Adaptive Landscapes in the Age of Synthetic Biology.

For nearly a century adaptive landscapes have provided overviews of the evolutionary process and yet they remain metaphors. We redefine adaptive landscapes in terms of biological processes rather than descriptive phenomenology. We focus on the underlying mechanisms that generate emergent properties such as epistasis, dominance, trade-offs and adaptive peaks. We illustrate the utility of landscapes in predicting the course of adaptation and the distribution of fitness effects. We abandon aged arguments concerning landscape ruggedness in favor of empirically determining landscape architecture. In so doing, we transform the landscape metaphor into a scientific framework within which causal hypotheses can be tested.

Haploids, polymorphisms and fluctuating selection.

I analyze the joint impact of directional and fluctuating selection with reversible mutation in finite bi-allelic haploid populations using diffusion approximations of the Moran and chemostat models. Results differ dramatically from those of the classic Wright-Fisher diffusion. There, a strong dispersive effect attributable to fluctuating selection dissipates nascent polymorphisms promoted by a relatively weak emergent frequency dependent selective effect. The dispersive effect in the Moran diffusion with fluctuations every birth-death event is trivial. The same frequency dependent selective effect now dominates and polymorphism is promoted. The dispersive effect in the chemostat diffusion with fluctuations every generation is identical to that in the Wright-Fisher diffusion. Nevertheless, polymorphism is again promoted because the emergent frequency dependent effect is doubled, an effect attributable to geometric reproduction within generations. Fluctuating selection in the Moran and chemostat diffusions can also promote bi-allelic polymorphisms when one allele confers a net benefit. Rapid fluctuations within generations are highly effective at promoting polymorphism in large populations. The bi-allelic distribution is approximately Gaussian but becomes uniform and then U-shaped as the frequency of environmental fluctuations decreases to once a generation and then once every multiple generations. Trade-offs (negative correlations in fitness) help promote polymorphisms but are not essential. In all three models the frequency dependent effect raises the probability of ultimate fixation of new alleles, but less effectively in the Wright-Fisher diffusion. Individual-based forward simulations confirm the calculations.

Evolution of a Catalytic Mechanism.

The means by which superfamilies of specialized enzymes arise by gene duplication and functional divergence are poorly understood. The escape from adaptive conflict hypothesis, which posits multiple copies of a gene encoding a primitive inefficient and highly promiscuous generalist ancestor, receives support from experiments showing that resurrected ancestral enzymes are indeed more substrate-promiscuous than their modern descendants. Here, we provide evidence in support of an alternative model, the innovation-amplification-divergence hypothesis, which posits a single-copied ancestor as efficient and specific as any modern enzyme. We argue that the catalytic mechanisms of plant esterases and descendent acetone cyanohydrin lyases are incompatible with each other (e.g., the reactive substrate carbonyl must bind in opposite orientations in the active site). We then show that resurrected ancestral plant esterases are as catalytically specific as modern esterases, that the ancestor of modern acetone cyanohydrin lyases was itself only very weakly promiscuous, and that improvements in lyase activity came at the expense of esterase activity. These observations support the innovation-amplification-divergence hypothesis, in which an ancestor gains a weak promiscuous activity that is improved by selection at the expense of the ancestral activity, and not the escape from adaptive conflict in which an inefficient generalist ancestral enzyme steadily loses promiscuity throughout the transition to a highly active specialized modern enzyme.

Catalytic Promiscuity of Ancestral Esterases and Hydroxynitrile Lyases.

Catalytic promiscuity is a useful, but accidental, enzyme property, so finding catalytically promiscuous enzymes in nature is inefficient. Some ancestral enzymes were branch points in the evolution of new enzymes and are hypothesized to have been promiscuous. To test the hypothesis that ancestral enzymes were more promiscuous than their modern descendants, we reconstructed ancestral enzymes at four branch points in the divergence hydroxynitrile lyases (HNL's) from esterases ∼ 100 million years ago. Both enzyme types are α/ß-hydrolase-fold enzymes and have the same catalytic triad, but differ in reaction type and mechanism. Esterases catalyze hydrolysis via an acyl enzyme intermediate, while lyases catalyze an elimination without an intermediate. Screening ancestral enzymes and their modern descendants with six esterase substrates and six lyase substrates found higher catalytic promiscuity among the ancestral enzymes (P < 0.01). Ancestral esterases were more likely to catalyze a lyase reaction than modern esterases, and the ancestral HNL was more likely to catalyze ester hydrolysis than modern HNL's. One ancestral enzyme (HNL1) along the path from esterase to hydroxynitrile lyases was especially promiscuous and catalyzed both hydrolysis and lyase reactions with many substrates. A broader screen tested mechanistically related reactions that were not selected for by evolution: decarboxylation, Michael addition, γ-lactam hydrolysis and 1,5-diketone hydrolysis. The ancestral enzymes were more promiscuous than their modern descendants (P = 0.04). Thus, these reconstructed ancestral enzymes are catalytically promiscuous, but HNL1 is especially so.

