Adaptive mutations in bacteria: high rate and small effects

Evolution by natural selection is driven by the continuous generation of adaptive mutations. We measured the genomic mutation rate that generates beneficial mutations and their effects on fitness in Escherichia coli under conditions in which the effect of competition between lineages carrying different beneficial mutations is minimized. We found a rate on the order of 10–5 per genome per generation, which is 1000 times as high as previous estimates, and a mean selective advantage of 1%. Such a high rate of adaptive evolution has implications for the evolution of antibiotic resistance and pathogenicity.

Phenotypic effect of mutations in evolving populations of RNA molecules

Background. The secondary structure of folded RNA sequences is a good model to map phenotype onto genotype, as represented by the RNA sequence. Computational studies of the evolution of ensembles of RNA molecules towards target secondary structures yield valuable clues to the mechanisms behind adaptation of complex populations. The relationship between the space of sequences and structures, the organization of RNA ensembles at mutation-selection equilibrium, the time of adaptation as a function of the population parameters, the presence of collective effects in quasispecies, or the optimal mutation rates to promote adaptation all are issues that can be explored within this framework. Results. We investigate the effect of microscopic mutations on the phenotype of RNA molecules during their in silico evolution and adaptation. We calculate the distribution of the effects of mutations on fitness, the relative fractions of beneficial and deleterious mutations and the corresponding selection coefficients for populations evolving under different mutation rates. Three different situations are explored: the mutation-selection equilibrium (optimized population) in three different fitness landscapes, the dynamics during adaptation towards a goal structure (adapting population), and the behavior under periodic population bottlenecks (perturbed population). Conclusions. The ratio between the number of beneficial and deleterious mutations experienced by a population of RNA sequences increases with the value of the mutation rate µ at which evolution proceeds. In contrast, the selective value of mutations remains almost constant, independent of µ, indicating that adaptation occurs through an increase in the amount of beneficial mutations, with little variations in the average effect they have on fitness. Statistical analyses of the distribution of fitness effects reveal that small effects, either beneficial or deleterious, are well described by a Pareto distribution. These results are robust under changes in the fitness landscape, remarkably when, in addition to selecting a target secondary structure, specific subsequences or low-energy folds are required. A population perturbed by bottlenecks behaves similarly to an adapting population, struggling to return to the optimized state. Whether it can survive in the long run or whether it goes extinct depends critically on the length of the time interval between bottlenecks. © 2010 Stich et al; licensee BioMed Central Ltd.

Evolutionary dynamics on rugged fitness landscapes: Exact dynamics and information theoretical aspects

The parallel mutation-selection evolutionary dynamics, in which mutation and replication are independent events, is solved exactly in the case that the Malthusian fitnesses associated to the genomes are described by the random energy model (REM) and by a ferromagnetic version of the REM. The solution method uses the mapping of the evolutionary dynamics into a quantum Ising chain in a transverse field and the Suzuki-Trotter formalism to calculate the transition probabilities between configurations at different times. We find that in the case of the REM landscape the dynamics can exhibit three distinct regimes: pure diffusion or stasis for short times, depending on the fitness of the initial configuration, and a spin-glass regime for large times. The dynamic transition between these dynamical regimes is marked by discontinuities in the mean-fitness as well as in the overlap with the initial reference sequence. The relaxation to equilibrium is described by an inverse time decay. In the ferromagnetic REM, we find in addition to these three regimes, a ferromagnetic regime where the overlap and the mean-fitness are frozen. In this case, the system relaxes to equilibrium in a finite time. The relevance of our results to information processing aspects of evolution is discussed.

Improving binding affinity and stability of peptide ligands by substituting glycines with D-amino acids.

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer-related protease urokinase-type plasminogen activator (uPA), we observed a glycine residue that has a positive ϕ dihedral angle when bound to the target. We hypothesized that replacing it with a D-amino acid, which favors positive ϕ angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D-serine in the bicyclic peptide indeed improved inhibitory activity (1.75-fold) and stability (fourfold). X-ray-structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ϕ angles are found in many protein-bound peptides. These results suggest that the glycine-to-D-amino acid mutagenesis strategy could be broadly applied.

Demogenetics in metapopulations

SUMMARY : Human-induced habitat fragmentation constitutes a major threat to biodiversity. Small and isolated populations suffer from increased stochasticity and from limited rescue effects. These two factors may be sufficient to cause local extinctions but fragmentation induces some genetic consequences that can also contribute significantly to extinction risks. Increased genetic drift reduces the effectiveness of selection against deleterious mutations, leading to their progressive accumulation. Drift also decreases both the standing genetic variation and the rate of fixation of beneficial mutations, limiting the evolutionary potential of isolated populations. Demography and genetics further interact and feed back on each other, progressively driving fragmented populations into "extinction vortices". The aim of the thesis was to better understand the processes occurring in fragmented populations. For this, I combined simulation studies and empirical data from three species that live in structured habitats. Chapter 1 and 2 investigate the demography of two shrew species in fragmented habitats. I showed that connectivity and habitat quality strongly affect the demography of the greater white-tooted shrew, although demographic stochasticity was extremely high. I also demonstrated that habitat fragmentation is one of the leading factors allowing the local coexistence of two competing shrew species. Chapter 3 and 4 focus on measuring connectivity in fragmented populations based on genetic data. In particular, I showed that genetic data can be used to detect the landscape elements impeding dispersal. In Chapter 5 that deals with the accumulation of deleterious mutations in fragmented populations, I demonstrated that mutation accumulation, as well a time to extinction, can be predicted from simple demographic and genetic measures. In the last two chapters, I monitored individual reproductive success in an isolated tree frogs population. These data allowed quantifying the effective population size, a measure closely linked to population evolutionary potential. To conclude, this thesis brings some new insights into the processes occurring in fragmented populations, and I hope it will contribute to the improvement of the management and conservation of fragmented populations.

