Imidazole. Final preparations of ClpX protein were dialyzed against the storage buffer (25 mM HEPES-NaOH pH 7.2, 0.1 mM EDTA, 1 mM DTT, 50 mM NaCl, and 10% glycerol). This ClpX refolding protocol yielded protein concentrations in the range of 7 to 8 mM and the protein obtained was used in various experiments described in this study. Our innumerable attempts to obtain a more concentrated ClpX protein stock were, however, not successful. Size exclusion chromatography of purified ClpX on Superdex 200 10/300 GL column revealed that the majority of ClpX was hexameric (data not shown). Unless otherwise mentioned, the molar concentrations of ClpX in the various assays are based on monomer molecular weight. FtsZ polymerization assays Light scatter assays. FtsZ polymerization was examined by the right angle light scatter assay [27]. Briefly, various concentrations of FtsZ were incubated without or with ClpX protein (molarity based on monomer concentration) in 50 mM MES buffer pH 6.5 containing 100 mM KCl, 5 mM MgCl2 and 1 mM ATP at 30uC. Polymerization was initiated by the addition of GTP to 1 mM. The change in light scatter was monitored in FP6500 fluorimeter at 400 nM using a 1 nM slit. The data points were collected every 5 s for 15 min and plotted using Excel. Buffer controls for polymerization assays contained ClpX storage buffer lacking ClpX protein and the scatter data obtained were used for data normalization. Sedimentation assay. Polymerization reactions containing FtsZ in a final volume of 50 ml were incubated at 30uC for 10 min and the polymerized FtsZ was collected by centrifugation at 80K for 10 min at 4uC. As needed, various concentrations of ClpX were added to the reaction. Supernatant and pellet fractions were separated on sodium dodecyl sulfate polyacrylamide (SDS-PA) gels, stained with Coomassie blue and scanned in a BioRad Molecular Imager using the QuantityOne software. Standard curve prepared with pure FtsZ protein was used for quantitating the FtsZ in the pellet fractions of the sedimentation assay. In some sedimentation assays 39-(N-Methyl-anthraniloyl)-29- deoxy-guanosine-59-triphosphate, GMPCPP (Jena Biosciences, Germany), was used at a final concentration of 1 mM [30,31]. As for light scatter assays, controls reactions contained an equivalent volume of ClpX storage buffer instead of the ClpX protein. Protein-protein interaction assays Pull-down assays: Experiments with pure proteins. Equimolar amounts of tag-free FtsZ and His-ClpX were mixed and incubated for 20 min, applied onto Ni-NTA resin in 1X binding buffer (50 mM Sodium Phosphate pH 7.8, 10% Glycerol, 5 mM 2-Mercaptoethanol, 150 mM NaCl, 0.1% CHAPS (3-[(3- Cholamidopropyl)dimethylammonio]-1- propanesulfonate) and 1% Nonidet P-40), washed 7X with 400 ml of 1X binding buffer containing 20 mM imidazole and eluted with 0.3 M imidazole. For assessing the strength of FtsZ-ClpX interactions, pull-down assays were performed in the presence of 0.2 or 0.5 M NaCl. Samples run on duplicate gels were probed with either a-FtsZ or a-ClpXBS antibodies as described above. We noted that the eluted FtsZ is not stoichimetric to His-ClpX, presumably due to loss during extensive washing steps. Experiments with cell lysates. E. coli strains expressing full length His-ClpX (pRD21) together with S-tag-FtsZ (pLR66; Table S2) under the T7 promoter were induced, clear cellular lysates were prepared in 1X binding buffer and applied to Ni-NTA resin. Bound proteins were eluted and loaded on NuPAGE gels and analyzed by immunoblotting as described above. M. tuberculosis ClpX and FtsZ Solid Phase assays. Four micrograms of purified ClpX or BSA was allowed to adsorb overnight onto the wells of a microtiter