<i>M. tuberculosis</i> encodes an α-ketoglutarate: ferredoxin oxidoreductase.

<p>(A) Genetic map of the <i>M. tuberculosis Rv2454c</i>-<i>Rv2455c</i> (<i>korAB</i>) region. Conserved γ, α and β domains are indicated by brackets. The bar labeled Δ<i>korAB</i> 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 Δ<i>korAB</i> 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 (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a>). Alignments were made by the ClustalW method, trees were reconstructed by the Neighbor Joining method using the European Bioinformatics Institute server (<a href="http://www.ebi.ac.uk/Tools/clustalw2/index.html" target="_blank">www.ebi.ac.uk/Tools/clustalw2/index.html</a>), graphics were generated using TreeView X (<a href="http://darwin.zoology.gla.ac.uk/~rpage/treeviewx/" target="_blank">darwin.zoology.gla.ac.uk/~rpage/treeviewx/</a>). 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.</p

<i>M. tuberculosis</i> KOR activity is tolerant to O₂ exposure.

<p>Whole cell lysates of <i>M. tuberculosis</i> and <i>B. fragilis</i> were exposed to air at room temperature and remaining KOR (<i>M. tuberculosis</i>, squares; <i>B. fragilis</i>, circles) and POR (<i>B. fragilis</i>, diamonds) activities were assessed under anaerobic conditions. Percent activity remaining was calculated by dividing the rate of methyl viologen reduction at time_x by that at time₀ (% activity remaining  = rate_t = x/rate_t = 0×100). Data shown represent the mean ± standard deviation of three independent determinations.</p

CoA-dependent α-ketoglutarate: MV oxidoreductase activity in <i>M. tuberculosis</i>, <i>M. bovis</i> and <i>M. smegmatis</i>.

*<p>Not measured.</p>†<p>A fraction of pyruvate: MV oxidoreductase activity from <i>M. smegmatis</i> mc²155 was CoA-independent (shown in parentheses).</p

<i>korAB</i> is essential for growth of <i>M. tuberculosis</i> in the absence of sufficient levels of CO₂.

<p>(A) Serial dilutions of <i>M. tuberculosis</i> mc²7000 (wild type), Δ<i>korAB</i> 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 <i>M. tuberculosis</i> mc²7000 (squares), Δ<i>korAB</i> (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).</p

<i>korAB</i> is essential for the formation of succinyl-CoA from α-ketoglutarate and CoA by <i>M. tuberculosis</i>.

<p>Reaction mixtures containing CoA, KG, MV, MgCl₂ and cell extracts from <i>M. tuberculosis</i> mc²7000 (wild type), the Δ<i>korAB</i> 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.</p

KGD and KOR are differentially required for growth of <i>M. tuberculosis</i>.

<p><i>M. tuberculosis</i> strains mc²7000 (wild type, squares), Δ<i>korAB</i> (circles), Δ<i>kgd</i> (diamonds) and Δ<i>korAB</i> Δ<i>kgd</i> (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 <i>M. tuberculosis</i> Δ<i>kgd</i> (diamonds) and Δ<i>korAB</i> Δ<i>kgd</i> (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.</p

<i>M. tuberculosis</i> strains and primers used in this study.

<p><i>M. tuberculosis</i> strains and primers used in this study.</p

Conditional essentiality of <i>korAB</i> for growth of <i>M. tuberculosis</i>.

<p>(A) Serial dilutions of single cell suspensions of <i>M. tuberculosis</i> mc²7000 (wild type) and the Δ<i>korAB</i> 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 <i>M. tuberculosis</i> mc²7000 (squares), Δ<i>korAB</i> (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 <i>M. tuberculosis</i> mc²7000 (squares), Δ<i>korAB</i> (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).</p

Integrated model of routes and regulation in the <i>M. tuberculosis</i> TCA cycle.

<p>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.</p

Model for redox control of respiration in <i>M. tuberculosis</i>.

<p>As an obligate aerobe, <i>M. tuberculosis</i> relies on oxidative phosphorylation to meet energy demands during growth phases. The organism accomplishes this by shuttling electrons into the ETC via the NADH dehydrogenases (Complex I) and succinate dehydrogenases. In aerobic conditions this electron flux both powers the electrochemical gradient and generates sufficient ATP to power cell division. As oxygen becomes restricted, Sdh1 (the primary aerobic succinate dehydrogenase) is inhibited and succinate accumulates intracellularly, thus depriving the ETC of a substantial source of electrons. Menaquinol is not replenished and oxygen reduction by the terminal cytochrome oxidases is diminished. Importantly, this process begins when DO concentrations drop below 30% - long before oxygen becomes limiting for growth. If Sdh1 is inhibited, intracellular succinate accumulates prematurely and the succinate oxidation must be catalyzed by Sdh2 or Frd. This results in unregulated menaquinone reduction which proceeds (even in oxygen replete conditions) to furnish the terminal oxidases with a substrate for oxygen reduction and respiration continues. (IV  =  Complex IV).</p