Loss-of-function mutations are main drivers of adaptations during short-term evolution.

We noticed that during short-term experimental evolution and carcinogenesis, mutations causing gene inactivation (i.e., nonsense mutations or frameshifts) are frequent. Our meta-analysis of 65 experiments using modified dN/dS statistics indicated that nonsense mutations are adaptive in different experimental conditions and we empirically confirmed this prediction. Using yeast S. cerevisiae as a model we show that fixed or highly frequent gene loss-of-function mutations are almost exclusively adaptive in the majority of experiments.

Apoptotic Factors Are Evolutionarily Conserved Since Mitochondrial Domestication.

The mechanisms initiating apoptotic programmed cell death in diverse eukaryotes are very similar. Basically, the mitochondrial permeability transition activates apoptotic proteases, DNases, and flavoproteins such as apoptosis-inducing factors (AIFs). According to the hypothesis of the endosymbiotic origin of apoptosis, these mechanisms evolved during mitochondrial domestication. Various phylogenetic analyses, including ours, have suggested that apoptotic factors were eubacterial protomitochondrial toxins used for killing protoeukaryotic hosts. Here, we tested whether the function of yeast Saccharomyces cerevisiae apoptotic proteases (metacaspases Mca1 and Nma111), DNase Nuc1, and flavoprotein Ndi1 can be substituted with orthologs from remotely related eukaryotes such as plants, protists, and eubacteria. We found that orthologs of remotely related eukaryotic and even eubacterial proteins can initiate apoptosis in yeast when triggered by chemical stresses. This observation suggests that apoptotic mechanisms have been maintained since mitochondrial domestication, which occurred approximately 1,800 Mya. Additionally, it supports the hypothesis that some of these apoptotic factors could be modified eubacterial toxins.

Detection of positive selection acting on protein surfaces at the whole-genome scale in the human malaria parasite Plasmodium falciparum.

The host-parasite evolutionary arms race is a fundamental process with medical implications. During this process, the host develops parasite resistance, and the parasite develops host immune evasion strategies. Thus, this process accelerates relevant protein evolution. This study test hypothesizes that proteins subject to sequence evolution structural constraints play a crucial role and that these constraints hinder the modification of such proteins in this process. These hypotheses were tested using Plasmodium falciparum model and evaluated protein structures predicted for the entire proteome by the AlphaFold method. Based on dN/dS test results and P. falciparum and P. reichenowi comparisons, the presented approach identified proteins subject to purifying selection acting on the whole sequence and buried residues (dN < dS) and positive selection on nonburied residues. Of the 26 proteins, some known antigens (ring-exported protein 3, RAP protein, erythrocyte binding antigen-140, and protein P47) targeted by the host immune system are promising vaccine candidates. The set also contained 11 enzymes, including FIKK kinase, which modifies host proteins. This set was compared with genes for which the dN/dS test suggested that positive selection acts on the whole gene (i.e., dN > dS). The present study found that such genes encode enzymes and antigenic vaccine candidates less frequently than genes for which evolution is not subject to selection constraints and positive selection acts on only exposed residues. The analysis was repeated comparing P. falciparum with P. alderi, which is more distantly related. The study discusses the potential implications of the presented methodology for rational vaccine design and the parasitology and evolutionary biology fields.

Genetic interaction network has a very limited impact on the evolutionary trajectories in continuous culture-grown populations of yeast.

