Despite efforts to broadly implement effective treatment strategies, the global burden of tuberculosis remains extremely high, and the incidence of multidrug-resistant tuberculosis is increasing in many countries. Insight into mechanisms by which M. tuberculosis adapts to the environment of the host during infection may provide new opportunities for the treatment or prevention or tuberculosis, including multidrug-resistant tuberculosis. A striking finding of the M. tuberculosis genome sequence was the presence of genes encoding several eukaryotic-like serine-threonine kinases. All but two of these appear to be receptor type kinases that likely interact with the extracellular environment and transmit signals by reversible phosphorylation of intracellular substrates, resulting in changes in bacterial physiology. Two of these genes, pknA and pknB are essential for M. tuberculosis viability, and are linked in an operon that also includes orthologues of genes involved in cell wall synthesis and cell shape control. We hypothesize that the proteins encoded by these genes mediate signaling pathways that regulate cell division in mycobacteria. The goal of this research is to characterize the function and signaling pathways of PknA and PknB in M. tuberculosis. To achieve this goal, we propose four specific aims: 1) To complete the characterization of the optimal substrate recognition motif of PknA and PknB, 2) To identify the in vivo phosphorylation targets of PknA and PknB, 3) To investigate the expression and function of PknA and PknB in mycobacterial cell physiology, and 4) To begin to investigate the molecules that interact with the extracellular domains of PknA and PknB. [unreadable] [unreadable]