Date of Award
9-12-2023
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
The mycobacterial family contains many human pathogens, including Mycobacterium tuberculosis, Mycobacterium abcessus, and Mycobacterium leprae. Mycobacterium tuberculosis, the causative agent of the disease tuberculosis (TB), is a major public health problem. The emergence of increasing numbers of antibiotic resistant cases of TB has led to a push for novel drug development. A popular target for antibiotic development is DNA replication, as this is an essential process in bacterial cell development. Further understanding of the DNA replication process in mycobacteria will be key to drug development efforts. The process of DNA replication has been well studied in Escherichia coli and Bacillus subtilis, however, mycobacteria lack many factors present in the replication processes in these bacteria. Most relevant to this thesis is the lack of any homolog to the DnaC/DnaI AAA+ ATPase helicase loaders present in E. coli and B. subtilis, respectively. In fact, many bacteria do not encode homologs to these proteins. Instead, most bacteria, including mycobacteria, encode a homolog to the protein DciA. DciA (DnaC/DnaI Antecedent) is an essential protein in mycobacteria, and its depletion leads to longer cells with decreased nucleoid occupancy and decreased DNA synthesis rates, phenotypes indicative of a defect in cell cycle progression. Similar phenotypes have also been observed for a depletion of dciA from Pseudomonas aeruginosa and Caulobacter vibrioides. DciA from both mycobacterial and proteobacterial species have been shown to interact with the replicative helicase DnaB, further suggesting that the function of DciA is analogous to the role of helicase loaders DnaC/DnaI. However, prior to my thesis, we had no information about the DciA protein family outside of the few bacteria it has been studied in (M. tuberculosis, Mycobacterium smegmatis, Vibrio cholera, P. aeruginosa, C. vibrioides). Further, we did not understand the mechanism of DciA, and how it might function as a helicase operator. E. coli and B. subtilis helicase loaders are AAA+ ATPases, and their ATPase activity is critical for helicase loading. However, DciA does not contain an ATPase domain, and therefore must use a distinct mechanism from previously described helicase loaders. In collaboration with the Ravi lab at Michigan State University, I performed a large-scale bioinformatic analysis of the entire DciA protein family to identify how DciA proteins from different phyla compare to one another. I found that DciA proteins diverge in domain architecture as well as protein length, with some DciA proteins having sequence extensions N- or C-terminal to the conserved DciA domain, and some proteins made up only of the DciA domain. Though DciA domains varied in domain organization, we predicted that they would have similar structural motifs that would contribute to their mechanism. In agreement with our model, when I used the structural prediction software AlphaFold to analyze predicted structural motifs in DciA proteins with varying domain organizations, we did indeed find conserved structural elements. These data raised the question – how do different predicted structural motifs in DciA contribute to its function in mycobacteria, and how does this compare to other previously studied DciA proteins? To answer this question, I took a genetic approach, using truncations and point mutants in different structural regions of M. tuberculosis DciA. I used this suite of mutants to analyze how expression of DciA mutants affect a strain of M. smegmatis with the native copy of DciA deleted. I showed that different regions of DciA have variable effects on cell morphology, leading us to a model about how different DciA structural regions contribute to M. tuberculosis DciA function. Throughout my thesis, I also undertook troubleshooting efforts to understand the biochemical relationship between M. tuberculosis DnaB and DciA. Overall, my efforts to understand if M. tuberculosis DciA enhances the helicase activity of DnaB were unsuccessful, however, we were able to learn a lot about DnaB protein purification and biochemical helicase assays. We have also reflected on further troubleshooting assays that we could undertake to better answer our questions biochemically. My thesis work has added insight into the DciA protein family as well as revealed the presence of multiple conserved structural motifs in DciA proteins from distant phyla. My work is also the first to demonstrate the diversity of DciA proteins throughout the entire bacterial kingdom. My research has also revealed a possible mechanism for DciA in mycobacteria, and lays out future directions with which to continue to probe the mechanism of DciA during replication initiation.
Language
English (en)
Chair and Committee
Christina Stallings
Recommended Citation
Blaine, Helen Catherine, "Investigating the role of DciA during the cell cycle in Mycobacterium" (2023). Arts & Sciences Electronic Theses and Dissertations. 3169.
https://openscholarship.wustl.edu/art_sci_etds/3169