Models of the functions and structures of TA in S. pneumoniae. (A) TA (multicolored rods) attached to the membrane (M) or to the peptidoglycan (PG) exclude each other to maintain a periplasmic space (PS). Additional functions arise from the presentation of various choline-binding proteins (blue). (B) Polymerized TA attached to undecaprenyl (UndPr)-pyrophosphate are transferred at the cell surface onto PG by the phosphotransferase LytR to form WTA; or onto DGlcp-DAG by TacL to form LTA. The enzymes are depicted by their AlphaFold models. The most abundant TA species have 6 repeating units (n = 4).

Electron micrographs of WT, ΔtacL and ΔlytR strains. (A) CEMOVIS micrographs allowing full view (white scale bar, 100 nm) and zoom (black scale bar, 10 nm) of WT and mutant strains. O and I signal the outer and inner sides of the cell envelope. A periplasmic space can be observed in all three strains. A granular layer is seen as a thin black line in the periplasmic space (arrowheads) of WT cells, whereas it is not observed in the ΔlytR and ΔtacL strains. (B) Corresponding pixel intensity profile of the cell envelope is shown for each strain. The intensity profiles were measured perpendicularly to the cell surface in regions boxed in white, and show variations in the density of the envelope ultrastructure. CW, cell wall; P, periplasm; GL, granular layer; M, membrane.

Dimensions of cell envelope structures measured on CEMOVIS images

Super-resolved dSTORM imaging of TA incorporated in growing WT, ΔtacL and ΔlytR cells after a 5 min pulse of metabolic labeling with 1.5 mM aCho followed by a chase, and subsequent secondary fluorescent labeling by click chemistry using DBCO-AF647. For comparison, the same pulse-chase procedure was applied to reveal the newly synthesized peptidoglycan (PG) with a metabolic labeling using 2 mM azido-D-Ala-D-Ala. Scale bars are 1 µm. Corresponding conventional and bright field images are shown in SI Fig. S5A.

(A) Polyacrylamide gel electrophoresis of fluorescently labeled TA demonstrating separation of LTA and WTA by centrifugation. Labeled compounds were revealed by UV-transillumination. TA were labeled by growing WT cells in C-medium in the presence of 200 µM aCho. Cells were lysed overnight with lysozyme, mutanolysine and LytA and the azido-groups were modified by reaction with 25 µM DBCO-AF488. The lysate was centrifuged for 2 min at 1,250, 5,000 or 20,000 g. The pellets were resuspended in the initial volume. An aliquot of the 20,000 g supernatant was treated with alkaline phosphatase. The buffer with the lysing enzymes, the medium with and without aCho and the culture supernatant were similarly incubated with 25 µM DBCO-AF488. B, lysis buffer with enzymes; M-, C-medium without aCho; M+, C-medium with aCho; MC, culture supernatant; L, lysate; P, pellet; S, supernatant; AP, alkaline phosphatase-treated. The control samples (B, M-, M+ and MC were loaded at one-fourth the volume of the cellular samples). Labels on the right side of the gel identify species coupled to the fluorophore AF488. (B-C) Titration of cellular TA. WT cells were grown for two generation in the presence of aCho prior to cell lysis. DBCO-AF488 (1.9 µM) was incubated for 24 h with varying amounts of cell lysate corresponding to up to 2.9 108 cells mL-1. The remaining DBCO-AF488 was blocked by addition of 100 mM aCho, and the various species were separated by polyacrylamide (17%) gel electrophoresis (B). The bands were quantified and the relative amount of the various species were plotted against the cell concentration (C). Black circles, blocked DBCO-AF488; open circle, phosphocholine; open triangle, TA. A linear regression of the DBCO-AF488 points at low cellular concentration was applied to obtain the titration point as the intercept of the cell concentration axis.

Evolution of TA during growth phases. (A) WT cells grown in C-medium containing 200 µM aCho were harvested during the exponential growth phase (Ex), at the onset of the stationary phase (St) and during the autolysis (Ly). TA were fluorescently click-labeled with DBCO-AF488 and cells were completely lysed by the addition of PG hydrolases prior to centrifugation. LTA are found in the pellet (P) whereas WTA are observed in the supernatant (S). The amounts of cells at the different culture stages were normalized. The LTA samples are 8-fold concentrated compared to the WTA samples. Fluorescently labeled TA were revealed by UV-transillumination after SDS-polyacrylamide electrophoresis. (B) Densitometric profiles of the fluorescent intensities of exponential (blue) and stationary phase samples (orange) shown in (A). (C) Electrophoretic analysis of TA of WT cells grown in BHI and pulse-labeled for 5 min with 1.5 mM aCho, and chased by further growth in the same medium without added aCho for the indicated duration.