Bounded population sizes, fluctuating selection and the tempo and mode of coexistence.

Existing theory predicts competitors (species or genetic clones) cannot coexist in a fluctuating environment unless relative fitness is negatively frequency-dependent (relative fitness declines as the frequency of a competitor increases). We develop simple theory to show coexistence does not require frequency-dependent selection, and we confirm this prediction by direct experiment. The conditions for coexistence in a fluctuating environment are precisely the same as those for coexistence in a spatially variable environment, conditions that arise naturally whenever population abundances are bounded. Simulations show the likelihood of coexistence increases with environmental uncertainty. The capacity of temporally variable environments to maintain biological diversity is far broader than generally envisaged.

Tipping the mutation-selection balance: Limited migration increases the frequency of deleterious mutants.

Typical mutation-selection models assume well-mixed populations, but dispersal and migration within many natural populations is spatially limited. Such limitations can lead to enhanced variation among locations as different types become clustered in different places. Such clustering weakens competition between unlike types relative to competition between like types; thus, the rate by which a fitter type displaces an inferior competitor can be affected by the spatial scale of movement. In this paper, we use a birth-death model to show that limited migration can affect asexual populations by creating competitive refugia. We use a moment closure approach to show that as population structure is introduced by limiting migration, the equilibrial frequency of deleterious mutants increases. We support and extend the model through stochastic simulation, and we use a spatially explicit cellular automaton approach to corroborate the results. We discuss the implications of these results for standing variation in structured populations and adaptive valley crossing in Wright's "shifting balance" process.

Evolution of a transition state: role of Lys100 in the active site of isocitrate dehydrogenase.

An active site lysine essential to catalysis in isocitrate dehydrogenase (IDH) is absent from related enzymes. As all family members catalyze the same oxidative ß-decarboxylation at the (2R)-malate core common to their substrates, it seems odd that an amino acid essential to one is not found in all. Ordinarily, hydride transfer to a nicotinamide C4 neutralizes the positive charge at N1 directly. In IDH, the negatively charged C4-carboxylate of isocitrate stabilizes the ground state positive charge on the adjacent nicotinamide N1, opposing hydride transfer. The critical lysine is poised to stabilize-and perhaps even protonate-an oxyanion formed on the nicotinamide 3-carboxamide, thereby enabling the hydride to be transferred while the positive charge at N1 is maintained. IDH might catalyze the same overall reaction as other family members, but dehydrogenation proceeds through a distinct, though related, transition state. Partial activation of lysine mutants by K(+) and NH4 (+) represents a throwback to the primordial state of the first promiscuous substrate family member.

Description of a riboflavin biosynthetic gene variant prevalent in the phylum Proteobacteria.

Riboflavin (vitamin B2) is the precursor of flavin mononucleotide and flavin adenine dinucleotide, which are cofactors essential for a host of intracellular redox reactions. Microorganisms synthesize flavins de novo to fulfill nutritional requirements, but it is becoming increasingly clear that flavins play a wider role in cellular physiology than was previously appreciated. Flavins mediate diverse processes beyond the cytoplasmic membrane, including iron acquisition, extracellular respiration, and interspecies interactions. While investigating the regulation of flavin electron shuttle biosynthesis in the Gram-negative gammaproteobacterium Shewanella oneidensis, we discovered that a riboflavin biosynthetic gene (ribBA) annotated as encoding a bifunctional 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase/GTP cyclohydrolase II does not possess both functions. The novel gene, renamed ribBX here, encodes an amino-terminal DHBP synthase domain. The carboxy-terminal end of RibBX not only lacks GTP cyclohydrolase II activity but also has evolved a different function altogether in S. oneidensis, regulating the activity of the DHBP synthase domain. Phylogenetic analysis revealed that the misannotation of ribBX as ribBA is rampant throughout the phylum Proteobacteria (40% of 2,173 annotated ribBA genes) and that ribBX emerged early in the evolution of this group of microorganisms. We examined the functionality of representative ribBX genes from Beta-, Gamma-, and Epsilonproteobacteria and found that, consistent with sequence-based predictions, the encoded GTP cyclohydrolase II domains lack catalytic activity. The persistence of ribBX in the genomes of so many phylogenetically divergent bacterial species lends weight to the argument that ribBX has evolved a function which lends a selective advantage to the host.

