Figure 1.
Structure of GBC and proposed scheme of GBC synthesis.
(A) The multiantennary GBC is shown linked to an N-acetyl muramic (NAM) moiety, a component of PG. (B) The figure depicts the first steps of GBC synthesis where GbcO is proposed to catalyze the transfer of UDP-GlcNAc to a lipid phosphate carrier.
Figure 2.
GbcO is required for GBC synthesis.
(A) IFM of bacteria harvested in stationary phase and labeled with anti-GBC serum and Wheat Germ Agglutinin lectin to detect GBC (red) or PG (green), respectively. The representative views show the GBC antigen exposed at the surface of NEM316 WT and complemented (ΔgbcOpTCVΩgbcO) strains but not at the surface of the ΔgbcO mutant. (B) Immunoelectron microscopy (IEM) of NEM316 WT, ΔgbcO mutant and complemented (ΔgbcO pTCVΩgbcO) strains. The subcellular localization of GBC was analyzed using IEM on thin sections (<100 nm) of frozen cells; labelled with anti-GBC serum and revealed with colloidal gold particles (black dots). Black dots are clearly visible on the periphery and septa of cells of WT and complemented strains; no labeling can be detected with the ΔgbcO strain. (C) Immunodetection of GBC in mutanolysin cell wall extracts obtained from cultures harvested in stationary phase. Cell wall extracts were treated with pronase, separated on SDS-PAGE, transferred on nitrocellulose and membrane incubated with anti-GBC serum. In this experiment, the GBC-associated signal appeared as a single band that was undetectable in ΔgbcO cell wall extracts. As a loading control, cell wall extracts before pronase treatment were probed with the biotinylated MalII lectin. (D) Analysis of muramic acid, phosphate, and rhamnose content of the cell wall of WT (black bars), ΔgbcO (light gray bars), and complemented (dark gray bars) strains harvested in stationary phase (see Text S1 in supporting information). For each compound the GC-MS analysis result is presented as a percentage of the WT value. Rhamnose, the main GBC sugar, was not detected in the cell wall of the ΔgbcO strain. Error bars represent ± S.E. of two independent experiments.
Figure 3.
Decreased growth rate and lack of tunicamycin sensitivity of ΔgbcO mutant.
(A) Growth curves of NEM316 WT (solid squares), ΔgbcO mutant (circles) and ΔgbcOpTCVΩgbcO (empty squares) strains. Cultures were performed in TH medium without antibiotics at 37°C in 96 wells plates in triplicate. Optical densities were recorded at 600 nm in a Tecan M200 apparatus with 5 sec agitation before measure. Average values of a typical experiment are presented. (B) Effect of various concentrations of tunicamycin on the growth rate of WT (solid squares), ΔgbcO (black circles) and ΔgbcOpTCVΩgbcO (empty squares) strains. Tunicamycin, a general inhibitor of UDP-GlcNAc:lipid phosphate carrier transferase activities, inhibits the growth of WT and complemented strains but not that of ΔgbcO mutant suggesting that GbcO carries this activity. Experiments were performed in triplicate and results are reported as a percentage of the growth rate in absence of tunicamycin. Error bars represent ± S.E. of triplicate experiments.
Figure 4.
GbcO functionally complement TarO of S. aureus.
(A) S. agalactiae ΔgbcO or S. aureus ΔtarO strains does not take Gram staining. In both species, the Gram staining and morphological phenotypes are restored by introduction of the plasmid pTCVΩgbcO carrying a functional S. agalactiae gbcO gene. (B) PAGE analysis of WTA extracted from S. aureus visualized with the alcyan blue-silver staining protocol. The gel shows the production of WTA in RN4220WT (first lane), the absence of WTA in the S. aureus ΔtarO strain (second lane) and the restoration of the WTA synthesis when the tarO deficiency is complemented in trans with the streptococcal gbcO gene (third lane). The arrowhead indicates the bromophenol blue migration front.
Figure 5.
Flocculation and aggregation phenotypes of ΔgbcO mutant.
(A) Overnight cultures showing the non-flocculating NEM316 WT and complemented strain (ΔgbcOpTCVΩgbcO) and the flocculating ΔgbcO mutant. (B) Phase contrast views illustrating the morphological switch from small individual chains to large bacterial clusters characteristic of ΔgbcO mutant (Scale bar, 5 µm).
Figure 6.
Electron microscopy imaging of NEM316 WT, ΔgbcO mutant, and complemented strains.
Bacteria were harvested in mid-log phase (OD600 nm = 0.5), fixed, and prepared as described in Supporting Materials and Methods (see Text S1) (A) Representative views of scanning electron microcopy analysis illustrating the morphological alterations (size, form, and cell division abnormalities) due to gbcO inactivation. (B, C) Transmission electron microscopy views of uranyl acetate stained thin cryosections at two magnifications (see scale bars). The presence of the pellicle (electron dense outer layer) at the surface of WT and complemented strains observed at the higher magnification is highlighted with black arrows. An open triangle depicts the equatorial ring (EqR), a zone of active peptidoglycan synthesis seen in almost all WT and complemented cells but absent in the ΔgbcO mutant cells.
Figure 7.
Alterations of the murein sacculus properties and of the PG synthesis in GBC-depleted cells.
(A,B) Cell lysis assays: exponentially growing cells were harvested and resuspended in PBS buffer (OD600 nm = 1) containing (A) 1 mg/ml lysozyme or (B) 20 units/ml mutanolysin. Lysis of NEM316 WT (black circles), ΔgbcO mutant (white squares), and ΔgbcOpTCVΩgbcO complemented (black diamonds) strains was recorded spectrophotometrically at 600 nm. Error bars represent ± S.E. of three independent experiments (C) Comparative analysis of the muropeptides resulting from mutanolysin-digested peptidoglycan (see Text S1) of NEM316 WT (upper panel), ΔgbcO (median panel), and complemented (bottom panel) strains. Muropeptides were separated by RP-HPLC and the peaks were collected and analyzed by MALDI-TOF. (D) Fluorescent vancomycin staining of exponentially growing NEM316 WT, ΔgbcO mutant and complemented strains. Fluorescent vancomycin (2 mg/ml) was added to exponential-phase cultures for 10 min at 37°C. Cells were harvested, transferred to glass slides, fixed, and observed by fluorescent microscopy as described in Supporting Materials and Methods (see Text S1).
Figure 8.
Fluorescent immunolocalization of the putative peptidoglycan hydrolase PcsB.
Exponentially growing NEM316 WT, ΔgbcO mutant and ΔgbcOpTCVΩgbcO complemented strains were harvested, transferred to glass slide, and fixed. IFM with anti-PcsB serum and DAPI staining were performed as described in Materials and Methods.