On Protein Loops, Prior Molecular States and Common Ancestors of Life.

The principle of continuity demands the existence of prior molecular states and common ancestors responsible for extant macromolecular structure. Here, we focus on the emergence and evolution of loop prototypes - the elemental architects of protein domain structure. Phylogenomic reconstruction spanning superkingdoms and viruses generated an evolutionary chronology of prototypes with six distinct evolutionary phases defining a most parsimonious evolutionary progression of cellular life. Each phase was marked by strategic prototype accumulation shaping the structures and functions of common ancestors. The last universal common ancestor (LUCA) of cells and viruses and the last universal cellular ancestor (LUCellA) defined stem lines that were structurally and functionally complex. The evolutionary saga highlighted transformative forces. LUCA lacked biosynthetic ribosomal machinery, while the pivotal LUCellA lacked essential DNA biosynthesis and modern transcription. Early proteins therefore relied on RNA for genetic information storage but appeared initially decoupled from it, hinting at transformative shifts of genetic processing. Urancestral loop types suggest advanced folding designs were present at an early evolutionary stage. An exploration of loop geometric properties revealed gradual replacement of prototypes with α-helix and ß-strand bracing structures over time, paving the way for the dominance of other loop types. AlphFold2-generated atomic models of prototype accretion described patterns of fold emergence. Our findings favor a ?processual' model of evolving stem lines aligned with Woese's vision of a communal world. This model prompts discussing the 'problem of ancestors' and the challenges that lie ahead for research in taxonomy, evolution and complexity.

The tree of life describes a tripartite cellular world.

The canonical view of a 3-domain (3D) tree of life was recently challenged by the discovery of Asgardarchaeota encoding eukaryote signature proteins (ESPs), which were treated as missing links of a 2-domain (2D) tree. Here we revisit the debate. We discuss methodological limitations of building trees with alignment-dependent approaches, which often fail to satisfactorily address the problem of ''gaps.'' In addition, most phylogenies are reconstructed unrooted, neglecting the power of direct rooting methods. Alignment-free methodologies lift most difficulties but require employing realistic evolutionary models. We argue that the discoveries of Asgards and ESPs, by themselves, do not rule out the 3D tree, which is strongly supported by comparative and evolutionary genomic analyses and vast genomic and biochemical superkingdom distinctions. Given uncertainties of retrodiction and interpretation difficulties, we conclude that the 3D view has not been falsified but instead has been strengthened by genomic analyses. In turn, the objections to the 2D model have not been lifted. The debate remains open. Also see the video abstract here: https://youtu.be/-6TBN0bubI8.

Phylogenetic profiling, an untapped resource for the prediction of secreted proteins and its complementation with sequence-based classifiers in bacterial type III, IV and VI secretion systems.

In the establishment and maintenance of the interaction between pathogenic or symbiotic bacteria with a eukaryotic organism, protein substrates of specialized bacterial secretion systems called effectors play a critical role once translocated into the host cell. Proteins are also secreted to the extracellular medium by free-living bacteria or directly injected into other competing organisms to hinder or kill. In this work, we explore an approach based on the evolutionary dependence that most of the effectors maintain with their specific secretion system that analyzes the co-occurrence of any orthologous protein group and their corresponding secretion system across multiple genomes. We compared and complemented our methodology with sequence-based machine learning prediction tools for the type III, IV and VI secretion systems. Finally, we provide the predictive results for the three secretion systems in 1606 complete genomes at http://www.iib.unsam.edu.ar/orgsissec/.

Tracing protein and proteome history with chronologies and networks: folding recapitulates evolution.