BACKGROUND: The impact of genetic interaction networks on evolution is a fundamental issue. Previous studies have demonstrated that the topology of the network is determined by the properties of the cellular machinery. Functionally related genes frequently interact with one another, and they establish modules, e.g., modules of protein complexes and biochemical pathways. In this study, we experimentally tested the hypothesis that compensatory evolutionary modifications, such as mutations and transcriptional changes, occur frequently in genes from perturbed modules of interacting genes. RESULTS: Using Saccharomyces cerevisiae haploid deletion mutants as a model, we investigated two modules lacking COG7 or NUP133, which are evolutionarily conserved genes with many interactions. We performed laboratory evolution experiments with these strains in two genetic backgrounds (with or without additional deletion of MSH2), subjecting them to continuous culture in a non-limiting minimal medium. Next, the evolved yeast populations were characterized through whole-genome sequencing and transcriptome analyses. No obvious compensatory changes resulting from inactivation of genes already included in modules were identified. The supposedly compensatory inactivation of genes in the evolved strains was only rarely observed to be in accordance with the established fitness effect of the genetic interaction network. In fact, a substantial majority of the gene inactivations were predicted to be neutral in the experimental conditions used to determine the interaction network. Similarly, transcriptome changes during continuous culture mostly signified adaptation to growth conditions rather than compensation of the absence of the COG7, NUP133 or MSH2 genes. However, we noticed that for genes whose inactivation was deleterious an upregulation of transcription was more common than downregulation. CONCLUSIONS: Our findings demonstrate that the genetic interactions and the modular structure of the network described by others have very limited effects on the evolutionary trajectory following gene deletion of module elements in our experimental conditions and has no significant impact on short-term compensatory evolution. However, we observed likely compensatory evolution in functionally related (albeit non-interacting) genes.

Slow Adaptive Response of Budding Yeast Cells to Stable Conditions of Continuous Culture Can Occur without Genome Modifications.

Continuous cultures assure the invariability of environmental conditions and the metabolic state of cultured microorganisms, whereas batch-cultured cells undergo constant changes in nutrients availability. For that reason, continuous culture is sometimes employed in the whole transcriptome, whole proteome, or whole metabolome studies. However, the typical method for establishing uniform growth of a cell population, i.e., by limited chemostat, results in the enrichment of the cell population gene pool with mutations adaptive for starvation conditions. These adaptive changes can skew the results of large-scale studies. It is commonly assumed that these adaptations reflect changes in the genome, and this assumption has been confirmed experimentally in rare cases. Here we show that in a population of budding yeast cells grown for over 200 generations in continuous culture in non-limiting minimal medium and therefore not subject to selection pressure, remodeling of transcriptome occurs, but not as a result of the accumulation of adaptive mutations. The observed changes indicate a shift in the metabolic balance towards catabolism, a decrease in ribosome biogenesis, a decrease in general stress alertness, reorganization of the cell wall, and transactions occurring at the cell periphery. These adaptive changes signify the acquisition of a new lifestyle in a stable nonstressful environment. The absence of underlying adaptive mutations suggests these changes may be regulated by another mechanism.

Symbiotic Origin of Apoptosis.

The progress of evolutionary biology has revealed that symbiosis played a basic role in the evolution of complex eukaryotic organisms, including humans. Mitochondria are actually simplified endosymbiotic bacteria currently playing the role of cellular organelles. Mitochondrial domestication occurred at the very beginning of eukaryotic evolution. Mitochondria have two different basic functions: they produce energy using oxidative respiration, and they initiate different forms of apoptotic programmed/regulated cell death. Apoptotic programmed cell death may have different cytological forms. Mechanisms of apoptotic programmed cell death exist even in the unicellular organisms, and they play a basic role in the development of complex multicellular organisms, such as fungi, green plants, and animals. Multicellularity was independently established many times among eukaryotes. There are indications that apoptotic programmed cell death is a trait required for the establishment of multicellularity. Regulated cell death is initiated by many different parallel biochemical pathways. It is generally accepted that apoptosis evolved during mitochondrial domestication. However, there are different hypothetical models of the origin of apoptosis. The phylogenetic studies of my group indicate that apoptosis probably evolved during an evolutionary arms race between host ancestral eukaryotic predators and ancestral prey mitochondria (named protomitochondria). Protomitochondrial prey produced many different toxins as a defense against predators. From these toxins evolved extant apoptotic factors. There are indications that aerobic respiration and apoptosis co-evolved and are functionally linked in extant organisms. Perturbations of apoptosis and oxidative respiration are frequently observed during neoplastic transition. Our group showed that perturbations of apoptosis in yeasts also cause perturbations of oxidative respiration.

An Apoptotic and Endosymbiotic Explanation of the Warburg and the Inverse Warburg Hypotheses.

