Sources of spontaneous mutagenesis in bacteria

Abstract

Mutations in an organism's genome can arise spontaneously, that is, in the absence of exogenous stress and prior to selection. Mutations are often neutral or deleterious to individual fitness but can also provide genetic diversity driving evolution. Mutagenesis in bacteria contributes to the already serious and growing problem of antibiotic resistance. However, the negative impacts of spontaneous mutagenesis on human health are not limited to bacterial antibiotic resistance. Spontaneous mutations also underlie tumorigenesis and evolution of drug resistance. To better understand the causes of genetic change and how they may be manipulated in order to curb antibiotic resistance or the development of cancer, we must acquire a mechanistic understanding of the major sources of mutagenesis. Bacterial systems are particularly well-suited to studying mutagenesis because of their fast growth rate and the panoply of available experimental tools, but efforts to understand mutagenic mechanisms can be complicated by the experimental system employed. Here, we review our current understanding of mutagenic mechanisms in bacteria and describe the methods used to study mutagenesis in bacterial systems.

Keywords: Spontaneous mutagenesis; evolution; fluctuation test; mutation accumulation; mutation reporter; selection.

Figure 1

Fluctuation tests, mutation reporters, mutation accumulation experiments, and maximum-depth sequencing. (A) An illustration of the fluctuation test. Mutants (black circles) arise during exponential growth of independent cultures. If selection is lethal to non-mutants, only mutants that arose during culture growth will form colonies (black colonies). However, if selection is for growth, slow growth during selection may give rise to additional mutants that form colonies during selection (red colonies). (B) The rationale behind reverse and forward mutation reporters is illustrated. For reverse mutation reporters, a reversion assay may be performed (top), in which a functional product allows survival and growth under selection. In the other class of reverse mutation reporter (middle) an active gene product is produced before and after mutagenesis, but the mutant gene product remains functional during selection, in contrast to non-mutant gene products, which do not function during selection. Forward mutation reporters (bottom) have a larger target size, in that any mutation eliminating expression or gene product activity results in growth or survival under selection. (C) A table of data from Luria and Delbruck’s 1943 paper (Luria and Delbruck, 1943) with examples of jackpot cultures indicated by rows shaded in gray. (D) An example mutation accumulation line. Mutations accumulate in the line’s genome (shown below the example mutation accumulation line) as it is passaged. After the desired number of generations, genomic DNA from each line is purified and sequenced to identify mutations that arose during the experiment. (E) The schematic workflow of maximum depth sequencing. A unique molecular identifier (UMI) is appended to the 3′ end of the original molecule of the region of interest (ROI), followed by linear amplification of the ROI molecules with their associated UMIs. The consensus sequence for each ROI/UMI pool is determined and the proportion of ROI/UMI pools with mutations at a given position is determined in order to calculate mutation rate at the position. A color version of this figure is available online.

Figure 2

Common sources of spontaneous mutagenesis in bacteria. Cellular processes that promote spontaneous mutagenesis are listed at the top of the schematic with arrows indicating the types of mutations with which they are associated.

Figure 3

Nucleobase modifications involved in mutagenesis. (A) Base pairing interactions between rare tautomeric forms of the nucleobases in DNA are shown for comparison to canonical base pairs. (B) Interactions between deoxyadenosine and syn-8-oxo-dG. (C) Cytosine deamination leads to uracil, causing C∙G→T∙A transitions, and adenine deamination leads to hypoxanthine, yielding A∙T→G∙C transitions (see section: Replication-transcription conflict). Structures were illustrated using ChemDraw software.