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Multi subunit protein projections termed pili are found on the surface of Gram-negative and Gram-positive bacteria. They are necessary for bacterial motility, biofilm formation, host adhesion/invasion etc. The assembly of pili in Gram-positiveMoreMulti subunit protein projections termed pili are found on the surface of Gram-negative and Gram-positive bacteria. They are necessary for bacterial motility, biofilm formation, host adhesion/invasion etc. The assembly of pili in Gram-positive bacteria is not well understood, hindering the development of prophylactic/therapeutic strategies to counter important human pathogens. A characteristic feature of pilus assembly in Gram-positive bacteria is the involvement of cysteine transpeptidases called sortases. Corynebacterium diphtheriae, the model organism utilized in this thesis has six sortase enzymes (Srt A, B, C, D, E and F) and assembles three distinct pilus structures designated as the SpaABC, SpaDEF and SpaHIG type pili. With the exception of the presumed housekeeping sortase SrtF, the other five pilus-specific sortases are organized into gene clusters along with the pilin substrates they help polymerize. Current models of pilus assembly in Gram-positive bacteria propose a specific biochemical basis for sortase-substrate interaction. In this model, the active site cysteine of sortase attacks the Thr-Gly junction of the LPXTG sorting signal motif of the substrate pilin causing the formation of a thioester linkage between the two proteins. The resulting sortase-substrate complex is referred to as the acyl enzyme intermediate and it is speculated to serve as the pilus assembly core. However, experimental evidence for this complex was lacking. Pilus assembly models also suggest that pilus polymers that are synthesized on different pilus assembly cores are ultimately transferred to a housekeeping sortase which then anchors them to the cell wall. However, systematic biochemical and genetic investigation of the housekeeping sortases function in pilus assembly was also lacking. The thesis work presented herein addresses the role of the housekeeping sortase SrtF and the mechanistic basis of the SrtA-SpaA reaction and their relevance to pilus assembly in C. diphtheria.