Fig. 10.

Dual-protein 2D and 3D-SIM IFM showing similar localization of MreC and MltG in equators and septa of dividing pneumococcal cells. Strain IU7580 (mreC-L-FLAG³ mltG-HA) was grown to mid-exponential phase in BHI broth and processed for dual-protein 2D and 3D-SIM IFM with DAPI labeling of DNA as described in Experimental procedures. (A) Representative field of 2D-IFM images with phase images. (B) Representative 3D-SIM IFM images of cells at division stages 2–4. DNA (DAPI) staining is pseudo-colored white or blue in column 1 or 5, respectively. MreC or MltG are pseudo-colored green or red respectively, and overlapping signal is colored yellow. Images in the first row of each panel were captured in the XY plane, and the second-row images were obtained by rotating a section of the midcell region around the X or Y axis. Images are representative of >20 examined cells in different division stages from one experiment. (scale bar = 1 μm). (C) Averaged 2D IFM images and fluorescence intensity traces. Cells were binned into division stages 1–4, and images from the indicated number of cells (n) were averaged and quantified as described for Figure 9, with the addition of the DNA nucleoid (DAPI) locations (row 2). (D) Scatter plot of labeled MreC width versus MltG width at midcell equators and septa of cells at division stages 1 to 4 in (C) (see Fig 9). NS, difference between the two widths was not significant (p>0.05). Data points in (C) and (D) were obtained from one experiment in which >100 cells were analyzed. Similar 3D-SIM IFM images and quantitation of 2D IFM images were obtained for strain IU7582 (mreC-L-FLAG³ mltG-Myc), which expresses MltG-Myc instead of MltG-HA (data not shown).