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Molecular characterization of Mycobacterium tuberculosis PKNB Drews, Steven Jeffrey

Abstract

The Mycobacterium tuberculosis genome contains eleven genes encoding for proteinserine/ threonine kinases (PSTKs). The putative PSTK gene pknB was chosen for study because the location of this gene within the chromosome led us to suspect that pknB encodes for a protein that may control mycobacterial growth. Recombinant M. tuberculosis PknB was expressed in Eschericia coli and was shown to be a functional kinase that is autophosphorylated on serine and threonine residues during in vitro kinase assays. In vitro kinase assays also showed that PknB phosphorylates myelin basic protein (MBP). A putative pknB promoter was cloned as a transcriptional fusion to a green fluorescence protein gene (gfp) within an epichromosomal mycobacterial shuttle vector. This putative promoter was active during log phase growth in M. smegmatis cultures, with attenuated activity in stationary phase growth. Anti-PknB antibodies were used to show that M. tuberculosis PknB is a membrane-localized protein. PknB protein expression also increased during log phase growth, with maximal expression evident during stationary phase growth. Attempts to knock out the pknB gene within M. tuberculosis H37Rv were unsuccessful due to a very low efficiency of electroporation and illegitimate recombination of the knock out construct. The pSJD202 vector was constructed by cloning pknB, with its putative promoter, into the integrative vector pGint. M. tuberculosis strains containing pSJD202 grew faster and produced relatively more PknB than pGINT strains. On-going experiments in our laboratory will use the anti-PknB antibodies as well as the pSJD202 vector as tools for future pknB disruption. The protein kinase inhibitor l-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) inhibited the growth of both the slow growing Mycobacterium bovis BCG, and the fast-growing saprophyte M. smegmatis mc 155, in a dose dependent manner. Furthermore, micromolar concentrations of H7 compound induced a significant decrease in the activity of the Mycobacterium tuberculosis protein-serine/threonine kinase (PSTK) PknB. This thesis outlines a variety of approaches used to study the function of pknB as well as other kinases of M. tuberculosis. The combination of information gained in these experiments provides a picture of PknB as a membrane-associated PSTK that is differentially expressed during mycobacterial growth and may play a role in both cell wall biosynthesis and growth.

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