Big-Benefit Mutations in a Bacteriophage Inhibited with Heat

J. J. Bull2,*{dagger}, M. R. Badgett* and H. A. Wichman{dagger}

*Department of Integrative Biology and
{dagger}Institute of Cellular and Molecular Biology, University of Texas at Austin; and
{ddagger}Department of Biological Sciences, University of Idaho


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
High temperature inhibits the growth of the wild-type bacteriophage {phi}X174. Three different point mutations were identified that each recovered growth at high temperature. Two affected the major capsid protein (residues F188 and F242), and one affected the internal scaffolding protein (B114). One of the major capsid mutations (F242) is located in a ß strand that contacts B114 in the procapsid during viral maturation, whereas the other capsid mutation (F188) is involved in subunit interactions at the threefold axis of symmetry. Selective coefficients of these mutations ranged from 13.9 to 3.8 in the inhibitory, hot environment, but all mutations reduced fitness at normal temperature. The selective effect of one of the mutations (F242) was evaluated at high temperature in four different genetic backgrounds and exhibited epistasis of diminishing returns: as log fitness of the background genotype increased from -0.1 to 4.1, the fitness boost provided by the F242 mutation decreased from 3.9 to 0.8. These results support a model in which viral fitness is bounded by an upper limit and the benefit of a mutation is scaled according to the remaining opportunity for fitness improvement in the genome.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Many disciplines within evolutionary biology regard natural selection as the chief determinant of adaptation—that is, in the long term, phenotypes will be molded to their selective optima, and the underlying genetic details can be ignored. This approach lends itself to predicting specific matches between the ecological determinants of selection and the morphology, behavior, or other phenotypes of a species (Fisher 1930Citation ; Williams 1966Citation ; Maynard Smith 1989Citation ). An alternative emphasis in studying adaptation is that evolution depends on the process by which a population responds to selection. According to this view, the population can proceed down alternative pathways, and stochastic elements in genetic drift, mutation, and environment determine which pathway is followed and what constraints apply to future adaptation (Wright 1977Citation ).

Until recently, the perspective emphasizing genetic details has been the more difficult to pursue, if only because the genetic basis of adaptation could be only loosely inferred. Now, however, the methods of molecular biology facilitate identifying genetic bases of phenotypes in many different classes of organisms, and it has become possible to determine the nucleotide changes responsible for some adaptations, as well as understand constraints on those processes at molecular levels. For example, a multiplicity of adaptive pathways have been revealed in viruses subjected to strong selection, indicating that even small genomes can respond in a variety of ways (Yin 1993Citation ; Kellam et al. 1994Citation ; Nijhuis et al. 1998Citation ; Escarmis, Davila, and Domingo 1999Citation ; Wichman et al. 1999Citation ; Crill, Wichman, and Bull 2000Citation ). Adaptive trajectories and the ruggedness of fitness landscapes have been explored for nucleic acid molecules selected to bind protein targets in vitro (Beaudry and Joyce 1992Citation ; Jhaveri et al. 1997Citation ). Constraints on evolutionary reversibility have even assumed a sinister significance in medicine: drug resistance in bacteria is not lost when antibiotic use is discontinued, because compensatory changes during the initial phase of adaptation create a fitness "valley" which opposes the reverse evolutionary process (Schrag, Perrot, and Levin 1997Citation ; Bjorkman, Hughes, and Andersson 1998Citation ).

A topic with broad relevance to the genetic basis of adaptation is the importance of mutations with large effect versus those with small effect. Earlier arguments concluded that most evolutionary change should result from mutations of small effect, both because those of large beneficial effect would be rare and because they would be overwhelmed by the more common mutations of small effect (Fisher 1930Citation ; Lande 1983Citation ). Recent empirical and theoretical evidence favors a more prominent role of major-effect mutations in adaptation (Orr 1998Citation ). Most fundamental to this question is whether mutations of large benefit arise. If they do occur, a variety of other questions follow, such as those regarding the nature of their pleiotropic effects and their effect in combination with other beneficial mutations.

The work presented in this paper was undertaken to address the biology of major-effect beneficial mutations. The bacteriophage {phi}X174 is strongly inhibited by high temperatures, yet mutants can be isolated that overcome this inhibition (Dowell 1980Citation ). In the present study, a large number of independent mutations were isolated that allowed this phage to plate at high temperature. The sequence changes were identified, and fitness effects of individual mutations were estimated at both high and normal temperatures and in combinations with other mutations.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
{phi}X174 Biology
{phi}X174 is a small icosahedral bacteriophage with a single-stranded DNA genome of 5,386 bases (Hayashi et al. 1988Citation ). The genome contains nine essential and two nonessential genes; genes overlap in approximately 20% of the genome. The mature virus particle consists of 60 copies of the major capsid protein (F), which together form the shell of the virion, 60 copies of the spike protein (G), which form 12 spikes on this shell, and 12 copies of the pilot protein (H), one in the core of each vertex. Sixty copies of the small DNA-binding protein (J) inside the virion help package the genome. The precursor to the mature particle is the procapsid, which consists of the empty virion plus 60 copies of the internal scaffolding protein (B) and 240 copies of the external scaffolding protein (D). Both sets of scaffolding proteins are eliminated as the virus particle matures.

