Evolutionary barriers to horizontal gene transfer in macrophage-associated Salmonella.

Horizontal gene transfer (HGT) is a powerful evolutionary force facilitating bacterial adaptation and emergence of novel phenotypes. Several factors, including environmental ones, are predicted to restrict HGT, but we lack systematic and experimental data supporting these predictions. Here, we address this gap by measuring the relative fitness of 44 genes horizontally transferred from Escherichia coli to Salmonella enterica in infection-relevant environments. We estimated the distribution of fitness effects in each environment and identified that dosage-dependent effects across different environments are a significant barrier to HGT. The majority of genes were found to be deleterious. We also found longer genes had stronger negative fitness consequences than shorter ones, showing that gene length was negatively associated with HGT. Furthermore, fitness effects of transferred genes were found to be environmentally dependent. In summary, a substantial fraction of transferred genes had a significant fitness cost on the recipient, with both gene characteristics and the environment acting as evolutionary barriers to HGT.

The Role of the Environment in Horizontal Gene Transfer.

Gene-by-environment interactions play a crucial role in horizontal gene transfer by affecting how the transferred genes alter host fitness. However, how the environment modulates the fitness effect of transferred genes has not been tested systematically in an experimental study. We adapted a high-throughput technique for obtaining very precise estimates of bacterial fitness, in order to measure the fitness effects of 44 orthologs transferred from Salmonella Typhimurium to Escherichia coli in six physiologically relevant environments. We found that the fitness effects of individual genes were highly dependent on the environment, while the distributions of fitness effects across genes were not, with all tested environments resulting in distributions of same shape and spread. Furthermore, the extent to which the fitness effects of a gene varied between environments depended on the average fitness effect of that gene across all environments, with nearly neutral and nearly lethal genes having more consistent fitness effects across all environments compared to deleterious genes. Put together, our results reveal the unpredictable nature of how environmental conditions impact the fitness effects of each individual gene. At the same time, distributions of fitness effects across environments exhibit consistent features, pointing to the generalizability of factors that shape horizontal gene transfer of orthologous genes.

Transcriptomic profiling of Escherichia coli K-12 in response to a compendium of stressors.

Environmental perturbations impact multiple cellular traits, including gene expression. Bacteria respond to these stressful situations through complex gene interaction networks, thereby inducing stress tolerance and survival of cells. In this paper, we study the response mechanisms of E. coli when exposed to different environmental stressors via differential expression and co-expression analysis. Gene co-expression networks were generated and analyzed via Weighted Gene Co-expression Network Analysis (WGCNA). Based on the gene co-expression networks, genes with similar expression profiles were clustered into modules. The modules were analysed for identification of hub genes, enrichment of biological processes and transcription factors. In addition, we also studied the link between transcription factors and their differentially regulated targets to understand the regulatory mechanisms involved. These networks validate known gene interactions and provide new insights into genes mediating transcriptional regulation in specific stress environments, thus allowing for in silico hypothesis generation.

Predicting bacterial promoter function and evolution from random sequences.

Predicting function from sequence is a central problem of biology. Currently, this is possible only locally in a narrow mutational neighborhood around a wildtype sequence rather than globally from any sequence. Using random mutant libraries, we developed a biophysical model that accounts for multiple features of σ70 binding bacterial promoters to predict constitutive gene expression levels from any sequence. We experimentally and theoretically estimated that 10-20% of random sequences lead to expression and ~80% of non-expressing sequences are one mutation away from a functional promoter. The potential for generating expression from random sequences is so pervasive that selection acts against σ70-RNA polymerase binding sites even within inter-genic, promoter-containing regions. This pervasiveness of σ70-binding sites implies that emergence of promoters is not the limiting step in gene regulatory evolution. Ultimately, the inclusion of novel features of promoter function into a mechanistic model enabled not only more accurate predictions of gene expression levels, but also identified that promoters evolve more rapidly than previously thought.

Experimental determination of evolutionary barriers to horizontal gene transfer.

