The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo.
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.
Cowley
Siobhan
S
Department of Medicine, Division of Infectious Diseases, University of British Columbia, 2733 Heather St., Vancouver, British Columbia, Canada V5Z 3J5.
Ko
Mary
M
Pick
Neora
N
Chow
Rayken
R
Downing
Katrina J
KJ
Gordhan
Bhavna G
BG
Betts
Joanna C
JC
Mizrahi
Valerie
V
Smith
Debbie A
DA
Stokes
Richard W
RW
Av-Gay
Yossef
Y
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D018698:Glutamic Acid / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D016513:Mice, SCID; D016254:Mutagenesis, Insertional; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism / Q000472:pathogenicity; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014376:Tuberculosis / Q000401:mortality; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005973:Glutamine; D018698:Glutamic Acid; D017346:Protein Serine-Threonine Kinases