Otto Warburg, a Nobel prize winner, observed that cancer cells typically "switch" from aerobic to anaerobic respiration. He hypothesized that mitochondrial damage induces neoplastic transformation. In contrast, pathological aging is observed mainly in neuron cells in neurodegenerative diseases. Oxidative respiration is particularly active in neurons. There is inverse comorbidity between cancer and neurodegenerative diseases. This led to the creation of the "inverse Warburg hypothesis", according to which excessive mitochondrial activity induces pathological aging. The findings of our studies suggest that both the Warburg effect and the "inverse Warburg hypothesis" can be elucidated by the activation or suppression of apoptosis through oxidative respiration. The key outcome of our phylogenetic studies was the discovery that apoptosis and apoptosis-like cell death evolved due to an evolutionary "arms race" conducted between "prey" protomitochondrion and "predator" primitive eukaryotes. The ancestral protomitochondrial machinery produces and releases toxic mitochondrial proteins. Extant apoptotic factors evolved from these toxins. Our experiments indicate that the mitochondrial machinery is directly involved in adaptation to aerobic conditions. Additionally, our hypothesis is supported by the fact that different apoptotic factors are directly involved in respiration.

Ancestral State Reconstruction of the Apoptosis Machinery in the Common Ancestor of Eukaryotes.

Apoptotic cell death is a type of eukaryotic cell death. In animals, it regulates development, is involved in cancer suppression, and causes cell death during pathological aging of neuronal cells in neurodegenerative diseases such as Alzheimer's. Mitochondrial apoptotic-like cell death, a form of primordial apoptosis, also occurs in unicellular organisms. Here, we ask the question why the apoptosis machinery has been acquired and maintained in unicellular organisms and attempt to answer it by performing ancestral state reconstruction. We found indications of an ancient evolutionary arms race between protomitochondria and host cells, leading to the establishment of the currently existing apoptotic pathways. According to this reconstruction, the ancestral protomitochondrial apoptosis machinery contained both caspases and metacaspases, four types of apoptosis induction factors (AIFs), both fungal and animal OMI/HTR proteases, and various apoptotic DNases. This leads to the prediction that in extant unicellular eukaryotes, the apoptotic factors are involved in mitochondrial respiration and their activity is needed exclusively in aerobic conditions. We test this prediction experimentally using yeast and find that a loss of the main apoptotic factors is beneficial under anaerobic conditions yet deleterious under aerobic conditions in the absence of lethal stimuli. We also point out potential medical implications of these findings.

Host-parasite interactions and ecology of the malaria parasite-a bioinformatics approach.

Malaria remains one of the highest mortality infectious diseases. Malaria is caused by parasites from the genus Plasmodium. Most deaths are caused by infections involving Plasmodium falciparum, which has a complex life cycle. Malaria parasites are extremely well adapted for interactions with their host and their host's immune system and are able to suppress the human immune system, erase immunological memory and rapidly alter exposed antigens. Owing to this rapid evolution, parasites develop drug resistance and express novel forms of antigenic proteins that are not recognized by the host immune system. There is an emerging need for novel interventions, including novel drugs and vaccines. Designing novel therapies requires knowledge about host-parasite interactions, which is still limited. However, significant progress has recently been achieved in this field through the application of bioinformatics analysis of parasite genome sequences. In this review, we describe the main achievements in 'malarial' bioinformatics and provide examples of successful applications of protein sequence analysis. These examples include the prediction of protein functions based on homology and the prediction of protein surface localization via domain and motif analysis. Additionally, we describe PlasmoDB, a database that stores accumulated experimental data. This tool allows data mining of the stored information and will play an important role in the development of malaria science. Finally, we illustrate the application of bioinformatics in the development of population genetics research on malaria parasites, an approach referred to as reverse ecology.

Control of seed dormancy in Arabidopsis by a cis-acting noncoding antisense transcript.

Seed dormancy is one of the most crucial process transitions in a plant's life cycle. Its timing is tightly controlled by the expression level of the Delay of Germination 1 gene (DOG1). DOG1 is the major quantitative trait locus for seed dormancy in Arabidopsis and has been shown to control dormancy in many other plant species. This is reflected by the evolutionary conservation of the functional short alternatively polyadenylated form of the DOG1 mRNA. Notably, the 3' region of DOG1, including the last exon that is not included in this transcript isoform, shows a high level of conservation at the DNA level, but the encoded polypeptide is poorly conserved. Here, we demonstrate that this region of DOG1 contains a promoter for the transcription of a noncoding antisense RNA, asDOG1, that is 5' capped, polyadenylated, and relatively stable. This promoter is autonomous and asDOG1 has an expression profile that is different from known DOG1 transcripts. Using several approaches we show that asDOG1 strongly suppresses DOG1 expression during seed maturation in cis, but is unable to do so in trans Therefore, the negative regulation of seed dormancy by asDOG1 in cis results in allele-specific suppression of DOG1 expression and promotes germination. Given the evolutionary conservation of the asDOG1 promoter, we propose that this cis-constrained noncoding RNA-mediated mechanism limiting the duration of seed dormancy functions across the Brassicaceae.