BACKGROUND: Horizontal gene transfer, the acquisition of genes across species boundaries, is a major source of novel phenotypes that enables microbes to rapidly adapt to new environments. How the transferred gene alters the growth - fitness - of the new host affects the success of the horizontal gene transfer event and how rapidly the gene spreads in the population. Several selective barriers - factors that impact the fitness effect of the transferred gene - have been suggested to impede the likelihood of horizontal transmission, however experimental evidence is scarce. The objective of this study was to determine the fitness effects of orthologous genes transferred from Salmonella enterica serovar Typhimurium to Escherichia coli to identify the selective barriers using highly precise experimental measurements. RESULTS: We found that most gene transfers result in strong fitness costs. Previously identified evolutionary barriers - gene function and the number of protein-protein interactions - did not predict the fitness effects of transferred genes. In contrast, dosage sensitivity, gene length, and the intrinsic protein disorder significantly impact the likelihood of a successful horizontal transfer. CONCLUSION: While computational approaches have been successful in describing long-term barriers to horizontal gene transfer, our experimental results identified previously underappreciated barriers that determine the fitness effects of newly transferred genes, and hence their short-term eco-evolutionary dynamics.

Evolutionary potential of transcription factors for gene regulatory rewiring.

Gene regulatory networks evolve through rewiring of individual components-that is, through changes in regulatory connections. However, the mechanistic basis of regulatory rewiring is poorly understood. Using a canonical gene regulatory system, we quantify the properties of transcription factors that determine the evolutionary potential for rewiring of regulatory connections: robustness, tunability and evolvability. In vivo repression measurements of two repressors at mutated operator sites reveal their contrasting evolutionary potential: while robustness and evolvability were positively correlated, both were in trade-off with tunability. Epistatic interactions between adjacent operators alleviated this trade-off. A thermodynamic model explains how the differences in robustness, tunability and evolvability arise from biophysical characteristics of repressor-DNA binding. The model also uncovers that the energy matrix, which describes how mutations affect repressor-DNA binding, encodes crucial information about the evolutionary potential of a repressor. The biophysical determinants of evolutionary potential for regulatory rewiring constitute a mechanistic framework for understanding network evolution.

CRISPR-based herd immunity can limit phage epidemics in bacterial populations.

Herd immunity, a process in which resistant individuals limit the spread of a pathogen among susceptible hosts has been extensively studied in eukaryotes. Even though bacteria have evolved multiple immune systems against their phage pathogens, herd immunity in bacteria remains unexplored. Here we experimentally demonstrate that herd immunity arises during phage epidemics in structured and unstructured Escherichia coli populations consisting of differing frequencies of susceptible and resistant cells harboring CRISPR immunity. In addition, we develop a mathematical model that quantifies how herd immunity is affected by spatial population structure, bacterial growth rate, and phage replication rate. Using our model we infer a general epidemiological rule describing the relative speed of an epidemic in partially resistant spatially structured populations. Our experimental and theoretical findings indicate that herd immunity may be important in bacterial communities, allowing for stable coexistence of bacteria and their phages and the maintenance of polymorphism in bacterial immunity.

Regulatory network structure determines patterns of intermolecular epistasis.

Most phenotypes are determined by molecular systems composed of specifically interacting molecules. However, unlike for individual components, little is known about the distributions of mutational effects of molecular systems as a whole. We ask how the distribution of mutational effects of a transcriptional regulatory system differs from the distributions of its components, by first independently, and then simultaneously, mutating a transcription factor and the associated promoter it represses. We find that the system distribution exhibits increased phenotypic variation compared to individual component distributions - an effect arising from intermolecular epistasis between the transcription factor and its DNA-binding site. In large part, this epistasis can be qualitatively attributed to the structure of the transcriptional regulatory system and could therefore be a common feature in prokaryotes. Counter-intuitively, intermolecular epistasis can alleviate the constraints of individual components, thereby increasing phenotypic variation that selection could act on and facilitating adaptive evolution.

On the mechanistic nature of epistasis in a canonical cis-regulatory element.

Understanding the relation between genotype and phenotype remains a major challenge. The difficulty of predicting individual mutation effects, and particularly the interactions between them, has prevented the development of a comprehensive theory that links genotypic changes to their phenotypic effects. We show that a general thermodynamic framework for gene regulation, based on a biophysical understanding of protein-DNA binding, accurately predicts the sign of epistasis in a canonical cis-regulatory element consisting of overlapping RNA polymerase and repressor binding sites. Sign and magnitude of individual mutation effects are sufficient to predict the sign of epistasis and its environmental dependence. Thus, the thermodynamic model offers the correct null prediction for epistasis between mutations across DNA-binding sites. Our results indicate that a predictive theory for the effects of cis-regulatory mutations is possible from first principles, as long as the essential molecular mechanisms and the constraints these impose on a biological system are accounted for.