(A) Electrophoretic analysis of TA from the WT strain, or deleted of the gene encoding the glycosyltransferase TacL, or the phosphotransferase LytR. Cells were grown in C-medium with no choline and 200 µM aCho, prior to cell lysis with PG hydrolases and secondary labeling with DBCO-AF488. Lysates were fractionated by centrifugation and the pellets were resuspended in one fourth the initial volume: P, pellet; S, supernatant. (B-E) Identification of sedimented TA from lysates of ΔtacL cells as undecaprenyl-pyrophosphate bound precursors. (B) Pulse-chase labeling experiment of TA in strain ΔtacL. Cells grown in BHI were exposed to 1.5 mM aCho for 10 min (pulse), prior to washing and further incubation in the same media without aCho (chase) for various time prior to cell lysis and secondary labeling with DBCO-AF488. Samples were treated with alkaline phosphatase prior to electrophoresis. (C) Pellets containing labeled TA from WT and ΔtacL cells were resuspended in a Tris- or ammonium acetate-buffered solution at pH 8 or pH 4.2 and incubated for 3 h at room temperature or 110°C prior to electrophoresis. (D) Labeled TA from WT and ΔtacL cells were submitted to the first steps of the traditional procedure of LTA isolation: boiling in 4% SDS in a citrate-buffered solution at pH 4.7, lyophilization and ethanol wash of the lyophilizate, butanol extraction and retention of the aqueous fraction. (E) Labeled sedimented TA from WT and ΔtacL cells were incubated overnight without or with various concentrations of recombinant P. aeruginosa colicin M prior to electrophoretic separation. (F) To probe on which side of the plasma membrane the TA are positioned, secondary click-labeling with DBCO-AF488 was performed prior or after cell lysis of WT and ΔtacL cells grown in C-medium in the presence of 0.2 mM azido-choline. L, whole lysate; S, supernatant of 20,000 g 2 min centrifugation; P, pellet resuspended in one-fourth initial volume.

S. pneumoniae strains used in this study

Chemical structure of (A) LTA, (B) WTA, and (C) the membrane-bound precursor. The constituent of the repeating unit are in green the acetamido-4-amino-6-deoxygalactopyranose (AATGalpNAc), in yellow the glucopyranose (DGlcp), in pink the ribitol-5-phosphate, in cyan the N-acetylgalactosamine (DGalpNAc), in purple the phosphocholine. One or both phosphocholine residues can be absent on the terminal unit. Some units may also lack the phosphocholine on the proximal DGalpNAc. The most abundant species have 6 repeating units (n=4). LTA are β-1-liked to monoglucosyldiacylglycerol (blue). WTA are α-1-linked via a phosphodiester to position 6 of a N-acetyl muramic acid of the peptidoglycan (PG, gray). The precursors remain attached to an undecaprenyl pyrophosphate (orange). The one-letter nomenclature of constituents proposed by Gisch (1) is given in magenta around the LTA.

Low-magnification CEMOVIS images of S. pneumoniae ΔtacL. (A) The ribbon spans the entire EM grid. Scale bar, 100 µm. (B) Stitching of several acquisitions areas showing one cryo-section (1) and half of a second one (2) inside the ribbon. Scale bar, 5 µm. (C). Cells are uniformly distributed in the section. Representative acquisition areas, where cells are located at the center of a hole in the carbon, are pointed with a white arrowhead. Scale bar, 1 µm. Black arrowheads: knife marks; asterisks: ice contamination.

(A-B) CETOVIS of a ΔtacL cell section. (A) Raw image of a tilt series. (B) Full view (scale bar, 100 nm) and zoom (scale bar, 20 nm) of a slice through the cryo-electron tomogram. Yellow arrow, membrane; magenta arrow; periplasmic space; cyan bracket, peptidoglycan. The reduction of the periplasmic space and peptidoglycan layers thickness is observed in the whole volume. No granular layer is detected in the volume. (C) TEM micrographs of stained freeze-substituted thin sections of the cell envelope of WT, ΔlytR and ΔtacL cells. The appearance varies depending on the angle between the cell envelope and the section plane. Two examples are shown for each strain. No consistent difference could be discerned between the strains. Scale bar 100 nm.

Fluorescence microscopy of R800 WT, ΔtacL and ΔlytR cells grown in BHI and pulse-labeled with 1.5 mM azido-choline for 5 min prior to fixation and secondary click-labeling with DBCO-AF647. The three top rows are with the same imaging settings. The fluorescent signal of the bottom row was enhanced. Scale bars are 2 µm.

(A) Corresponding conventional and bright field microscopy images of the pulse-chase experiments shown by dSTORM imaging in Fig. 3. New TA were revealed in growing R800 WT, ΔtacL and ΔlytR cells by a 5 min pulse of metabolic labeling with 1.5 mM azido-choline followed by a chase, and subsequent secondary fluorescent labeling with clickable DBCO-AF647. For comparison, the same pulse-chase procedure was applied to reveal the new peptidoglycan with a metabolic labeling using 1.5 mM azido-D-Ala-D-Ala. (B) dSTORM imaging (with corresponding conventional and bright field images at half scale) of sacculi prepared from cells pulse-labeled during 5 min and chased for 15 min. Labeled TA in sacculi were revealed by secondary fluorescent labeling with clickable DBCO-AF647. Scale bars are 1 µm.

