Figure 1.
Schematic depiction of the rv1129 (prpR) region on the M. tuberculosis H37Rv chromosome.
prpD and prpC genes encode methylcitrate dehydratase and methylcitrate synthase, respectively. The bent arrow represents the putative transcription start sites of prpR (see Fig. S6) and prpD genes [21].
Figure 2.
PrpR interacts with the promoter region of the prpD gene.
(A) EMSA. Oligonucleotides corresponding to the prpD (260 bp) or mtrA (251 bp, negative control) promoter region were incubated with increasing amounts of 6HisPrpRMt protein; nucleoprotein complexes were analyzed on 4% polyacrylamide gels. (B) SPR. Sensograms were obtained by binding 6HisPrpRMt to biotinylated DNA fragment corresponding to the promoter region of the prpD gene (pprpDR, 260 bp), amplified with biotinylated p1129_Fw and p1129_Rv primers and immobilized on a streptavidin-coated chip in the BIAcore apparatus. (C) DNase I footprinting. A 32P-labeled, 260-bp DNA fragment was incubated with increasing amounts of 6HisPrpRMt protein and then subjected to DNase I digestion. Lanes: T, G, C, and A represent sequencing reactions for the pprpRD fragment. Radiolabeled p1129_Rv primer was used to perform sequencing reactions.
Figure 3.
PrpR interacts with the promoter region of the icl1 gene (EMSA).
The icl1 (284 bp) promoter region was incubated with increasing amounts of 6HisPrpRMt protein; nucleoprotein complexes were analyzed on 4% polyacrylamide gels.
Figure 4.
PrpR binds the promoter region of prpDC and icl1 genes in intact M. tuberculosis cells.
Identification of intracellular PrpR-DNA complex using immunoprecipitation. PrpR-DNA complexes cross-linked with glutaraldehyde were immunoprecipitated with anti-6HisPrpRMt polyclonal antibodies (sample 1). PCR was carried out with the primer pairs, p1129_Fw and p1129_Rv (pprpDR)(A); picl_Fw and picl_Rv (picl1)(B); and pmtrA_Fw and pmtrA_Rv (pmtrA, negative control)(C). Negative control (2) consisted of DNA template extracted from the cells subjected to immunoprecipitation, but nucleoprotein complexes were not previously cross-linked. Positives controls (+) were also performed using template obtained from strains subjected only to cross-linking (3) or total DNA extracted from the cells (4).
Figure 5.
PrpR interacts with the promoter region of the ramB gene.
(A) EMSA. An oligonucleotide (250 bp) corresponding to the ramB promoter region was incubated with increasing amounts of 6HisPrpRMt protein; nucleoprotein complexes were analyzed on 4% polyacrylamide gels. (B) SPR. Sensograms were obtained by binding 6HisPrpRMt to a biotinylated DNA fragment (250 bp) corresponding to the promoter region of the ramB gene (pramB), amplified with biotinylated pramB_Fw and pramB_Rv primers and immobilized on a streptavidin-coated chip in the BIAcore apparatus. (C) DNase I footprinting. A 32P-labeled, 250 bp DNA fragment was incubated with increasing amounts of 6HisPrpRMt protein and then subjected to DNase I digestion. Lanes: T, G, C, and A represent sequencing reactions for the pramB fragment. Radiolabeled pramB_Rv primer was used to perform sequencing reactions.
Figure 6.
The prpR-deletion mutant of M. tuberculosis H37Rv exhibits impaired growth on propionate as a sole carbon source.
Comparison of wild-type (H37Rv), ΔprpR (prpR-deletion mutant) and ΔprpR+pMVprpR (complementation of ΔprpR strain) strains of M. tuberculosis H37Rv, grown on different carbon sources. Growth was monitored in M9 minimal medium supplemented with glucose (A), acetate (B), or propionate (C) (0.5% each). Wild-type and deletion strain growth curves were significantly different (p<0.05), but wild-type and complementation strain growth curves were similar (p>0.05).
Figure 7.
Influence of carbon source on the expression of prpR and ramB genes in M. tuberculosis H37Rv.
Total RNA was extracted from a culture growing in 7H9+OADC broth (rich medium) or M9 minimal medium containing either acetate or propionate (0.5%) until reaching an OD600 value of 0.6–0.8. For minimal media, cells were incubated for an additional 48 hours. The primer pairs for analysis of genes by qPCR are listed in Table S5. mRNA levels of target genes, prpR (A), ramB (B), were normalized internally to that of the constitutively expressed housekeeping gene, sigA. Means were calculated from three independent experiments and three determinations per experiment. The error bars indicate standard deviations of triplicate samples. Statistical significance was calculated by the Student's t-test.
Figure 8.
PrpR promotes transcription of prpDC and icl1 and represses ramB during growth on propionate.
Indicated M. tuberculosis strains were grown in M9 minimal medium containing propionate and total RNA was extracted. The primer pairs for analysis of genes by qPCR are listed in Table S5. mRNA levels of target genes, prpDC (A), icl1 (B), and ramB (C) were normalized internally to that of the constitutively expressed housekeeping gene, sigA. Means were calculated from three independent experiments and three determinations per experiment. The error bars indicate standard deviations of triplicate samples. Statistical significance was calculated by the Student's t-test.
Figure 9.
PrpR promotes transcription of both prpDC and icl1 in M. tuberculosis during growth in rich 7H9+OADC broth, but does not influence ramB expression.
Analyzed M. tuberculosis strains were grown in 7H9+OADC broth (rich medium) until reaching an OD600 value of 0.6–0.8 and total RNA was extracted. The primer pairs for analysis of genes by qPCR are listed in Table S5. mRNA levels of target genes, prpDC (A), icl1 (B), and ramB (C) were normalized internally to sigA. Means were calculated from three independent experiments and three determinations per experiment. The error bars indicate standard deviations of triplicate samples. Statistical significance was calculated by the Student's t-test.
Figure 10.
PrpR interacts with the promoter region of the kstR gene and activates its transcription during M. tuberculosis growth in rich medium.
(A) EMSA. An oligonucleotide (240 bp) corresponding to the kstR promoter region was incubated with increasing amounts of 6HisPrpRMt protein; nucleoprotein complexes were analyzed on 4% polyacrylamide gels. (B) SPR. Sensograms were obtained by binding 6HisPrpRMt to a biotinylated DNA fragment (240 bp) corresponding to the promoter region of the kstR gene (pkstR), amplified with biotinylated pkstR_Fw and pkstR_Rv primers and immobilized on a streptavidin-coated chip in the BIAcore apparatus.(C) qPCR. Total RNA was extracted from indicated M. tuberculosis strains growing in 7H9+OADC broth (rich medium). mRNA levels of kstR gene were normalized internally to sigA. Means were calculated from three independent experiments and three determinations per experiment. The error bars indicate standard deviations of triplicate samples. Statistical significance was calculated by the Student's t-test.
Figure 11.
PrpR regulates the expression of genes involved in methylcitrate and glyoxylate cycles in M. tuberculosis.
Perpendicular lines indicate negative regulation. Grey perpendicular lines from Micklinghoff et al. [11].