Strains and General Methods
LB broth was used to grow cells, with or without phage. LB broth was mixed as 10 g NaCl, 10 g Bacto-tryptone, and 5 g yeast extract per liter of water, supplemented to 2mM CaCl2 after autoclaving. Agar was added at 15 g/l for plates and at 7 g/l for overlay (soft agar). Borate-EDTA (BE), used for long-term storage of phage, was 80 mM aqueous sodium borate and 80 mM EDTA, adjusted to pH 9.5 with NaOH. Cells used for propagation of phage were Escherichia coli C.

We employed two bacteriophage strains, one that was a wild-type strain, A, not specifically adapted to E. coli C, and another, Af, that was adapted to E. coli C through 66 flask culture serial passages at 37°C (as described below for fitness assays). The complete genome sequence of A has been published (GenBank accession number AF176027; Bull et al. 1997Citation ); the sequence of Af differed from that of A at four nucleotide positions: 756C, 873C, 1033C, and 4706G.

Plate Selection
Spontaneous mutations beneficial to high-temperature growth were isolated as 45°C plaques from parental stocks consisting of resuspended 37°C plaques. (A single 45°C plaque was chosen from each 37°C plaque suspension to ensure that each 45°C isolate had an mutational origin independent of all other 45°C isolates.) Except for phenotype confirmation, plaque purification and subsequent growth of high-temperature mutants was done only at 37°C to suppress the accumulation of secondary mutations beneficial to high temperatures.

Fitness Assays
Assays of phage fitness were conducted at 37°C and at 44°C. A 10-ml volume of LB supplemented with 2 mM CaCl2 was placed in a 125-ml flask in an orbital shaking water bath at 200 rpm. An aliquot of E. coli frozen in LB with 20% glycerol was added and grown for 1 h to a concentration of ~2 x 108 to 4 x 108. Phages were added, and a sample was taken over chloroform at 35 min for an assay at 37°C (40 min for an assay at 44°C); the multiplicity of infection at the end of the assay was typically 0.1 or less (occasionally near unity), so that phages were at low density relative to cells throughout the assay. Fitness was calculated as the number of doublings of phage concentration per 15 min, the latter taken as an approximate generation time of the phage: log2(titer at end of assay/titer at outset)/(duration of assay in quarter hours).

Our fitness estimate may be considered a general measure of phage population growth rate under the assay conditions, subsuming properties of lag time, burst size, and adsorption rate into an intrinsic rate of increase. However, interpretation of our fitness measure in this fashion requires that certain conditions be satisfied. Phage reproduction is highly discontinuous: a single phage infects a cell and for 15 min may fail to yield any viable progeny, whereas minutes later it may release 100 new phages. Consequently, a culture of many phages started in synchrony can exhibit strong cycles in phage number, as the cells adsorb and then release phages in unison. Over time, variation in lag time and adsorption of different individual infections causes this synchrony to decay until a stable age-of- infection distribution is obtained (Levin, Bull, and Stewart 1996Citation ). Our fitness measure represents a time-independent rate of phage growth only if the second sample is taken after an approximate stable age-of-infection distribution has been achieved. To test the validity of this assumption, fitness assays were carried out (at 44°C) and sampled at 35, 37.5, 40, and 45 min for sequential estimates from the same cultures; these tests were conducted on phage genomes representing high, middle, and low fitnesses. The fitness estimates across this 10-min interval were sufficiently close to each other to justify our interpretation of these assays as time-independent measures of phage growth rate (spanning less than 0.25 on the log fitness scale).

Phage stocks for fitness assays were stored in LB broth, as storage in BE caused a reduction in estimated fitness. (Presumably, the stored phage experienced a delay in adsorption when initially added to the culture). Our interest in genotypes A.1565.3843 and A.1565.3843.1727 concerned only the effect of mutations 1727T at 44°C, so these genotypes were not assayed at 37°C.

The fitness measure here is log2 of absolute growth rate (doublings of phage number per 15 min). A fitness of 0 means that no change in phage numbers occurs, whereas a negative fitness means that phage numbers decline. The relative fitness of one genotype versus another (w or 1 + s in population genetics notation) is simply the ratio of the two absolute growth rates, which may be seen as follows. Let N1 be the starting number of genotype 1 (fitness X1) and N2 be the starting number of genotype 2 (fitness X2). At the end of a generation, there will be 2X1N1 individuals of genotype 1 (2X2N2 of genotype 2). If we let p be the relative frequency of genotype 1, then the frequency of genotype 1 in the next generation is simply


Sequencing
DNA sequences were obtained from chain-termination reactions using an automated machine (ABI377). The DNA samples sequenced were viral-positive strands from PCR products of phage samples amplified from plaque isolates or from phage samples taken from lysates. The entire phage genome was amplified as two overlapping PCR products. Mutations 28A, 1565G, and 1727T were confirmed for many of the strains by sequencing multiple isolates and by screening with oligos (see below).

