MOLECULAR BASIS OF PKNB ESSENTIALITY IN MYCOBACTERIA

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Mycobacteria are a group of versatile microorganisms which include medically important pathogens and environmental bacteria. Mycobacteria have developed distinct mechanisms enabling their prolonged survival in hostile conditions and adaptation to a wide range of environmental niches. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a slow-growing bacterium with a complex lipid-rich cell wall that uses multiple signaling mechanisms to coordinate division with the biosynthesis of cellular components. Eleven serine/threonine protein kinases are particularly important for regulation of cellular processes in Mtb. The serine/threonine protein kinase B (PknB) is essential for mycobacterial growth, although the reasons for this are unknown. Previous attempts to deplete or over-produce PknB resulted in rapid death of mycobacteria. We have recently discovered special conditions, which support growth of mycobacteria that do not produce PknB, and compared phosphorylated proteins in mycobacteria producing and missing PknB. This novel approach has aided the identification of proteins that are phosphorylated by PknB, which is the first step in establishing the molecular mechanisms of PknB essentiality. Our findings and previously published results suggest that PknB phosphorylates enzymes involved in biosynthesis and remodelling of peptidoglycan, the major component of bacterial cell wall, however the precise role of phosphorylation of these proteins remains elusive. Within this project we propose to investigate the mechanisms of PknB-mediated regulation of peptidoglycan biosynthesis by measuring the activities of phosphorylated and non-phosphorylated enzymes, their localization in cells and export onto the cell surface, and the role of phosphorylation in the interaction of enzymes with other proteins. We will work on two genetically related organisms, Mtb and Mycobacterium smegmatis, and generate mutants missing the PknB substrates or expressing their altered forms which cannot be phosphorylated by PknB. The growth of these mutants in various media will be assessed. We will also use methods established by us to identify partners interacting with PknB-phosphorylated proteins and to elucidate the mechanistic details of peptidoglycan biosynthesis in mycobacteria and its regulation by PknB. In separate experiments we will study how PknB phosphorylation contributes to the formation of dormant mycobacteria and their resuscitation. The expected outcomes of this project will improve our understanding of fundamental cellular processes in mycobacteria and will stimulate the development of novel approaches to target Mtb in different physiological states. Our results will also contribute to deciphering the function of serine/threonine protein kinases in prokaryotes.