Downloaded from http://cshperspectives.cshlp.org/ on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Mutation—The Engine of Evolution: Studying Mutation and Its Role in the Evolution of Bacteria Ruth Hershberg Rachel & Menachem Mendelovitch Evolutionary Processes of Mutation & Natural Selection Research Laboratory, Department of Genetics and Developmental Biology, The Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel Correspondence: [email protected] Mutation is the engine of evolution in that it generates the genetic variation on which the evolutionary process depends. To understand the evolutionary process we must therefore characterize the rates and patterns of mutation. Starting with the seminal Luria and Delbruck fluctuation experiments in 1943, studies utilizing a variety of approaches have revealed much about mutation rates and patterns and about how these may vary between different bacterial strains and species along the chromosome and between different growth condi- tions. This work provides a critical overview of the results and conclusions drawn from these studies, of the debate surrounding some of these conclusions, and of the challenges faced when studying mutation and its role in bacterial evolution. enetic variation is a prerequisite to evolu- eral of our decades-old assumptions were shown Gtionary change. In the absence of such var- to be mistaken, in light of newly available data. iation, no subsequent change can be achieved. Genetic variation is ultimately all generated by MUTATIONS VERSUS SUBSTITUTIONS mutation. It is therefore clear that mutation is a major evolutionary force that must be studied It is important to note that, in this article, and understood to understand evolution. Yet, we will only be considering de novo point mu- often mutation is set aside and thought of as a tations. We will not discuss large insertions or random generator of variation that follows very deletions or horizontal gene transfer events. To simple and predictable rules. proceed, we must define some terms. Many reviews of mutation deal with the For the purpose of this article, we will define molecular mechanisms of mutation and repair “DNA mutations” as single nucleotide changes (e.g., Modrich 1991; Smith 1992; Lieber 2010). in the DNA sequence of an individual organ- This work, in contrast, relates to mutation as ism. These will be the end result of the molec- an evolutionary force, focusing on bacteria. ular DNA change, and of the fact that this DNA We will show that mutation is extremely diffi- change was not repaired by the cellular repair cult to study, that we do not know nearly systems. Once a mutation occurs and is present enough about mutation and that recently sev- within an individual, it will either increase in Editor: Howard Ochman Additional Perspectives on Microbial Evolution available at www.cshperspectives.org Copyright # 2015 Cold Spring Harbor Laboratory Press; all rights reserved Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a018077 1 Downloaded from http://cshperspectives.cshlp.org/ on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press R. Hershberg frequency within the population, or will vanish once it occurs (Fig. 1). Note that our definition from the population. The ultimate fate of mu- of substitutions differs somewhat from that of tations depends on a combination of natural others that sometimes define substitutions as selection and stochastic forces, such as genetic either mutations that have fixed (e.g., Gillespie drift. 1998) or a specific class of base-change muta- Wewill define “DNA substitutions” as those tion (e.g., Graur and Li 2000). mutations that we can directly observe when Wewill define a phenotypic, or marker mu- we consider DNA sequence data. The substitu- tation, as a phenotypic change occurring in an tions we observe may reflect the mutations that individual. For example, an antibiotic resistance have occurred for better or worse, depending on phenotypic mutation causes an individual bac- how natural selection has affected them. For terium to become resistant to an antibiotic. example, if when comparing sequences we ob- Similarly, we can define a phenotypic, or marker serve that a certain substitution type (e.g., C to substitution, as a phenotypic change we are able T transitions) occurs more frequently within to observe, for example, an increase in the fre- our data, this could either mean that this mu- quency of resistant mutants within a bacterial tation type occurs more frequently, or that nat- population. Such an increase can occur because ural selection tends to favor this mutation type the resistance mutation occurs more frequently A Normal levels of selection Mutations Substitutions Selective sieve B Relaxed selection Mutations Substitutions Selective sieve Figure 1. Different types of mutations (represented by differently colored arrows) occur at different frequencies (represented by arrow thickness). Selection acts as a sieve and allows only a subset of these mutations to persist and become the differences we see between genomes. Such differences are referred to as substitutions. Various types of mutations have different fitness effect distributions, and will be differently affected by selection. (A) Under normal levels of selection, selection will introduce its own biases into patterns of variation. Thus, biases in the patterns of observable substitutions between genomes are likely not to reflect mutational biases. (B) When selection is extremely relaxed, it is expected to affect patterns of variation to a much lesser extent, because it will affect only mutations with very high-fitness effects. Under such conditions, observed substitutions between genomes approximate a random sample of the mutations that have occurred. Because of this, when selection is relaxed, biases in the patterns of substitutions observed between genomes will better approximate mutational biases. 2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a018077 Downloaded from http://cshperspectives.cshlp.org/ on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Bacterial Mutation as an Evolutionary Force or because of natural selection favoring the re- Luria and Delbruck modeled the variance sistant mutant. expected in the number of resistant mutants Often, mutation is studied by assuming that under both these scenarios (Luria and Delbruck certain types of DNA mutations (e.g., synony- 1943). Their models showed that a much higher mous mutations) or certain marker mutations variance would be expected if the emergence of (e.g., antibiotic resistance mutations when a resistance were caused by mutations occurring bacterium is not exposed to antibiotics) evolve before exposure to viruses. If mutation is a Pois- entirely neutrally. If there is absolutely no selec- son process and if mutations occur after and in tion acting on an observed class of substitu- response to viral exposure, one would expect the tions, their patterns and rates will indeed be a number of resistant mutants following exposure derivative of the patterns and rates of mutation. to be distributed around a certain mean, with However, as we will see later in this article, it the variance equal to the mean (a known char- is rare to find cases in which DNA or marker acteristic of the Poisson distribution). If, how- mutations are totally unaffected by selection. ever, mutations occur before exposure, they can Determining mutational patterns and rates is occur in any generation of growth. Mutations therefore a tricky business that requires one to occurring in earlier generations will rise to find creative ways to eliminate or minimize the higher frequencies by the end of an experiment, effects of natural selection on observed substi- compared with mutations occurring in later tutions. generations. Therefore, the number of resistant mutants at the end of an experiment will de- pend not only on the number of mutations that LURIA AND DELBRUCK—ESTIMATING have occurred, but also on when these muta- MUTATION RATES CAN BE A NOISY tions occurred. This should greatly enhance BUSINESS the variance in the numbers of resistant mu- In their seminal 1943 “fluctuation experi- tants observed between different experiments. ments,” Luria and Delbruck showed that even Indeed, Luria and Delbruck then went on to if mutational markers truly did evolve neutrally, show that in different experiments they saw a estimates of mutation rates based on such variance that was much higher than the mean markers would be extremely noisy (Luria and number of resistant mutants. This provided Delbruck 1943). Luria and Delbruck were at- the first ever demonstration that mutations oc- tempting to understand the following phenom- curred before selection for their outcome (Luria enon. When a pure bacterial culture is exposed and Delbruck 1943). to a bacteriophage, the culture will disappear In addition to showing for the first time that because of destruction of cells sensitive to the mutation precedes selection, the Luria and Del- virus. After further incubation, the culture will bruck study also shed light on the great variance often become turbid again because of growth of in substitution rates one can expect to observe a variant that is resistant to the phage. Once the when considering phenotypic markers (Luria variant is isolated, it often remains resistant and Delbruck