Testing optimality with experimental evolution: lysis time in a bacteriophage

Optimality models collapse the vagaries of genetics into simple trade-offs to calculate phenotypes expected to evolve by natural selection. Optimality approaches are commonly criticized for this neglect of genetic details, but resolution of this disagreement has been difficult. The importance of genetic details may be tested by experimental evolution of a trait for which an optimality model exists and in which genetic details can be studied. Here we evolved lysis time in bacteriophage T7, a virus of Escherichia coli. Lysis time is equivalent to the age of reproduction in an organism that reproduces once and then dies. Delaying lysis increases the number of offspring but slows generation time, and this trade-off renders the optimum sensitive to environmental conditions: earlier lysis is favored when bacterial hosts are dense, later lysis is favored when hosts are sparse. In experimental adaptations, T7 evolved close to the optimum in conditions favoring early lysis but not in conditions favoring late lysis. One of the late lysis adaptations exhibited no detectable phenotypic evolution despite genetic evolution; the other evolved only partly toward the expected optimum. Overall, the lysis time of the adapted phages remained closer to their starting values than predicted by the model. From the perspective of the optimality model, the experimental conditions were expected to select changes only along the postulated trade-off, but a trait outside the trade-off evolved as well. Evidence suggests that the model's failure ultimately stems from a violation of the trade-off, rather than a paucity of mutations.     

Roles of bacteriophage T4 gene 5 and gene s products in cell lysis.

Previous studies indicated that (i) T4 gene s product (gps) protects infected cells from superinfection lysis from without, (ii) the absence of gps in infected cells also leads to lysis from within even when T4 e lysozyme is absent, (iii) T4 gene 5 product (gp5), a polypeptide of the virion baseplate, may be responsible for inducing lysis from without, and (iv) altered gp5 of the T4 mutant 5ts1 can replace e lysozyme to cause lysis from within. Results of this study showed that (i) wild-type gp5 in infected cells lacking e lysozyme was responsible for lysis from within in the absence of gps, and (ii) gps did not protect infected cells from superinfection lysis from without by 5ts1 phage. We prpose that gps normally prevents functional expression of wild-type gp5 activity from either side of the cell wall, whereas the 5ts1 form of gp5 is insensitive to the gps barrier and induces lysis from either side of the cell wall.     

Control of Lysis of T4-infected Escherichia coli

The lysis of Escherichia coli B/5 infected with T4Dr48 could be delayed by addition of 9-aminoacridine (9AA). Infected cells showed an early period of maximal response followed by a decline in sensitivity. The ultimate rate of lysis was also affected by the dye. Deoxyribonucleic acid (DNA), protein, and lysozyme synthesis began at the normal time in complexes inhibited by 9AA addition. The rates of synthesis of these macromolecules were lower in the presence of the dye, with DNA and lysozyme synthesis being more strongly affected than total protein synthesis. Penicillin-sensitive cell wall synthesis stopped at about 10 min after infection. Inhibition of oxidative metabolism by early potassium cyanide addition prevented lysis in the presence of intracellular lysozyme. The cyanide-sensitive event occurred at about 20 min in normal infections, and between 30 and 40 min in 9AA-inhibited infections. 9AA could alter both the time at which the cyanide-sensitive event occurred and the time of lysis. Addition of chloramphenicol did not prevent lysis once intracellular lysozyme was present. Lysis from without of infected cells consisted of three phases: an initial sensitivity, followed by a short period of resistance, and then a return to sensitivity in normal infections. The demonstration of the late return to sensitivity depended on the presence of intracellular lysozyme, and could be delayed by 9AA addition.     

