Abstract

Thiopeptides are a family of antimicrobial peptides that are characterized for having sulfur-containing heterocycles, and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The antimicrobial peptide micrococcin is a thiopeptide that is synthesized by the ribosome and undergoes several post-translational modifications (PTMs). Micrococcin is formed from a precursor peptide, TclE. TclE comprises an N-terminal leader (35-AA) that is crucial for recognition of the PTM machinery, alongside a C-terminal core sequence (14-AA) that undergoes multiple PTMs to acquire its antimicrobial activity. In the first series of modifications, the scaffold protein TclI binds the leader of TclE and presents the core of TclE to the modifying enzymes TclJ and TclN, facilitating the conversion of 6 cysteine residues into thiazoles. The work of this dissertation focuses on understanding the key interactions between the TclIJN protein complex and the precursor peptide TclE. By carrying out mutagenesis analysis on the leader peptide, I determined a minimal region of TclE that is required for thiazole installation. By doing bioinformatic analysis and copurification experiments, I determined that the TclI scaffold protein binds to the enzymes TclJ and TclN one at a time in dynamic equilibrium. I also further characterized the region of TclI that is important for coordinating these interactions and determined key residues that play a role for binding to its enzymatic partners. During my PhD, I had the opportunity to work on a few side projects that came up as I was working on plasmid construction for the Tcl project and working as a teaching assistant for the Microbial Genetics class (MMBIO360). During plasmid construction for protein expression of Tcl proteins, we recognized that there was room for improvement on transcriptional terminators, especially for the widely used T7 RNA polymerase. We engineered a set T7 terminators that are shorter and more efficient compared to previously reported T7 terminators, both in vivo and in vitro. As a teaching assistant for the MMBIO360 class, I had the opportunity to coordinate the work of undergraduate students in research-driven projects. We used the genetically tractable organism Agrobacterium fabrum to investigate flagellar motility. We carried out a near-saturating screen that led to the finding of four previously undescribed genes that are essential for motility in this organism. Another side project that also emerged from this class is investigating the genetics of streptomycin resistance in A. fabrum. Once paper from each of these three side projects are reprinted in Chapters 3, 4 and 5, respectively.

Degree

PhD

College and Department

Life Sciences; Microbiology and Molecular Biology

Rights

https://lib.byu.edu/about/copyright/

Date Submitted

2023-11-20

Document Type

Dissertation

Handle

http://hdl.lib.byu.edu/1877/etd12991

Keywords

thiopeptide, thiazole, post-translational modification

Language

english

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