The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo

Mol Microbiol. 2004 Jun;52(6):1691-702. doi: 10.1111/j.1365-2958.2004.04085.x.

Abstract

The function of the Mycobacterium tuberculosis eukaryotic-like protein serine/threonine kinase PknG was investigated by gene knock-out and by expression and biochemical analysis. The pknG gene (Rv0410c), when cloned and expressed in Escherichia coli, encodes a functional kinase. An in vitro kinase assay of the recombinant protein demonstrated that PknG can autophosphorylate its kinase domain as well as its 30 kDa C-terminal portion, which contains a tetratricopeptide (TPR) structural signalling motif. Western analysis revealed that PknG is located in the cytosol as well as in mycobacterial membrane. The pknG gene was inactivated by allelic exchange in M. tuberculosis. The resulting mutant strain causes delayed mortality in SCID mice and displays decreased viability both in vitro and upon infection of BALB/c mice. The reduced growth of the mutant was more pronounced in the stationary phase of the mycobacterial growth cycle and when grown in nutrient-depleted media. The PknG-deficient mutant accumulates glutamate and glutamine. The cellular levels of these two amino acids reached approximately threefold of their parental strain levels. Higher cellular levels of the amine sugar-containing molecules, GlcN-Ins and mycothiol, which are derived from glutamate, were detected in the DeltapknG mutant. De novo glutamine synthesis was shown to be reduced by 50%. This is consistent with current knowledge suggesting that glutamine synthesis is regulated by glutamate and glutamine levels. These data support our hypothesis that PknG mediates the transfer of signals sensing nutritional stress in M. tuberculosis and translates them into metabolic adaptation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Blotting, Western
  • Cell Membrane / chemistry
  • Cloning, Molecular
  • Cytoplasm / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Deletion
  • Gene Expression Regulation, Bacterial*
  • Genes, Bacterial
  • Glutamic Acid / metabolism*
  • Glutamine / metabolism*
  • Mice
  • Mice, Inbred BALB C
  • Mice, SCID
  • Mutagenesis, Insertional
  • Mycobacterium tuberculosis / genetics
  • Mycobacterium tuberculosis / growth & development*
  • Mycobacterium tuberculosis / metabolism
  • Mycobacterium tuberculosis / pathogenicity
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Tuberculosis / mortality
  • Virulence / genetics

Substances

  • Bacterial Proteins
  • Recombinant Proteins
  • Glutamine
  • Glutamic Acid
  • Protein-Serine-Threonine Kinases