Apoptosis: its origin, history, maintenance and the medical implications for cancer and aging.

Programmed cell death is a basic cellular mechanism. Apoptotic-like programmed cell death (called apoptosis in animals) occurs in both unicellular and multicellular eukaryotes, and some apoptotic mechanisms are observed in bacteria. Endosymbiosis between mitochondria and eukaryotic cells took place early in the eukaryotic evolution, and some of the apoptotic-like mechanisms of mitochondria that were retained after this event now serve as parts of the eukaryotic apoptotic machinery. Apoptotic mechanisms have several functions in unicellular organisms: they include kin-selected altruistic suicide that controls population size, sharing common goods, and responding to viral infection. Apoptotic factors also have non-apoptotic functions. Apoptosis is involved in the cellular aging of eukaryotes, including humans. In addition, apoptosis is a key part of the innate tumor-suppression mechanism. Several anticancer drugs induce apoptosis, because apoptotic mechanisms are inactivated during oncogenesis. Because of the ancient history of apoptosis, I hypothesize that there is a deep relationship between mitochondrial metabolism, its role in aerobic versus anaerobic respiration, and the connection between apoptosis and cancer. Whereas normal cells rely primarily on oxidative mitochondrial respiration, most cancer cells use anaerobic metabolism. According to the Warburg hypothesis, the remodeling of the metabolism is one of the processes that leads to cancer. Recent studies indicate that anaerobic, non-mitochondrial respiration is particularly active in embryonic cells, stem cells, and aggressive stem-like cancer cells. Mitochondrial respiration is particularly active during the pathological aging of human cells in neurodegenerative diseases. According to the reversed Warburg hypothesis formulated by Demetrius, pathological aging is induced by mitochondrial respiration. Here, I advance the hypothesis that the stimulation of mitochondrial metabolism leads to pathological aging.

Seed Dormancy in Arabidopsis Is Controlled by Alternative Polyadenylation of DOG1.

DOG1 (Delay of Germination 1) is a key regulator of seed dormancy in Arabidopsis (Arabidopsis thaliana) and other plants. Interestingly, the C terminus of DOG1 is either absent or not conserved in many plant species. Here, we show that in Arabidopsis, DOG1 transcript is subject to alternative polyadenylation. In line with this, mutants in RNA 3' processing complex display weakened seed dormancy in parallel with defects in DOG1 proximal polyadenylation site selection, suggesting that the short DOG1 transcript is functional. This is corroborated by the finding that the proximally polyadenylated short DOG1 mRNA is translated in vivo and complements the dog1 mutant. In summary, our findings indicate that the short DOG1 protein isoform produced from the proximally polyadenylated DOG1 mRNA is a key player in the establishment of seed dormancy in Arabidopsis and characterizes a set of mutants in RNA 3' processing complex required for production of proximally polyadenylated functional DOG1 transcript.

Dominance from the perspective of gene-gene and gene-chemical interactions.

In this study, we used genetic interaction (GI) and gene-chemical interaction (GCI) data to compare mutations with different dominance phenotypes. Our analysis focused primarily on Saccharomyces cerevisiae, where haploinsufficient genes (HI; genes with dominant loss-of-function mutations) were found to be participating in gene expression processes, namely, the translation and regulation of gene transcription. Non-ribosomal HI genes (mainly regulators of gene transcription) were found to have more GIs and GCIs than haplosufficient (HS) genes. Several properties seem to lead to the enrichment of interactions, most notably, the following: importance, pleiotropy, gene expression level and gene expression variation. Importantly, after these properties were appropriately considered in the analysis, the correlation between dominance and GI/GCI degrees was still observed. Strikingly, for the GCIs of heterozygous strains, haploinsufficiency was the only property significantly correlated with the number of GCIs. We found ribosomal HI genes to be depleted in GIs/GCIs. This finding can be explained by their high variation in gene expression under different genetic backgrounds and environmental conditions. We observed the same distributions of GIs among non-ribosomal HI, ribosomal HI and HS genes in three other species: Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens. One potentially interesting exception was the lack of significant differences in the degree of GIs between non-ribosomal HI and HS genes in Schizosaccharomyces pombe.