Site-Directed Mutations
In two cases, genomes with two or more mutations were combined into a single genome. A mutagenic oligo containing one of the mutations was annealed to a genome carrying the second mutation, and the oligo was extended using T4 DNA polymerase, essentially as in Sambrook, Fritsch, and Maniatis (1989)Citation . Isolates with the desired changes were first identified by oligo screening (see below) and then by sequencing of the entire genome.

Screening for Changes
To screen large numbers of isolates for specific changes, individual plaques were picked into separate wells of microtiter plates, replica-plated, blotted, and hybridized with radiolabeled oligos as in Crill, Wichman, and Bull (2000).

Verification of Genotypes in Fitness Assays
The high-temperature environment is not only inhibitory to {phi}X174 but, as shown here, allows major fitness gains from single-nucleotide changes. In estimating the fitness of a genotype carrying the mutation X, a potential problem is that a second beneficial mutation (Y), already at moderate frequency in the stock (e.g., 3%), may ascend to high frequency during the fitness assay such that the final culture is predominantly the double-mutant XY, instead of just X. The apparent fitness of X will thus be higher than its true fitness. This problem was foreshadowed by the fact that plaque isolates obtained from an isolate of known consensus genotype often carried second changes; hence, we were led to evaluate consensus genotypes in the cultures obtained from fitness assays. The problem appeared to be most pronounced for a couple of mutations in the A genome background and was so severe that the effect of one of the mutations (1565G) was evaluated only in the Af background. For three genotypes (A.1727T, A.28A, and Af), genotype verification was done by obtaining consensus sequences of the entire phage genome in the culture of a fitness assay completed at 44°C. In some other cases, sequences were verified after a series of passages on a host (at 37°C or 44°C). In several cases, we were interested only in obtaining an upper limit on fitness, so genotype verification at the completion of those fitness assays was not attempted. Although not every genotype was confirmed at the completion of a fitness assay, the main conclusions of this paper are robust to any upward biases that may have remained.

The isolate of one genotype (Af.1565G.3843T.1727T, created partly by site-directed mutagenesis) was stored as a plug in BE and showed a 2–3-log drop in titer between its isolation and the verification of its sequence. A drop in titer of this magnitude could easily bias the genetic pool of surviving phages, so DNA was recovered from the plug and electroporated; the DNA from the formerly infectious phage particles should have been stable in the 80 mM EDTA of the BE. The stock of this genotype used in fitness assays was then recreated by suspending 50–100 plaques obtained from the electroporation. This was one case in which our interest lay in obtaining merely an upper limit on fitness, so no attempt was made to verify genotype at the completion of fitness assays.

Statistical Tests
Average fitnesses (doublings per hour) were compared between two or, in some cases, four isolates. In a comparison of two isolates, the question was simply whether the null hypothesis of equality could be rejected, and those tests employed standard t-tests for the difference of two means (Snedecor and Cochran 1980Citation ). Comparisons of four isolates used the following test. Let Xibe the fitness estimate of genotype i, and let it have a normal distribution with mean µi and variance {sigma}2. In a comparison of four genotypes (i {1, 2, 3, 4}), the question was whether the difference between µ1 and µ2 was the same as the difference between µ3 and µ4, i.e., whether {delta} {equiv}2 - µ1) - (µ4 - µ3) = 0. Representing the sample averages as (X1, X2, X3, X4), each average is ni, {sigma}2/mi), where mi is the sample size. Under the null model, = X2 - X1 - (X4 - X3) obeys a normal distribution with mean zero and variance v2 = {sigma}2(1/m1 + 1/m2 + 1/m3 + 1/m4). The ratio (/v)/s/{sigma} follows a t distribution with m1 + m2 + m3 + m4 - 4 degrees of freedom, where s2 is the estimate of {sigma}2.

In one case (that of fig. 3 ), an analysis of variance was applied to assess the significance of the heterogeneity observed among four differences involving eight genotypes (Y1 = X2 - X1, Y2 = X4 - X3, Y3 = X6 - X5, Y4 = X8 - X7). Because of highly unequal sample sizes used to estimate the various Xi values, the distribution of the heterogeneity statistic H = {Sigma}i (Yi - )2 was determined numerically. Using a 95% upper confidence limit of the pooled estimate of the variance of X1, normal random values were drawn numerically to reconstitute the sample sizes obtained in the actual data; these drawings assumed that the true means were identical among the Yi values. The proportion of trials in which the simulated H values equaled or exceeded the observed H value was considered the significance level of the observed H value.



