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24 PROCEEDINGS OF INTERNATIONAL SCIENTIFIC CONFERENCE ON APPLIED BIOTECHNOLOGYonto 7H11 agar just prior to addition of resazurin and Tween 80 to the test wells. The cultures were washed with phosphatebuffered saline (PBS) once to remove test compounds prior to plating. TheMBCwas defined as the lowest concentration reducing CFU by 99% relative to the zero time inoculum. MBCs for nonreplicating (NR) cultures were determined by subculture from the low-oxygen recovery assay (LORA) (15) plates onto 7H11 agar. M. tuberculosis isolates had been exposed for 10 days to test compounds in a low-oxygen environment prior to subculture. The MBC was defined as the lowest concentration reducing CFU by 99% relative to the zero time inoculum. The reported MBC results were from one representative experiment of three that were independently performed.2.4. Identification of the target protein by generation of ecumicin resistant M. tuberculosis mutants Selection of ecumicin-resistant strains of M. tuberculosis was carried on the 7H11 agar plates containing ecumicin at 1X, 2X, 4X, and 8X MIC (determined on 7H11 agar) against M. tuberculosis. A total of 109 CFU of M. tuberculosis H37Rv were plated onto 7H11 agar plates containing 1X MIC ecumicin and incubated at 37%u00b0C for 3 to 4 weeks. The colonies from the selection plates were phenotypically characterized and the genome were sequenced to compared with wild type M. tuberculosis. 2.5. Effect of ecumicin and rufomycin on the activity of ClpC1/P1/P2The effect of ecumicn and rufomycin on the activity of ClpC1/P1/P2 was determined by the measurement of the ATPase activity and proteolytic activity according to the previous report [7]. 2.6. Toxicity of ecumicin and rufomycin in mammalian cellsCompounds were tested for cytotoxicity using Vero cells (ATCC CRL-1586). After 72 h of exposure, viability was assessed