Evolutionary interplay between structure, energy and epistasis in the coat protein of the ϕX174 phage family.

Viral capsids are structurally constrained by interactions among the amino acids (AAs) of their constituent proteins. Therefore, epistasis is expected to evolve among physically interacting sites and to influence the rates of substitution. To study the evolution of epistasis, we focused on the major structural protein of the ϕX174 phage family by first reconstructing the ancestral protein sequences of 18 species using a Bayesian statistical framework. The inferred ancestral reconstruction differed at eight AAs, for a total of 256 possible ancestral haplotypes. For each ancestral haplotype and the extant species, we estimated, in silico, the distribution of free energies and epistasis of the capsid structure. We found that free energy has not significantly increased but epistasis has. We decomposed epistasis up to fifth order and found that higher-order epistasis sometimes compensates pairwise interactions making the free energy seem additive. The dN/dS ratio is low, suggesting strong purifying selection, and that structure is under stabilizing selection. We synthesized phages carrying ancestral haplotypes of the coat protein gene and measured their fitness experimentally. Our findings indicate that stabilizing mutations can have higher fitness, and that fitness optima do not necessarily coincide with energy minima.

Epistatic Interactions in the Arabinose Cis-Regulatory Element.

Changes in gene expression are an important mode of evolution; however, the proximate mechanism of these changes is poorly understood. In particular, little is known about the effects of mutations within cis binding sites for transcription factors, or the nature of epistatic interactions between these mutations. Here, we tested the effects of single and double mutants in two cis binding sites involved in the transcriptional regulation of the Escherichia coli araBAD operon, a component of arabinose metabolism, using a synthetic system. This system decouples transcriptional control from any posttranslational effects on fitness, allowing a precise estimate of the effect of single and double mutations, and hence epistasis, on gene expression. We found that epistatic interactions between mutations in the araBAD cis-regulatory element are common, and that the predominant form of epistasis is negative. The magnitude of the interactions depended on whether the mutations are located in the same or in different operator sites. Importantly, these epistatic interactions were dependent on the presence of arabinose, a native inducer of the araBAD operon in vivo, with some interactions changing in sign (e.g., from negative to positive) in its presence. This study thus reveals that mutations in even relatively simple cis-regulatory elements interact in complex ways such that selection on the level of gene expression in one environment might perturb regulation in the other environment in an unpredictable and uncorrelated manner.

Motif depletion in bacteriophages infecting hosts with CRISPR systems.

BACKGROUND: CRISPR is a microbial immune system likely to be involved in host-parasite coevolution. It functions using target sequences encoded by the bacterial genome, which interfere with invading nucleic acids using a homology-dependent system. The system also requires protospacer associated motifs (PAMs), short motifs close to the target sequence that are required for interference in CRISPR types I and II. Here, we investigate whether PAMs are depleted in phage genomes due to selection pressure to escape recognition. RESULTS: To this end, we analyzed two data sets. Phages infecting all bacterial hosts were analyzed first, followed by a detailed analysis of phages infecting the genus Streptococcus, where PAMs are best understood. We use two different measures of motif underrepresentation that control for codon bias and the frequency of submotifs. We compare phages infecting species with a particular CRISPR type to those infecting species without that type. Since only known PAMs were investigated, the analysis is restricted to CRISPR types I-C and I-E and in Streptococcus to types I-C and II. We found evidence for PAM depletion in Streptococcus phages infecting hosts with CRISPR type I-C, in Vibrio phages infecting hosts with CRISPR type I-E and in Streptococcus thermopilus phages infecting hosts with type II-A, known as CRISPR3. CONCLUSIONS: The observed motif depletion in phages with hosts having CRISPR can be attributed to selection rather than to mutational bias, as mutational bias should affect the phages of all hosts. This observation implies that the CRISPR system has been efficient in the groups discussed here.

Evolutionary interactions between haemagglutinin and neuraminidase in avian influenza.