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Fig. 3.—Diminishing-returns epistasis. The contribution of 1727T to fitness at 44°C was evaluated in four genomes. The fitness of the background genome in the absence of 1727T is given on the X axis (horizontal), whereas the improvement in fitness by adding 1727T is given by the value on the Y axis (vertical). If the effect of 1727T were independent of the background genome fitness, the line connecting the points would be horizontal. The benefit of 1727T thus declines as the baseline fitness increases. Total fitness of a genome with 1727T is given by adding the X and Y values of the point. Total fitness is significantly heterogeneous across these genotypes (P < 0.01, F3,20 = 78). Likewise, the contributions of 1727T are also significantly heterogeneous between backgrounds (P < 0.0001, numerical test)

 

    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Three distinct mutations were recovered that individually conferred a large-plaque phenotype at 45°C on E. coli C (table 1 ). Two of these changes (mutations 1565G and 1727T) caused inferred amino acid substitutions in the major capsid gene F (ThrF188Ala, LeuF242Phe). (By convention, amino acid positions for F are the codon numbers minus 1, because the leading methionine is removed by the cell.) The other change was missense only in the internal scaffolding protein (mutation 28A, ValB114Ile). All three mutations were transitions, but they were not recovered with equal frequency (table 1 ). Based on comparisons of plaque numbers at 44°C and 37°C, the efficiency of plating was near unity for all three mutations, suggesting that the unequal recovery was not due to different rates of plaque formation by these mutants.


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Table 1 Mutations Recovered in 45°C Plaques

 
Structural Locations
These mutations were localized in available structural models of the {phi}X174 virion and procapsid (fig. 1 ; McKenna, Ilag, and Rossman 1994Citation ; Dokland et al. 1999Citation ). Mutations at 1565 (F188) and 1727 (F242) are located in different regions of the major capsid protein (McKenna, Ilag, and Rossman 1994Citation ). F242 resides in the ßF strand of the barrel and is slightly exposed on the virion's inner surface. F188 is, instead, located at the threefold axis of symmetry and is a site of F-F interaction between subunits. Although 28A affects the internal scaffolding protein instead of the capsid protein, the affected residue (B114) contacts the ßF strand of protein F (Dokland et al. 1999Citation ). By these structural considerations, therefore, the 1727T and 28A mutations may be causing similar changes during procapsid formation (see Discussion).



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Fig. 1.—Location of mutations in the structural model of the major capsid protein F as observed in the procapsid (Dokland et al. 1999Citation ). The view here is from the inside of the virion, looking out at one copy of protein F, and includes the single residue B114 from the internal scaffolding protein. The protein is shown as a ribbon model, with the residues of the four mutations shown in space-filling mode (the wild-type amino acids are depicted). The approximate position of protein G is at the right extremity, and five G-F units would then be radially arranged (with the five subunits of G central) to form a pentameric unit of the virus. F242 and F188 are the two capsid mutations observed to confer phage growth at high temperature. Residue F242 resides in the ß-barrel, central in the folded protein and exposed on the inner surface of the virion. Residue F188 is near one extremity of the folded protein, at the threefold axis of symmetry. The change at F87 is also shown (it is compensatory for F188 at 37°C but abolishes growth at 44°C). F87 is distant from both F242 and F188, and it does not interact with these residues between different F subunits (McKenna, Ilag, and Rossman 1994Citation ). Residue B114 lies slightly inside the plane of F (toward the reader) and is not in contact with the portion of F shown overlapping in this projection (Dokland et al. 1999Citation )

 
Fitness Effects of Single Mutations
The plaque assay offers a convenient way to identify beneficial mutations in the high-temperature environment, but this environment is difficult to control and changes during the course of plaque formation as cell density increases on the plate. Fitness effects of plaque-isolated mutations were therefore quantified in a standardized high-temperature liquid environment that was reproducible and sensitive to fitness differences. Fitness in this liquid environment is not necessarily equivalent to fitness on plates or to fitness in any other high-temperature environment (e.g., Dykhuizen 1990Citation ), but it offers a way of assessing and comparing fitness in a standardized high-temperature environment.

All three mutations conferred large gains in fitness at 44°C, but fitness losses at 37°C (fig. 2 ). The largest increase was conferred by 1727T in the A background, with a net improvement of 3.9 doublings per generation over the slightly negative fitness of A. The relative fitness of A.1727 over A is thus 23.9 = 14.9 per generation (table 2 ). Thus, the mutant would outgrow the ancestor with a selective coefficient of s = 13.9 (relative fitness w = 1 + s). The benefit conferred by 1727T in the Af background is also large (s = 6.7) but is significantly less than that in the A background (t28 = 3.56, P < 0.002). Mutations 28A and 1565G conferred significantly lower benefits at 44°C than did 1727T, but both still represent large effects in evolutionary terms (t11 = 9.5, P < 0.0001 for equal effects of 28A vs. 1727T in the A background; t5 = 5.4, P < 0.005 for equal effects of 1565G vs. 1727T in the Af background).



