Article Figures & Data
Figures
- Fig 1
prrA and prrB transcription and PrrA levels during in vitro growth. (A) Quantitation of prrA and prrB transcripts during M. tuberculosis H37Rv in vitro exponential growth in supplemented Middlebrook 7H9 broth with glycerol. The optical density at 600 nm of the cultures is indicated by the solid line and scaled on the right y axis. (B) qRT-PCR comparison of prrA and prrB expression in logarithmic and stationary phases of M. tuberculosis growth in supplemented Middlebrook 7H9 broth without glycerol. Normalized expression of prrA and prrB with respect to 16S rRNA expression is presented as the mean ± SD of data from three independent experiments. The decrease in prrA and prrB expression during stationary phase is presented relative to the logarithmic-phase expression, which is set at 1.0. ∗∗∗, P < 0.0001. (C) Western blot analyses of PrrA in total M. tuberculosis H37Rv protein (20 μg) collected from logarithmic (lane 1) and stationary (lane 2) growth phases and detected using anti-PrrA antibodies. Lane 3 represents the purified His-tagged PrrA protein (40 ng). (D) Northern analysis of M. tuberculosis H37Rv with prrA and prrB single-stranded RNA probes. RNA samples were collected from exponential-phase cultures of M. tuberculosis H37Rv grown in Middlebrook 7H9 broth with glycerol. (E) Diagram of the prrA-prrB region of the chromosome, drawn to scale and showing the prrA and prrB gene lengths.
- Fig 2
M. tuberculosis prrA and prrB are differentially transcribed and translated upon exposure to in vitro environmental stress conditions. (A) qRT-PCR comparison of prrA and prrB expression upon M. tuberculosis H37Rv exposure to low pH (pH 5.5), carbon starvation, or nitrogen-limiting environments. The fold change in prrA and prrB expression in H37Rv grown under environmental stress conditions with respect to H37Rv grown in Middlebrook 7H9-Tween-ADS medium (untreated control) (baseline expression set to 1.0) is presented as the mean ± SD of data from three independent experiments. ∗∗, prrA P value = 0.0013 or prrB P value = 0.0032. d, days. (B) qRT-PCR analysis of prrA and prrB expression, comparing logarithmic growth and 24- or 48-h exposure to hypoxic conditions. Normalized expression of prrA and prrB with respect to 16S rRNA expression is presented as the mean ± SD of data from three independent experiments. The decrease in prrAB transcript levels in hypoxia is presented relative to logarithmic-phase expression, which is set at 1.0. ∗∗∗, prrA P value = 0.0002 by ANOVA or prrB P value < 0.0001 by ANOVA. (C) Western blot analysis of PrrA during M. tuberculosis H37Rv logarithmic growth, stationary phase, and hypoxia. Total protein (40 μg) was separated via SDS-PAGE and detected using polyclonal anti-PrrA antibodies with purified His-tagged PrrA protein (40 ng) serving as a positive control. Lanes: 1, aerobic growth control; 2, hypoxia, 24 h; 3, hypoxia, 48 h; 4, logarithmic growth, 3 days; 5, stationary phase, 30 days; 6, purified PrrA protein. (D) Western blot analysis of PrrA during M. tuberculosis H37Rv nitrogen limitation, logarithmic growth, stationary phase, and hypoxia. Total protein (40 μg) was separated via SDS-PAGE and detected using polyclonal anti-PrrA antibodies, with the purified His-tagged PrrA protein (20 ng) serving as a positive control. Lanes: 1, MSX exposure, 4 h; 2, MSX exposure, 4 days; 3, purified PrrA protein.
- Fig 3
Southern blot analysis of M. tuberculosis chromosomal DNA from wild-type H37Rv, STS16 (H37Rv::prrAB), and STS22 (prrAB-complemented ΔprrAB::hyg deletion mutant), confirming generation of a wild-type ΔprrAB::hyg mutation in the presence of an ectopic copy of prrAB. Genomic DNA was digested with HpaI and SpeI and hybridized with the prrA gene and the hygromycin (hyg) resistance cassette. Expected sizes of the hybridized DNA fragments: wild-type prrA, 6,823 bp; episomal copy of prrA, 3,048 bp; hyg, 6,730 bp. Molecular size markers (in kilobases) are shown on the left.
- Fig 4
Characterization of prrA from M. tuberculosis Mt103 wild-type and Mt21D3 prrA transposon mutant strains. (A) Genomic DNA from Mt103 and Mt21D3 was digested with BamHI, which is present within Tn5367 (7), and hybridized with the prrA gene or the Km resistance (aph) cassette. Based on BamHI sites located within Tn5367 and flanking the prrA and prrB genes (7), the expected sizes of the hybridized DNA fragments are as follows: prrA (Mt103), 7,801 bp; prrA with Tn5367 inserted in the promoter (Mt21D3), 6,607 bp; and aph (Mt21D3), 4,292 bp. Molecular size markers (in kilobases) are shown on the left. (B) qRT-PCR analysis of prrA and prrB expression in Mt103 and Mt21D3. Results were normalized with respect to 16S rRNA expression and are shown as expression differences from results for the Mt103 control sample, which were assigned a value of 1. The data represent the means ± SD of data from three independent experiments. ∗, prrA P value = 0.0378; ∗∗, prrB P value = 0.0037. (C) Western blot analysis of whole-cell lysates from M. tuberculosis Mt103 (1×, 25 μg) and Mt21D3 (1×, 25 μg; 2×, 50 μg) strains probed with polyclonal anti-PrrA antibody, with the purified His-tagged PrrA protein (40 ng) serving as a control.