BACKGROUND: Reassortment between the RNA segments encoding haemagglutinin (HA) and neuraminidase (NA), the major antigenic influenza proteins, produces viruses with novel HA and NA subtype combinations and has preceded the emergence of pandemic strains. It has been suggested that productive viral infection requires a balance in the level of functional activity of HA and NA, arising from their closely interacting roles in the viral life cycle, and that this functional balance could be mediated by genetic changes in the HA and NA. Here, we investigate how the selective pressure varies for H7 avian influenza HA on different NA subtype backgrounds. RESULTS: By extending Bayesian stochastic mutational mapping methods to calculate the ratio of the rate of non-synonymous change to the rate of synonymous change (d(N)/d(S)), we found the average d(N)/d(S) across the avian influenza H7 HA1 region to be significantly greater on an N2 NA subtype background than on an N1, N3 or N7 background. Observed differences in evolutionary rates of H7 HA on different NA subtype backgrounds could not be attributed to underlying differences between avian host species or virus pathogenicity. Examination of d(N)/d(S) values for each subtype on a site-by-site basis indicated that the elevated d(N)/d(S) on the N2 NA background was a result of increased selection, rather than a relaxation of selective constraint. CONCLUSIONS: Our results are consistent with the hypothesis that reassortment exposes influenza HA to significant changes in selective pressure through genetic interactions with NA. Such epistatic effects might be explicitly accounted for in future models of influenza evolution.

Complete Genome Sequence of the Novel Phage MG-B1 Infecting Bacillus weihenstephanensis.

Here, we describe a novel virulent bacteriophage that infects Bacillus weihenstephanensis, isolated from soil in Austria. It is the first phage to be discovered that infects this species. Here, we present the complete genome sequence of this podovirus.

Probabilistic models for CRISPR spacer content evolution.

BACKGROUND: The CRISPR/Cas system is known to act as an adaptive and heritable immune system in Eubacteria and Archaea. Immunity is encoded in an array of spacer sequences. Each spacer can provide specific immunity to invasive elements that carry the same or a similar sequence. Even in closely related strains, spacer content is very dynamic and evolves quickly. Standard models of nucleotide evolution cannot be applied to quantify its rate of change since processes other than single nucleotide changes determine its evolution. METHODS: We present probabilistic models that are specific for spacer content evolution. They account for the different processes of insertion and deletion. Insertions can be constrained to occur on one end only or are allowed to occur throughout the array. One deletion event can affect one spacer or a whole fragment of adjacent spacers. Parameters of the underlying models are estimated for a pair of arrays by maximum likelihood using explicit ancestor enumeration. RESULTS: Simulations show that parameters are well estimated on average under the models presented here. There is a bias in the rate estimation when including fragment deletions. The models also estimate times between pairs of strains. But with increasing time, spacer overlap goes to zero, and thus there is an upper bound on the distance that can be estimated. Spacer content similarities are displayed in a distance based phylogeny using the estimated times.We use the presented models to analyze different Yersinia pestis data sets and find that the results among them are largely congruent. The models also capture the variation in diversity of spacers among the data sets. A comparison of spacer-based phylogenies and Cas gene phylogenies shows that they resolve very different time scales for this data set. CONCLUSIONS: The simulations and data analyses show that the presented models are useful for quantifying spacer content evolution and for displaying spacer content similarities of closely related strains in a phylogeny. This allows for comparisons of different CRISPR arrays or for comparisons between CRISPR arrays and nucleotide substitution rates.

Ancient DNA extracted from Danish aurochs (Bos primigenius): genetic diversity and preservation.

We extracted DNA from 39 Danish aurochs specimens and successfully amplified and sequenced a 252 base pair long fragment of the multivariable region I of the mitochondrial control region from 11 specimens. The sequences from these specimens dated back to 9830-2865 14Cyr BP and represent the first study of genetic variation of Danish aurochs. In addition, for all specimens we address correlations between the ability to obtain DNA sequences and various parameters such as the age of the sample, the collagen content, the museum storage period, Danish geography and whether the specimens were found in an archeological or geological context. We find that aurochs from southern Scandinavia display a star-shaped population genetic structure, that is indicative of a local and relatively recent diversification from a few ancestral haplotypes that may have originated in the ancestral Western European population before migration northwards during the retreat of the glaciers. Scenarios suggesting several invasions of genetically distinct aurochs are not supported by these analyses. Rather, our results suggest that a single continuous migration northward occurred. Our findings also suggest, although with only limited support, that aurochs in Northwestern Europe underwent a population expansion beginning shortly after the retreat of the glacial ice from Denmark and had a stable population size until the population decline that must have occurred prior to extinction. The absence of haplotypes similar to modern domestic cattle in our aurochs suggests that introgression between these species must have been limited, if it occurred at all. We found that the successful recovery of genetic material for PCR amplification correlates with sample age and local geographic conditions. However, contrary to other studies, we found no significant correlation between length of time in museum storage or the type of the locality in which a specimen was discovered (archeological or geological) and amplification success. Finally, we found large variances in our estimates of collagen content preventing an evaluation of this as an indicator of preservation quality.