Pervasive cryptic epistasis in molecular evolution.

The functional effects of most amino acid replacements accumulated during molecular evolution are unknown, because most are not observed naturally and the possible combinations are too numerous. We created 168 single mutations in wild-type Escherichia coli isopropymalate dehydrogenase (IMDH) that match the differences found in wild-type Pseudomonas aeruginosa IMDH. 104 mutant enzymes performed similarly to E. coli wild-type IMDH, one was functionally enhanced, and 63 were functionally compromised. The transition from E. coli IMDH, or an ancestral form, to the functional wild-type P. aeruginosa IMDH requires extensive epistasis to ameliorate the combined effects of the deleterious mutations. This result stands in marked contrast with a basic assumption of molecular phylogenetics, that sites in sequences evolve independently of each other. Residues that affect function are scattered haphazardly throughout the IMDH structure. We screened for compensatory mutations at three sites, all of which lie near the active site and all of which are among the least active mutants. No compensatory mutations were found at two sites indicating that a single site may engage in compound epistatic interactions. One complete and three partial compensatory mutations of the third site are remote and lie in a different domain. This demonstrates that epistatic interactions can occur between distant (>20Å) sites. Phylogenetic analysis shows that incompatible mutations were fixed in different lineages.

Local migration promotes competitive restraint in a host-pathogen 'tragedy of the commons'.

Fragmented populations possess an intriguing duplicity: even if subpopulations are reliably extinction-prone, asynchrony in local extinctions and recolonizations makes global persistence possible. Migration is a double-edged sword in such cases: too little migration prevents recolonization of extinct patches, whereas too much synchronizes subpopulations, raising the likelihood of global extinction. Both edges of this proverbial sword have been explored by manipulating the rate of migration within experimental populations. However, few experiments have examined how the evolutionary ecology of fragmented populations depends on the pattern of migration. Here, we show that the migration pattern affects both coexistence and evolution within a community of bacterial hosts (Escherichia coli) and viral pathogens (T4 coliphage) distributed across a large network of subpopulations. In particular, different patterns of migration select for distinct pathogen strategies, which we term 'rapacious' and 'prudent'. These strategies define a 'tragedy of the commons': rapacious phage displace prudent variants for shared host resources, but prudent phage are more productive when alone. We find that prudent phage dominate when migration is spatially restricted, while rapacious phage evolve under unrestricted migration. Thus, migration pattern alone can determine whether a de novo tragedy of the commons is resolved in favour of restraint.

Survival of the first rather than the fittest in a Shewanella electrode biofilm.

For natural selection to operate there must exist heritable variation among individuals that affects their survival and reproduction. Among free-living microbes, where differences in growth rates largely define selection intensities, competitive exclusion is common. However, among surface attached communities, these dynamics become less predictable. If extreme circumstances were to dictate that a surface population is immortal and all offspring must emigrate, the offspring would be unable to contribute to the composition of the population. Meanwhile, the immortals, regardless of reproductive capacity, would remain unchanged in relative abundance. The normal cycle of birth, death, and competitive exclusion would be broken. We tested whether conditions required to set up this idealized scenario can be approximated in a microbial biofilm. Using two differentially-reproducing strains of Shewanella oneidensis grown on an anode as the sole terminal electron acceptor - a system in which metabolism is obligately tied to surface attachment - we found that selection against a slow-growing competitor is drastically reduced. This work furthers understanding of natural selection dynamics in sessile microbial communities, and provides a framework for designing stable microbial communities for industrial and experimental applications.

Transcription, translation, and the evolution of specialists and generalists.

