Hidden suppressive interactions are common in higher-order drug combinations.

The rapid increase of multi-drug resistant bacteria has led to a greater emphasis on multi-drug combination treatments. However, some combinations can be suppressive-that is, bacteria grow faster in some drug combinations than when treated with a single drug. Typically, when studying interactions, the overall effect of the combination is only compared with the single-drug effects. However, doing so could miss "hidden" cases of suppression, which occur when the highest order is suppressive compared with a lower-order combination but not to a single drug. We examined an extensive dataset of 5-drug combinations and all lower-order-single, 2-, 3-, and 4-drug-combinations. We found that a majority of all combinations-54%-contain hidden suppression. Examining hidden interactions is critical to understanding the architecture of higher-order interactions and can substantially affect our understanding and predictions of the evolution of antibiotic resistance under multi-drug treatments.

Adaptive Processes Change as Multiple Functions Evolve.

Epistasis influences the gene-environment interactions that shape bacterial fitness through antibiotic exposure, which can ultimately affect the availability of certain resistance phenotypes to bacteria. The substitutions present within blaTEM-50 confer both cephalosporin and ß-lactamase inhibitor resistance. We wanted to compare the evolution of blaTEM-50 with that of another variant, blaTEM-85, which differs in that blaTEM-85 contains only substitutions that contribute to cephalosporin resistance. Differences between the landscapes and epistatic interactions of these TEM variants are important for understanding their separate evolutionary responses to antibiotics. We hypothesized the substitutions within blaTEM-50 would result in more epistatic interactions than for blaTEM-85 As expected, we found more epistatic interactions between the substitutions present in blaTEM-50 than in blaTEM-85 Our results suggest that selection from many cephalosporins is required to achieve the full potential resistance to cephalosporins but that a single ß-lactam and inhibitor combination will drive evolution of the full potential resistance phenotype. Surprisingly, we also found significantly positive increases in growth rates as antibiotic concentration increased for some of the strains expressing blaTEM-85 precursor genotypes but not the blaTEM-50 variants. This result further suggests that additive interactions more effectively optimize phenotypes than epistatic interactions, which means that exposure to numerous cephalosporins actually increases the ability of a TEM enzyme to confer resistance to any single cephalosporin.

Genotype network intersections promote evolutionary innovation.

Evolutionary innovations are qualitatively novel traits that emerge through evolution and increase biodiversity. The genetic mechanisms of innovation remain poorly understood. A systems view of innovation requires the analysis of genotype networks-the vast networks of genetic variants that produce the same phenotype. Innovations can occur at the intersection of two different genotype networks. However, the experimental characterization of genotype networks has been hindered by the vast number of genetic variants that need to be functionally analyzed. Here, we use high-throughput sequencing to study the fitness landscape at the intersection of the genotype networks of two catalytic RNA molecules (ribozymes). We determined the ability of numerous neighboring RNA sequences to catalyze two different chemical reactions, and we use these data as a proxy for a genotype to fitness map where two functions come in close proximity. We find extensive functional overlap, and numerous genotypes can catalyze both functions. We demonstrate through evolutionary simulations that these numerous points of intersection facilitate the discovery of a new function. However, the rate of adaptation of the new function depends upon the local ruggedness around the starting location in the genotype network. As a consequence, one direction of adaptation is more rapid than the other. We find that periods of neutral evolution increase rates of adaptation to the new function by allowing populations to spread out in their genotype network. Our study reveals the properties of a fitness landscape where genotype networks intersect and the consequences for evolutionary innovations. Our results suggest that historic innovations in natural systems may have been facilitated by overlapping genotype networks.

Negative Epistasis in Experimental RNA Fitness Landscapes.

Mutations and their effects on fitness are a fundamental component of evolution. The effects of some mutations change in the presence of other mutations, and this is referred to as epistasis. Epistasis can occur between mutations in different genes or within the same gene. A systematic study of epistasis requires the analysis of numerous mutations and their combinations, which has recently become feasible with advancements in DNA synthesis and sequencing. Here we review the mutational effects and epistatic interactions within RNA molecules revealed by several recent high-throughput mutational studies involving two ribozymes studied in vitro, as well as a tRNA and a snoRNA studied in yeast. The data allow an analysis of the distribution of fitness effects of individual mutations as well as combinations of two or more mutations. Two different approaches to measuring epistasis in the data both reveal a predominance of negative epistasis, such that higher combinations of two or more mutations are typically lower in fitness than expected from the effect of each individual mutation. These data are in contrast to past studies of epistasis that used computationally predicted secondary structures of RNA that revealed a predominance of positive epistasis. The RNA data reviewed here are more similar to that found from mutational experiments on individual protein enzymes, suggesting that a common thermodynamic framework may explain negative epistasis between mutations within macromolecules.

A genome wide dosage suppressor network reveals genomic robustness.

Genomic robustness is the extent to which an organism has evolved to withstand the effects of deleterious mutations. We explored the extent of genomic robustness in budding yeast by genome wide dosage suppressor analysis of 53 conditional lethal mutations in cell division cycle and RNA synthesis related genes, revealing 660 suppressor interactions of which 642 are novel. This collection has several distinctive features, including high co-occurrence of mutant-suppressor pairs within protein modules, highly correlated functions between the pairs and higher diversity of functions among the co-suppressors than previously observed. Dosage suppression of essential genes encoding RNA polymerase subunits and chromosome cohesion complex suggests a surprising degree of functional plasticity of macromolecular complexes, and the existence of numerous degenerate pathways for circumventing the effects of potentially lethal mutations. These results imply that organisms and cancer are likely able to exploit the genomic robustness properties, due the persistence of cryptic gene and pathway functions, to generate variation and adapt to selective pressures.