Genomic evolution during a 10,000-generation experiment with bacteria

Molecular methods are used widely to measure genetic diversity within populations and determine relationships among species. However, it is difficult to observe genomic evolution in action because these dynamics are too slow in most organisms. To overcome this limitation, we sampled genomes from populations of Escherichia coli evolving in the laboratory for 10,000 generations. We analyzed the genomes for restriction fragment length polymorphisms (RFLP) using seven insertion sequences (IS) as probes; most polymorphisms detected by this approach reflect rearrangements (including transpositions) rather than point mutations. The evolving genomes became increasingly different from their ancestor over time. Moreover, tremendous diversity accumulated within each population, such that almost every individual had a different genetic fingerprint after 10,000 generations. As has been often suggested, but not previously shown by experiment, the rates of phenotypic and genomic change were discordant, both across replicate populations and over time within a population. Certain pivotal mutations were shared by all descendants in a population, and these are candidates for beneficial mutations, which are rare and difficult to find. More generally, these data show that the genome is highly dynamic even over a time scale that is, from an evolutionary perspective, very brief.

A structural view of evolutionary divergence

Two directed evolution experiments on p-nitrobenzyl esterase yielded one enzyme with a 100-fold increased activity in aqueous-organic solvents and another with a 17°C increase in thermostability. Structures of the wild type and its organophilic and thermophilic counterparts are presented at resolutions of 1.5 Å, 1.6 Å, and 2.0 Å, respectively. These structures identify groups of interacting mutations and demonstrate how directed evolution can traverse complex fitness landscapes. Early-generation mutations stabilize flexible loops not visible in the wild-type structure and set the stage for further beneficial mutations in later generations. The mutations exert their influence on the esterase structure over large distances, in a manner that would be difficult to predict. The loops with the largest structural changes generally are not the sites of mutations. Similarly, none of the seven amino acid substitutions in the organophile are in the active site, even though the enzyme experiences significant changes in the organization of this site. In addition to reduction of surface loop flexibility, thermostability in the evolved esterase results from altered core packing, helix stabilization, and the acquisition of surface salt bridges, in agreement with other comparative studies of mesophilic and thermophilic enzymes. Crystallographic analysis of the wild type and its evolved counterparts reveals networks of mutations that collectively reorganize the active site. Interestingly, the changes that led to diversity within the α/β hydrolase enzyme family and the reorganization seen in this study result from main-chain movements.

Computational method to reduce the search space for directed protein evolution

We introduce a computational method to optimize the in vitro evolution of proteins. Simulating evolution with a simple model that statistically describes the fitness landscape, we find that beneficial mutations tend to occur at amino acid positions that are tolerant to substitutions, in the limit of small libraries and low mutation rates. We transform this observation into a design strategy by applying mean-field theory to a structure-based computational model to calculate each residue's structural tolerance. Thermostabilizing and activity-increasing mutations accumulated during the experimental directed evolution of subtilisin E and T4 lysozyme are strongly directed to sites identified by using this computational approach. This method can be used to predict positions where mutations are likely to lead to improvement of specific protein properties.

The evolution of RNA viruses: A population genetics view

RNA viruses are excellent experimental models for studying evolution under the theoretical framework of population genetics. For a proper justification of this thesis we have introduced some properties of RNA viruses that are relevant for studying evolution. On the other hand, population genetics is a reductionistic theory of evolution. It does not consider or make simplistic assumptions on the transformation laws within and between genotypic and phenotypic spaces. However, such laws are minimized in the case of RNA viruses because the phenotypic space maps onto the genotypic space in a much more linear way than on higher DNA-based organisms. Under experimental conditions, we have tested the role of deleterious and beneficial mutations in the degree of adaptation of vesicular stomatitis virus (VSV), a nonsegmented virus of negative strand. We also have studied how effective population size, initial genetic variability in populations, and environmental heterogeneity shapes the impact of mutations in the evolution of vesicular stomatitis virus. Finally, in an integrative attempt, we discuss pros and cons of the quasispecies theory compared with classic population genetics models for haploid organisms to explain the evolution of RNA viruses.

Mutations in NR2E3 can cause dominant or recessive retinal degenerations in the same family.

