In my dissertation work, I investigated bacterial mutagenesis. I asked: where do mutations occur in the bacterial genome? What types of mutations occur? What factors promote or prevent mutations? And lastly, how do the answers to these questions change depending on the bacterial life cycle (When)? I used Bacillus subtilis, a Gram-positive bacterium belonging to the phylum Firmicutes, and Escherichia coli, a Gram-negative bacterium belonging to the phylum Proteobacteria, to answer these questions. B. subtilis and E. coli are model systems that have been used to gain insights into fundamental processes of mutations and evolution for nearly ~8 decades. Here, each one of my three research chapters adds to our understanding of bacterial mutagenesis.
In my 1st research chapter (chapter 2) I explored if non-B DNA motifs are mutagenic hotspots in B. subtilis. Non-B DNA-motifs are sequences that can adopt structures that deviate from the canonical right- handed Watson and Crick B-structure. Non-B DNA correlate with genomic instability in eukaryotes but their role in bacterial mutagenesis is understudied. My findings show that sequences predicted to form non-B DNA structures accumulated more mutations compared to sequences that were not predicted to form any stable structures. Importantly, this effect was only seen in stationary-phase cells, as such a response did not occur in growing conditions, and it was dependent on the DNA repair factor Mfd. This research chapter gives us insight into where mutations occur (at non-B motifs), when (during stationary phase), and what factors promote and prevent mutations (Mfd promotes mutations at these sites).
In my 2nd research chapter (chapter 3), I investigated if out-of-frame stop codons (OSCs)have a function in bacterial genomes. Out-of-frame stop codons (OSC), a.k.a. hidden stops or iii premature stops, are created when two consecutive amino acid codons form one of three translational stop codons in an alternate reading frame of the coding sequence (e.g., CTG AAA). The function of OSCs in Bacteria is unclear, but they are speculated to protect against translational frameshifts that could result in energy loss through the production of aberrant proteins, however in vivo data is limited. Results from my experiments show that the loss of OSCs in a DNA region that codes for a flexible loop region in a protein led to an increase in insertion and deletion (indels) mutagenic events. Interestingly, the effect was seen in both growing and stationary-phase cells and loss of the DNA repair factor Mfd decreased mutations. This research chapter gives us insight into why we observe certain types of mutations like indels at specific places in the genome (regions that code for protein loops) and what factors promote and prevent mutations (OSCs may limit indel appearance while Mfd promotes the formation of indels).
In my last research (chapter 4) I integrated the strengths of Maximum Depth Sequencing and a gain-of-function system to advance our understanding of mutagenesis that occurs in the presence and absence of selection of a region of interest (ROI). Maximum Depth Sequencing (MDS) was developed to measure the strand-specific mutation rate/frequency of any region of interest in a bacterial genome through error-corrected, high-throughput sequencing independent of selection. In a gain-of-function system, a mutation, induced by a mutagen or engineered into a gene, renders the bacterium unable to carry out a function needed for growth, therefore mutations that restore the function encoded by that gene also restore growth and allow bacterial colonies to form and be counted on a Petri plate. My research showed that there were discrepancies in the frequency or rate of mutations between the methods used to measure mutagenesis. Because MDS detects transient genetic changes that may or may not get fixed into mutations, my analysis suggests that the frequency of a transient DNA change does not predict its frequency of mutation fixation. Furthermore, my experiments showed that DNA repair factors and stress change the mutagenic spectrum, this study addresses what factors prevent and promote mutations and when.
By describing new life-cycle-dependent mutation hotspots and signatures along with the factors that drive them, my research continues to add to our understanding of where mutations occur, what type of mutations occur when, and what factors promote and prevent mutations in Bacteria. The answers to these basic questions about mutations in the smallest forms of life have already proven to have a large impact on human health and our understanding evolution