Bacteria diversity and arsenic mobilization in rock biofilm from an ancient gold and arsenic mine.

In this paper we characterize the biofilm community from an ancient Zloty Stok gold and arsenic mine. Bacterial diversity was examined using a culture-independent technique based on 16S rRNA gene amplification, cloning and sequencing. We show that unexpectedly the microbial diversity of this community was extremely high (more than 190 OTUs detected), with the most numerous members from Rhizobiales (α-Proteobacteria). Although the level of rock biofilm diversity was similar to the microbial mat community we have previously characterized in the same adit, its taxonomic composition was completely different. Detailed analysis of functional arrA and aioA genes, chemical properties of siderophores found in pore water as well as the biofilm chemical composition suggest that the biofilm community contributes to arsenic pollution of surrounding water in a biogeochemical cycle similar to the one observed in bacterial mats. To interpret our results concerning the biological arsenic cycle, we applied the theory of ecological pyramids of Charles Elton.

A kinetic model of the evolution of a protein interaction network.

BACKGROUND: Known protein interaction networks have very particular properties. Old proteins tend to have more interactions than new ones. One of the best statistical representatives of this property is the node degree distribution (distribution of proteins having a given number of interactions). It has previously been shown that this distribution is very close to the sum of two distinct exponential components. In this paper, we asked: What are the possible mechanisms of evolution for such types of networks? To answer this question, we tested a kinetic model for simplified evolution of a protein interactome. Our proposed model considers the emergence of new genes and interactions and the loss of old ones. We assumed that there are generally two coexisting classes of proteins. Proteins constituting the first class are essential only for ecological adaptations and are easily lost when ecological conditions change. Proteins of the second class are essential for basic life processes and, hence, are always effectively protected against deletion. All proteins can transit between the above classes in both directions. We also assumed that the phenomenon of gene duplication is always related to ecological adaptation and that a new copy of a duplicated gene is not essential. According to this model, all proteins gain new interactions with a rate that preferentially increases with the number of interactions (the rich get richer). Proteins can also gain interactions because of duplication. Proteins lose their interactions both with and without the loss of partner genes. RESULTS: The proposed model reproduces the main properties of protein-protein interaction networks very well. The connectivity of the oldest part of the interaction network is densest, and the node degree distribution follows the sum of two shifted power-law functions, which is a theoretical generalization of the previous finding. The above distribution covers the wide range of values of node degrees very well, much better than a power law or generalized power law supplemented with an exponential cut-off. The presented model also relates the total number of interactome links to the total number of interacting proteins. The theoretical results were for the interactomes of A. thaliana, B. taurus, C. elegans, D. melanogaster, E. coli, H. pylori, H. sapiens, M. musculus, R. norvegicus and S. cerevisiae. CONCLUSIONS: Using these approaches, the kinetic parameters could be estimated. Finally, the model revealed the evolutionary kinetics of proteome formation, the phenomenon of protein differentiation and the process of gaining new interactions.

The evolutionary rate of antibacterial drug targets.