NR2E3, a photoreceptor-specific nuclear receptor (PNR), represses cone-specific genes and activates several rod-specific genes. In humans, mutations in NR2E3 have been associated with the recessively-inherited enhanced short-wavelength sensitive S-cone syndrome (ESCS) and, recently, with autosomal dominant (ad) retinitis pigmentosa (RP) (adRP). In the present work, we describe two additional families affected by adRP that carry a heterozygous c.166G>A (p.G56R) mutation in the NR2E3 gene. Functional analysis determined the dominant negative activity of the p.G56R mutant protein as the molecular mechanism of adRP. Interestingly, in one pedigree, the most common causal variant for ESCS (p.R311Q) cosegregated with the adRP-linked p.G56R mutation, and the compound heterozygotes exhibited an ESCS-like phenotype, which in 1 of the 2 cases was strikingly "milder" than the patients carrying the p.G56R mutation alone. Impaired repression of cone-specific genes by the corepressors atrophin-1 (dentatorubral-pallidoluysian atrophy [DRPLA] gene product) and atrophin-2 (arginine-glutamic acid dipeptide repeat [RERE] protein) appeared to be a molecular mechanism mediating the beneficial effect of the p.R311Q mutation. Finally, the functional dominance of the p.R311Q variant to the p.G56R mutation is discussed.

Emergence of secretion-defective sublines of Pseudomonas aeruginosa PAO1 resulting from spontaneous mutations in the vfr global regulatory gene.

Pseudomonas aeruginosa undergoes spontaneous mutation that impairs secretion of several extracellular enzymes during extended cultivation in vitro in rich media, as well as during long-term colonization of the cystic fibrosis lung. A frequent type of strong secretion deficiency is caused by inactivation of the quorum-sensing regulatory gene lasR. Here we analyzed a spontaneously emerging subline of strain PAO1 that exhibited moderate secretion deficiency and partial loss of quorum-sensing control. Using generalized transduction, we mapped the secretion defect to the vfr gene, which is known to control positively the expression of the lasR gene and type II secretion of several proteases. We confirmed this secretion defect by sequencing and complementation of the vfr mutation. In a reconstruction experiment conducted with a 1:1 mixture of wild-type strain PAO1 and a vfr mutant of PAO1, we observed that the vfr mutant had a selective advantage over the wild type after growth in static culture for 4 days. Under these conditions, spontaneous vfr emerged in a strain PAO1 population after four growth cycles, and these mutants accounted for more than 40% of the population after seven cycles. These results suggest that partial or complete loss of quorum sensing and secretion can be beneficial to P. aeruginosa under certain environmental conditions.

Egfr Activating Mutations And Their Association With Response To Platinum-doublet Chemotherapy In Brazilian Non-small Cell Lung Cancer Patients.

The aim of the study was to analyze the frequency of epidermal growth factor receptor (EGFR) mutations in Brazilian non-small cell lung cancer patients and to correlate these mutations with response to benefit of platinum-based chemotherapy in non-small cell lung cancer (NSCLC). Our cohort consisted of prospective patients with NSCLCs who received chemotherapy (platinum derivates plus paclitaxel) at the [UNICAMP], Brazil. EGFR exons 18-21 were analyzed in tumor-derived DNA. Fifty patients were included in the study (25 with adenocarcinoma). EGFR mutations were identified in 6/50 (12 %) NSCLCs and in 6/25 (24 %) adenocarcinomas; representing the frequency of EGFR mutations in a mostly self-reported White (82.0 %) southeastern Brazilian population of NSCLCs. Patients with NSCLCs harboring EGFR exon 19 deletions or the exon 21 L858R mutation were found to have a higher chance of response to platinum-paclitaxel (OR 9.67 [95 % CI 1.03-90.41], p = 0.047). We report the frequency of EGFR activating mutations in a typical southeastern Brazilian population with NSCLC, which are similar to that of other countries with Western European ethnicity. EGFR mutations seem to be predictive of a response to platinum-paclitaxel, and additional studies are needed to confirm or refute this relationship.

Mucopolysaccharidosis Type Ii: Identification Of 30 Novel Mutations Among Latin American Patients.

In this study, 103 unrelated South-American patients with mucopolysaccharidosis type II (MPS II) were investigated aiming at the identification of iduronate-2-sulfatase (IDS) disease causing mutations and the possibility of some insights on the genotype-phenotype correlation The strategy used for genotyping involved the identification of the previously reported inversion/disruption of the IDS gene by PCR and screening for other mutations by PCR/SSCP. The exons with altered mobility on SSCP were sequenced, as well as all the exons of patients with no SSCP alteration. By using this strategy, we were able to find the pathogenic mutation in all patients. Alterations such as inversion/disruption and partial/total deletions of the IDS gene were found in 20/103 (19%) patients. Small insertions/deletions/indels (<22 bp) and point mutations were identified in 83/103 (88%) patients, including 30 novel mutations; except for a higher frequency of small duplications in relation to small deletions, the frequencies of major and minor alterations found in our sample are in accordance with those described in the literature.

Compound Heterozygous Rag2 Mutations Mimicking Hyper Igm Syndrome.

34