with other proteins. And while colocalization of two proteins is not a direct indication of interaction, these data combined with bacterial two-hybrid and MPFC assays suggest that increased ClpX localization at the cell poles helps to disassemble the remnants of FtsZ polymers from new cell poles and to restrict polar ring formation. It is known that the MinCDE system is critical for targeting FtsZ to midcell sites [3]. The M. tuberculosis genome lacks identifiable homologs of the MinCDE system [15]. Perhaps in the absence of the MinCDE system, ClpX-FtsZ interactions could restrict Z-ring assembly to midcell sites in M. tuberculosis. Materials and Methods Strains and bacterial growth conditions E. coli strains were grown in Luria-Bertani (LB) broth or LB agar supplemented with ampicillin (Amp, 50 mg ml21) or kanamycin (Km, 50 mg ml21) or hygromycin (Hyg, 200 mg ml21). M. tuberculosis strains were propagated in Middlebrook 7H9 broth supplemented with OADC (oleic acid, albumin, dextrose, catalase with sodium chloride), 0.05% tween 80 and appropriate antibiotics (Km at 25 mg ml21; Hyg at 50 mg ml21). When needed, strains were plated on 7H10 agar (BD biosciences) plates containing OADC and appropriate antibiotics. Growth was monitored by measuring absorbance at 600 nm and viability by determining the colony forming units on Middlebrook 7H10 agar plates without or with anhydrotetracycline. Recombinant M. smegmatis or M. tuberculosis strains with an additional copy of extrachromosomal or integrated clpX are referred to as merodiploid strains. Integrating and replicating plasmids were confirmed by bead- beating followed, respectively by PCR or restriction digestion of the recovered DNA as described [5,6]. Molecular techniques Oligonucleotide primers used in this study are listed in Supplementary Table S1. The clpX coding region was cloned in the sense orientation using primers ClpX-PacI and ClpX-SwaI or antisense orientation using asClpX-PacI and asClpX-SwaI under the inducible tetracycline (tet) promoter in pLR52, a replicating E. coli - Mycobacterium shuttle vector (Tables S1 and S2). As needed, cfp was cloned in-frame downstream of the clpX gene using CFP-XbaI and CFP-SwaI primers (Table S1). For some experiments, ftsZ-gfp or ftsZ-yfp fusions were cloned downstream of the tet or amidase promoter in an integrating vector (Table S2). Plasmids pUAB100 and pUAB200 (kind gift from Dr. Adries Steyn, UAB) were used for creating pMR118 and pMR119 (Table S2). For overproduc- tion and purification of the recombinant ClpX protein, the clpX coding region was cloned in pET-19b vector (Novagen) and the recombinant His-ClpX fusion protein was purified on Ni-NTA affinity columns. A truncated ftsQ gene coding for the N-term 100 aa was also cloned in pET-19b vector using primers FtsQ-NdeI and FtsQN100-BamHI (see T1-S) and the recombinant protein was purified. All constructs were verified by sequencing. Recombinant plasmids were used to transform E. coli, M. smegmatis or M. tuberculosis as described [50]. Constructs used for Bacterial Adenylate Cyclase-based Two-Hybrid (BACTH) and Mycobacte- rial Protein Fragment Complementation (M-PFC) assays were produced using the primers described in supplementary Table S1. For M-PFC assay, ftsZ and clpX were cloned under the inducible tet promoter as expression from the constitutive hsp60 promoter caused growth defects in the host strain (Table S2; [50]). Recombinant M. tuberculosis and M. smegmatis strains were confirmed by PCR of genomic DNA or restriction digestion of recovered plasmid DNA as described [50]. M. tuberculosis ClpX and FtsZ Western blotting Intracellular lysates of M. tuberculosis strains were prepared, the ClpX and FtsZ levels were quantitated by immunoblotting and normalized to SigA as described [26]. Cell lysates were resolved on NuPAGE polyacrylamide gels, transferred to PVDF membrane and probed with anti- FtsZ [6], anti- His or anti- B. subtilis ClpX antibodies (a-ClpXBS, kind gift from Dr. Ulf Gerth, Germany), diluted to 1:1000, 1:2000 and 1:25,000, respectively. Anti-s70 antibodies (Neoclone Biotechnology, Madison, WI) that recognize SigA and anti- His-tag antibodies (Genscript) were used as recommended by the manufacturer [26]. Immunoblots were processed with the ECF Western blotting kit (GE life sciences, Piscataway, NJ) and scanned on a Bio-Rad Molecular Imager. For quantitative immunoblotting, known amounts of FtsZ or ClpX were quantified by volume analysis function of the QuantityOne software and standard curves were plotted. Lysates loaded on the same gel as the standards were then quantitated using the standard curve. Microscopy Wild type (WT) and recombinant M. tuberculosis strains were grown for various periods of time with shaking, harvested by centrifugation, washed in phosphate buffered saline, fixed in 4% paraformaldehyde (PAF), and stored at 4uC until further use. DAPI staining was done after washing off the PAF and incubating with 0.25 mg/ml DAPI for 15 min at RT. Excess stain was removed by centrifugation, cells were resuspended in PBS and viewed immediately. Brightfield and fluorescence imaging was done on a Nikon Eclipse 600 microscope equipped with a 100X Nikon Plan Fluor oil immersion objective with a numerical aperture of 1.4. The following filter sets were used for microscopy: GFP - standard fluorescein isothiocyanate filter set (Ex484–499, Em459–509, Chroma Technology); CFP (Ex426–446, Em460–500, Nikon); YFP (Ex490–510, Em520–550, Nikon); DAPI- DAPI filter set (Ex325–375, Em435–485, Chroma Technology). All images were acquired with a Photometrics Coolsnap ES camera and Meta- morph 6.2 imaging software (Universal Imaging Corporation) and optimized with Adobe Photoshop 7.0. It is noteworthy that bright focused localization was obtained in some strains but not in others. Since all strains were grown and processed under similar conditions, we tend to favor an argument that diffuse signals in particular strains are related to the experimental context and are not an artifact of microscopy. Purification of FtsZ and ClpX His-tag fusion recombinant proteins FtsZ (pSAR1), FtsZG103S (pRR3) or FtsZD210G (pRR7), FtsZD374-76A (pLR12), FtsZDC21 (pMK13) were purified under soluble conditions on Ni-NTA columns as described [28]. As needed, His-tag was removed by incubating the recombinant proteins with thrombin for 8 h at 4uC and the tag-free protein was recovered in the flow-through fractions on Ni-NTA column. The ClpX (pRD21) protein was prepared by isolation and subsequent solubilization of inclusion bodies using 8 M Urea. The resulting ClpX was then refolded by addition of the protein to a volume of refolding buffer containing 0.4 M L-arginine and 2% glycine to yield a final protein concentration of 0.5 mg/ml. The refolded ClpX was dialyzed and applied to a DEAE Sephacel column in column buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl and 10% glycerol), washed with column buffer, and eluted with the same buffer containing 1 M NaCl. Peak ClpX fractions were pooled, dialyzed and applied to a Ni-NTA column, washed, and eluted with 1X binding buffer (50 mM Sodium Phosphate pH 7.8, 500 mM NaCl, 10% Glycerol, 5 mM 2-Mercaptoethanol) containing I M PLoS ONE | www.plosone.org 11 July 2010 | Volume 5 | Issue 7 | e11058