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. 2009 Dec;191(24):7545-53.
doi: 10.1128/JB.00619-09. Epub 2009 Oct 9.

Global effects of inactivation of the pyruvate kinase gene in the Mycobacterium tuberculosis complex

Sivagamisundaram Chavadi  1 , Esen Wooff, Nicholas G Coldham, Manjula Sritharan, R Glyn Hewinson, Stephen V Gordon, Paul R Wheeler
Affiliations

Global effects of inactivation of the pyruvate kinase gene in the Mycobacterium tuberculosis complex

Sivagamisundaram Chavadi et al. J Bacteriol. 2009 Dec.

Abstract

To better understand the global effects of "natural" lesions in genes involved in the pyruvate metabolism in Mycobacterium bovis, null mutations were made in the Mycobacterium tuberculosis H37Rv ald and pykA genes to mimic the M. bovis situation. Like M. bovis, the M. tuberculosis DeltapykA mutant yielded dysgonic colonies on solid medium lacking pyruvate, whereas colony morphology was eugonic on pyruvate-containing medium. Global effects of the loss of the pykA gene, possibly underlying colony morphology, were investigated by using proteomics on cultures grown in the same conditions. The levels of Icd2 increased and those of Icl and PckA decreased in the DeltapykA knockout. Proteomics suggested that the synthesis of enzymes involved in fatty acid and lipid biosynthesis were decreased, whereas those involved in beta-oxidation were increased in the M. tuberculosis DeltapykA mutant, as confirmed by direct assays for these activities. Thus, the loss of pykA from M. tuberculosis results in fatty acids being used principally for energy production, in contrast to the situation in the host when carbon from fatty acids is conserved through the glyoxylate cycle and gluconeogenesis; when an active pykA gene was introduced into M. bovis, the opposite effects occurred. Proteins involved in oxidative stress-AhpC, KatG, and SodA-showed increased synthesis in the DeltapykA mutant, and iron-regulated proteins were also affected. Ald levels were decreased in the DeltapykA knockout, explaining why an M. tuberculosis DeltapykA Deltaald double mutant showed little additional phenotypic effect. Overall, these data show that the loss of the pykA gene has powerful, global effects on proteins associated with central metabolism.

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Figures

FIG. 1.
FIG. 1.
Pathways of carbon metabolism possible in strains with or without pyruvate kinase (PykA). Boxes denote substrates and/or products where arrows are used to denote pathways. Arrows to and from boxes are pathways; other arrows show reactions catalyzed by a single enzyme. Substrates are in text with serifs; pathways and enzymes in plain text. Colored arrows are used to denote glycolysis or gluconeogenesis in red, the tricarboxylic acid cycle in blue, and the glyoxylate cycle in magenta.
FIG. 2.
FIG. 2.
Comparison of the colony morphology of the M. tuberculosis wild type, the M. tuberculosis ΔpykA knockout mutant, the knockout complemented with pLK102 containing the pykA gene, and M. bovis. The strains are arranged from left to right, each grown on Middlebrook 7H11 plates with glycerol plus OADC (g), OADC only (o), and pyruvate plus OADC (p).
FIG. 3.
FIG. 3.
Comparison of the colony morphology of the M. tuberculosis wild type with the M. tuberculosis Δald mutant, the M. tuberculosis ΔpykA mutant, and the M. tuberculosis ΔpykA Δald (double-knockout) mutant. The strains are arranged from left to right, each grown on Middlebrook 7H11 plates with OADC only (o), pyruvate plus OADC (p), and pyruvate plus alanine plus OADC (pa). The same images of colonies of the pykA mutant on rows o and p are shown in both figures so that comparisons can be made within each figure.
See this image and copyright information in PMC

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