INTRODUCTION: While the origin and evolution of proteins remain mysterious, advances in evolutionary genomics and systems biology are facilitating the historical exploration of the structure, function and organization of proteins and proteomes. Molecular chronologies are series of time events describing the history of biological systems and subsystems and the rise of biological innovations. Together with time-varying networks, these chronologies provide a window into the past. AREAS COVERED: Here, we review molecular chronologies and networks built with modern methods of phylogeny reconstruction. We discuss how chronologies of structural domain families uncover the explosive emergence of metabolism, the late rise of translation, the co-evolution of ribosomal proteins and rRNA, and the late development of the ribosomal exit tunnel; events that coincided with a tendency to shorten folding time. Evolving networks described the early emergence of domains and a late 'big bang' of domain combinations. EXPERT OPINION: Two processes, folding and recruitment appear central to the evolutionary progression. The former increases protein persistence. The later fosters diversity. Chronologically, protein evolution mirrors folding by combining supersecondary structures into domains, developing translation machinery to facilitate folding speed and stability, and enhancing structural complexity by establishing long-distance interactions in novel structural and architectural designs.

The origin and evolution of viruses inferred from fold family structure.

The canonical frameworks of viral evolution describe viruses as cellular predecessors, reduced forms of cells, or entities that escaped cellular control. The discovery of giant viruses has changed these standard paradigms. Their genetic, proteomic and structural complexities resemble those of cells, prompting a redefinition and reclassification of viruses. In a previous genome-wide analysis of the evolution of structural domains in proteomes, with domains defined at the fold superfamily level, we found the origins of viruses intertwined with those of ancient cells. Here, we extend these data-driven analyses to the study of fold families confirming the co-evolution of viruses and ancient cells and the genetic ability of viruses to foster molecular innovation. The results support our suggestion that viruses arose by genomic reduction from ancient cells and validate a co-evolutionary 'symbiogenic' model of viral origins.

Archaea-First and the Co-Evolutionary Diversification of Domains of Life.

The origins and evolution of the Archaea, Bacteria, and Eukarya remain controversial. Phylogenomic-wide studies of molecular features that are evolutionarily conserved, such as protein structural domains, suggest Archaea is the first domain of life to diversify from a stem line of descent. This line embodies the last universal common ancestor of cellular life. Here, we propose that ancestors of Euryarchaeota co-evolved with those of Bacteria prior to the diversification of Eukarya. This co-evolutionary scenario is supported by comparative genomic and phylogenomic analyses of the distributions of fold families of domains in the proteomes of free-living organisms, which show horizontal gene recruitments and informational process homologies. It also benefits from the molecular study of cell physiologies responsible for membrane phospholipids, methanogenesis, methane oxidation, cell division, gas vesicles, and the cell wall. Our theory however challenges popular cell fusion and two-domain of life scenarios derived from sequence analysis, demanding phylogenetic reconciliation. Also see the video abstract here: https://youtu.be/9yVWn_Q9faY.

Genetic Structure of Ralstonia solanacearum and Ralstonia pseudosolanacearum in Brazil.

Bacterial wilt-causing Ralstonia threaten numerous crops throughout the world. We studied the population structure of 196 isolates of Ralstonia solanacearum and 39 isolates of Ralstonia pseudosolanacearum, which were collected from potato- and tomato-growing areas in 19 states of Brazil. Regardless of the species, three groups of isolates were identified. One group encompassed R. pseudosolanacearum isolates. The other two groups comprise isolates of R. solanacearum (phylotype II) split according to geographic regions, one made of isolates from the North and Northeast and the other made of isolates from the Central, Southeast, and South regions (CSS). Among the isolates collected in CSS, those from tomato were genetically distinct from the potato isolates. The genetic variability in the population of R. pseudosolanacearum was lower than that of R. solanacearum, suggesting that the former was introduced in Brazil. Conversely, the high genetic variability of R. solanacearum in all regions, hosts, and times supports the hypothesis that this species is autochthonous in South America, more precisely in Brazil and Peru. For R. solanacearum, higher variability and lower migration rates were observed when tomato isolates were analyzed, indicating that the variability is caused mainly by the differences of the local, native soil population. The North subpopulation was distinct from all others, possibly because of differences in environmental features of this region. The proximity of some geographic regions and the movement of potato tubers could have facilitated migration and therefore low genetic differentiation between geographic regions. Finally, geography, which also influences host distribution, affects the structure of the population of R. solanacearum in Brazil. Despite quarantine procedures in Brazil, increasing levels of trade are a threat to biosecurity, and these results emphasize the need for improving our regional efforts to prevent the dispersal of pathogens.