Compensatory evolution in response to a novel RNA polymerase: orthologous replacement of a central network gene

A bacteriophage genome was forced to evolve a new system of regulation by replacing its RNA polymerase (RNAP) gene, a central component of the phage developmental pathway, with that of a relative. The experiment used the obligate lytic phage T7 and the RNAP gene of phage T3. T7 RNAP uses 17 phage promoters, which are responsible for all middle and late gene expression, DNA replication, and progeny maturation, but the enzyme has known physical contacts with only 2 other phage proteins. T3 RNAP was supplied in trans by the bacterial host to a T7 genome lacking its own RNAP gene and the phage population was continually propagated on naive bacteria throughout the adaptation. Evolution of the T3 RNAP gene was thereby prevented, and selection was for the evolution of regulatory signals throughout the phage genome. T3 RNAP transcribes from T7 promoters only at low levels, but a single mutation in the promoter confers high expression, providing a ready mechanism for reevolution of gene expression in this system. When selected for rapid growth, fitness of the engineered phage evolved from a low of 5 doublings/h to 33 doublings/h, close to the expected maximum of 37 doublings/h. However, the experiment was terminated before it could be determined accurately that fitness had reached an obvious plateau, and it is not known whether further adaptation could have resulted in complete recovery of fitness. More than 30 mutations were observed in the evolved genome, but changes were found in only 9 of the 16 promoters, and several coding changes occurred in genes with no known contacts with the RNAP. Surprisingly, the T7 genome adapted to T3 RNAP also maintained high fitness when using T7 RNAP, suggesting that the extreme incompatibility of T7 elements with T3 RNAP is not an invariant property of divergence in these expression systems.     

Fitness recovery and compensatory evolution in natural mutant lines of C. elegans

Deleterious mutation accumulation plays a central role in evolutionary genetics, conservation biology, human health, and evolutionary medicine (e.g., methods of viral attenuation for live vaccines). It is therefore important to understand whether and how quickly populations with accumulated deleterious mutational loads can recover fitness through adaptive evolution. We used laboratory experimental evolution with four long-term mutation-accumulation (MA) lines of Caenorhabditis elegans nematodes to study the dynamics of such fitness evolution. We previously showed that when homozygous mutant populations are evolved in large population sizes, they can rapidly achieve wild-type fitness through the accumulation of new beneficial or compensatory epistatic mutations. Here, we expand this approach to demonstrate that when replicate lineages are initiated from the same mutant genotype, phenotypic evolution is only sometimes repeatable. MA genotypes that recovered ancestral fitness in the previous experiment did not always do so here. Further, the pattern of adaptive evolution in independently evolved replicates was contingent upon the MA genotype and varied among fitness-related traits. Our findings suggest that new beneficial mutations can drive rapid fitness evolution, but that the adaptive process is rendered somewhat unpredictable by its susceptibility to chance events and sensitivity to the evolutionary history of the starting population.     

Statistical optimization of the lysis agents for Gram-negative bacterial cells in a microfluidic device

Through statistically designed experiments, lysis agents were optimized to effectively disrupt bacterial cells in a microfluidic device. Most surfactants caused the efficient lysis of Gram-positive microbes, but not of Gram-negative bacteria. A Plackett-Burman design was used to select the components that increase the efficiency of the lysis of the Gram-negative bacteriaEscherichia coli. Using this experimental design, both lysozyme and benzalkonium chloride were shown to significantly increase the cell lysis efficiency, and ATP was extracted in proportion to the lysis efficiency. Benzalkonium chloride affected the cell membrane physically, while lysozyme destroyed the cell wall, and the amount of ATP extracted increased through the synergistic interaction of these two components. The two-factor response-surface design method was used to determine the optimum concentrations of lysozyme and benzalkonium chloride, which were found to be 202 and 99 ppm, respectively. The lysis effect was further verified by microscopic observations in the microchannels. These results indicate that Gram-negative cells can be lysed efficiently in a microfluidic device, thereby allowing the rapid detection of bacterial cells using a bioluminescence-based assay of the released ATP.     

