Characterization of the Mycobacterium tuberculosis iniBAC promoter, a promoter that responds to cell wall biosynthesis inhibition

Abstract

The cell wall provides an attractive target for antibiotics against Mycobacterium tuberculosis. Agents such as isoniazid and ethambutol that work by inhibiting cell wall biosynthesis are among the most highly effective antibiotics against this pathogen. Although considerable progress has been made identifying the targets for cell wall active antibiotics, little is known about the intracellular mechanisms that are activated as a consequence of cell wall injury. These mechanisms are likely to have an important role in growth regulation and in the induction of cell death by antibiotics. We previously discovered three isoniazid-induced genes (iniB, iniA, and iniC) organized in tandem on the M. tuberculosis genome. Here, we investigate the unique features of the putative iniBAC promoter. This promoter was specifically induced by a broad range of inhibitors of cell wall biosynthesis but was not inducible by other conditions that are toxic to mycobacteria via other mechanisms. Induction required inhibitory concentrations of antibiotics and could be detected only in actively growing cells. Analysis of the iniBAC promoter sequence revealed both a regulatory element upstream and a potential repressor binding region downstream of the transcriptional start site. The induction phenotype and structure of the iniBAC promoter suggest that a complex intracellular response occurs when cell wall biosynthesis is inhibited in M. tuberculosis and other mycobacteria.

FIG. 1

Effects of different compounds on iniBAC promoter activity as measured by luciferase assays of integrated transcriptional fusion plasmids. Induction is shown after incubation with compounds for 24 h (grey bars) or 48 h (black bars). Final concentrations are indicated in micrograms per milliliter. Error bars represent 95% confidence intervals. (A) INH-susceptible BCG^S(pG4697-6) containing the iniBAC promoter fused to lacZ and fflux genes [-Pro indicates assays performed with BCG^S(pG1697-3), which contains the same construct, except that the transcriptional fusion was performed by inserting the promoter in the opposite orientation from the coding region]. (B) INH-susceptible BCG(pMKB15) containing the L5 phage p_L promoter fused to the fflux gene. (C) INH-resistant BCG^R(pG4697-6) containing the iniBAC promoter fused to lacZ and fflux genes.

FIG. 1

Effects of different compounds on iniBAC promoter activity as measured by luciferase assays of integrated transcriptional fusion plasmids. Induction is shown after incubation with compounds for 24 h (grey bars) or 48 h (black bars). Final concentrations are indicated in micrograms per milliliter. Error bars represent 95% confidence intervals. (A) INH-susceptible BCG^S(pG4697-6) containing the iniBAC promoter fused to lacZ and fflux genes [-Pro indicates assays performed with BCG^S(pG1697-3), which contains the same construct, except that the transcriptional fusion was performed by inserting the promoter in the opposite orientation from the coding region]. (B) INH-susceptible BCG(pMKB15) containing the L5 phage p_L promoter fused to the fflux gene. (C) INH-resistant BCG^R(pG4697-6) containing the iniBAC promoter fused to lacZ and fflux genes.

FIG. 2

Induction of the iniBAC promoter in BCG^S(pG4697-6) after incubation with INH for 24 (■) and 48 (□) h and with ethambutol for 24 (▴) and 48 (▵) h.

FIG. 3

Induction of the iniBAC promoter in BCG strain BCG^S(pG4697-6) after incubation with INH (1 μg/ml; ■) and ethambutol (5 μg/ml; ▵) as a function of incubation time.

FIG. 4

Induction in different phases of growth. BCG^S(pG4697-6) was subcultured by performing a 1:100 dilution of an actively growing culture. Serial aliquots were removed at increasing OD₅₉₀ and split into paired subcultures. One subculture of each pair was not treated with antibiotics (●); the other subculture of each pair was treated with ethambutol at a final concentration of 5 μg/ml (□). After an additional 24 h, β-galactosidase activity was measured. β-Galactosidase units are shown as a function of the OD₅₉₀ of the paired subcultures at the time that they were removed from the parent culture.

FIG. 5

Testing of the iniBAC operon in different mycobacterial species. Mycobacterial species were tested by low-stringency Southern blot hybridization for the presence of the iniA and iniB genes: M. marinum (lane 1), H37ra (lane 2), M. tuberculosis strains Erdman (lane 3) and H37Rv (lane 4), M. avium (lane 5), BCG (lane 6), M. smegmatis (lane 7), M. nonchromogenicum (lane 8), and M. microti (lane 9). (A) Agarose gel showing PvuII digests of chromosomal DNA. (B) Southern blot of the gel in panel A hybridized with the iniA gene probe. (C) Hybridization with the M. tuberculosis 16S RNA gene probe. (D) Hybridization with the iniB gene.

FIG. 6

Mapping the transcriptional start sites of the iniBAC operon. Primer extension experiments were performed with end-labeled iniBprimer ext-3 using either no RNA or RNA isolated from BCG cultured in the absence of INH (INH−) or in the presence of INH at a final concentration of 1 μg/ml (INH+). Sequencing reactions were performed with the identical primer and run alongside the primer extension reactions.

FIG. 7

Effect of promoter deletions on antibiotic induction. Serial 5′ and 3′ deletions of the 211-bp sequence immediately upstream of the translational start site of iniB were fused to the lacZ gene in the integrating plasmid pCV125 and transformed into M. smegmatis strain mc²155. (A) Schematic of promoter deletions and induction after 4 h of treatment with either INH (100 μg/ml) or ethambutol (EMB; 5 μg/ml). The dotted line indicates the position of a 19-bp spacer that is unrelated to the sequence it replaced; induction represents mean results from at least three experiments. Numbering corresponds to the distance from the translational start site. (B) β-Galactosidase activity of promoter deletions without (grey) or with (black) 4 h of incubation with INH (100 μg/ml). Values represent means of at least three experiments. Error bars represent 95% confidence intervals.

FIG. 8

Proposed organization of iniBAC promoter region. Regions found to be essential for induction are underlined with solid lines; the dotted underline indicates the sequence whose deletion leads to increased induction. Also shown are the transcriptional start site (arrowhead), 6-bp inverted repeats (solid arrows), 8-bp direct repeats (dotted arrows), the position of the primer iniBprimer ext-3 used in the primer extension experiments (striped arrow), and possible −10 sequence and ribosomal binding site (rbs). The asterisk marks an alternate translational start site as annotated by Cole et al. (; NCBI database).