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Fig. 2.—Viral fitness of different phage genotypes. Fitness (vertical axis) is measured as the number of doublings of phage concentration per 15 min under standard conditions (see Materials and Methods); the horizontal axis indicates genotype. Isolates are grouped according to genetic background (A and Af). Top, Fitness at the inhibitory temperature of 44°C. The mutations (1727T, 1565G, 28A) all confer large increases. Bottom, Fitness at the normal temperature of 37°C. The three mutations beneficial at high temperature all lower fitness at 37°C. Af is the most fit genotype at 37°C, reflecting its previous adaptation to these conditions. The change at 1263 is compensatory for the lower fitness of 1565G at 37°C but abolishes the benefit of 1565G at 44°C

 

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Table 2 Fitness Effects of Mutations at 44°C

 
Diminishing-Returns Epistasis
The benefit of 1727T at 44°C was compared in four different backgrounds: A, Af, A.28, and Af.1565.3843 (table 2 and fig. 3 ). In two of these backgrounds, the mutation 1727T was recovered from a plaque at 45°C; the other two genomes were created in part by site-directed mutagenesis. Since the A.1565G genome was prone to acquire additional changes, we took the precautionary step of passaging Af.1565G through 15 flask cycles at 44°C. It acquired the additional change 3843 G->T. Site-directed mutagenesis was used to add 1727T to this genotype.

The fitnesses of all four genomes improved with 1727T and showed the same ranking of total fitness after inclusion of 1727T. However, the benefit provided by 1727T declined monotonically with background fitness. Thus, 1727T offered the most to those genomes that had the most to gain.

Compensation
The mutants Af.1727 and Af.1565 were each propagated in liquid at 37°C under conditions similar to those used to adapt the Af genotype to 37°C. The fact that both high-temperature mutations lowered fitness at 37°C (fig. 2 ) meant that further adaptation at 37°C was possible, via reversions if not also via compensatory changes. Thirteen passages of Af.1727T failed to evolve any change that could be detected in the consensus sequence of the lysate (transfer size was typically between 104 and 106). After four transfers of the Af.1565G line, approximately 90% of the phage failed to plate at high temperature. A plaque isolated at 37°C that failed to plate at high temperature was chosen for further work. It retained the 1565G mutation but exhibited another substitution in gene F: 1263A->G (AspF87Gly). In the structural model, F87 does not directly interact with F188, either within the protein subunit or between subunits, but it is adjacent to a site of twofold F-F interaction (F86).

As evidence of compensatory evolution, 1263G boosted the fitness of Af.1565G at 37°C significantly (fig. 2 ; t7 = 13, P < 0.0001 for equality of fitness). The recovery was only partial, because the fitness of Af.1565G/1263G at 37°C was still less than that of Af (fig. 2 ; t6 = 9.0, P < 0.0002), but it approximately matched the fitness of Af.1727, which had failed to acquire compensatory changes in 13 passages. In contrast to the compensatory effect at 37°C, 1263G abolished the benefit of 1565G at 44°C: the fitness of Af.1565/1263 dropped significantly and was now indistinguishable from that of Af (fig. 2 ; t5 = 14.6, P < 0.0001 for equality of Af.1565 and Af.1565/1263; t9 = 1.8, P ~ 0.1 for equality of Af and Af.1565/1263, both at 44°C). Loss of the original phenotype during compensatory evolution has also been seen with bacterial antibiotic resistance (Bjorkman, Hughes, and Andersson 1998Citation ).


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
The goal of this study was to identify and characterize any mutations that conferred a large benefit to {phi}X174 grown at high temperature. Three big-benefit missense point mutations were recovered. Two were located in the major capsid gene F, and the third affected the amino acid sequence of the internal scaffolding protein (gene B). At the inhibitory temperature of 44°C, the estimated selection coefficients of these mutations relative to their genetic backgrounds ranged from 3.2 to 13.9. These effects are indeed large, as it is usually regarded that selective coefficients in the range of 0.01–0.1 are large.

The assay used to identify these mutations was plaque formation at 45°C on E. coli C, which recovers only mutations of large effect. Thus, the study demonstrates that big-benefit mutations exist, but it leaves open the question of the number of mutations of lesser benefit, and it does not resolve how the phage would adapt during long-term exposure to high temperature. The skewed distribution of mutations observed here (57 of 1727T, 9 of 1565G, and 1 of 28A) further emphasizes that other point mutations may confer a large benefit but were too rare to be recovered in our sample. In phage, the same base substitution can exhibit orders-of-magnitude differences in rate between sites (Ronen and Rahat 1976Citation ), so most of the big-benefit mutations in {phi}X174 may remain to be discovered.

Major-Effect Mutations in Evolution
Theoretical arguments have questioned the importance of mutations with large fitness effects in evolution for two reasons. First, mutations with large beneficial effects are thought to be rare (Fisher 1930Citation ). Second, it has been assumed that most natural selection is weak (Lande 1983Citation ). Nonetheless, direct evidence is accumulating that mutations of large effect are important in evolution (Golding and Dean 1998Citation ; Orr 1998Citation ). Although the strength-of-selection issue has not been resolved, comparisons between species have shown that major differences (morphological, biochemical) are often due to few amino acid changes or to few quantitative trait loci (QTLs). For example, in monkey flowers, major morphological differences between two incipient species are attributable to single QTLs for each of several traits (Bradshaw et al. 1998Citation ). Analysis of the molecular bases of species differences in certain enzymes, using both a structural and a site-directed mutagenesis approach, has shown that profound differences can be attributed to single or few amino acid differences (reviewed in Golding and Dean 1998Citation ). Indeed, an emerging view of molecular evolution is that major shifts in enzyme function occur from one or a few changes of major effect, with early fine-tuning provided by a few other substitutions; subsequent changes serve largely to track small environmental changes (Golding and Dean 1998Citation ). This view has been bolstered by experiments in which organisms have been adapted to new environments (Hall 1984Citation ) and by experiments in which enzyme function has been altered by site-directed metagenesis (e.g., Dean 1988Citation ; Dean and Golding 1997Citation ; Wu et al. 1999Citation ). This model of molecular evolution overlaps with that of Lande (1983)Citation in attributing most substitutions to weak effects, while contrasting with Lande's model in predicting that major differences in function will be due to very few amino acid differences.