Effect of the concentration of added recombinant LytA on the sedimentation of LTA. R800 WT cells grown in C-medium with 200 µM azido-choline were click-labeled with DBCO-AF488 and lysed overnight with mutanolysine, lysozyme and various concentration of recombinant LytA. Lysates were centrifuged at 20,000 g for 2 min. The pellets were resuspended in one-fourth the initial volume and samples were analyzed by gel electrophoresis. Fluorescently labeled compounds were revealed by UV trans-illumination.

Polyacrylamide gel electrophoresis of click-labeled TA, azido-choline (zCho) and the clickable fluorophore DBCO-AF488 with (A) 12% (w/v) and (B) 17% acrylamide. Three lanes on the left of the gels: R800 cells were grown in C-medium in the presence of 0.2 µM azido-choline prior to lysis and secondary labeling with 10 µM DBCO-AF488 overnight at room temperature. Samples were further incubated 4 h with either 1000 U.mL-1 alkaline phosphatase (AP), nothing (-), or 10 mM additional azido-choline. Six lanes on the right of gels. In either 50 mM Tris pH 8, 150 mM NaCl or C-medium, 20 µM DBCO-AF488 were incubated at room temperature for 4 h with either 0, 10 mM or 200 µM azido-choline. Prior to gel loading, samples were supplemented with 8% glycerol, 0.8% SDS, 8 mM DTT and trace of bromophenol blue. The migration position of the fluorophore-triazole-linked species are indicated on the left or on the gels by symbols (PCho^, phospho-choline; Cho*, choline). The position of the DBCO-AF488 reagent is indicated on the right or on the gel with the symbol #.

Protein Coomassie staining of TA analysis gels presented in Figs 4A, 5A, 5B, 6A, 6B, 6E.

Characterization of the cell lysate fractionation by sedimentation. (A) Iodine vapor-stained thin layer chromatography of lipids extracted from R800 cell lysate, the resuspended low-speed centrifugation pellet and the supernatant (Spnt) of the lysate. Lipids were tentatively identified according (2). GlcDAG, mono-glucosyl-diacyl-glycerol (DGlcp-DAG); GalGlcDAG, galactosyl-glucosyl-diacyl-glycerol (DGalp-DGlcp-DAG). (B) Negative stain electron micrograph of the resuspended low-speed centrifugation pellet. Magnification was 4800x. Scale bar is 1 µm.

NMR spectra of S. pneumoniae LTA obtained by low speed centrifugation of cell lysate. Main is 13C-1H HSQC (constant-time) spectrum; 13C nuclei that are coupled to either zero or two other carbons (red) and those coupled to one or three other carbons (dark blue), exhibit opposite signs. Inset is 31P 1D spectrum. The peak nomenclature is that proposed by Gisch et al. (1). Peaks A1 to A6, GRO1 GRO3 are originating from the lipid anchor DGlcp-DAG of LTA.

(A-B) Titration of cellular TA. R800 cells were grown for two generation in the presence of azido-choline. DBCO-AF488 (3.7 µM) was incubated for 24 h with varying amount of cell lysate corresponding to up to 2.9 108 cells mL-1. The remaining DBCO-AF488 was blocked by addition of 100 mM azido-choline, and the various species were separated by polyacrylamide gel electrophoresis. (A) The gel was imaged by trans-illumination with UV light. (B) The bands were quantified and the relative amount of the various species were plotted against the cell concentration. Black circles, blocked DBCO-AF488; open circle, phospho-choline; open triangle, TA. A linear regression of the DBCO-AF488 points at low cellular concentration was applied to obtain the titration point as the intercept of the cell concentration axis.

Evolution of TA during growth. (A) ΔlytA cells grown in C-medium containing 200 µM azido-choline were harvested during the exponential growth phase (Ex), at the onset of the stationary phase (St) and during the autolysis (Ly). TA were fluorescently click-labeled with DBCO-AF488 and cells were completely lysed by the addition of peptidoglycan hydrolases prior to centrifugation. LTA are found in the pellet (P) whereas WTA are observed in the supernatant (S). The amounts of cells at the different culture stages were normalized. The LTA samples are 8-fold concentrated compared to the WTA samples. Fluorescently labeled TA were revealed by UV-transillumination after SDS-polyacrylamide electrophoresis. (B) Electrophoretic analysis of TA of WT cells grown in C-medium supplemented with 0.1% yeast extract and pulse-labeled for 5 min with the addition of 0.2 mM azido-choline, and chased by further growth in the same medium without added azido-choline for the indicated duration.

Growth of the S. pneumoniae WT (black), ΔlytA (gray), ΔtacL (red) and ΔlytR (blue) strains in BHI (A) and C-medium 200 µM choline (B).