Figure 2.

Transmission electron microscopy (TEM) study of cell walls in thin sections of Synechococcus 7002. (a) Wild type (WT). (b) cesA mutant. (c) WT cells incubated in a cellulase solution (200 μg/ml in the presence of 2 mM EDTA) for 30 min at 37 °C. The arrow in a indicates the electron-dense layer, and the arrows in b and c indicate its absence. Note that there is more cell-surface-associated glycocalyx material in a than in b or c. (d, e) TEM images of thin sections of Synechococcus 7002 cells stained with ruthenium red showing the changes in the cell wall and glycocalyx of WT (d) and a cesA deletion mutant (e), respectively. The black arrows indicate the possibly laminated, CesA-dependent cellulose layer, and the white arrows indicate the thin peptidoglycan layer. Although it was easier to see on lower-magnification images (data not shown), these images show less ruthenium red staining for the cesA mutant (e), more staining for the WT (d), and much more staining for a strain overproducing CesA (see Figure 4c). These data show that the cesA mutant had the least surface-associated polysaccharides and that the strain overproducing CesA had the most. An electron-opaque, laminated layer near the outer membrane in WT (black arrows in d) was missing in the cesA mutant (e). This layer apparently corresponds to the cellulose layer observed between the peptidoglycan (white arrows in d and e) and outer membrane layers in thin sections in a. (f) Confocal fluorescence microscopy showing spectrally deconvoluted, Z-stack image of Synechococcus 7002 cells expressing CesA–YFP. This image shows the absence of YFP fluorescence in the cytoplasm and thylakoid membranes, which could easily be visualized by fluorescence from chlorophyll a (data not shown). Scale bars, 100 nm (a-e) and 2 μm (f).