Figure 1.
Identification of a salicylate-inducible promoter in M. tuberculosis.
A. The genetic organization of Rv0561c and Rv0560c in M. tuberculosis. The regions tested for promoter activity are indicated as PRv0561c, and PRv0560c. B–E. Promoter activity was measured in M. tuberculosis transformants grown under aerobic growth conditions. B Activity in the absence of salicylate. C, D and E: Promoter activity of PRv0561c (C), PRv0560c (D and E), after 2 h treatment with varying concentrations of salicylate. Results are the average and standard deviation of three independent transformants assayed in duplicate. Activity is given in Miller Units. A significant difference compared using Student's t-test to the untreated control is marked by an * for p<0.05) ** for p<0.01, *** for p<0.0001.
Figure 2.
PRv0560c induction kinetics after exposure to salicylate in M. tuberculosis.
A and B. Promoter activity was measured in M. tuberculosis transformants grown under aerobic growth conditions exposed to 0.4 mM salicylate (A and B), or 0.2 mM or 0.4 mM salicylate (C) or with the use of LacZ tagged for degradation(D). Results are the average and standard deviation of three independent transformants assayed in duplicate. Activity is given in Miller Units. LacZ-ASV was tagged with AANDENYAASV; LacZ-LAA was tagged with AANDENYALAA.
Figure 3.
PRv0560c induction by structural analogs of salicylate in M. tuberculosis.
Promoter activity was measured in M. tuberculosis transformants grown under aerobic growth conditions. A. Promoter activity of PRv0560c after treatment with 0.4 mM of compound for 3 d. Results are the average and standard deviation of three independent transformants assayed in duplicate. Activity is given in Miller Units. B. Chemical structures of compounds of interest. A significant difference compared using Student's t-test to the untreated control is marked by an * for p<0.05) ** for p<0.01, *** for p<0.0001.
Figure 4.
PRv0560c off kinetics in salicylate-free or iron-free medium in M. tuberculosis.
M. tuberculosis transformants were grown under aerobic growth conditions in the presence of 0.4 mM salicylate. Cultures were washed and inoculated into salicylate-free medium A. Washed cells. Transformants were washed and resuspended in salicylate-free medium. B. Cells were washed and inoculated into salicylate-free medium; cultures were passaged into fresh salicylate-free medium at a dilution of 1/10. C. Cells were washed and inoculated in low iron minimal medium (MMT); cultures were passaged into fresh MMT medium at a dilution of 1/100. Results are the average and standard deviation of three independent transformants assayed in duplicate. Activity is given in Miller Units.
Figure 5.
Identification of the promoter and regulatory elements.
A. DNA sequence of the PRv0560c region. The predicted translation start site of Rv0560c according to TubercuList is marked with **. Protein sequences of Rv0561c and Rv0560c are shown. Potential −10 promoter elements (PM1, PM2, PM3) are underlined. The −35 and extended −10 element are in bold. A palindromic moitif is indicated by grey shading. B. Promoter activity following mutation of the promoter region. M. tuberculosis transformants were grown under aerobic growth conditions in the absence/presence of 0.4 mM salicylate. Results are the average and standard deviation of three independent transformants assayed in duplicate. Activity is given in Miller Units. A significant difference compared to the wild type is marked by an * (p<0.05).