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For genetic complementation analysis, a wild type copy of the pknG gene, transcriptionally fused to the M.
As seen in Figure 2a, the deletion of the C-terminal domain of PknG did not significantly affect the in vitro growth of M.
Immunofluorescence microscopy using an IgG [alpha]-PknG-GST showed a lower abundance of PknG in the mycobacteria envelope of the wild type strain in stationary phase, as compared to the one registered during the exponential phase.
To evaluate the effect of the PknG C-terminal domain deletion on colony morphology, undiluted aliquots of 0, 4, 7, 14 and 21 days old cultures (Figure 4) of the wild type, mutant and complemented strains were grown on solid medium.
Dimerization of PknG and interaction with its substrate
The TPR domain is implicated in protein-protein interactions, and in PknG it has been shown to be involved in the formation of dimers during the crystallization of the protein (Scherr et al., 2007).
Comparative analysis of amino acid sequences of mycobacterial PknG proteins has shown that they have similar kinase, Trx and TPR domains (Houben et al., 2009).
In order to determine the role of the C-terminal domain TPR motif in the function of the PknG protein, a mutant of M.
tuberculosis lacking a complete PknG gene (Cowley et al., 2004), which grows in 7H9 medium at a rate significantly lower than the parental strain, and whose wild type phenotype can be restored upon complementation.
As the deletion of the C-terminal domain of PknG leads to a decrease in the kinase activity (Tiwary et al., 2009), it could be that some phosphorylation cascade involving PknG is altered, and this in turn modifies the cell envelope structure.
However, since PknG had been previously proposed to interact with GarA through phosphothreonine residues located in the N-terminal domain of the protein (O'Hare et al., 2008; Scherr et al., 2009), the lack of interaction between PknG-[DELTA]Ct and GarA could be result of conformational changes in the mutant protein, brought about by deletion of the C-terminus, which would prevent the interaction of its N-terminal domain with GarA.
The present results show that deletion of the C-terminal domain of the PknG protein of M.
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- PKN gamma