. Cells were harvested by centrifugation (6,200 × g, 15 min, 4°C), and the pellet was resuspended in buffer I (25 mM Tris-HCl, 1 M NaCl [pH 7.5]). After addition of 200 μM PMSF, a 1-in-1,000 dilution of protease inhibitor cocktail (Sigma-Aldrich), and DNase, the cells were disrupted by sonication (Branson Digital), and the cell lysate was centrifuged (130,000 × g, 60 min, 4°C). For DamX-His, the supernatant was discarded, and the membrane pellet was resuspended in extraction buffer (25 mM Tris-HCl, 1 M NaCl, 2% Triton X-100 reduced, 10 mM MgCl₂, 10% glycerol [pH 7.5]) and incubated overnight with mixing at 4°C. Resuspended sample was centrifuged (130,000 × g, 60 min, 4°C), and the supernatant was incubated with Ni-NTA Superflow (Qiagen), washed, and eluted as described for RlpA using buffers containing 0.2% Triton X-100 reduced. The eluted protein was dialyzed against 3 liters of storage buffer (25 mM HEPES NaOH, 10 mM MgCl₂, 150 mM NaCl, 10% glycerol [pH 7.5]) and stored at −80°C. sDamX-His was purified from the supernatant of the cell lysate centrifugation using the protocol for DamX-His but omitting detergent in buffers. The eluted protein was dialyzed against 3 liters of dialysis buffer I (25 mM Tris-HCl, 200 mM NaCl, 10 mM MgCl₂ [pH 7.5]) for 1.5 h and against 3 liters of dialysis buffer II (10 mM sodium acetate, 200 mM NaCl, 10 mM MgCl₂ [pH 4.8]) overnight. The sample was diluted 1:1 with buffer no salt (10 mM sodium acetate, 10 mM MgCl₂ [pH 4.8]) and applied in AKTA A buffer (10 mM sodium acetate, 100 mM NaCl, 10 mM MgCl₂ [pH 4.8]) to a 5-ml HiTrap SP HP column using an ÄKTA Prime system (GE Healthcare Bio-Sciences) for cation-exchange chromatography (flow rate, 1 ml min⁻¹). The protein was eluted in a gradient from 100 mM to 2 M NaCl. Protein-containing fractions were dialyzed against storage buffer (25 mM HEPES NaOH, 150 mM NaCl, 10 mM MgCl₂, 10% glycerol [pH 7.5]) and stored at −80°C.

(iv) DedD. LOBSTR cells containing plasmid pPZW24 were grown in 2 liters of LB medium supplemented with kanamycin at 37°C to an OD₅₇₈ of 0.4 to 0.5. Protein overproduction was induced by addition of 0.5 mM IPTG to the cell culture, which was further incubated for 4 h at 37°C. Cells were harvested by centrifugation (6,200 × g, 15 min, 4°C) and the pellet was resuspended in buffer I (25 mM Tris-HCl, 1 M NaCl [pH 7.5]). After addition of 200 μM PMSF, a 1-in-1,000 dilution of protease inhibitor cocktail (Sigma-Aldrich) and DNase, the cells were disrupted by sonication (Branson Digital), and the cell lysate was centrifuged (130,000 × g, 60 min, 4°C). The supernatant was discarded, and the membrane pellet was resuspended in extraction buffer (25 mM Tris-HCl, 1 M NaCl, 2% Triton X-100 reduced, 10 mM MgCl₂, 10% glycerol [pH 7.5]) and incubated overnight with mixing at 4°C. Resuspended sample was centrifuged (130,000 × g, 60 min, 4°C), and the supernatant was incubated with Ni-NTA Superflow (Qiagen), washed, and eluted as described for RlpA using buffers containing 0.2% Triton X-100 reduced. Restriction-grade thrombin (4 U ml⁻¹; Merck Millipore) was added to the Ni-NTA-eluted protein to remove the oligohistidine tag during dialysis against 3 liters of dialysis buffer I (25 mM Tris-HCl, 1 M NaCl, 10 mM MgCl₂ [pH 7.5]) for 20 h at 4°C. Sample was dialyzed against 3 liters of dialysis buffer II (10 mM sodium acetate, 500 mM NaCl, 10 mM MgCl₂ [pH 4.8]) for 4 h at 4°C and 3 liters of dialysis buffer III (10 mM sodium acetate, 300 mM NaCl, 10 mM MgCl₂ [pH 4.8]) for 18 h at 4°C. The sample was diluted 1:1 with no-salt buffer (10 mM sodium acetate, 10 mM MgCl₂, 0.2% Triton X-100 reduced [pH 4.8]) and applied in AKTA A buffer (10 mM sodium acetate, 150 mM NaCl, 10 mM MgCl₂, 0.2% Triton X-100 reduced [pH 4.8]) to a 5-ml HiTrap SP HP column using an ÄKTA Prime system (GE Healthcare Bio-Sciences) for cation-exchange chromatography (flow rate, 1 ml min⁻¹). The protein eluted in a gradient from 150 mM to 2 M NaCl. Protein-containing fractions were dialyzed against storage buffer (25 mM HEPES NaOH, 150 mM NaCl, 10 mM MgCl₂, 10% glycerol [pH 7.5]) and stored at −80°C.

