The promoter of Rv0560c is induced by salicylate and structurally-related compounds in Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a major global health threat. During infection, bacteria are believed to encounter adverse conditions such as iron depletion. Mycobacteria synthesize iron-sequestering mycobactins, which are essential for survival in the host, via the intermediate salicylate. Salicylate is a ubiquitous compound which is known to induce a mild antibiotic resistance phenotype. In M. tuberculosis salicylate highly induces the expression of Rv0560c, a putative methyltransferase. We identified and characterized the promoter and regulatory elements of Rv0560c. P(Rv0560c) activity was highly inducible by salicylate in a dose-dependent manner. The induction kinetics of P(Rv0560c) were slow, taking several days to reach maximal activity, which was sustained over several weeks. Promoter activity could also be induced by compounds structurally related to salicylate, such as aspirin or para-aminosalicylic acid, but not by benzoate, indicating that induction is specific to a structural motif. The -10 and -35 promoter elements were identified and residues involved in regulation of promoter activity were identified in close proximity to an inverted repeat spanning the -35 promoter element. We conclude that Rv0560c expression is controlled by a yet unknown repressor via a highly-inducible promoter.

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 P_Rv0561c, and P_Rv0560c. 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 P_Rv0561c (C), P_Rv0560c (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. P_Rv0560c 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. P_Rv0560c 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 P_Rv0560c 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. P_Rv0560c 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 P_Rv0560c 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).