Testing Empirical Support for Evolutionary Models that Root the Tree of Life.

Trees of life (ToLs) can only be rooted with direct methods that seek optimization of character state information in ingroup taxa. This involves optimizing phylogenetic tree, model and data in an exercise of reciprocal illumination. Rooted ToLs have been built from a census of protein structural domains in proteomes using two kinds of models. Fully-reversible models use standard-ordered (additive) characters and Wagner parsimony to generate unrooted trees of proteomes that are then rooted with Weston's generality criterion. Non-reversible models directly build rooted trees with unordered characters and asymmetric stepmatrices of transformation costs that penalize gain over loss of domains. Here, we test the empirical support for the evolutionary models with character state reconstruction methods using two published proteomic datasets. We show that the reversible models match reconstructed frequencies of character change and are faithful to the distribution of serial homologies in trees. In contrast, the non-reversible models go counter to trends in the data they must explain, attracting organisms with large proteomes to the base of the rooted trees while violating the triangle inequality of distances. This can lead to serious reconstruction inconsistencies that show model inadequacy. Our study highlights the aprioristic perils of disposing of countering evidence in natural history reconstruction.

Genome-wide analysis of the MYB-CC gene family of maize.

The MYB-CC gene family encode proteins that harbor a combination of characteristic myeloblastosis (MYB) and coiled-coil (CC) domain structures. Some MYB-CC genes have been demonstrated to represent transcription factors regulating phosphate uptake and controlling the starvation response in plants. Despite their physiological importance, a systematic analysis of MYB-CC genes has not been reported in maize. In our study, we identified and characterized maize MYB-CC genes at whole-genome level. A total of 12 maize MYB-CC genes (ZmMYB-CC1 to ZmMYB-CC12) were identified located in six out of the 10 chromosomes of maize. Their gene structures showed similar splicing patterns and large variations of intron length. Multiple sequence alignments revealed that all MYB-CC proteins in maize shared conserved sequence cores corresponding to the MYB and CC domains, respectively. The family expanded in maize partly due to tandem and segmental duplication events. Phylogenetic analysis of MYB-CC genes indicated that the MYB-CC gene family can be divided into two subfamilies and that gene members with same functions were found in the same groups. Results provide a very useful reference for cloning and functional analysis of PHR-like genes in maize and suggest a method to predict and select appropriate candidate genes for functional genomic analysis of useful traits in crop plants.

Correction to: Genome-wide analysis of the MYB-CC gene family of maize.

In the original publication of the article, the incorrect version of Fig. 1 was mistakenly used. The correct version of the figure is provided in this correction.

Long-term evolution of viruses: A Janus-faced balance.

The popular textbook image of viruses as noxious and selfish genetic parasites greatly underestimates the beneficial contributions of viruses to the biosphere. Given the crucial dependency of viruses to reproduce in an intracellular environment, viruses that engage in excessive killing (lysis) can drive their cellular hosts to extinction and will not survive. The lytic mode of virus propagation must, therefore, be tempered and balanced by non-lytic modes of virus latency and symbiosis. Here, we review recent bioinformatics and metagenomic studies to argue that viral endogenization and domestication may be more frequent mechanisms of virus persistence than lysis. We use a triangle diagram to explain the three major virus persistence strategies that explain the global scope of virus-cell interactions including lysis, latency and virus-cell symbiosis. This paradigm can help identify novel directions in virology research where scientists could artificially gain control over switching lytic and beneficial viral lifestyles. Also see the Video Abstract: http://youtu.be/GwXWz4N8o8.

Genome-Wide Identification and Characterization of the Vacuolar H+-ATPase Subunit H Gene Family in Crop Plants.

