M. tuberculosis ClpX and FtsZ Figure 8. Localization of ClpX and FtsZ proteins in M. smegmatis and M. tuberculosis. (A) M. smegmatis, (B) M. tuberculosis. Recombinant mycobacterial strains producing ClpX-CFP were propagated in broth with 5 ng/ml anydrotetracycline for 1 h (M. smegmatis) or 24 h (M. tuberculosis). Brightfield and respective fluorescence images are shown. Arrow - midcell localization of ClpX; arrowhead - polar localization. (C) Colocalization of ClpX and FtsZ. M. smegmatis expressing Pami::ftsZ-gfp and Ptet::clpX-cfp was grown in broth containing 5 ng/ml anydrotetracycline and acetamide at a final concentration of 0.2% for 1 h. The levels of ClpX-CFP and FtsZ-YFP induced under these conditions did not lead to overproduction associated phenotypes. Individual brightfield and fluorescent images are shown. Bar - 5 mm. FtsZ-YFP - pseuocolored green, ClpX-CFP - pseudocolored red and their colocalization as yellow spots. Split arrow - colocalization of ClpX and FtsZ proteins. doi:10.1371/journal.pone.0011058.g008 dehyde fixation, we expressed Ptet::clpXTB-cfp in M. smegmatis, a rapidly growing and nonpathogenic mycobacterial member. Visualization of ClpX-CFP in M. smegmatis revealed distinct localization at the cell poles in the majority of cells and at midcell sites in a small fraction of cells (Fig. 8A). When quantified, these structures corresponded to 82.566.5% at the cell poles and 1162% at midcell sites (n = 124). Next, to address whether ClpX- CFP colocalizes with FtsZ, we transformed Pami::ftsZ-yfp into the M. smegmatis strain expressing Ptet::clpX-cfp and visualized ClpX and FtsZ following a 1-h induction with 5 ng of anhydrotetracy- cline and 0.2% acetamide. As expected, FtsZ-YFP structures were evident at midcell sites and poles (Fig. 8C, see panel FtsZ-YFP), similar to our earlier published data [34]. ClpX-CFP showed similar localization (Fig. 8C, see panel ClpX-CFP). The FtsZ-YFP structures were faint and tended to bleach easily. Even with this limitation, we were able to see a colocalization pattern (see Fig. 8, panel C Merge). ClpX-FtsZ colocalization was noted at the cell poles in 1262.8% of cells and at midcell sites in 3.562.1% of cells (n = 110). Discussion The results presented in this study show that M. tuberculosis ClpX interacts with FtsZ and a possible consequence of these interactions is modulation of Z-ring assembly at midcell sites in vivo and interference with GTP-dependent FtsZ polymerization activity in vitro (Figs. 7, 8 and 2). The critical concentration of FtsZ polymerization is increased in the presence of ClpX (Fig. 2B). The intracellular levels of FtsZ are, however, unaffected upon ClpX overproduction. These results, combined with the observa- tion that the intracellular ratio of ClpX to FtsZ is 1:2, are consistent with a notion that in M. tuberculosis ClpX interacts with FtsZ at stoichiometric levels. Such an interaction could lead to sequestration of the available pools of FtsZ required for catalyzing Z-ring assembly and cell division in M. tuberculosis. FtsZ localization at midcell sites and cell poles and the potential regulators affecting Z-ring assembly have been extensively studied in E. coli, B. subtilis and a handful of other bacteria (reviewed in [7]). In contrast to the situation in other bacteria, only a few proteins affecting Z-ring assembly in M. tuberculosis have been identified. One of these is ChiZ (Rv2719c), a cell wall hydrolase and a negative regulator of cell division [5]. The ChiZ protein has been shown to modulate Z-ring assembly by localizing its activity to midcell sites and cell poles, the same locations where FtsZ is found [5]). The ChiZ protein, however, does not interact with FtsZ; hence, the interference effects are rather indirect. The second protein is FtsW, a bona fide FtsZ-interacting partner [38] hypothesized to function as a positive regulator of Z-ring assembly PLoS ONE | www.plosone.org 9 July 2010 | Volume 5 | Issue 7 | e11058