Regardless of the absolute importance of major-effect mutations in adaptation, major-effect mutations may be expected to have a greater role in viruses and bacteria than in most plants and animals. First, the large population sizes and rapid reproductive rates of these microbes predispose them to evolution by mutations of large effect: the wide range of mutations realized in the large population sizes typical of microbes improves the chance that those with large benefit will occur, and the high reproductive rates allow very high fitness to be realized. Second, the largely asexual nature of many microbes means that beneficial genotypes compete with one another (Fisher 1930Citation ; Dykhuizen 1990Citation ; Gerrish and Lenski 1998Citation ; deVisser et al. 1999Citation ; Miralles et al. 1999Citation ). The selective sweep of one mutation will depress the standing variation of small-effect mutations, thereby giving an advantage to the next mutation of large effect (as per Lande's [1983Citation ] model). Third, the population structure of many microbes creates a selective sieve in favor of beneficial mutations of large effect. If populations periodically pass through bottlenecks, as with infectious microbes at transmission, beneficial mutations will be lost through genetic drift if they have not reached high enough frequency to be included in the colonizing population. Upon arising, major- effect mutations disproportionately increase relative to minor- effect mutations, so they are more apt to reach a frequency that will survive a bottleneck (e.g., Gerrish and Lenski 1998Citation ).

Epistasis
In {phi}X174, the fitnesses of mutations with large effects varied with genetic background, exhibiting a principle of diminishing returns: genetic backgrounds of higher initial fitness received lesser benefits from 1727T, although the more fit backgrounds also had the greater total fitness when combined with 1727T. These results point to the possibility of a maximum attainable fitness for a virus in a particular environment and to the possibility that a mutation which provides a large benefit when the virus is far from this upper limit can only offer a small benefit if the virus is close to this upper limit. If this is true, then adaptive trajectories might exhibit the property that the first beneficial mutations to arise provide the largest benefits, with later mutations providing lesser benefits, regardless of the identities of those mutations. Note that this model does not preclude other types of epistasis (such as antagonistic epistasis), but merely places an upper limit on fitness.

The model of diminishing-returns epistasis is not unique to {phi}X174 high-temperature adaptation. Indeed, it reflects a common view of interactions among multiple mutations that improve the same defect. Metabolic control theory predicts diminishing-returns epistasis a priori (Hartl, Dykhuizen, and Dean 1985Citation ; Dean 1994Citation ). An analogy lies in oiling a squeaky hinge: if one drop of oil stops the squeak, a second drop of oil does not make the hinge twice as quiet. Diminishing returns is also invoked to explain evolution toward an optimum, because the maximal possible fitness effect of a beneficial mutation necessarily decreases as the population average approaches the optimum (Fisher 1930Citation ; Orr 1998Citation ). What is perhaps unusual in the {phi}X174 case is that the example applies to structural interactions instead of enzymatic ones. Using an analogy of stabilizing a tabletop, a fourth leg may contribute much less to stability than does a third leg. Thus, these examples perhaps more generally belong to the category of multiple solutions to a single problem.

Diminishing-returns epistasis has previously been observed. Lenski (1988)Citation observed that a compensatory mutation in a bacterium which reduced the deleterious effect of phage-resistance provided more benefit to the low-fitness resistant strain than to the high-fitness wild-type strain. Bacterial resistance to some antibiotics is often achieved with any of several nucleotide changes, but combinations of multiple changes apparently do not achieve superiority far beyond that of the best single mutations (e.g., Bryan 1982Citation ). However, there is no obvious pattern of diminishing returns among AZT-resistance mutations in human immunodeficiency virus (HIV; Kellam et al. 1994Citation ).