The vacuolar H+-ATPase (V-ATPase) plays many important roles in cell growth and in response to stresses in plants. The V-ATPase subunit H (VHA-H) is required to form a stable and active V-ATPase. Genome-wide analyses of VHA-H genes in crops contribute significantly to a systematic understanding of their functions. A total of 22 VHA-H genes were identified from 11 plants representing major crops including cotton, rice, millet, sorghum, rapeseed, maize, wheat, soybean, barley, potato, and beet. All of these VHA-H genes shared exon-intron structures similar to those of Arabidopsis thaliana. The C-terminal domain of VHA-H was shorter and more conserved than the N-terminal domain. The VHA-H gene was effectively used as a genetic marker to infer the phylogenetic relationships among plants, which were congruent with currently accepted taxonomic groupings. The VHA-H genes from six species of crops (Gossypium raimondii, Brassica napus, Glycine max, Solanum tuberosum, Triticum aestivum, and Zea mays) showed high gene structural diversity. This resulted from the gains and losses of introns. Seven VHA-H genes in six species of crops (Gossypium raimondii, Hordeum vulgare, Solanum tuberosum, Setaria italica, Triticum aestivum, and Zea mays) contained multiple transcript isoforms arising from alternative splicing. The study of cis-acting elements of gene promoters and RNA-seq gene expression patterns confirms the role of VHA-H genes as eco-enzymes. The gene structural diversity and proteomic diversity of VHA-H genes in our crop sampling facilitate understanding of their functional diversity, including stress responses and traits important for crop improvement.

Evolution of macromolecular structure: a 'double tale' of biological accretion and diversification.

The evolution of structure in biology is driven by accretion and diversification. Accretion brings together disparate parts to form bigger wholes. Diversification provides opportunities for growth and innovation. Here, we review patterns and processes that are responsible for a 'double tale' of accretion and diversification at various levels of complexity, from proteins and nucleic acids to high-rise building structures in cities. Parts are at first weakly linked and associate variously. As they diversify, they compete with each other and are selected for performance. The emerging interactions constrain their structure and associations. This causes parts to self-organise into modules with tight linkage. In a second phase, variants of the modules evolve and become new parts for a new generative cycle of higher-level organisation. Evolutionary genomics and network biology support the 'double tale' of structural module creation and validate an evolutionary principle of maximum abundance that drives the gain and loss of modules.

A Dynamic Model for the Evolution of Protein Structure.

Domains are folded structures and evolutionary building blocks of protein molecules. Their three-dimensional atomic conformations, which define biological functions, can be coarse-grained into levels of a hierarchy. Here we build global dynamical models for the evolution of domains at fold and fold superfamily (FSF) levels. We fit the models with data from phylogenomic trees of domain structures and evaluate the distributions of the resulting parameters and their implications. The trees were inferred from a census of domain structures in hundreds of genomes from all three superkingdoms of life. The models used birth-death differential equations with the global abundances of structures as state variables, with one set of equations for folds and another for FSFs. Only the transitions present in the tree are assumed possible. Each fold or FSF diversifies in variants, eventually producing a new fold or FSF. The parameters specify rates of generation of variants and of new folds or FSFs. The equations were solved for the parameters by simplifying the trees to a comb-like topology, treating branches as emerging directly from a trunk. We found that the rate constants for folds and FSFs evolved similarly. These parameters showed a sharp transient change at about 1.5 Gyrs ago. This time coincides with a period in which domains massively combined in proteins and their arrangements distributed in novel lineages during the rise of organismal diversification. Our simulations suggest that exploration of protein structure space occurs through coarse-grained discoveries that undergo fine-grained elaboration.

Arguments Reinforcing the Three-Domain View of Diversified Cellular Life.