Protein coexpression, copurification, and Bocillin FL labeling.C43(DE3) cells transformed with pETDuet plasmids (Table S2) were grown in 500 ml Miller Luria-Bertani (LB) supplemented with ampicillin (100 μg ml⁻¹) at 37°C to an A₆₀₀ of 0.8. Protein expression was induced for 3.5 h by addition of 0.5 mM IPTG. Cells were collected by centrifugation at 4,000 × g for 20 min at 15°C and resuspended in a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and EDTA-free protease inhibitor cocktail (Roche). The cells were lysed by three passages through a cell homogenizer (Emulsiflex C3; Avestin). After centrifugation at 4,000 × g for 20 min at 4°C, the supernatant was recovered and spun down at 150,000 × g for 1 h at 4°C. The pelleted membranes were solubilized in 25 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10% (vol/vol) glycerol, 40 mM DDM (Inalco) and 1 tablet of complete EDTA-free protease inhibitors (Sigma) per 50 ml of buffer. The mixture was incubated for 1 h at room temperature under gentle agitation followed by centrifugation at 150,000 × g for 1 h at 4°C. The supernatant containing the solubilized membrane proteins was loaded onto a HisTrap column (GE HealthCare) conditioned in buffer B (25 mM Tris-HCl [pH 7.5], 500 mM NaCl, 4 mM DDM). After a wash with buffer B supplemented with 80 mM imidazole, the proteins were eluted in 0.5- to 1-ml fractions using a linear gradient of imidazole from 80 to 500 mM. A 15-μl portion of each fraction was incubated with 2 μM Bocillin FL for 30 min at 37°C. The fractions were analyzed by SDS-PAGE, followed by fluorescence imaging and Coomassie blue staining.

Protein expression for NMR.For labeled DedD (residues 28 to 220) purification, pYS001 was transformed into BL21(DE3). This strain was cultured in 1 liter of M9 containing 1 g/liter of ammonium chloride and 2 g/liter of glucose at 37°C to an OD₆₀₀ of 0.6, at which point 1 mM IPTG was added to induce protein overproduction overnight at 25°C. Harvested cell pellets were resuspended in lysis buffer (20 mM HEPES [pH 8.0], 300 mM NaCl, 10% glycerol) and lysed by processing twice with a homogenizer (15 kPa; Avestin). Cellular debris was pelleted by centrifugation at 125,000 × g for 1 h. The resultant supernatant was loaded onto 10 ml Ni²⁺-saturated Ni-NTA Superflow beads (Qiagen) and washed with 65 mM imidazole in 20 mM HEPES (pH 8.0)–300 mM NaCl, and the protein was eluted with 300 mM imidazole in the previous buffer. Fractions containing pure DedD were pooled and desalted into a buffer of 20 mM HEPES (pH 8.0) and 300 mM NaCl. Protein was frozen rapidly in liquid nitrogen and stored at −80°C until required.

Spot plate assay.Cells were grown overnight at 30°C, the optical density was normalized for each strain assayed in the plate, and the cells were spotted in a 10-fold dilution series on Lennox LB plates (1% tryptone, 0.5% yeast extract, 0.5% NaCl), which were incubated overnight at 30°C. Plates were supplemented with 20 μg ml⁻¹ chloramphenicol when strains carrying pKG110-derived plasmids were assayed. When appropriate, 10 μM sodium salicylate (inducer) was added to the plates. Unless stated otherwise, 30 μg ml⁻¹ cefsulodin was used.

Aztreonam susceptibility assay.Overnight cultures of the test strains were grown at 30°C in LB Lennox, reinoculated 1:100, and grown to an OD₅₇₈ of 0.3 to 0.4. A 1-ml portion of each strain was centrifuged for 1 min at 13,000 × g and resuspended in 1 ml of 0.9% NaCl. Resuspended cells were diluted to an OD₅₇₈ of 0.125 using 0.9% NaCl. Cells were distributed onto LB Lennox plates using a cotton swab soaked with the cell suspension. Once the plates were dried, an aztreonam MIC test strip (Liofilchem) was applied to each plate, and all the plates were incubated overnight at 30°C.