Evolutionary Trajectories
The isolation of three different large-effect mutations raises the question of the number of pathways and endpoints during adaptation to high temperature. The double mutant A.1727/28 had higher fitness than did either single mutant (as did Af.1565.3843/1727), so perhaps 28A, 1565G, and 1727T could all accumulate into one genome during prolonged adaptation to high temperature. If the triple-mutant genotype was in fact superior to all other combinations, adaptation might yield a common endpoint from different starting points. However, results from {phi}X174 adapted to E. coli C in chemostats at 43.5°C raise doubts about the likelihood of this outcome. In four chemostat passages of ancestor A on E. coli C, evolution of 1565G or 1727T was observed in all lineages, yet no lineage accumulated both substitutions (isolates C1, C2, SC1, and SC2 from Bull et al. [1997Citation ]). Interestingly, the combination of 1727T and 28A was observed in one of those lineages, consistent with our observation that A.1727T/28A had higher flask fitness at 44°C than did either single mutant. Of course, fitness in a chemostat is not equivalent to fitness in the flask assays used here, but the fact that chemostats initiated with A nonetheless accumulated at least one of 28A, 1565G, or 1727T suggests that these mutations are beneficial in chemostats as well. Instead, it may be that each of these mutations is better as the first mutation than as a later one (i.e., diminishing returns).

The Defect at High Temperature
The fact that the combination of different mutations exhibited strong diminishing-returns epistasis is consistent with a single defect at high temperature. Dowell (1980)Citation studied {phi}X174 development and virion stability at high temperatures (42°C, 44°C) for a wild-type virus and two mutants that plaqued at high temperature. Those mutants were not mapped or characterized at the sequence level, and both may have carried the same mutation. The mutant virons exhibited greater stability than did the wild type at an extreme temperature (56°C), suggesting that the mutants possessed an altered capsid. At high temperatures, wild-type virus infections exhibited normal levels of double-stranded DNA but reduced levels of single-stranded DNA, which is synthesized late. From these combined observations, Dowell (1980)Citation concluded that virion assembly was disrupted at high temperatures and that mutations restoring growth would be located either in capsid protein genes or in genes contributing to virion assembly but absent from the mature particle.

The results presented here provide a remarkable confirmation of both possibilities suggested by Dowell (1980)Citation . Two of the mutations affect the major capsid protein (F188, F242), and the other affects the internal scaffolding protein (B114). F242 is in a ß strand that contacts B114 (Dokland et al. 1999Citation ), so both F242 and B114 may contribute to procapsid stability in the same way. The change at F188 has no obvious association with the other two changes, however. It lies at the threefold axis of symmetry (the pore at which the DNA is loaded into the capsid) and is a site of interaction between subunits of F.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We thank Ben Fane and Ian Molineux for advice on {phi}X174 biology and Tony Dean for discussions on molecular evolution and epistasis. Two anonymous reviewers contributed to the revision of the final draft. This work was supported by grant GM 57756 from the NIH. J.J.B. was supported as the Miescher Regents Professor at the University of Texas.


    Footnotes
 
Antony Dean, Reviewing Editor

1 Keywords: {phi}X174 beneficial mutations microbial evolution epistasis inhibitor adaptation Back

2 Address for correspondence and reprints: J. J. Bull, Department of Integrative Biology, University of Texas, Austin, Texas 78712-1023. E-mail: bull{at}bull.biosci.utexas.edu . Back


    literature cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 

    Beaudry, A. A., and G. F. Joyce. 1992. Directed evolution of an RNA enzyme. Science 257:635–641.

    Bjorkman, J., D. Hughes, and D. I. Andersson. 1998. Virulence of antibiotic-resistant Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 95:3949–3953.

    Bradshaw, H. D. Jr., K. G. Otto, B. E. Frewen, J. K. McKay, and D. W. Schemske. 1998. Quantitative trait loci affecting differences in floral morphology between two species of monkeyflowers (Mimulus). Genetics 149:367–382.

    Bryan, L. E. 1982. Bacterial resistance and susceptibility to chemotherapeutic agents. Cambridge University Press, Cambridge, England.

    Bull, J. J., M. R. Badgett, H. A. Wichman, J. P. Huelsenbeck, D. M. Hillis, A. Gulati, C. Ho, and I. J. Molineux. 1997. Exceptional convergent evolution in a virus. Genetics 147:1497–1507.

    Crill, W. D., H. A. Wichman, and J. J. Bull. 2000. Evolutionary reversals during viral adaptation to alternating hosts. Genetics 154:27–37.

    Dean, A. M. 1988. Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli. Mol. Biol. Evol. 5:469–485.[Abstract]

    ———. 1994. Fitness, flux, and phantoms in temporally variable environments. Genetics 136:1481–1495.

    Dean, A. M., and G. B. Golding. 1997. Protein engineering reveals ancient adaptive replacements in isocitrate dehydrogenase. Proc. Natl. Acad. Sci. USA 94:3104–3109.

    deVisser, J. A. G. M., C. W. Zeyl, P. J. Gerrish, J. L. Blanchard, and R. E. Lenski. 1999. Diminishing returns from mutation supply rate in asexual populations. Science 283:404–406.

    Dokland, T., R. A. Bernal, A. Burch, S. Pletnev, B. A. Fane, and M. G. Rossmann. 1999. The role of scaffolding proteins in the assembly of the small, single-stranded DNA virus {phi}X174. J. Mol. Biol. 288:595–608.[ISI][Medline]

    Dowell, C. E. 1980. Growth of bacteriophage {phi}X-174 at elevated temperatures. J. Gen. Virol. 49:41–50.[Abstract]

    Dykhuizen, D. E. 1990. Experimental studies of natural selection in bacteria. Annu. Rev. Ecol. Syst. 21:373–389.[ISI]

    Escarmis, C., M. Davila, and E. Domingo. 1999. Multiple molecular pathways for fitness recovery of an RNA virus debilitated by operation of Muller's ratchet. J. Mol. Biol. 285:495–505.[ISI][Medline]

    Fisher, R. A. 1930. The genetical theory of natural selection. Oxford University Press, Oxford, England.