The archaeal ancestor scenario (AAS) for the origin of eukaryotes implies the emergence of a new kind of organism from the fusion of ancestral archaeal and bacterial cells. Equipped with this "chimeric" molecular arsenal, the resulting cell would gradually accumulate unique genes and develop the complex molecular machineries and cellular compartments that are hallmarks of modern eukaryotes. In this regard, proteins related to phagocytosis and cell movement should be present in the archaeal ancestor, thus identifying the recently described candidate archaeal phylum "Lokiarchaeota" as resembling a possible candidate ancestor of eukaryotes. Despite its appeal, AAS seems incompatible with the genomic, molecular, and biochemical differences that exist between Archaea and Eukarya. In particular, the distribution of conserved protein domain structures in the proteomes of cellular organisms and viruses appears hard to reconcile with the AAS. In addition, concerns related to taxon and character sampling, presupposing bacterial outgroups in phylogenies, and nonuniform effects of protein domain structure rearrangement and gain/loss in concatenated alignments of protein sequences cast further doubt on AAS-supporting phylogenies. Here, we evaluate AAS against the traditional "three-domain" world of cellular organisms and propose that the discovery of Lokiarchaeota could be better reconciled under the latter view, especially in light of several additional biological and technical considerations.

Genomic characterization of plant cell wall degrading enzymes and in silico analysis of xylanases and polygalacturonases of Fusarium virguliforme.

BACKGROUND: Plant cell wall degrading enzymes (PCWDEs) are a subset of carbohydrate-active enzymes (CAZy) produced by plant pathogens to degrade plant cell walls. To counteract PCWDEs, plants release PCWDEs inhibitor proteins (PIPs) to reduce their impact. Several transgenic plants expressing exogenous PIPs that interact with fungal glycoside hydrolase (GH)11-type xylanases or GH28-type polygalacturonase (PG) have been shown to enhance disease resistance. However, many plant pathogenic Fusarium species were reported to escape PIPs inhibition. Fusarium virguliforme is a soilborne pathogen that causes soybean sudden death syndrome (SDS). Although the genome of F. virguliforme was sequenced, there were limited studies focused on the PCWDEs of F. virguliforme. Our goal was to understand the genomic CAZy structure of F. viguliforme, and determine if exogenous PIPs could be theoretically used in soybean to enhance resistance against F. virguliforme. RESULTS: F. virguliforme produces diverse CAZy to degrade cellulose and pectin, similar to other necrotorphic and hemibiotrophic plant pathogenic fungi. However, some common CAZy of plant pathogenic fungi that catalyze hemicellulose, such as GH29, GH30, GH44, GH54, GH62, and GH67, were deficient in F. virguliforme. While the absence of these CAZy families might be complemented by other hemicellulases, F. virguliforme contained unique families including GH131, polysaccharide lyase (PL) 9, PL20, and PL22 that were not reported in other plant pathogenic fungi or oomycetes. Sequence analysis revealed two GH11 xylanases of F. virguliforme, FvXyn11A and FvXyn11B, have conserved residues that allow xylanase inhibitor protein I (XIP-I) binding. Structural modeling suggested that FvXyn11A and FvXyn11B could be blocked by XIP-I that serves as good candidate for developing transgenic soybeans. In contrast, one GH28 PG, FvPG2, contains an amino acid substitution that is potentially incompatible with the bean polygalacturonase-inhibitor protein II (PvPGIP2). CONCLUSIONS: Identification and annotation of CAZy provided advanced understanding of genomic composition of PCWDEs in F. virguliforme. Sequence and structural analyses of FvXyn11A and FvXyn11B suggested both xylanases were conserved in residues that allow XIP-I inhibition, and expression of both xylanases were detected during soybean roots infection. We postulate that a transgenic soybean expressing wheat XIP-I may be useful for developing root rot resistance to F. virguliforme.

Ancestral Insertions and Expansions of rRNA do not Support an Origin of the Ribosome in Its Peptidyl Transferase Center.