BACKGROUND: One of the major issues in the fight against infectious diseases is the notable increase in multiple drug resistance in pathogenic species. For that reason, newly acquired high-throughput data on virulent microbial agents attract the attention of many researchers seeking potential new drug targets. Many approaches have been used to evaluate proteins from infectious pathogens, including, but not limited to, similarity analysis, reverse docking, statistical 3D structure analysis, machine learning, topological properties of interaction networks or a combination of the aforementioned methods. From a biological perspective, most essential proteins (knockout lethal for bacteria) or highly conserved proteins (broad spectrum activity) are potential drug targets. Ribosomal proteins comprise such an example. Many of them are well-known drug targets in bacteria. It is intuitive that we should learn from nature how to design good drugs. Firstly, known antibiotics are mainly originating from natural products of microorganisms targeting other microorganisms. Secondly, paleontological data suggests that antibiotics have been used by microorganisms for million years. Thus, we have hypothesized that good drug targets are evolutionary constrained and are subject of evolutionary selection. This means that mutations in such proteins are deleterious and removed by selection, which makes them less susceptible to random development of resistance. Analysis of the speed of evolution seems to be good approach to test this hypothesis. RESULTS: In this study we show that pN/pS ratio of genes coding for known drug targets is significantly lower than the genome average and also lower than that for essential genes identified by experimental methods. Similar results are observed in the case of dN/dS analysis. Both analyzes suggest that drug targets tend to evolve slowly and that the rate of evolution is a better predictor of drugability than essentiality. CONCLUSIONS: Evolutionary rate can be used to score and find potential drug targets. The results presented here may become a useful addition to a repertoire of drug target prediction methods. As a proof of concept, we analyzed GO enrichment among the slowest evolving genes. These may become the starting point in the search for antibiotics with a novel mechanism.

Why are male malaria parasites in such a rush?: Sex-specific evolution and host-parasite interactions.

BACKGROUND: Disease-causing organisms are notorious for fast rates of molecular evolution and the ability to adapt rapidly to changes in their ecology. Sex plays a key role in evolution, and recent studies, in humans and other multicellular organisms, document that genes expressed principally or exclusively in males exhibit the fastest rates of adaptive evolution. However, despite the importance of sexual reproduction for many unicellular taxa, sex-biased gene expression and its evolutionary implications have been overlooked. METHODS: We analyse genomic data from multiple malaria parasite (Plasmodium) species and proteomic data sets from different parasite life cycle stages. RESULTS: The accelerated evolution of male-biased genes has only been examined in multicellular taxa, but our analyses reveal that accelerated evolution in genes with male-specific expression is also a feature of unicellular organisms. This 'fast-male' evolution is adaptive and likely facilitated by the male-biased sex ratio of gametes in the mating pool. Furthermore, we propose that the exceptional rates of evolution we observe are driven by interactions between males and host immune responses. CONCLUSIONS: We reveal a novel form of host-parasite coevolution that enables parasites to evade host immune responses that negatively impact upon fertility. The identification of parasite genes with accelerated evolution has important implications for the identification of drug and vaccine targets. Specifically, vaccines targeting males will be more vulnerable to parasite evolution than those targeting females or both sexes.

Analysis of immunogenicity of different protein groups from malaria parasite Plasmodium falciparum.

It was observed that pressure of host immune system leads to diversifying selection (which can be measured in terms of pN/pS ratio). In this research we checked whether Plasmodium falciparum proteins containing experimentally evident epitopes from the IEDB database are subject to diversifying selection. We also investigated which life stage of this parasite and which proteins are subject to the strongest immune pressure. To answer these questions we used information about experimentally evident epitopes from P. falciparum, that interact with human immune system and sequences of different isolates of P. falciparum obtained from PlasmoDB. We confirmed the expectations that proteins containing IEDB epitopes are subject to stronger diversifying selection which is evidenced by higher pN/pS ratio. A stage characterized by the highest average pN/pS ratio is that of the sporozoite. The greatest fraction of putative antigens is also present at this stage. We also found that the sporozoite stage is particularly interesting for further analysis as it potentially contains the highest number of unidentified epitopes.

The contribution of microbial mats to the arsenic geochemistry of an ancient gold mine.

The ancient Zloty Stok (SW Poland) gold mine is such an environment, where different microbial communities, able to utilize inorganic arsenic species As(III) and As(V), are found. The purpose of the present study was to (i) estimate prokaryotic diversity in the microbial mats in bottom sediments of this gold mine, (ii) identify microorganisms that can metabolize arsenic, and (iii) estimate their potential role in the arsenic geochemistry of the mine and in the environment. The oxidation/reduction experiments showed that the microbial mat community may significantly contribute to arsenic contamination in groundwater. The presence of both arsenite oxidizing and dissimilatory arsenate reducing bacteria in the mat was confirmed by the detection of arsenite oxidase and dissimilatory arsenate reductase genes, respectively. This work also demonstrated that microorganisms utilizing other compounds that naturally co-occur with arsenic are present within the microbial mat community and may contribute to the arsenic geochemistry in the environment.