Dominance Effects of Deleterious and Beneficial Mutations in a Single Gene of the RNA Virus ϕ6

Citation: Joseph SB, Peck KM, Burch CL ( Dominance Effects of Deleterious and Beneficial Mutations in a Single Gene of the RNA Virus w6 Sarah B. Joseph 0 1 Kayla M. Peck 0 1 Christina L. Burch 0 1 Yury E. Khudyakov, Centers for Disease Control and Prevention, United States of America 0 Current address: Lineberger Comprehensive Cancer Center, University of North Carolina , Chapel Hill, North Carolina , United States of America 1 Department of Biology, University of North Carolina , Chapel Hill, North Carolina , United States of America Most of our knowledge of dominance stems from studies of deleterious mutations. From these studies we know that most deleterious mutations are recessive, and that this recessivity arises from a hyperbolic relationship between protein function (i.e., protein concentration or activity) and fitness. Here we investigate whether this knowledge can be used to make predictions about the dominance of beneficial and deleterious mutations in a single gene. We employed a model system - the bacteriophage w6 - that allowed us to generate a collection of mutations in haploid conditions so that it was not biased toward either dominant beneficial or recessive deleterious mutations. Screening for the ability to infect a bacterial host that does not permit infection by the wildtype w6, we generated a collection of mutations in P3, a gene involved in attachment to the host and in phage particle assembly. The resulting collection contained mutations with both deleterious and beneficial effects on fitness. The deleterious mutations in our collection had additive effects on fitness and the beneficial mutations were recessive. Neither of these observations were predicted from previous studies of dominance. This pattern is not consistent with the hyperbolic (diminishing returns) relationship between protein function and fitness that is characteristic of enzymatic genes, but could have resulted from a curve of increasing returns. - Funding: This work was funded by National Institutes of Health grants F32-GM080086-01A1 to S.B.J. and R01-GM067940 to C.L.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Nearly 150 years after Mendel first observed recessive traits in pea plants [1], empirical studies have shown that most deleterious mutations are recessive [2,3,4]. The most widely accepted theory for why mutations should be recessive is the Physiological Theory [5,6], which argues that dominance is a natural result of the physiological mechanics of protein function. For mutations in enzymatic genes, the dominance of the wildtype over most deleterious mutations results, simply, from the hyperbolic relationship between enzyme concentration and flux through a metabolic pathway (see Figure 1). Empirical investigations of mutational effects in enzymes have confirmed that enzyme concentration is hyperbolically related to flux [5], and also to fitness [7]. If we consider the Physiological Theory more generally, the exceptions seem to prove the rule. In cases like Huntingtons disease, where deleterious mutations are dominant, they typically occur in non-enzymatic genes (reviewed in [4]). While many studies have examined the dominance of deleterious mutations, the rarity of beneficial mutations makes it difficult to perform analogous studies on them without inadvertently selecting for dominant mutations (Haldanes sieve; [8,9]). In light of these limitations, it is worth considering whether studies of deleterious mutations can inform our knowledge of beneficial mutations. In the specific example described above, if the recessivity of most deleterious mutations is explained by the hyperbolic (diminishing returns) relationship between protein concentration and function that characterizes enzymatic genes, does that mean that most beneficial mutations are also governed by that hyperbolic relationship, causing their effects to be dominant (see Figure 1)? More generally, if the dominance effects of deleterious mutations in a particular gene were known whether recessive, additive, or dominant could that knowledge be used to predict the dominance effects of beneficial mutations in the same gene? In this study, we examine the dominance and selection coefficients of a collection of spontaneous mutations in the bacteriophage w6. Our collection differs from those of earlier studies in several important ways the mutations occur primarily in a single gene, span a wide range of fitness effects, and include an unbiased sample of deleterious and beneficial mutations. Thus, we are able to test whether deleterious mutations in this gene are recessive, and beneficial mutations are dominant, as would be predicted by a hyperbolic relationship between protein function and fitness (Figures 1A and 1C). Materials and Methods Ancestor Strain, Culture Conditions and Archiving In this study we used two laboratory strains of the doublestranded RNA bacteriophage w6, both descended from the original isolate [10]. The first strain, w6mindich, was reconstructed from cloned genome segments [11]. The bacteria and plasmids used to construct this strain were supplied by Leonard Mindich (Public Health Research Institute of New Jersey Medical School). The second strain, w637F-41, was obtained from Lin Chao (University of California, San Diego). w637F-41 has served as the ancestor for previous evolution experiments [12,13,14,15] and was used here because it generates a wide array of host range mutations during the 5 generations that elapse during formation of single plaques [15]. w637F-41 has a higher fitness than w6mindich, probably because of differences in laboratory passage. We employed two host bacteria, the standard laboratory host Pseudomonas syringae pathovar phaseolicola strain HB10Y, obtained from the American Type Culture Collection (ATCC no. 21781), and a novel host Pseudomonas syringae pathovar glycinea strain R4a, obtained from Jeff Dangl (University of North Carolina). Bacteriophage and their hosts were cultured and titered in standard LC media (5 g yeast extract, 5 g NaCl, and 10 g Bactotryptone per liter H2O) [13]. Phage were grown on plates by overlaying a mixture of phage, 200 mL of an overnight culture of bacteria, and 3.5 mL top agar (LC+0.7% agar) onto solid media (LC+1.5% agar). Bacteriophage and bacteria were incubated for growth at 25uC, and archived in 40% glycerol at 220uC and 2 80uC, respectively. Host Range Mutants We isolated host range mutants capable of growth on Pseudomonas syringae pathovar glycinea, an alternative host that w6mindich and w637F-41 cannot infect. The ancestor phage was plated on the standard laboratory host P. phaseolicola to obtain isolated plaques. Phage were harvested from randomly chosen isolated plaques and plated on the alternative bacterial host P. glycinea. After 24 hours of growth, (...truncated)