Phylogenetic reconstruction of ribosomal history suggests that the ribonucleoprotein complex originated in structures supporting RNA decoding and ribosomal mechanics. A recent study of accretion of ancestral expansion segments of rRNA, however, contends that the large subunit of the ribosome originated in its peptidyl transferase center (PTC). Here I re-analyze the rRNA insertion data that supports this claim. Analysis of a crucial three-way junction connecting the long-helical coaxial branch that supports the PTC to the L1 stalk and its translocation functions reveals an incorrect branch-to-trunk insertion assignment that is in conflict with the PTC-centered accretion model. Instead, the insertion supports the ancestral origin of translocation. Similarly, an insertion linking a terminal coaxial trunk that holds the L7-12 stalk and its GTPase center to a seven-way junction of the molecule again questions the early origin of the PTC. Unwarranted assumptions, dismissals of conflicting data, structural insertion ambiguities, and lack of phylogenetic information compromise the construction of an unequivocal insertion-based model of macromolecular accretion. Results prompt integration of phylogenetic and structure-based models to address RNA junction growth and evolutionary constraints acting on ribosomal structure.

The natural history of biocatalytic mechanisms.

Phylogenomic analysis of the occurrence and abundance of protein domains in proteomes has recently showed that the α/ß architecture is probably the oldest fold design. This holds important implications for the origins of biochemistry. Here we explore structure-function relationships addressing the use of chemical mechanisms by ancestral enzymes. We test the hypothesis that the oldest folds used the most mechanisms. We start by tracing biocatalytic mechanisms operating in metabolic enzymes along a phylogenetic timeline of the first appearance of homologous superfamilies of protein domain structures from CATH. A total of 335 enzyme reactions were retrieved from MACiE and were mapped over fold age. We define a mechanistic step type as one of the 51 mechanistic annotations given in MACiE, and each step of each of the 335 mechanisms was described using one or more of these annotations. We find that the first two folds, the P-loop containing nucleotide triphosphate hydrolase and the NAD(P)-binding Rossmann-like homologous superfamilies, were α/ß architectures responsible for introducing 35% (18/51) of the known mechanistic step types. We find that these two oldest structures in the phylogenomic analysis of protein domains introduced many mechanistic step types that were later combinatorially spread in catalytic history. The most common mechanistic step types included fundamental building blocks of enzyme chemistry: "Proton transfer," "Bimolecular nucleophilic addition," "Bimolecular nucleophilic substitution," and "Unimolecular elimination by the conjugate base." They were associated with the most ancestral fold structure typical of P-loop containing nucleotide triphosphate hydrolases. Over half of the mechanistic step types were introduced in the evolutionary timeline before the appearance of structures specific to diversified organisms, during a period of architectural diversification. The other half unfolded gradually after organismal diversification and during a period that spanned ∼2 billion years of evolutionary history.

Global patterns of protein domain gain and loss in superkingdoms.

Domains are modules within proteins that can fold and function independently and are evolutionarily conserved. Here we compared the usage and distribution of protein domain families in the free-living proteomes of Archaea, Bacteria and Eukarya and reconstructed species phylogenies while tracing the history of domain emergence and loss in proteomes. We show that both gains and losses of domains occurred frequently during proteome evolution. The rate of domain discovery increased approximately linearly in evolutionary time. Remarkably, gains generally outnumbered losses and the gain-to-loss ratios were much higher in akaryotes compared to eukaryotes. Functional annotations of domain families revealed that both Archaea and Bacteria gained and lost metabolic capabilities during the course of evolution while Eukarya acquired a number of diverse molecular functions including those involved in extracellular processes, immunological mechanisms, and cell regulation. Results also highlighted significant contemporary sharing of informational enzymes between Archaea and Eukarya and metabolic enzymes between Bacteria and Eukarya. Finally, the analysis provided useful insights into the evolution of species. The archaeal superkingdom appeared first in evolution by gradual loss of ancestral domains, bacterial lineages were the first to gain superkingdom-specific domains, and eukaryotes (likely) originated when an expanding proto-eukaryotic stem lineage gained organelles through endosymbiosis of already diversified bacterial lineages. The evolutionary dynamics of domain families in proteomes and the increasing number of domain gains is predicted to redefine the persistence strategies of organisms in superkingdoms, influence the make up of molecular functions, and enhance organismal complexity by the generation of new domain architectures. This dynamics highlights ongoing secondary evolutionary adaptations in akaryotic microbes, especially Archaea.