Premium
The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo
Author(s) -
Cowley Siobhan,
Ko Mary,
Pick Neora,
Chow Rayken,
Downing Katrina J.,
Gordhan Bhavna G.,
Betts Joanna C.,
Mizrahi Valerie,
Smith Debbie A.,
Stokes Richard W.,
AvGay Yossef
Publication year - 2004
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2004.04085.x
Subject(s) - biology , glutamine , biochemistry , serine , kinase , mutant , threonine , glutamine synthetase , protein kinase a , microbiology and biotechnology , amino acid , phosphorylation , gene
Summary 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 pkn G 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 pkn G 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 Δ pknG 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.