Transition from positive to neutral in mutation fixation along with continuing rising fitness in thermal adaptive evolution

It remains to be determined experimentally whether increasing fitness is related to positive selection, while stationary fitness is related to neutral evolution. Long-term laboratory evolution in Escherichia coli was performed under conditions of thermal stress under defined laboratory conditions. The complete cell growth data showed common continuous fitness recovery to every 2°C or 4°C stepwise temperature upshift, finally resulting in an evolved E. coli strain with an improved upper temperature limit as high as 45.9°C after 523 days of serial transfer, equivalent to 7,560 generations, in minimal medium. Two-phase fitness dynamics, a rapid growth recovery phase followed by a gradual increasing growth phase, was clearly observed at diverse temperatures throughout the entire evolutionary process. Whole-genome sequence analysis revealed the transition from positive to neutral in mutation fixation, accompanied with a considerable escalation of spontaneous substitution rate in the late fitness recovery phase. It suggested that continually increasing fitness not always resulted in the reduction of genetic diversity due to the sequential takeovers by fit mutants, but caused the accumulation of a considerable number of mutations that facilitated the neutral evolution.     

Evolutionary design on a budget: robustness and optimality of bacteriophage T7

Exploring how biological systems have been 'designed' by evolution to achieve robust behaviours is now a subject of increasing research effort. Yet, it still remains unclear how environmental factors may contribute to this process. This issue is addressed by employing a detailed computer model for the intracellular growth of phage T7. More than 150 000 in silico T7 mutants were generated and the rates and efficiencies of their growth in two host environments, namely, a realistic environment that offered finite host resources for the synthesis of phage functions and a hypothetical environment where the phage was supplied infinite host resources, were evaluated. Results revealed two key properties of phage T7. First, T7 growth was overall robust with respect to perturbations in its parameters, but fragile with respect to changes in the ordering of its genetic elements. Secondly, the wild-type T7 had close to optimal fitness in the finite environment. Furthermore, a strong correlation was found between fitness and growth efficiency in the finite environment. The results underscore the potential importance of the environment in shaping robust design of a biological system. In particular, the strong correlation between fitness and growth efficiency suggests that T7 may have evolved to maximise its growth rate by minimising waste of finite resources.     

Placing single-molecule T4 lysozyme enzymes on a bacterial cell surface: toward probing single-molecule enzymatic reaction in living cells

The T4 lysozyme enzymatic hydrolyzation reaction of bacterial cell walls is an important biological process, and single-molecule enzymatic reaction dynamics have been studied under physiological condition using purified Escherichia coli cell walls as substrates. Here, we report progress toward characterizing the T4 lysozyme enzymatic reaction on a living bacterial cell wall using a combined single-molecule placement and spectroscopy. Placing a dye-labeled single T4 lysozyme molecule on a targeted bacterial cell wall by using a hydrodynamic microinjection approach, we monitored single-molecule rotational motions during binding, attachment to, and dissociation from the cell wall by tracing single-molecule fluorescence intensity time trajectories and polarization. The single-molecule attachment duration of the T4 lysozyme to the cell wall during enzymatic reactions was typically shorter than the photobleaching time under physiological conditions. Applying single-molecule fluorescence polarization measurements to characterize the binding and motions of the T4 lysozyme molecules, we observed that the motions of wild-type and mutant T4 lysozyme proteins are essentially the same whether under an enzymatic reaction or not. The changing of the fluorescence polarization suggests that the motions of the T4 lysozyme are associated with orientational rotations. This observation also suggests that the T4 lysozyme binding-unbinding motions on cell walls involve a complex mechanism beyond a single-step first-order rate process.     

Effects of genotype on rates of substitution during experimental evolution

Rates of molecular evolution may vary widely between populations, yet the causes of this variation are still incompletely understood. Genetic differences between populations may make an important contribution to variation in rates of evolution, owing to differences in fitness, population size, mutation rates, or in the distribution of fitness effects (DFEs) of available beneficial mutations. By whole genome sequencing of Escherichia coli populations experimentally evolved in the presence of a quinolone antibiotic, we found that rates of substitution varied by genotype, with evidence for a contribution from a genotype's starting fitness. Subsequent targeted sequencing showed that genotypes with high average substitution rates were more likely to undergo the simultaneous fixation of several mutations, consistent with theoretical models of multiple mutation dynamics. Moreover, patterns of substitution were indicative of epistatic relationships between known resistance mutations.     