We used DNA microarrays to measure transcription and iTRAQ 2D liquid chromatography-mass spectrometry/mass spectrometry (a mass-tag labeling proteomic technique) to measure protein expression in 14 strains of Escherichia coli adapted for hundreds of generations to growth-limiting concentrations of either lactulose, methylgalactoside, or a 72:28 mixture of the two. The two ancestors, TD2 and TD10, differ only in their lac operons and have similar transcription and protein expression profiles. Changes in transcription and protein expression are observed at 30-250 genes depending on the evolved strain. Lactulose specialists carry duplications of the lac operon and show increased transcription and translation at lac. Methylgalactoside specialists are galS(-) and so constitutively transcribe and translate mgl, which encodes a transporter of methylgalactoside. However, there are two strains that carry lac duplications, are galS(-), and show increased transcription and translation at both operons. One is a generalist, the other a lactulose specialist. The generalist fails to sweep to fixation because its lac(+), galS(+) competitor expresses the csg adhesin and sticks to the chemostat wall, thereby preventing complete washout. Transcription and translation are sometimes decoupled. Lactulose-adapted strains show increased protein expression at fru, a fructose transporter, without evidence of increased transcription. This suggests that fructose, produced by the action of beta-galactosidase on lactulose, may leach from cells before being recouped. Reduced expression, at "late" flagella genes and the constitutive gat operon, is an adaptation to starvation. A comparison with two other long-term evolution experiments suggests that certain aspects of adaptation are predictable, some are characteristic of an experimental system, whereas others seem erratic.

Evolution of enzymatic activities of testis-specific short-chain dehydrogenase/reductase in Drosophila.

The testis-specific gene Jingwei (jgw) is a newly evolved short-chain dehydrogenase/reductase in Drosophila. Preliminary substrate screening indicated that JGW prefers long-chain primary alcohols as substrates, including several exotic alcohols such as farnesol and geraniol. Using steady-state kinetics analyses and molecular docking, we not only confirmed JGW's substrate specificity, but also demonstrated that the new enzymatic activities of JGW evolved extensively after exon-shuffling from a preexisting enzyme. Analysis of JGW orthologs in sister species shows that subsequent evolutionary changes following the birth of JGW altered substrate specificities and enzyme stabilities. Our results lend support to a general mechanism for the evolution of a new enzyme, in which catalytic chemistry evolves first followed by diversification of substrate utilization.

An empirical test of the concomitantly variable codon hypothesis.

A central assumption of models of molecular evolution, that each site in a sequence evolves independently of all other sites, lacks empirical support. We investigated the extent to which sites evolve codependently in triosephosphate isomerase (TIM), a ubiquitous glycolytic enzyme conserved in both structure and function. Codependencies among sites, or concomitantly variable codons (covarions), are evident from the reduced function and misfolding of hybrid TIM proteins. Although they exist, we find covarions are relatively rare, and closely related proteins are unlikely to have developed them. However, the potential for covarions increases with genetic distance so that highly divergent proteins may have evolved codependencies between many sites. The evolution of covarions undermines a key assumption in phylogenetics and calls into question our ability to disentangle ancient relationships among major taxonomic groups.

Stochasticity-induced stabilization in ecology and evolution: a new synthesis.

The ability of random environmental variation to stabilize competitor coexistence was pointed out long ago and, in recent years, has received considerable attention. Analyses have focused on variations in the log abundances of species, with mean logarithmic growth rates when rare, Er , used as metrics for persistence. However, invasion probabilities and the times to extinction are not single-valued functions of Er and, in some cases, decrease as Er increases. Here, we present a synthesis of stochasticity-induced stabilization (SIS) phenomena based on the ratio between the expected arithmetic growth µ and its variance g . When the diffusion approximation holds, explicit formulas for invasion probabilities and persistence times are single-valued, monotonic functions of µ/g . The storage effect in the lottery model, together with other well-known examples drawn from population genetics, microbiology, and ecology (including discrete and continuous dynamics, with overlapping and non-overlapping generations), are placed together, reviewed, and explained within this new, transparent theoretical framework. We also clarify the relationships between life-history strategies and SIS, and study the dynamics of extinction when SIS fails.

Resistance of South American opossums to vWF-binding venom C-type lectins.