Evolutionary genomics of Staphylococcus aureus reveals insights into the origin and molecular basis of ruminant host adaptation.

Phenotypic biotyping has traditionally been used to differentiate bacteria occupying distinct ecological niches such as host species. For example, the capacity of Staphylococcus aureus from sheep to coagulate ruminant plasma, reported over 60 years ago, led to the description of small ruminant and bovine S. aureus ecovars. The great majority of small ruminant isolates are represented by a single, widespread clonal complex (CC133) of S. aureus, but its evolutionary origin and the molecular basis for its host tropism remain unknown. Here, we provide evidence that the CC133 clone evolved as the result of a human to ruminant host jump followed by adaptive genome diversification. Comparative whole-genome sequencing revealed molecular evidence for host adaptation including gene decay and diversification of proteins involved in host-pathogen interactions. Importantly, several novel mobile genetic elements encoding virulence proteins with attenuated or enhanced activity in ruminants were widely distributed in CC133 isolates, suggesting a key role in its host-specific interactions. To investigate this further, we examined the activity of a novel staphylococcal pathogenicity island (SaPIov2) found in the great majority of CC133 isolates which encodes a variant of the chromosomally encoded von Willebrand-binding protein (vWbp(Sov2)), previously demonstrated to have coagulase activity for human plasma. Remarkably, we discovered that SaPIov2 confers the ability to coagulate ruminant plasma suggesting an important role in ruminant disease pathogenesis and revealing the origin of a defining phenotype of the classical S. aureus biotyping scheme. Taken together, these data provide broad new insights into the origin and molecular basis of S. aureus ruminant host specificity.

Parallel genetic evolution within and between bacteriophage species of varying degrees of divergence.

Parallel evolution is the acquisition of identical adaptive traits in independently evolving populations. Understanding whether the genetic changes underlying adaptation to a common selective environment are parallel within and between species is interesting because it sheds light on the degree of evolutionary constraints. If parallel evolution is perfect, then the implication is that forces such as functional constraints, epistasis, and pleiotropy play an important role in shaping the outcomes of adaptive evolution. In addition, population genetic theory predicts that the probability of parallel evolution will decline with an increase in the number of adaptive solutions-if a single adaptive solution exists, then parallel evolution will be observed among highly divergent species. For this reason, it is predicted that close relatives-which likely overlap more in the details of their adaptive solutions-will show more parallel evolution. By adapting three related bacteriophage species to a novel environment we find (1) a high rate of parallel genetic evolution at orthologous nucleotide and amino acid residues within species, (2) parallel beneficial mutations do not occur in a common order in which they fix or appear in an evolving population, (3) low rates of parallel evolution and convergent evolution between species, and (4) the probability of parallel and convergent evolution between species is strongly effected by divergence.

Estimation of 2Nes from temporal allele frequency data.

We develop a new method for estimating effective population sizes, Ne, and selection coefficients, s, from time-series data of allele frequencies sampled from a single diallelic locus. The method is based on calculating transition probabilities, using a numerical solution of the diffusion process, and assuming independent binomial sampling from this diffusion process at each time point. We apply the method in two example applications. First, we estimate selection coefficients acting on the CCR5-delta 32 mutation on the basis of published samples of contemporary and ancient human DNA. We show that the data are compatible with the assumption of s = 0, although moderate amounts of selection acting on this mutation cannot be excluded. In our second example, we estimate the selection coefficient acting on a mutation segregating in an experimental phage population. We show that the selection coefficient acting on this mutation is approximately 0.43.