Sequence-based Reconstruction Research Articles

Proteome-wide 3D structure prediction provides insights into the ancestral metabolism of ancient archaea and bacteria

Ancestral metabolism has remained controversial due to a lack of evidence beyond sequence-based reconstructions. Although prebiotic chemists have provided hints that metabolism might originate from non-enzymatic protometabolic pathways, gaps between ancestral reconstruction and prebiotic processes mean there is much that is still unknown. Here, we apply proteome-wide 3D structure predictions and comparisons to investigate ancestorial metabolism of ancient bacteria and archaea, to provide information beyond sequence as a bridge to the prebiotic processes. We compare representative bacterial and archaeal strains, which reveal surprisingly similar physiological and metabolic characteristics via microbiological and biophysical experiments. Pairwise comparison of protein structures identify the conserved metabolic modules in bacteria and archaea, despite interference from overly variable sequences. The conserved modules (for example, middle of glycolysis, partial TCA, proton/sulfur respiration, building block biosynthesis) constitute the basic functions that possibly existed in the archaeal-bacterial common ancestor, which are remarkably consistent with the experimentally confirmed protometabolic pathways. These structure-based findings provide a new perspective to reconstructing the ancestral metabolism and understanding its origin, which suggests high-throughput protein 3D structure prediction is a promising approach, deserving broader application in future ancestral exploration.

An Analysis of Methylome Evolution in Primates.

Although the investigation of the epigenome becomes increasingly important, still little is known about the long-term evolution of epigenetic marks and systematic investigation strategies are still lacking. Here, we systematically demonstrate the transfer of classic phylogenetic methods such as maximum likelihood based on substitution models, parsimony, and distance-based to interval-scaled epigenetic data. Using a great apes blood data set, we demonstrate that DNA methylation is evolutionarily conserved at the level of individual CpGs in promotors, enhancers, and genic regions. Our analysis also reveals that this epigenomic conservation is significantly correlated with its transcription factor binding density. Binding sites for transcription factors involved in neuron differentiation and components of AP-1 evolve at a significantly higher rate at methylation than at the nucleotide level. Moreover, our models suggest an accelerated epigenomic evolution at binding sites of BRCA1, chromobox homolog protein 2, and factors of the polycomb repressor 2 complex in humans. For most genomic regions, the methylation-based reconstruction of phylogenetic trees is at par with sequence-based reconstruction. Most strikingly, phylogenetic reconstruction using methylation rates in enhancer regions was ineffective independently of the chosen model. We identify a set of phylogenetically uninformative CpG sites enriched in enhancers controlling immune-related genes.

Hexokinase as a versatile molecular genetic marker for Microsporidia.

Hexokinase (HK) is a core glycolytic enzyme of Microsporidia which regulates host cell metabolic processes. The goal of the present study was to test for the utility of HK for molecular phylogenetics, species identification and molecular detection of microsporidia in infected insects. HK sequence-based reconstructions were essentially similar to those based upon largest subunit RNA polymerase (RPB1) gene sequences, as well as previously published rRNA gene and genome-based trees. Comparing HK sequences allowed clear differentiation of closely related taxa, such as Nosema bombycis and Nosema pyrausta. In Nosema ceranae, unique SNPs were found for an isolate from wild colonies of the Burzyan dark honey bee as compared with the isolates from domesticated European honey bee. Similarly, in Encephalitozoon cuniculi, HK was as effective as RPB1 for discrimination of isolates belonging to different ITS genotypes. Amplification using species-specific primers flanking short fragments at the 3'-end of HK gene showed the presence of infection in insect tissues infected with N. pyrausta, Nosema ceranae and Paranosema (Antonospora) locustae. For the latter parasite species, HK expression was also demonstrated at early stages of infection using total mRNA extracts of locust larvae. These results indicate the suitability of HK as a novel tool for molecular genetic studies of Microsporidia.

