Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA.

In Mycobacterium tuberculosis, the genes Rv1954A-Rv1957 form an operon that includes Rv1955 and Rv1956 which encode the HigB toxin and the HigA antitoxin respectively. We are interested in the role and regulation of this operon, since toxin-antitoxin systems have been suggested to play a part in the formation of persister cells in mycobacteria. To investigate the function of the higBA locus, effects of toxin expression on mycobacterial growth and transcript levels were assessed in M. tuberculosis H37Rv wild type and in an operon deletion background. We show that expression of HigB toxin in the absence of HigA antitoxin arrests growth and causes cell death in M. tuberculosis. We demonstrate HigB expression to reduce the abundance of IdeR and Zur regulated mRNAs and to cleave tmRNA in M. tuberculosis, Escherichia coli and Mycobacterium smegmatis. This study provides the first identification of possible target transcripts of HigB in M. tuberculosis

Mycobacterium tuberculosis ClpP Proteases Are Co-transcribed but Exhibit Different Substrate Specificities

PMCID: PMC3613350This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

The Promoter of Rv0560c Is Induced by Salicylate and Structurally-Related Compounds in Mycobacterium tuberculosis

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. PRv0560c activity was highly inducible by salicylate in a dose-dependent manner. The induction kinetics of PRv0560c 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

P_Rv0560c induction kinetics after exposure to salicylate in <i>M. tuberculosis</i>.

<p>A and B. Promoter activity was measured in <i>M. tuberculosis</i> 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.</p

P_Rv0560c induction by structural analogs of salicylate in <i>M. tuberculosis</i>.

<p>Promoter activity was measured in <i>M. tuberculosis</i> 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.</p

Identification of the promoter and regulatory elements.

<p>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. <i>M. tuberculosis</i> 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).</p

P₂₇₈ promoter activity was not induced by stress treatments.

<p>A) P₂₇₈ promoter activity in standing liquid cultures. Treatments were: 50 µg/mL of chlorpromazine for 3 h, 10 µg/mL of menadione for 3 h, 10 µg/mL of valinomycin for 3h, 6 µg/mL of vancomycin for 90 min. B) Promoter activity in rolling cultures. Treatments were: 42°C for 1 h, 10 mM diamide for 1 h C) Promoter activity in response to diamide treatment in <i>M. tuberculosis</i> CDC1551. D) Promoter activity in response to diamide and vancomycin treatments in presence or absence of streptomycin selection. Stress treatments were 10 mM diamide for 1 h or 6 µg/mL of vancomycin for 90 min. The average and standard deviation of three independent transformants assayed in duplicate is given. ß-galactosidase activity is given in Miller Units - measured as nmol of O-nitrophenol produced over time (min) per mg of protein. Activity is given in Miller Units (MU)- measured as nmol of O-nitrophenol produced per min per mg of protein. The background activity from pSM128 (control vector) was 6±3 MU under the different conditions tested.</p

Identification of key residues in a regulatory region of the <i>clpP1P2</i> operon.

<p>A) Sequence of the region upstream of <i>clpP1P2</i>. The <i>tig</i> stop codon, −10 promoter element, and <i>clpP1</i> start codons are underlined. The 18 nucleotides that constitute the regulatory region are boxed in grey. The CGC region mutated is underlined in bold. B) Identification of a regulatory region. The CGC motif (underlined bold) was mutated to AAA. C) Mapping of a regulatory binding site. Single nucleotide substitutions in P₂₇₈ were made by SDM. Residues A or T were mutated to G or and residues C or G were mutated to A. Results are the average activity of three independent transformants assayed in duplicate ± standard deviation. Activity is given in Miller Units- measured as nmol of O-nitrophenol produced per min per mg of protein. A significant difference of activity compared to wild-type P₂₇₈ is marked by an *(p<0.05) using the student’s t-test (unpaired, two sided).</p

Promoter activity during aerobic growth, hypoxia, and reaeration in <i>M. tuberculosis.</i>

<p>A) <i>M. tuberculosis</i> transformants harbouring P₂₇₈ were grown in aerobic culture. Results are the average activity of three transformants against average OD₅₈₀. A significant difference, measured by the student’s t-test (unpaired, two sided), compared to promoter activity at OD₅₈₀ = 0.15 is marked by an *(p<0.05). B) P₂₇₈ promoter activity in the Wayne model of hypoxia. <i>M. tuberculosis</i> liquid cultures were inoculated to a theoretical starting OD₅₈₀ of 0.004 in DTA medium. A significant difference compared to activity at day 0 is marked by an *(p<0.05) using the student’s t-test (unpaired, two sided). C) P₂₇₈ promoter activity after reaeration. Long term hypoxic cultures (12 weeks) were used to inoculate medium and grown in aerobic rolling cultures. Cell-free extracts were prepared once the cultures reached an OD₅₈₀ of 0.3. Results are the average activity of three independent transformants assayed in duplicate ± standard deviation. Activity is given in Miller Units- measured as nmol of O-nitrophenol produced per min per mg of protein.</p