Figure 1.
M. tuberculosis encodes an α-ketoglutarate: ferredoxin oxidoreductase.
(A) Genetic map of the M. tuberculosis Rv2454c-Rv2455c (korAB) region. Conserved γ, α and β domains are indicated by brackets. The bar labeled ΔkorAB denotes the region that was replaced by a hygromycin cassette using specialized transduction. The bar labeled compl. represents the region of the genome that was used for complementation of the ΔkorAB strain. (B) Phylogenetic tree of the α subunits of characterized members of the α-ketoic acid: ferredoxin oxidoreductase family. Sequences were acquired from the NCBI protein database (www.ncbi.nlm.nih.gov). Alignments were made by the ClustalW method, trees were reconstructed by the Neighbor Joining method using the European Bioinformatics Institute server (www.ebi.ac.uk/Tools/clustalw2/index.html), graphics were generated using TreeView X (darwin.zoology.gla.ac.uk/~rpage/treeviewx/). α-ketoic acid substrates utilized by members of each clade are indicated to the right. The scale represents substitutions per residue. (C) Phylogenetic tree of α subunits of the α-ketoic acid: ferredoxin oxidoreductase found in several Actinobacteria. Alignments and trees were generated as described in B.
Table 1.
CoA-dependent α-ketoglutarate: MV oxidoreductase activity in M. tuberculosis, M. bovis and M. smegmatis.
Figure 2.
korAB is essential for the formation of succinyl-CoA from α-ketoglutarate and CoA by M. tuberculosis.
Reaction mixtures containing CoA, KG, MV, MgCl2 and cell extracts from M. tuberculosis mc27000 (wild type), the ΔkorAB strain, and the complemented strain (compl.) were separated by ion exchange chromatography and CoA species were detected by UV absorbance (260 nm) following elution. CoA and succinyl-CoA were run as standards.
Figure 3.
M. tuberculosis KOR activity is tolerant to O2 exposure.
Whole cell lysates of M. tuberculosis and B. fragilis were exposed to air at room temperature and remaining KOR (M. tuberculosis, squares; B. fragilis, circles) and POR (B. fragilis, diamonds) activities were assessed under anaerobic conditions. Percent activity remaining was calculated by dividing the rate of methyl viologen reduction at timex by that at time0 (% activity remaining = ratet = x/ratet = 0×100). Data shown represent the mean ± standard deviation of three independent determinations.
Figure 4.
korAB is essential for growth of M. tuberculosis in the absence of sufficient levels of CO2.
(A) Serial dilutions of M. tuberculosis mc27000 (wild type), ΔkorAB and the complemented strain (compl.) were spotted on supplemented 7H10 medium containing glycerol (0.5%), dextrose (0.2%), oleic acid (60 nl ml−1) and Tween 80 (0.05%). Plates were incubated under atmospheres with indicated amounts of CO2 for 20 days. (B–D) Growth of M. tuberculosis mc27000 (squares), ΔkorAB (circles) and complemented strain (diamonds) in supplemented 7H9 medium containing glycerol (0.5%), dextrose (0.2%), oleic acid (60 nl ml−1) and Tween 80 (0.05%) under an atmosphere containing 0.04% CO2 (B), 0.08% CO2 (C) or 5% CO2 (D).
Figure 5.
Conditional essentiality of korAB for growth of M. tuberculosis.
(A) Serial dilutions of single cell suspensions of M. tuberculosis mc27000 (wild type) and the ΔkorAB strain were spotted on supplemented 7H10 medium containing glycerol (0.5%), dextrose (0.2%) and Tween 80 (0.05%) with or without 200 µM 3NP. Plates were incubated under atmospheres with indicated amounts of CO2 for 20 days. (B) Growth of M. tuberculosis mc27000 (squares), ΔkorAB (circles) and complemented strain (diamonds) in supplemented 7H9 medium with glycerol and dextrose without oleic acid or Tween 80 under an atmosphere containing 0.04% CO2. (C–E) Growth of M. tuberculosis mc27000 (squares), ΔkorAB (circles; filled circles, 0.1% succinate) and complemented strain (diamonds) in supplemented 7H9 medium with Tween 80 (0.5%) as the sole carbon source under atmospheres containing 5% (C), 0.08% (D), and 0.04% CO2 (E).
Figure 6.
KGD and KOR are differentially required for growth of M. tuberculosis.
M. tuberculosis strains mc27000 (wild type, squares), ΔkorAB (circles), Δkgd (diamonds) and ΔkorAB Δkgd (triangles) were grown under a 5% CO2 (A–D) or 0.08% CO2 (E) atmosphere in supplemented 7H9 medium containing glycerol and dextrose (carbs, A–E) with 0.05% Tween 80 (A & B) or tyloxapol (C–E). 200 µM 3-nitropropionate (B & D) and 0.1% succinate (E, solid symbols) were added to the growth media. (F) Strains M. tuberculosis Δkgd (diamonds) and ΔkorAB Δkgd (triangles) were grown under a 5% CO2 (open symbols) or 0.08% CO2 (solid symbols) atmosphere in supplemented 7H9 medium containing 0.5% Tween 80 as the sole carbon source.
Figure 7.
Integrated model of routes and regulation in the M. tuberculosis TCA cycle.
The glyoxylate cycle (inner cycle), canonical TCA cycle (medial cycle), and variant TCA cycle (outer cycle) are depicted. Blue lines indicate pathways that are utilized concurrently with β-oxidation, green lines indicate pathways that are utilized during growth on carbohydrates as the sole carbon source, and black lines indicate pathways that are common to both modes of growth. Red lines indicate blocks imposed by 3NP on isocitrate lyase (ICL), PknG on GarA, and GarA on KGD. The dotted red lines represent the putative blocks imposed by glyoxylate on SSA dehydrogenase and PknG.
Table 2.
M. tuberculosis strains and primers used in this study.