results suggest that altered levels of ClpX do not affect intracellular M. tuberculosis FtsZ levels. We also found that M. tuberculosis sense and antisense clpX strains showed a moderate reduction in growth (Fig. 6B) and viability (supplementary Fig. S8). These defects can possibly be attributed to interruptions in the ClpX chaperone and protein degradation functions due to reduction in ClpX levels. The merodiploids overexpressing clpX were elongated, with an ,50% increase in cell length (3.3 mm 60.25) as compared to WT (2.2 mm 60.58; Fig. 6C, compare brightfield panels WT and ClpX). Visualization of nucleoid staining with DAPI revealed elongated cells with multiple nucleoids (Fig. 6C, see arrows in ClpX panels). In contrast, the WT cells had one or two distinct nucleoids per cell (Fig. 6C, WT panels). A decrease in ClpX levels caused only a modest 7% increase in cell length (2.36 mm 60.11), and the DAPI staining patterns were similar to WT (Fig. 6C, asClpX panels). To determine whether the observed morphological changes associated with ClpX overproduction were due to defects in FtsZ ring assembly, we created an M. tuberculosis strain expressing Ptet::ftsZ-gfp and Ptet::clpX, visualized FtsZ structures and compared the results with the control strain expressing Ptet::ftsZ-gfp. Incubation of the control strain with 5 ng of anhydrotetracycline for 24 h revealed distinct bright FtsZ-structures at the midcell position (Fig. 7D, see arrow 1) and in some cases, at the cell poles (Fig. 7D, arrow 2). The midcell rings accounted for 16.861.4% of cells (n = 627). The polar FtsZ localization, believed to be a remnant from the previous cell division, accounted for approxi- mately 6.5% of cells. These results are consistent with our earlier published data [26]. In contrast, M. tuberculosis cells expressing Ptet::ftsZ-gfp and Ptet::clpX were elongated, with an average cell length of 5.561.0 mms (Fig. 7, compare panel A with C). Unlike the WT cells (2.160.6 mms), many elongated cells were devoid of bright Z-rings and had diffuse fluorescence along the entire cell length. Approximately 10.862.2% of cells had less-fluorescent M. tuberculosis ClpX and FtsZ midcell Z-rings, which tended to bleach quickly (see discussion). Polar FtsZ structures were rarely present (Fig. 7, panel F), and some elongated cells had aberrant FtsZ-GFP localization (Fig. 7, panel F, see arrowhead). The average length of FtsZ-GFP ring containing cells increased by 52% under ClpX overproduction conditions. Increased cell length and diffuse midcell Z-rings are consistent with the idea that increased ClpX levels delay FtsZ assembly and cell division in M. tuberculosis. The antisense clpX cells showed FtsZ structures at midcell sites and cell poles, as in the WT strain, but these structures were less vibrant (Fig. 7, asClpX panels). Since all strains were grown and processed under similar conditions, we believe that the diffuse fluorescence of FtsZ-GFP rings in asClpX strain is not an artifact of microscopy. Given that clpX is an essential gene [42], lowered ClpX levels might therefore affect the activities of other proteins and/or other potential regulators of cell division. ClpX-CFP localizes to the cell poles and to midcell sites and colocalizes with FtsZ Visualization of fewer and fainter FtsZ-structures under ClpX overproduction conditions suggested that fewer FtsZ molecules were in the midcell Z-ring. This could be due to sequestration of FtsZ protomers by ClpX thereby limiting the available pools of FtsZ for polymerization. Alternatively, ClpX association with FtsZ in the midcell Z-rings could lead to the latter’s disassembly. Both possibilities are not mutually exclusive. To address the latter possibility, we attempted to visualize ClpX structures by creating an M. tuberculosis strain expressing Ptet::clpX-cfp and determining whether ClpX colocalizes with FtsZ. The ClpX-CFP structures in M. tuberculosis were less distinct and generally more diffuse (Fig. 8, panel B). It is presumed that the fluorescence quenching is due to the paraformaldehyde fixation step required in processing samples with the virulent strain. To visualize ClpX without paraformal- Figure 7. Altered ClpX levels affect FtsZ ring formation in M. tuberculosis. M. tuberculosis Ptet::ftsZ-gfp strains producing either elevated (Ptet::clpX) (ClpX) or decreased (asClpX) (Ptet::asclpX) levels of ClpX were examined by fluorescence microscopy. Control strain is M. tuberculosis Ptet::ftsZ-gfp (WT). These strains were induced with 5 ng/ml anhydrotetracycline for 24 h and cells were visualized by microscopy. A, B, and C are brightfield images and D, E, and F are the corresponding fluorescence images. Arrows and arrowheads represent FtsZ-GFP structures. Arrow 1 and arrow 2 represent midcell and polar localizations, respectively, whereas arrowhead shows aberrant localization. Bar - 5 mm. doi:10.1371/journal.pone.0011058.g007 PLoS ONE | www.plosone.org 8 July 2010 | Volume 5 | Issue 7 | e11058