time PCR (TaqMan chemistry) was carried out in a BioRad ICycler using the Taq DNA polymerase (NEB). The calculated threshold cycle (Ct) value for clpX was normalized to the Ct value for 16S rRNA and the fold expression was calculated using the formula: Fold change = 2D(DCt) [51]. No RT RNA samples were included as negative controls. Expression data are average from 3 independent RNA preparations, each reverse transcribed and quantitated by real time PCR in triplicate. Real-time PCR conditions: initial activation at 95uC for 3 minutes; followed by 40 cycles of 95uC for 10 sec and 55uC for 30 seconds. Supporting Information Figure S1 FtsZ assembly is not affected by RecA: Light scatter assay for FtsZ polymerization in the presence or absence of RecA. Reactions containing 7.5 mM FtsZ mixed with storage buffer alone or storage buffer containing 3 mM RecA were initiated with GTP to a final concentration of 1 mM and followed for 15 minutes. Neither the rate and nor the extent of FtsZ polymerization was affected by RecA. Found at: doi:10.1371/journal.pone.0011058.s001 (0.17 MB TIF) Figure S2 ClpX inhibition of FtsZ assembly: FtsZ polymeriza- tion was examined by the sedimentation assay. Reactions contained varying amounts of FtsZ polymerized without (A) or with a fixed concentration of ClpX at 2 mM (B). Polymerized FtsZ was collected by centrifugation and supernatant (S) and pellet (P) fractions were loaded on SDS-PA and visualized by Coomassie staining. Found at: doi:10.1371/journal.pone.0011058.s002 (0.15 MB TIF) Figure S3 Addition of ClpX interferes to the preformed FtsZ polymers: Sedimentation assay was performed as described above with 5.4 mM FtsZ and 2 mM ClpX. For reactions 2 and 3, ClpX or storage buffer, respectively, was added at the same time as FtsZ. For reactions 4 and 5, FtsZ polymers were preformed for 5 minutes and then ClpX or storage buffer was added. Some fraction of ClpX in the pellet fractions is presumably due to its oligomerization/aggregation [20]. Found at: doi:10.1371/journal.pone.0011058.s003 (0.21 MB TIF) Figure S4 Characterization of ClpX-FtsZ interaction: (A) Strength of FtsZ-ClpX interaction. Pull-down assay using His- ClpX and tag-free FtsZ was performed in buffers containing 0.2 M and 0.5 M NaCl (see materials and methods for details). Load (L), wash (W) and elution (E) fractions were analyzed by immunoblotting. (B) FtsZ-ClpX complex isolated from cell free lysates. Cellular lysates from E. coli strain expressing his-clpXTB and ftsZTB-S-tag were loaded on NiNTA column and pull-down assay performed as described in the text. Load (L), wash (W) and elution (W) fractions were analyzed by immunoblotting as described above. Found at: doi:10.1371/journal.pone.0011058.s004 (1.41 MB TIF) References 1. Gandhi NR, Moll A, Sturm AW, Pawinski R, Govender T, et al. (2006) Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet 368: 1575–1580. 2. Smith I (2003) Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin Microbiol Rev 16: 463–496. 3. Margolin W (2000) Themes and variations in prokaryotic cell division. FEMS Microbiol Rev 24: 531–548. 4. Romberg L, Levin PA (2003) Assembly dynamics of the bacterial cell division protein FTSZ: poised at the edge of stability. Annu Rev Microbiol 57: 125–154. 5. Chauhan A, Lofton H, Maloney E, Moore J, Fol M, et al. (2006) Interference of Mycobacterium tuberculosis cell division by Rv2719c, a cell wall hydrolase. Mol Microbiol 62: 132–147. M. tuberculosis ClpX and FtsZ Figure S5 ClpXDN200 does not interact with FtsZ in BACTH (A) and MPFC (B) assays. ClpXDN200 was cloned in BACTH or MPFC vectors (Table 1) and used along with respective FtsZ constructs in the two hybrid assays as described for figures 4A and 4B. Found at: doi:10.1371/journal.pone.0011058.s005 (0.24 MB PDF) Figure S6 ClpX expression in M.tuberculosis antisense ClpX strain. Antisense ClpX strain was propagated in Middlebrook 7H9 broth, RNA was extracted and clpX mRNA levels were determined by quantitative real time PCR. clpX mRNA levels were normalized to 16S rRNA and data are expressed with respect to levels in WT strain grown in broth. Mean 6 SD from two independent experiments are shown. Found at: doi:10.1371/journal.pone.0011058.s006 (0.14 MB TIF) Figure S7 Overproduction of ClpX does not affect intracellular FtsZ levels. FtsZ levels in M. tuberculosis clpX overexpression (A) and underexpression (B) strains were examined by quantitative immunoblotting. M. tuberculosis cellular lysates were resolved on a 10% NuPage gel, transferred to PVDF membrane and probed with a-FtsZ or a-sigma70 antibodies. FtsZ and SigA bands were quantitated using the volume analysis function of the QuantityOne Software. Found at: doi:10.1371/journal.pone.0011058.s007 (0.24 MB TIF) Figure S8 The viability of M. tuberculosis merodiploids overpro- ducing ClpX (ClpX) and depleted in ClpX (asClpX). Broth-grown strains were spread on the plates containing 100 ng/ml anhy- drotetracycline. Grown colonies were counted and data plotted using Microsoft Excel. Mean 6 SD from three independent experiments are shown. Found at: doi:10.1371/journal.pone.0011058.s008 (0.13 MB TIF) Table S1 Found at: doi:10.1371/journal.pone.0011058.s009 (0.07 DOC) Table S2 Found at: doi:10.1371/journal.pone.0011058.s010 (0.11 DOC) MB MB Acknowledgments We thank the members of the Madiraju and Rajagopalan labs for helpful discussions and Krishna Sarva, Maha Al Zayer, Hava Lofton and Ashwini Chauhan for technical assistance. We also thank Dr. Ulf Gerth for the kind gift of antibodies to B. subtilis ClpX and Dr. A. Steyn for the MPFC system. Author Contributions Conceived and designed the experiments: RD JD MVVM MR. Performed the experiments: RD MK PP MZ AB MM IV MVVM MR. Analyzed the data: RD MK PP MZ AB MM JD MVVM MR. Wrote the paper: RD MVVM MR. 6. Dziadek J, Rutherford SA, Madiraju MV, Atkinson MA, Rajagopalan M (2003) Conditional expression of Mycobacterium smegmatis ftsZ, an essential cell division gene. Microbiology 149: 1593–1603. 7. Errington J, Daniel RA, Scheffers DJ (2003) Cytokinesis in bacteria. Microbiol Mol Biol Rev 67: 52–65, table of contents. 8. Del Sol R, Mullins JG, Grantcharova N, Flardh K, Dyson P (2006) Influence of CrgA on assembly of the cell division protein FtsZ during development of Streptomyces coelicolor. J Bacteriol 188: 1540–1550. 9. Frees D, Savijoki K, Varmanen P, Ingmer H (2007) Clp ATPases and ClpP proteolytic complexes regulate vital biological processes in low GC, Gram- positive bacteria. Mol Microbiol 63: 1285–1295. 10. Gottesman S (2003) Proteolysis in bacterial regulatory circuits. Annu Rev Cell Dev Biol 19: 565–587. PLoS ONE | www.plosone.org 13 July 2010 | Volume 5 | Issue 7 | e11058