    Gerrish, P. J., and R. E. Lenski. 1998. The fate of competing beneficial mutations in an asexual population. Genetica 102/103:127–144.

    Golding, G. B., and A. M. Dean. 1998. The structural basis of molecular adaptation. Mol. Biol. Evol. 15:355–369.[Abstract]

    Hall, B. G. 1984. The evolved ß-galactosidase system of Escherichia coli. Pp. 165–185 in R. P. Mortlock, ed. Microorganisms as model systems for studying evolution. Plenum Press, New York.

    Hartl, D. L., D. E. Dykhuizen, and A. M. Dean. 1985. Limits of adaptation: the evolution of selective neutrality. Genetics 111:655–674.

    Hayashi, M., A. Aoyama, D. L. Richardson, and M. N. Hayashi. 1988. Biology of the bacteriophage {Phi}X174. Pp. 1–17 in R. Calendar, ed. The bacteriophages. Vol. 2. Plenum Press, New York.

    Jhaveri, S. D., I. Hiral, S. Ball, K. W. Uphoff, and A. D. Ellington. 1997. Landscapes for molecular evolution: lessons from in vitro selection experiments with nucleic acids. Annu. Rep. Combinatorial Chem. Mol. Diversity 1:169–191.

    Kellam, P., C. A. Boucher, J. M. Tijnagel, and B. A. Larder. 1994. Zidovudine treatment results in the selection of human immunodeficiency virus type 1 variants whose genotypes confer increasing levels of drug resistance. J. Gen. Virol. 75:341–51.[Abstract]

    Lande, R. 1983. The response to selection on major and minor mutations affecting a metrical trait. Heredity 50:47–65.

    Lenski, R. E. 1988. Experimental studies of pleiotropy and epistasis in Escherichia coli II. compensation for maladaptive effects associated with resistance to virus T4. Evolution 42:433–440.

    Levin, B. R., J. J. Bull, and F. M. Stewart. 1996. The intrinsic rate of increase of HIV/AIDS: epidemiological and evolutionary implications. Math. Biosci. 132:69–96.[ISI][Medline]

    McKenna, R., L. L. Ilag, and M. G. Rossmann. 1994. Analysis of the single-stranded DNA bacteriophage phi X174, refined at a resolution of 3.0 A. J. Mol. Biol. 237:517–543.[ISI][Medline]

    Maynard Smith, J. 1989. Evolutionary genetics. Oxford University Press, Oxford, England.

    Miralles, R., P. J. Gerrish, A. Moya, and S. F. Elena. 1999. Clonal interference and the evolution of RNA viruses. Science 285:1745–1747.

    Nijhuis, M., C. A. Boucher, P. Schipper, T. Leitner, R. Schuurman, and J. Albert. 1998. Stochastic processes strongly influence HIV-1 evolution during suboptimal protease-inhibitor therapy. Proc. Natl. Acad. Sci. USA 95:14441–14446.

    Orr, H. A. 1998. The population genetics of adaptation: the distribution of factors fixed during adaptive evolution. Evolution 52:935–949.

    Ronen, A., and A. Rahat. 1976. Mutagen specificity and position effects on mutation in T4rII nonsense sites. Mutat. Res. 34:21–34.[ISI][Medline]

    Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd edition. Cold Spring Harbor Laboratory Press, New York.

    Schrag, S. J., V. Perrot, and B. R. Levin. 1997. Adaptation to the fitness costs of antibiotic resistance in Escherichia coli. Proc. R. Soc. Lond. B Biol. Sci. 264:1287–1291.[ISI][Medline]

    Snedecor, G. W., and W. G. Cochran. 1980. Statistical methods. 7th edition. Iowa State University Press, Ames, Iowa.

    Wichman, H. A., M. R. Badgett, L. A. Scott, C. M. Boulianne, and J. J. Bull. 1999. Different trajectories of parallel evolution during viral adaptation. Science 285:422–424.

    Williams, G. C. 1966. Adaptation and natural selection. Princeton University Press, Princeton, N.J.

    Wright, S. 1977. Evolution and the genetics of natural populations. Vol. 3. University of Chicago Press, Chicago.

    Wu, G., A. Fiser, B. Ter Kuile, A. Sai, and M. Muller. 1999. Convergent evolution of Trichomonas vaginalis lactate dehydrogenase from malate dehydrogenase. Proc. Natl. Acad. Sci. USA 96:6285–6290.

    Yin, J. 1993. Evolution of bacteriophage T7 in a growing plaque. J. Bacteriol. 175:1272–1277.[Abstract]

Accepted for publication February 23, 2000.