Modeling Sequence-Space Exploration and Emergence of Epistatic Signals in Protein Evolution

During their evolution, proteins explore sequence space via an interplay between random mutations and phenotypic selection. Here, we build upon recent progress in reconstructing data-driven fitness landscapes for families of homologous proteins, to propose stochastic models of experimental protein evolution. These models predict quantitatively important features of experimentally evolved sequence libraries, like fitness distributions and position-specific mutational spectra. They also allow us to efficiently simulate sequence libraries for a vast array of combinations of experimental parameters like sequence divergence, selection strength, and library size. We showcase the potential of the approach in reanalyzing two recent experiments to determine protein structure from signals of epistasis emerging in experimental sequence libraries. To be detectable, these signals require sufficiently large and sufficiently diverged libraries. Our modeling framework offers a quantitative explanation for different outcomes of recently published experiments. Furthermore, we can forecast the outcome of time- and resource-intensive evolution experiments, opening thereby a way to computationally optimize experimental protocols.     

Reducing the cost of resistance; experimental evolution in the filamentous fungus Aspergillus nidulans

We have studied compensatory evolution in a fludioxonil resistant mutant of the filamentous fungus Aspergillus nidulans. In an evolution experiment lasting for 27 weeks (about 3000 cell cycles) 35 parallel strains of this mutant evolved in three different environmental conditions. Our results show a severe cost of resistance (56%) in the absence of fludioxonil and in all conditions the mutant strain was able to restore fitness without loss of the resistance. In several cases, the evolved strain reached a higher fitness than the original sensitive ancestor. Fitness compensation occurred in one, two or three discrete steps. Genetic analysis of crosses between different evolved strains and between evolved and ancestral strains revealed interaction between compensatory mutations and provided information on the number of loci involved in fitness compensation. In addition, we discuss the opportunities for the experimental study of evolutionary processes provided by the filamentous fungus A. nidulans.     

Experimental Evolution of an Oncolytic Vesicular Stomatitis Virus with Increased Selectivity for p53-Deficient Cells

Experimental evolution has been used for various biotechnological applications including protein and microbial cell engineering, but less commonly in the field of oncolytic virotherapy. Here, we sought to adapt a rapidly evolving RNA virus to cells deficient for the tumor suppressor gene p53, a hallmark of cancer cells. To achieve this goal, we established four independent evolution lines of the vesicular stomatitis virus (VSV) in p53-knockout mouse embryonic fibroblasts (p53−/− MEFs) under conditions favoring the action of natural selection. We found that some evolved viruses showed increased fitness and cytotoxicity in p53−/− cells but not in isogenic p53+/+ cells, indicating gene-specific adaptation. However, full-length sequencing revealed no obvious or previously described genetic changes associated with oncolytic activity. Half-maximal effective dose (EC₅₀) assays in mouse p53-positive colon cancer (CT26) and p53-deficient breast cancer (4T1) cells indicated that the evolved viruses were more effective against 4T1 cells than the parental virus or a reference oncolytic VSV (MΔ51), but showed no increased efficacy against CT26 cells. In vivo assays using 4T1 syngeneic tumor models showed that one of the evolved lines significantly delayed tumor growth compared to mice treated with the parental virus or untreated controls, and was able to induce transient tumor suppression. Our results show that RNA viruses can be specifically adapted typical cancer features such as p53 inactivation, and illustrate the usefulness of experimental evolution for oncolytic virotherapy.     