PG synthesis assays.[¹⁴C]GlcNAc-labeled lipid II (46), dansylated lipid II (47), and ATTO 550 lipid II (45, 48) were prepared as previously published. Continuous fluorescence GTase assays was performed as described elsewhere (49), using 0.5 μM PBP1A or PBP1B and a 2 μM concentration of the SPOR domain proteins, in a buffer with a final concentration of 50 mM HEPES NaOH (pH 7.5), 150 mM NaCl, 25 mM MgCl₂ and 0.05% Triton X-100. Briefly, dansylated lipid II was added to start the reactions, and the decrease in fluorescence at 30°C was measured over time using a plate reader (excitation wavelength of 330 nm; emission wavelength of 520 nm). An endpoint GTase-TPase activity assay was performed as described elsewhere (50) using either 0.75 μM PBP1A and 1.8 μM SPOR domain proteins or 0.5 μM PBP1B and 2 μM SPOR domain proteins and a final concentration of 10 mM HEPES NaOH (pH 7.5), 150 mM NaCl, 10 mM MgCl₂ and 0.05% Triton X-100 in the reaction buffer. Briefly, 1.2 nmol (11,000 dpm) of [¹⁴C]GlcNAc-labeled lipid II was dried in a glass vial using a vacuum concentrator and resuspended in 5 μl of 0.2% Triton X-100. To start the reactions, the assayed proteins were added to the resuspended lipid II and further incubated for 60 min at 37°C with shaking (800 rpm). Reactions were stopped by boiling for 5 min, and further cellosyl digestion, reduction, and analysis by HPLC were performed as described in reference 50. The following protein concentrations were used in the assays with low concentrations of PBP1A or PBP1B (0.075 μM PBP1A and 0.038 μM PBP1B) and 0.75 μM SPOR domain proteins. In samples with low PG synthase activity (with abundant unused lipid II), the total radioactivity eluted from the HPLC column (C₁₈) differs between samples due to differences in peak 1, the phosphorylated disaccharide pentapeptide. Peak 1 is generated by acid hydrolysis of unused lipid II (or glycan strands ends carrying the C55-PP moiety) after the GTase-TPase reaction, because lipid II (without hydrolysis) does not elute from the C₁₈ HPLC column used to separate the muropeptides. In samples with abundant unused lipid II, peak 1 varies due to differences in the efficiency of the acid hydrolysis of lipid II between samples. This effect does not impair the quantification of other peaks (PG products). Tris-Tricine SDS-PAGE was used to separate glycan strands (51), using the same protein concentrations and reaction conditions as the TPase activity experiment at low PBP1A and PBP1B concentrations but in the presence of 1 mM ampicillin to inhibit the TPase activity.

NMR spectroscopy.NMR data were collected in 20 mM HEPES, 300 mM NaCl, 10% D₂O (pH 7.0), at 298 K on 1.08 mM ¹³C,¹⁵N-labeled DedD sample prepared in a 3-mm-diameter NMR tube. All NMR spectra for backbone, side chains, and NOE assignments were recorded on Bruker spectrometers operating at 700, 850, and 950 MHz ¹H NMR frequencies and equipped with ¹H,¹³C,¹⁵N-labeled cryoprobes.

Resonance assignments of the backbone was performed using two-dimensional (2D) ¹H,¹⁵N-BEST-TROSY (BT), 3D BT-HNCANH, 3D BT-HNCO, 3D BT-HNCACO, 3D BT-HNCACB and 3D BT-HN(CO)CACB spectra. Manual side chain assignment was then achieved with conventional 2D ¹H,¹³C-HSQC (gradient heteronuclear single quantum coherence), 3D (H)C(CCO)NH, 3D H(CCCO)NH, and 3D ¹⁵N-NOESY-HSQC, as well as 3D aliphatic and aromatic ¹³C-NOESY-HSQC experiments. Spectra were analyzed with CcpNmr Analysis 2.4.1.

For structural restraints, dihedral angles (phi and psi) were predicted from backbone chemical shift with the neural network program TALOS+, and distance constraints were determined after manual peak-picking and automatic assignment of the 3D NOESY-HSQC experiments reported above by Unio′10 version 2.0.2. Structures were subsequently calculated from these restraints by Aria 2.3.1, with 80 structures from runs 0 to 5, 200 for runs 6 and 7, and 600 for the last run. The 20 lowest-energy structures were further refined in water. Ramachandran analysis showed 86.1%, 13.9%, 0.0%, and 0.0% of the residues of DedD in most favored, additional allowed, generously allowed, and disallowed regions, respectively.

¹H-¹⁵N NOE relaxation data were collected at 25°C on Bruker spectrometers operating at 700 MHz and equipped with ¹H,¹³C,¹⁵N-labeled cryoprobes. ¹H-¹⁵N NOE values were determined by the comparison of the intensities of each amide resonance with and without a 3-s saturation period and using the BEST-HETNOE sequence (52). Standard deviations were calculated from errors on peak intensities.

Data availability.The 20 lowest-energy structures were deposited in the PDB with accession number 6ZTG. All other data supporting the findings of this study are included in the main text and supplemental material.