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. 2009 Mar 12;28(9):1208–1219. doi: 10.1038/emboj.2009.61

Figure 3.

Figure 3

Cell curvature is the result of a peptidoglycan growth differential around the cell circumference. (A) Strains lacking crescentin (CB15N ΔcreS), with wild-type levels of crescentin (CB15N), or overproducing crescentin (CB15N/pJS14creS). Bar, 2 μm. (B) DAPI staining (blue) overlaid with anti-crescentin immunofluorescence (red) of wild-type CB15N cells and cells overproducing crescentin (CB15N/pJS14creS). The line scan gives the fluorescence values along the yellow lines in the micrographs. Bar, 2 μm. (C) Transmission EM of uranyl acetate-contrasted peptidoglycan sacculi isolated from the three differently curved strains in panel A. As observed earlier (Poindexter and Hagenzieker, 1982), the isolated sacculi contained polyhydroxybutyrate (PHB) granules (arrow). Bar, 1 μm. (D) After digestion of the sacculi, muropeptides were subjected to HPLC to determine their relative composition. For the hypercurved CB15N/pJS14creS strain (CJW1430), which carries a plasmid encoding chloramphenicol resistance, we used a strain containing the empty vector (CB15N/pJS14; CJW1534) as a control; this strain exhibits wild-type curvature (data not shown). Major peaks 1–6 are proposed to have the following structures based on elution similarity to known E. coli muropeptides and to the published muropeptide profile from C. crescentus (Markiewicz et al, 1983): (1) disaccharide tetrapeptide, (2) disaccharide pentapeptide(Gly5), (3) disaccharide pentapeptide, (4) bis-disaccharide tetrapentapeptide(Gly5), (5) bis-disaccharide tetratetrapeptide and (6) bis-disaccharide tetrapentapeptide. (E) Schematic of how a cell length gradient along the long axis of the sidewall constitutes cell curvature. The sidewall displays a smooth gradient of lengths from the longest line, at the middle of the outer curvature (line a), through line b, at the middle of the gradient, to the shortest line, at the middle of the inner curvature (line c). (F) Transmission EM of peptidoglycan sacculi from a creS-overexpressing strain (CB15N/pJS14creS; CJW1430) and a ΔcreS strain carrying an empty vector (CB15N ΔcreS/pJS14; CJW2855) chased for 90 min after growth in D-Cys to reveal regions of new peptidoglycan insertion. D-Cys residues were biotinylated, then the sacculi were labelled with anti-biotin and gold-coupled anti-rabbit antibodies. Areas devoid of gold label show regions where new peptidoglycan material was incorporated during the chase. The antibodies used to detect the biotinylated D-Cys residues appeared to clump together. Measured lengths of the cleared region on each side of the sacculus are shown with broken lines. PHB, polyhydroxybutyrate granules. Bar, 0.5 μm.