Evolution of a single niche specialist in variable environments

The pattern (space versus time) and scale (relative to the lifetime of individuals) of environmental variation is thought to play a central role in governing the evolution of the ecological niche and the maintenance of genetic variance in fitness. To evaluate this idea, we serially propagated an initially genetically uniform population of the bacterium Pseudomonas fluorescens for a few hundred generations in environments that differed in both the pattern and scale at which two highly contrasted carbon substrates were experienced. We found that, contrary to expectations, populations often evolved into a single niche specialist adapted to the less-productive substrate in variable environments and that the genetic variance in fitness across different components of the environment was not generally higher in variable environments when compared with constant environments. We provide evidence to suggest that our results reflect a novel constraint on niche evolution imposed by the supply of beneficial mutations available to selection in variable environments.     

Slow fitness recovery in a codon-modified viral genome

Extensive synonymous codon modification of viral genomes appears to be an effective way of attenuating strains for use as live vaccines. An assumption of this method is that codon changes have individually small effects, such that codon-attenuated viruses will be slow to evolve back to high fitness (and thus to high virulence). The major capsid gene of the bacterial virus T7 was modified to have varying levels of suboptimal synonymous codons in different constructs, and fitnesses declined linearly with the number of changes. Adaptation of the most extreme design, with 182 codon changes, resulted in a slow fitness recovery by standards of previous experimental evolution with this virus, although fitness effects of substitutions were higher than expected from the average effect of an engineered codon modification. Molecular evolution during recovery was modest, and changes evolved both within the modified gene and outside it. Some changes within the modified gene evolved in parallel across replicates, but with no obvious explanation. Overall, the study supports the premise that codon-modified viruses recover fitness slowly, although the evolution is substantially more rapid than expected from the design principle.     

Mosaic evolution of ruminant stomach lysozyme genes

The genomes of ruminant artiodactyls, such as cow and sheep, have approximately 10 lysozyme genes, 4 of which are expressed in the stomach. Most of the duplications of the lysozyme genes occurred 40-50 million years ago, before the divergence of cow and sheep. Despite this, the coding regions of stomach lysozyme genes within a species (e.g., cow, sheep, or deer) are more similar to each other than to lysozyme genes in other ruminants. This observation suggests that the coding regions of the stomach lysozyme genes have evolved in a concerted fashion. Our previous characterization of 3 cow stomach lysozyme genes suggested that it was only the coding exons that had participated in concerted evolution. To determine whether the introns and flanking regions of ruminant stomach lysozyme genes are evolving in a concerted or a divergent fashion, we have isolated and characterized 2 sheep stomach lysozyme genes. Comparison of the sequences of the sheep and cow stomach lysozyme genes clearly shows that the introns and flanking regions have evolved, like the 3' untranslated region of the mRNAs, in a divergent manner. Thus, if the four coding exons are evolving by concerted evolution, then a mosaic pattern of concerted and divergent evolution is occurring in these genes. The independent concerted evolution of coding exons of the ruminant stomach lysozyme gene may have assisted in the accelerated adaptive evolution of the lysozyme to new function in the early ruminant.     

Delayed transmission selects for increased survival of vesicular stomatitis virus

Life-history theory predicts that traits for survival and reproduction cannot be simultaneously maximized in evolving populations. For this reason, in obligate parasites such as infectious viruses, selection for improved between-host survival during transmission may lead to evolution of decreased within-host reproduction. We tested this idea using experimental evolution of RNA virus populations, passaged under differing transmission times in the laboratory. A single ancestral genotype of vesicular stomatitis virus (VSV), a negative-sense RNA Rhabdovirus, was used to found multiple virus lineages evolved in either ordinary 24-h cell-culture passage, or in delayed passages of 48 h. After 30 passages (120 generations of viral evolution), we observed that delayed transmission selected for improved extracellular survival, which traded-off with lowered viral fecundity (slower exponential population growth and smaller mean plaque size). To further examine the confirmed evolutionary trade-off, we obtained consensus whole-genome sequences of evolved virus populations, to infer phenotype–genotype associations. Results implied that increased virus survival did not occur via convergence; rather, improved virion stability was gained via independent mutations in various VSV structural proteins. Our study suggests that RNA viruses can evolve different molecular solutions for enhanced survival despite their limited genetic architecture, but suffer generalized reproductive trade-offs that limit overall fitness gains.