PROJECT SUMMARY: N-terminal (Nt) acetylation is an understudied aspect of bacteriology. Nt-acetylation is the addition of an acetyl group to the amino group on the α-carbon of the first amino acid of a protein. The fundamental mechanisms promoting and regulating Nt-acetylation, and the consequences of this modification in bacteria remain undefined. The objective of this renewal application is to define the fundamental mechanisms underlying Nt-acetylation in mycobacteria. The central hypothesis is that protein Nt-acetylation is a dynamic, regulated process that directly impacts mycobacterial virulence. The central hypothesis will be tested by following these specific aims: 1) Define the enzymes promoting Nt-acetylation in mycobacteria. 2) Establish the mechanisms and consequences of vir- ulence factor Nt-acetylation in mycobacteria. 3) Determine the link between Nt-acetylation and mycobacterial metabolism. Under the first aim, the applicant proposes to use enrichment strategies combined with quantitative proteomics to determine the function and substrate specificity of conserved mycobacterial NATs. Under the sec- ond aim, in vitro biochemical assays will be combined with targeted and quantitative proteomics to identify the NATs that modify essential mycobacterial virulence factors. Genetic and molecular approaches will be used to define functional relationships between predicted NATs in mycobacteria. Under the third aim, the applicant will combine enrichment and proteomics approaches to investigate differential Nt-acetylation following growth of mycobacteria on host-relevant carbon sources. The applicant will use proximity-dependent labeling to identify potential regulators of NAT activity. The successful completion of this proposal will contribute a fundamental understanding of the mechanisms promoting Nt-acetylation and establish a link between NATs, Nt-acetylation and essential mycobacterial virulence pathways. These contributions will be significant because they will ad- vance our understanding of an understudied protein modification important for mycobacterial virulence, which may be applicable to other bacterial species. The topic of this proposal is conceptually innovative because Nt acetylation is an under-investigated protein modification in both areas of tuberculosis research and bacteriology. Furthermore, studying the regulation of Nt-acetylation by metabolism to identify Nt-acetylation events essential for mycobacterial virulence is an innovative idea. The proposal is technically innovative because the applicant combines biochemical screens, enrichment protocols with bioanalytical chemistry, and expertise in molecular and genetic manipulation of pathogenic mycobacteria. The applicant leverages both M. tuberculosis and M. marinum strains to optimize productivity. These studies in bacteria will lay a foundation for focused and informed studies in animal virulence models in the future. By rigorously studying the mechanisms and regulation ...