Figure 3.

Rv1717 expressed in M. smegmatis localizes to the CW and prefers the poles. (A) Western blot analysis of equal amounts (30 μg protein) of whole cell lysate (WCL), CW, cell membrane (CM), and cytosolic (CY) fractions of Msm^Rv1717. Rv1717 which was expressed with a C-terminal 6 × His tag was detected by anti-6 × His tag antibody. Lipoarabinomannan (LAM) and Hsp65 were detected using anti-M. smegmatis LAM monoclonal antibody NR-13798 (BEI Resources, Manassas, VA) and anti-Hsp65 monoclonal antibody, respectively. LAM was used as marker for the cell wall, while Hsp65 was used as major cytosolic marker. (B) A monomeric RFP was expressed in M. smegmatis either alone (Msm^rfp) or as a C-terminal fusion to Rv1717 (Msm^Rv1717-rfp). Both the strains were stained with BacLight Green⁽™⁾ fluorescent dye and subjected to confocal microscopy. Individual fluorescence of RFP and BacLight Green⁽™⁾ were recorded and merged. Unfused RFP fluorescence was distributed uniformly across the bacterium, while Rv1717-RFP fluorescence was localized more toward one of the poles.