Opossums in the clade Didelphini are well known to be resistant to snake venom due to endogenous circulating inhibitors which target metalloproteinases and phospholipases. However, the mechanisms through which these opossums cope with a variety of other damaging venom proteins are unknown. A protein involved in blood clotting (von Willebrand Factor) has been found to have undergone rapid adaptive evolution in venom-resistant opossums. This protein is a known target for a subset of snake venom C-type lectins (CTLs), which bind it and then induce it to bind platelets, causing hemostatic disruption. Several amino acid changes in vWF unique to these opossums could explain their resistance; however, experimental evidence that these changes disrupt venom CTL binding was lacking. We used platelet aggregation assays to quantify resistance to a venom-induced platelet response in two species of venom-resistant opossums (Didelphis virginiana, Didelphis aurita), and one venom-sensitive opossum (Monodelphis domestica). We found that all three species have lost nearly all their aggregation response to the venom CTLs tested. Using washed platelet assays we showed that this loss of aggregation response is not due to inhibitors in the plasma, but rather to the failure of either vWF or platelets (or both) to respond to venom CTLs. These results demonstrate the potential adaptive function of a trait previously shown to be evolving under positive selection. Surprisingly, these findings also expand the list of potentially venom tolerant species to include Monodelphis domestica and suggest that an ecological relationship between opossums and vipers may be a broader driver of adaptive evolution across South American marsupials than previously thought.

The cost of expression of Escherichia coli lac operon proteins is in the process, not in the products.

Transcriptional regulatory networks allow bacteria to express proteins only when they are needed. Adaptive hypotheses explaining the evolution of regulatory networks assume that unneeded expression is costly and therefore decreases fitness, but the proximate cause of this cost is not clear. We show that the cost in fitness to Escherichia coli strains constitutively expressing the lactose operon when lactose is absent is associated with the process of making the lac gene products, i.e., associated with the acts of transcription and/or translation. These results reject the hypotheses that regulation exists to prevent the waste of amino acids in useless protein or the detrimental activity of unnecessary proteins. While the cost of the process of protein expression occurs in all of the environments that we tested, the expression of the lactose permease could be costly or beneficial, depending on the environment. Our results identify the basis of a single selective pressure likely acting across the entire E. coli transcriptome.

Fluctuating Selection in the Moran.

Contrary to classical population genetics theory, experiments demonstrate that fluctuating selection can protect a haploid polymorphism in the absence of frequency dependent effects on fitness. Using forward simulations with the Moran model, we confirm our analytical results showing that a fluctuating selection regime, with a mean selection coefficient of zero, promotes polymorphism. We find that increases in heterozygosity over neutral expectations are especially pronounced when fluctuations are rapid, mutation is weak, the population size is large, and the variance in selection is big. Lowering the frequency of fluctuations makes selection more directional, and so heterozygosity declines. We also show that fluctuating selection raises dn /ds ratios for polymorphism, not only by sweeping selected alleles into the population, but also by purging the neutral variants of selected alleles as they undergo repeated bottlenecks. Our analysis shows that randomly fluctuating selection increases the rate of evolution by increasing the probability of fixation. The impact is especially noticeable when the selection is strong and mutation is weak. Simulations show the increase in the rate of evolution declines as the rate of new mutations entering the population increases, an effect attributable to clonal interference. Intriguingly, fluctuating selection increases the dn /ds ratios for divergence more than for polymorphism, a pattern commonly seen in comparative genomics. Our model, which extends the classical neutral model of molecular evolution by incorporating random fluctuations in selection, accommodates a wide variety of observations, both neutral and selected, with economy.

The argRB of Escherichia coli is rare in isolates obtained from natural sources.

A single nucleotide polymorphism between Escherichia coli strains K12 and B is known to alter the mechanism by which the arginine repressor regulates arginine biosynthesis, from a regulated system in E. coli K12 to a deregulated system in E. coli B. Laboratory experiments have demonstrated that the different regulatory strategies are selectively favored under different environmental conditions. In this study we analyzed 537 E. coli strains and show that the argR allele in E. coli B, which causes deregulation, is rare in isolates obtained from natural sources. Moreover, sequence analysis of 85 strains shows no evidence of selection at the arginine repressor locus. This illustrates that analysis of sequence data is insufficient to detect selection of uncommon alleles in rare environments.