A family of acr-coregulated Mycobacterium tuberculosis genes shares a common DNA motif and requires Rv3133c (dosR or devR) for expression

Abstract

Previous work has shown that the divergently transcribed Mycobacterium tuberculosis genes acr (hspX, Rv2031c) and acg (Rv2032) are induced under conditions of shallow standing culture and low oxygen and intracellularly within macrophages. We used a combination of computational and experimental methods to identify promoters for eight additional genes that are regulated in a similar manner and that comprise an acr-coregulated promoter (ACP) family. Transcriptional regulation of these ACP family members was evaluated by using a plasmid-based promoter-green fluorescent protein fusion system and flow cytometry. All promoters showed increased expression in shallow standing versus shaking cultures, in low- versus high-oxygen conditions, and intracellularly within macrophages versus extracellularly in tissue culture medium. However, there were quantitative differences in expression among promoters and among conditions for each promoter. A conserved 18-bp palindromic sequence motif was identified in all ACPs by Gibbs sampling-based computational analyses. Two such motifs overlap regions in the acr and acg promoters that were previously shown to be required for their expression. In addition, we found that 5% carbon dioxide was required for growth of Mycobacterium bovis BCG under microaerophilic (1.3% O(2)) culture conditions and fully prevented the growth cessation typically associated with rapid removal of oxygen. These findings are likely to be relevant to the in vivo environment and will contribute to our understanding of the pathogenesis of tuberculosis infection.

FIG. 1.

Relative expression of ACP family promoters in M. bovis BCG during growth under each of the AFR culture conditions. Fluorescence readings shown are the means per bacterium (in arbitrary fluorescence units) from the flow-cytometric analysis of recombinant promoter-GFP-expressing M. bovis BCG bacteria. Recombinant bacteria were grown either shaking or standing under ambient or hypoxic conditions (1.3% O₂) supplemented with 5% CO₂ for 7 days. Intracellular (IC) and extracellular (XC) expression levels were analyzed after 48 h of infection in J774.16 macrophages or tissue culture media alone. Bacteria for the IC and XC experiments were pregrown for 7 days under either shaking or standing ambient culture conditions. Note the different relative fluorescence scales for different promoters. Values shown are the means of three independent experiments, and error bars denote standard deviations for each experimental condition. Asterisks above ambient culture conditions indicate that the difference in fluorescence expression between shaking and standing cultures was statistically significant; asterisks above the hypoxic shaking condition indicate that the difference in fluorescence expression between shaking ambient cultures and shaking hypoxic cultures was statistically significant; asterisks above the macrophage culture condition indicate that the difference in fluorescence expression between XC and IC grown bacteria was statistically significant (t test for independent samples; *, P = 0.01 to 0.05; **, P = 0.001 to 0.01; ***, P < 0.001) (see Materials and Methods).

FIG. 2.

The ACP family motif. (A) Promoter sequences, with sites matching the motif at a P value of <0.05 in red and an additional site identified at a P value of 0.32 in blue. Start codons, the coding region, and stop codons are italicized, and the start codons for the differentially regulated genes are in boldface. Arrows, direction of transcription of the genes; boldface arrows, genes that are differentially regulated. For our promoter studies, we amplified the sequence upstream of Rv3127 that included part of the putative Rv3126c ORF. The DNA regions that we previously showed to be necessary for regulation of acr and acg are underlined (33). Rv1996 was included in the final alignment because preliminary data suggested that its promoter may also be an ACP family member. (B) Sequence logo of the ACP family motif. The logo represents the alignment of the sites from panel A that are in red (without regard to orientation), showing the relative frequency of each base at each position of the motif (35). The y axis indicates the information content measured in bits, and error bars represent standard deviations at each position due to the limited sample size. All sequences except those between Rv1738 and Rv1737c are listed in the orientation of the published H37Rv sequence. The reverse complement of the Rv1738-Rv1737c intergenic sequence is shown so that the two ACP family motifs with the 4-bp separation in this sequence are aligned with those of the other sequences.

FIG. 3.

Comparative expression of the ACP-GFP fusion constructs in M. tuberculosis H37Rv (solid bars) and H37Rv:ΔRv3133c (hatched bars). Recombinant bacteria were grown shaking under hypoxic conditions (1.3% O₂) supplemented with 5% CO₂ for 7 days. Fluorescence readings shown are the means per bacterium (in arbitrary fluorescence units) from the flow-cytometric analysis of recombinant promoter-GFP-expressing M. tuberculosis H37Rv and H37Rv:ΔRv3133c bacteria. Values are the means of two or three independent experiments, and error bars denote standard deviations for each experimental condition. Expression of each of the ACP family promoters was significantly reduced (Student's t test; P < 0.05) in M. tuberculosis H37Rv:ΔRv3133c compared to expression in M. tuberculosis H37Rv.

FIG. 4.

Effects of O₂ and CO₂ tension on the growth rates of M. bovis BCG in mycobacterial growth media during shallow standing or shaking conditions. Absorbance at 600 nm (A₆₀₀) was measured for shaking and standing M. bovis BCG liquid cultures grown in ambient (A), 20% O₂-5% CO₂ (B), 1.3% O₂ without CO₂ supplementation (C), and 1.3% O₂-5% CO₂ (D) conditions. Values shown are the means of three independent experiments. Error bars denote standard deviations.

FIG. 5.

Resuscitation of microaerophilic M. bovis BCG cultures that lacked CO₂ supplementation. Aliquots of 11-day standing (A) and shaking (B) cultures were subcultured (1:100) into flasks containing fresh mycobacterial growth media and grown shaking or standing under ambient or low-oxygen culture conditions supplemented with 5% CO₂. Mycobacterial growth was determined by measuring the optical absorbance (A₆₀₀) of individual tissue culture flasks for each time point. Data shown are from one of three representative experiments.

FIG. 6.

HPLC measurement of the consumption of glycerol (A and B) and glucose (C and D) by M. bovis BCG grown in shaking or standing cultures in ambient air, air supplemented with 5% CO₂, or 5% CO₂-supplemented microaerophilic (1.3% O₂) conditions. Values shown are the means of results from three independent experiments. Error bars denote standard deviations.

FIG.7.

Morphology of M. bovis BCG grown under AFR conditions. M. bovis BCG was grown for 7 (A to D) or 11 (E to H) days in ambient air in shaking cultures (A and E) or standing cultures (B and F) or in 1.3% O₂-5% CO₂ in shaking cultures (C and G) or standing cultures (D and H). Note the phenotypic changes, notably vacuolization, in bacteria grown in standing cultures at 1.3% O₂-5% CO₂ (D and H). Bars = 500 nm.