Date of Award
Fall 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Microbiology
First Advisor
Rego, Elizabeth
Abstract
Chromosomal mutations drive the evolution of antibiotic resistance in M. tuberculosis. Unlike many other pathogens, M. tuberculosis has no demonstrated ability for horizontal gene transfer, a major mechanism for developing antibiotic resistance. The error-prone translesion polymerase complex DnaE2/ImuA’/ImuB is one of the primary mediators of mutagenesis and antibiotic resistance in vitro and possibly in vivo. To catalyze error-prone DNA synthesis, DnaE2/ImuA’/ImuB must be able to access the DNA by binding the β2-sliding clamp of the replisome, and potentially compete with other polymerases, including the high-fidelity replicative polymerase DnaE1. However, the mechanisms that govern polymerase switching are not well understood, especially in mycobacteria. Here, I show that one of the major mycobacterial nucleoid-associated proteins, Lsr2, promotes dynamic exchange of the DnaE2/ImuA’/ImuB translesion and DnaE1 replicative polymerases. Specifically, using fluorescence recovery after photobleaching to quantify polymerase dynamics, I show that loss of lsr2 results in longer bound times for DnaE1 and shorter bound times for ImuB, suggesting reduced stability of DnaE2/ImuA’/ImuB binding to the DNA when Lsr2 is absent. Correspondingly, loss of lsr2 also results in decreased frequency of rifampicin-resistant mutants, suggesting increased reliance on the high-fidelity DnaE1 polymerase. Our Tn-seq data further suggest that Lsr2 may alter replisome dynamics by helping to prevent or resolve transcription-replication conflicts. As Lsr2 is capable of controlling transcription, I also evaluated the effect of lsr2 deletion on the expression of DNA repair machinery. Intriguingly, we observed that while Lsr2 negatively regulates the promoters of imuA’B and several other DNA damage-inducible genes, it does not alter the endogenous RNA levels. Further characterization using synthetic constructs harboring variations of Lsr2-regulated promoters reveals that Lsr2 transcriptional regulation is highly dependent on the sequence context and promoter architecture. Furthermore, I show that Lsr2’s ability to control transcription requires phosphorylation of its terminal threonine. Although the corresponding phosphoablative mutant is incapable of regulating transcription, it nevertheless complements certain Lsr2 growth defects, suggesting that it retains at least partial ability to perform other Lsr2 functions. This mutant may therefore be used in subsequent mechanistic studies to distinguish phenotypes originating from Lsr2’s ability to control transcription from those originating from its other functions. Overall, this work establishes a novel role for Lsr2 in DNA replication and mutagenesis and implicates Lsr2 as a potential factor in the emergence of mycobacterial antibiotic resistance.
Recommended Citation
Ng, Wei Lynn, "Uncovering a novel role for Lsr2 in mycobacterial mutagenesis and deciphering the determinants of Lsr2 transcriptional regulation" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1203.
https://elischolar.library.yale.edu/gsas_dissertations/1203
