M. tuberculosis ClpX and FtsZ Figure 6. Overproduction of ClpX delays growth and cell division of M. tuberculosis. (A) Intracellular levels of ClpX in WT M. tuberculosis: Ten and twenty micrograms of clear protein lysates from WT M. tuberculosis were resolved on a 10% NuPAGE gel, transferred to a PVDF membrane and probed with a-ClpXBS antibodies. Various known concentrations of recombinant ClpX were also resolved on the same gel and processed under the same conditions. ClpX bands were quantitated by volumetric analysis using QuantityOne software. A standard curve shown in the inset is prepared from pure protein standards and was used to determine the amount of ClpX in the lysates (see materials and methods for details). (B) Growth of M. tuberculosis clpX strains. M. tuberculosis strains with elevated (ClpX) or decreased ClpX levels (asClpX) were grown in broth in the presence of 100 ng/ml anhydrotetracycline. The M. tuberculosis H37Rv strain served as the control strain. Growth was monitored by absorbance at 600 nm. Mean 6 SD from three independent experiments are shown. Inset - ClpX levels in M. tuberculosis clpX strains. Cellular lysates (8 mg) were resolved on a 10% NuPAGE gel, transferred to a PVDF membrane and probed with a-ClpX or a-SigA antibodies. Bands were quantified using QuantityOne Software in a BioRad Imager. ClpX levels were normalized to SigA and fold change with respect to WT strain was determined (see text). (C) Effect of altered ClpX levels on M. tuberculosis cell and nucleoid morphology. Exponential cultures of ClpX sense and antisense M. tuberculosis strains were induced with 100 ng/ml anhydrotetracycline for 48 hrs. Cells were stained with DAPI as described in the text and examined by fluorescence microscopy. WT - wild type, ClpX - ClpX overproducing strain and asClpX - ClpX antisense strain. Images were optimized using Adobe Photoshop 7.0. Bar - 5 mm. Arrows - multiple nucleoids. doi:10.1371/journal.pone.0011058.g006 the reduction in ClpX levels in this strain (data not shown). As an alternative, we evaluated clpX transcript expression by qRT-PCR (supplementary Fig. S6). The clpX transcript expression relative to the housekeeping gene 16S rRNA was decreased by 12-fold in the asClpX strain (supplementary Fig. S6). We next examined if intracellular levels of FtsZ were altered in cells producing altered levels of ClpX. The FtsZ levels were neither decreased under ClpX overproduction condition nor increased under ClpX underproduction condition, i.e. in asClpX strain (supplementary Fig. S7, panels A and B). Together, these PLoS ONE | www.plosone.org 7 July 2010 | Volume 5 | Issue 7 | e11058