M. tuberculosis ClpX and FtsZ Figure 4. FtsZ interacts with ClpX in vivo. (A) BACTH assays showing FtsZ-ClpX interactions. clpX, ftsZ, ftsI and ftsQ fusions to T18 or T25 fragments of adenylate cyclase were cloned into various BACTH vectors and interactions were examined as described [35]. E. coli BTH101 recombinants bearing indicated combinations of plasmids were plated on MacConkey agar containing 1% maltose. Red colonies indicate strong positive interactions (Inset). FtsZ-FtsZ and GCN4-GCN4 served as positive controls and ClpX-FtsQ, FtsZ-FtsI, served as negative controls. b- galactosidase activity of the indicated recombinant strains was measured and is shown. Assay methodology is as described in the text. Mean 6 SD values from three independent experiments are shown. (B) M-PFC assay for detecting the interactions between FtsZ and ClpX proteins in M. smegmatis. Recombinant M. smegmatis strains producing FtsZ and ClpX fused to DHFR1, 2 or DHFR3 were grown on 7H11/Km/Hyg or 7H11/Km/Hyg/ Trim. Growth on Trim plates indicates strong positive protein-protein interaction. (C) Quantitation of M-PFC interactions using fluorescent alamar blue dye. M. smegmatis strains coexpressing the tested interaction pairs were propagated in 7H9 broth with 17.85 mg/ml trimethoprim and viability was determined using the fluorescent alamar blue assay [37]. Color change of alamar blue from non-fluorescent blue to a fluorescent pink indicates positive protein-protein interaction. Note GCN4[F1,2]/GCN4[F3] is a positive control whereas ClpX[F1,2]/GCN4[3] is negative control. The vector control strain DHFR[1,2]/DHFR[3] was used for background subtraction. Samples were analyzed in duplicate and mean 6 SD from two experiments are shown. doi:10.1371/journal.pone.0011058.g004 (supplementary Fig. S5, panel A; Table S2). Together, the above results validate the in vitro data showing interactions between the ClpX and FtsZ proteins. In vivo: M-PFC assay confirms ClpX and FtsZ interactions. We next examined the interactions between ClpX and FtsZ proteins in their native environment using the M-PFC assay [37]. This assay scores for trimethoprim (Trim) resistance due to the regeneration of functional murine dihydrofolate reductase (mDHFR) activity from two independent mDHFR protein fragment domains fused to two potential protein interaction partners. The strength of interaction is measured by monitoring the reduction of alamar blue in growth media containing Trim [37]. Accordingly, we fused full-length clpX and ftsZ to the 39 or 59 end of murine dhfr fragments 1–2 and 3 in bait and prey vectors, respectively, and expressed them from the tetracycline-inducible promoter in M. smegmatis (see methodology section and Table S2). The recombinant strains expressing Ptet::clpX-dhfr1,2 and Ptet::ftsZ-dhfr3 showed resistance to Trim (Fig. 4B), similar to those expressing ftsZ/ftsZ or gcn4/gcn4, the two positive controls. Negative control transformants expressing gcn4 and clpX did not show any growth on Trim plates (Fig. 4B). Evaluation of the extent of alamar blue reduction confirmed these results (Fig. 4C). Transformants expressing Ptet::clpXDN200-dhfr1,2 and Ptet::ftsZ-dhfr3 did not grow on Trim plates (Supplementary Fig. S5, panel B) corroborating the conclusion from BACTH data that the N-terminal 200 amino acids of ClpX are needed for interaction with FtsZ. PLoS ONE | www.plosone.org 5 July 2010 | Volume 5 | Issue 7 | e11058