Gene expression analysis of E. coli strains provides insights into the role of gene regulation in diversification.

Escherichia coli spans a genetic continuum from enteric strains to several phylogenetically distinct, atypical lineages that are rare in humans, but more common in extra-intestinal environments. To investigate the link between gene regulation, phylogeny and diversification in this species, we analyzed global gene expression profiles of four strains representing distinct evolutionary lineages, including a well-studied laboratory strain, a typical commensal (enteric) strain and two environmental strains. RNA-Seq was employed to compare the whole transcriptomes of strains grown under batch, chemostat and starvation conditions. Highly differentially expressed genes showed a significantly lower nucleotide sequence identity compared with other genes, indicating that gene regulation and coding sequence conservation are directly connected. Overall, distances between the strains based on gene expression profiles were largely dependent on the culture condition and did not reflect phylogenetic relatedness. Expression differences of commonly shared genes (all four strains) and E. coli core genes were consistently smaller between strains characterized by more similar primary habitats. For instance, environmental strains exhibited increased expression of stress defense genes under carbon-limited growth and entered a more pronounced survival-like phenotype during starvation compared with other strains, which stayed more alert for substrate scavenging and catabolism during no-growth conditions. Since those environmental strains show similar genetic distance to each other and to the other two strains, these findings cannot be simply attributed to genetic relatedness but suggest physiological adaptations. Our study provides new insights into ecologically relevant gene-expression and underscores the role of (differential) gene regulation for the diversification of the model bacterial species.

Trade-offs drive resource specialization and the gradual establishment of ecotypes.

BACKGROUND: Speciation is driven by many different factors. Among those are trade-offs between different ways an organism utilizes resources, and these trade-offs can constrain the manner in which selection can optimize traits. Limited migration among allopatric populations and species interactions can also drive speciation, but here we ask if trade-offs alone are sufficient to drive speciation in the absence of other factors. RESULTS: We present a model to study the effects of trade-offs on specialization and adaptive radiation in asexual organisms based solely on competition for limiting resources, where trade-offs are stronger the greater an organism's ability to utilize resources. In this model resources are perfectly substitutable, and fitness is derived from the consumption of these resources. The model contains no spatial parameters, and is therefore strictly sympatric. We quantify the degree of specialization by the number of ecotypes evolved and the niche breadth of the population, and observe that these are sensitive to resource influx and trade-offs. Resource influx has a strong effect on the degree of specialization, with a clear transition between minimal diversification at high influx and multiple species evolving at low resource influx. At low resource influx the degree of specialization further depends on the strength of the trade-offs, with more ecotypes evolving the stronger trade-offs are. The specialized organisms persist through negative frequency-dependent selection. In addition, by analyzing one of the evolutionary radiations in greater detail we demonstrate that a single mutation alone is not enough to establish a new ecotype, even though phylogenetic reconstruction identifies that mutation as the branching point. Instead, it takes a series of additional mutations to ensure the stable coexistence of the new ecotype in the background of the existing ones. CONCLUSIONS: Trade-offs are sufficient to drive the evolution of specialization in sympatric asexual populations. Without trade-offs to restrain traits, generalists evolve and diversity decreases. The observation that several mutations are required to complete speciation, even when a single mutation creates the new species, highlights the gradual nature of speciation and the importance of phyletic evolution.

Impact of epistasis and pleiotropy on evolutionary adaptation.

Evolutionary adaptation is often likened to climbing a hill or peak. While this process is simple for fitness landscapes where mutations are independent, the interaction between mutations (epistasis) as well as mutations at loci that affect more than one trait (pleiotropy) are crucial in complex and realistic fitness landscapes. We investigate the impact of epistasis and pleiotropy on adaptive evolution by studying the evolution of a population of asexual haploid organisms (haplotypes) in a model of N interacting loci, where each locus interacts with K other loci. We use a quantitative measure of the magnitude of epistatic interactions between substitutions, and find that it is an increasing function of K. When haplotypes adapt at high mutation rates, more epistatic pairs of substitutions are observed on the line of descent than expected. The highest fitness is attained in landscapes with an intermediate amount of ruggedness that balance the higher fitness potential of interacting genes with their concomitant decreased evolvability. Our findings imply that the synergism between loci that interact epistatically is crucial for evolving genetic modules with high fitness, while too much ruggedness stalls the adaptive process.

Repression and loss of gene expression outpaces activation and gain in recently duplicated fly genes.

Evolutionists widely acknowledge that regulatory genetic changes are of paramount importance for morphological and genomic evolution. Nevertheless, mechanistic complexity and a paucity of data from nonmodel organisms have prevented testing and quantifying universal hypotheses about the macroevolution of gene regulatory mechanisms. Here, we use a phylogenetic approach to provide a quantitative demonstration of a previously hypothesized trend, whereby the evolutionary rate of repression or loss of gene expression regions is significantly higher than the rate of activation or gain. Such a trend is expected based on case studies in regulatory evolution and under models of molecular evolution where duplicated genes lose duplicated expression patterns in a complementary fashion. The trend is important because repression of gene expression is a hypothesized mechanism for the origin of evolutionarily novel morphologies through specialization.