Concentration of intracellular glucose-6-phosphate and fructose-6-phosphate in aerobic <i>M. tuberculosis</i> strains.

<p>Concentration of intracellular metabolites of mid-log phase <i>M. tuberculosis</i> strains cultured in complete Dubos liquid medium or Dubos liquid medium without glucose. Each biological sample was measured in duplicate. The data represent the values obtained for each duplicate of each biological sample. This experiment was repeated as least once independently and comparable values and trends were observed.</p

Western blot analysis of PFKB expression in wild-type <i>M. tuberculosis</i> and mutants.

<p>(A) Detection of PFKB with rabbit-anti-PFKB antibodies. (B) Ponseus-S stained of the membrane showing equal loading of cell-free extracts. Lane 1: WT; 2: <i>ΔpfkB</i> ; 3: <i>pfkB</i>-complemented <i>ΔpfkA;</i> 4: <i>ΔpfkA ;</i> 5: purified His-PFKB as control.</p

Deletion of <i>pfkA</i> and <i>pfkB</i> genes in <i>M. tuberculosis</i>.

<p>Schematic representation of the genomic regions of (A) <i>pfkA</i> in WT and <b><i>Δ</i></b><i>pfkA</i> mutant, (C) <i>pfkB</i> in WT and <i>ΔpfkB</i> mutant, and location of restriction sites and probes. (B) and (D) are Southern blots confirming the knockout of <i>pfkA</i> and <i>pfkB</i> genes respectively. <i>res</i>: sites of resolvase; <i>hyg</i>: hygromycin resistance cassette.</p

<i>pfkA</i> encodes a functional phosphofructokinase.

<p>Fructose-6-phosphate kinase activity of cell-free extracts from <i>M. tuberculosis</i> strains was measured by coupling fructose-1,6-bisphosphate formation to oxidation of NADH with aldose, triosephosphate isomerase and α-glycerophosphate dehydrogenase. Each biological sample was measured in duplicate. The data represent the values obtained for each duplicate of each biological sample. ∧ Enzymatic assay of purified recombinant His-tagged PFKA and His-tagged PFKB of <i>M. tuberculosis</i> was performed in triplicates and results are expressed as mean ± SD. Each experiment was repeated as least once independently and comparable values and trends were observed. Legend: nd, not detectable.</p

<i>In vitro</i> growth kinetic of <i>ΔpfkA</i> and <i>ΔpfkB</i> mutants on various carbon sources.

<p>Growth in liquid medium with glucose, glycerol or acetate as sole carbon source (as indicated) was monitored for Wild-type, Δ<i>pfkA</i>, Δ<i>pfkA</i> complemented with <i>pfkA</i>, Δ<i>pfkA</i> complemented with <i>pfkB, and </i><b><i>Δ</i></b><i>pfkB M. tuberculosis</i> strains (as indicated). Bacterial growth was monitored by OD absorbance at 600 nm over time. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056037#s3" target="_blank">Results</a> are representative of at least two independent experiments.</p

Growth kinetic of <i>ΔpfkA</i> mutant under aerobic or hypoxic conditions.

<p>Growth in aerobic (A, B) or hypoxic (Wayne model) (C, D) conditions was monitored over time for wild-type (open circle), Δ<i>pfkA</i> (open square) and complemented Δ<i>pfkA</i> (black triangle) strains as determined by OD_{600 nm} (A, B) or CFU counts (mean ± SD of triplicates) (C, D) in Dubos medium with (A, C) or without (B, D) glucose. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056037#s3" target="_blank">Results</a> are representative of at least two independent experiments.</p

Growth kinetic of <i>M. tuberculosis</i> H37Rv under hypoxia in the presence or absence of glucose.

<p>Growth under hypoxia (Wayne model) of wild-type <i>M. tuberculosis</i> was monitored by determining the number of CFU at various time-point up to Day 60 in the presence (black square) or absence (open square) of glucose. Data are expressed as mean ± SD of triplicates. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056037#s3" target="_blank">Results</a> are representative of two independent experiments. Arrow heads mark the start of decolourization of methylene blue and full arrows mark the complete decolourization of methylene blue in culture medium with (black) or without (red) glucose.</p

Infection profile of <i>ΔpfkA</i> mutant in mouse.

<p>8-weeks old female BALB/c mice were nasally infected with the wild-type (black circle) or Δ<i>pfkA</i> (open circle) strains. Four animals per time point per group were used. Bacterial loads in lung (A) and spleen (B) were determined by CFU counts. Data are expressed in Log₁₀ CFU per organ as the mean ± SD of four mice per group.</p

Schematic representation of the glycolytic pathway.

<p>Key committing step of glycolysis is catalyzed by <i>pfkA/pfkB</i>.</p

ATP and nitrite production in the presence of exogenous nitrate.

<p><i>M. tuberculosis</i> viability (A), ATP production (B), membrane potential (C) and nitrite production (D) were assessed under hypoxic conditions at neutral (6.6) or mild acidic (5.5) pH, in the presence (black bar) or absence (open bar) of 20 mM nitrate after 4 and 10 days incubation period. Results are expressed as the means ± SD of triplicates. RLU, relative luminescence units; MFI, Mean fluorescence intensity. *, p<0.005.</p