plate. After washing the excess unbound protein with phosphate- buffered saline containing 0.5% Tween-20 (PBST), the wells were blocked with 1% BSA in PBST for 1 hr at room temperature and incubated for 2 hrs with varying concentrations of FtsZ protein. Unbound FtsZ was removed and wells washed 5X with PBST. FtsZ bound to ClpX was detected with anti-FtsZTB antibodies by ELISA (AnaSpec). Wells with BSA served as negative controls. Mycobacterial protein fragment complementation (M- PFC) assay. Recombinant M. smegmatis expressing ftsZ- dhfr[F1,2]/ftsZ-dhfr[F3]; clpX-dhfr [1,2]/ftsZ-dhfr[3]; gcn4-dhfr[1,2]/ gcn4-dhfr[3]; clpX-dhfr[1,2]/gcn4-dhfr[3]; dhfr[1,2]/dhfr[3] or clpXDN200-dhfr[1,2]/ftsZ-dhfr[3] were selected on 7H11 agar plates containing Hyg and Km. Single colonies were patched on 7H11 plates with appropriate antibiotics in the presence/absence of various concentrations of Trimethoprim (Trim). Growth on Trim plates indicated interaction. Positive interactions were further confirmed by alamar blue assay [37]. Briefly, M. smegmatis strains expressing interacting proteins were cultured in 0.1 ml of 7H9 broth in 96-well, untreated, white polystyrene Nunc plates. All strains except ftsZ-dhfr [F1,2]/ftsZ-dhfr[F3] were grown with 10 ng/ml anhydrotetracycline (tet) for 3 h. Various concentrations of Trim were added for an additional 24 hrs at 37uC and finally incubated with 5 ml of alamar blue for 6 hrs. The plates were read in a Varian Cary Eclipse Fluorescence Spectrophotometer (Ex530 and Em590). M. smegmatis with gcn4- dhfr[1,2]/gcn4-dhfr[3] served as control. Bacterial two hybrid (BACTH) assay. BACTH system [35] kit was purchased from Euromedex and used as recommended. E. coli BTH101 recombinants with various combinations of plasmids (see Table S2) were selected on MacConkey agar supplemented with 100 mg/ml Amp and 50 mg/ml Km at 30uC for 24 to 36 h. For evaluating the strength of interaction, b-galactosidase activity was measured with cells grown in LB broth and samples were processed as recommended by the supplier. Reactions were started by the addition of 0.25 ml of 0.4% o-nitrophenol-b-galactoside (ONPG) and the tubes incubated at 28uC for 5 min or until a visible yellow color developed. Reactions were stopped by the addition of 0.5 ml of 1 M Na2CO3 and OD420 was recorded [35]. The enzymatic activity was defined as units per milliliter: 2006 [(OD420 of the culture - OD420 in the control tube)/minutes of incubation] 6 dilution factor. The specific activity of b-galactosidase is defined as units/mg dry weight bacteria and 1 unit corresponds to 1 nmol of ONPG hydrolyzed per min at 28uC. At least 5-fold higher b- galactosidase activity than that measured for BTH101 carrying a single gene and an empty vector was considered indicative of an interaction. E. coli BTH101 transformants obtained with pKT25- zip and pUT18C-zip served as positive controls for complementation (Table S2). RNA extraction and RT-qPCR RNA was prepared from exponential cultures of M. tuberculosis strains grown in vitro or in macrophages essentially as described [51]. For some experiments RNA was isolated from WT M. tuberculosis exposed to 20 mg ml21 cephalexin and 30 mg ml21 lithium clavulanate for 24 hrs. For extraction of RNA from intracellular bacteria, macrophage monolayers were separated 3 days after infection, washed, suspended directly in RNAzol and processed as described [5,51,52]. Extraction of total RNA and synthesis of complementary strand DNA from mRNA specific to 16S rRNA and clpX using reverse transcription primers and iScript kit (BioRad) were as described [5,51,52]. Quantitative real PLoS ONE | www.plosone.org 12 July 2010 | Volume 5 | Issue 7 | e11058