Structural reconstruction of protein ancestry

Ancestral protein reconstruction allows the resurrection and characterization of ancient proteins based on computational analyses of sequences of modern-day proteins. Unfortunately, many protein families are highly divergent and not suitable for sequence-based reconstruction approaches. This limitation is exemplified by the antigen receptors of jawed vertebrates (B- and T-cell receptors), heterodimers formed by pairs of Ig domains. These receptors are believed to have evolved from an extinct homodimeric ancestor through a process of gene duplication and diversification; however molecular evidence has so far remained elusive. Here, we use a structural approach and laboratory evolution to reconstruct such molecules and characterize their interaction with antigen. High-resolution crystal structures of reconstructed homodimeric receptors in complex with hen-egg white lysozyme demonstrate how nanomolar affinity binding of asymmetrical antigen is enabled through selective recruitment and structural plasticity within the receptor-binding site. Our results provide structural evidence in support of long-held theories concerning the evolution of antigen receptors, and provide a blueprint for the experimental reconstruction of protein ancestry in the absence of phylogenetic evidence.

Patterns of species range evolution in Indo-Pacific reef assemblages reveal the Coral Triangle as a net source of transoceanic diversity.

The Coral Triangle in the Indo-Pacific is a region renowned for exceptional marine biodiversity. The area could have acted as a ‘centre of origin’ where speciation has been prolific or a ‘centre of survival’ by providing refuge during major environmental shifts such as sea-level changes. The region could also have acted as a ‘centre of accumulation’ for species with origins outside of the Coral Triangle, owing to it being at a central position between the Indian and Pacific oceans. Here, we investigated support for these hypotheses using population-level DNA sequence-based reconstructions of the range evolution of 45 species (314 populations) of Indo-Pacific reef-associated organisms. Our results show that populations undergoing the most ancient establishment were significantly more likely to be closer to the centre of the Coral Triangle than to peripheral locations. The data are consistent with the Coral Triangle being a net source of coral-reef biodiversity for the Indo-Pacific region, suggesting that the region has acted primarily as a centre of survival, a centre of origin or both. These results provide evidence of how a key location can influence the large-scale distributions of biodiversity over evolutionary timescales.

Genome-wide genotype and sequence-based reconstruction of the 140,000 year history of modern human ancestry.

We investigated ancestry of 3,528 modern humans from 163 samples. We identified 19 ancestral components, with 94.4% of individuals showing mixed ancestry. After using whole genome sequences to correct for ascertainment biases in genome-wide genotype data, we dated the oldest divergence event to 140,000 years ago. We detected an Out-of-Africa migration 100,000–87,000 years ago, leading to peoples of the Americas, east and north Asia, and Oceania, followed by another migration 61,000–44,000 years ago, leading to peoples of the Caucasus, Europe, the Middle East, and south Asia. We dated eight divergence events to 33,000–20,000 years ago, coincident with the Last Glacial Maximum. We refined understanding of the ancestry of several ethno-linguistic groups, including African Americans, Ethiopians, the Kalash, Latin Americans, Mozabites, Pygmies, and Uygurs, as well as the CEU sample. Ubiquity of mixed ancestry emphasizes the importance of accounting for ancestry in history, forensics, and health.

MGcV: the microbial genomic context viewer for comparative genome analysis

BackgroundConserved gene context is used in many types of comparative genome analyses. It is used to provide leads on gene function, to guide the discovery of regulatory sequences, but also to aid in the reconstruction of metabolic networks. We present the Microbial Genomic context Viewer (MGcV), an interactive, web-based application tailored to strengthen the practice of manual comparative genome context analysis for bacteria.ResultsMGcV is a versatile, easy-to-use tool that renders a visualization of the genomic context of any set of selected genes, genes within a phylogenetic tree, genomic segments, or regulatory elements. It is tailored to facilitate laborious tasks such as the interactive annotation of gene function, the discovery of regulatory elements, or the sequence-based reconstruction of gene regulatory networks. We illustrate that MGcV can be used in gene function annotation by visually integrating information on prokaryotic genes, like their annotation as available from NCBI with other annotation data such as Pfam domains, sub-cellular location predictions and gene-sequence characteristics such as GC content. We also illustrate the usefulness of the interactive features that allow the graphical selection of genes to facilitate data gathering (e.g. upstream regions, ID’s or annotation), in the analysis and reconstruction of transcription regulation. Moreover, putative regulatory elements and their corresponding scores or data from RNA-seq and microarray experiments can be uploaded, visualized and interpreted in (ranked-) comparative context maps. The ranked maps allow the interpretation of predicted regulatory elements and experimental data in light of each other.ConclusionMGcV advances the manual comparative analysis of genes and regulatory elements by providing fast and flexible integration of gene related data combined with straightforward data retrieval. MGcV is available at http://mgcv.cmbi.ru.nl.

Bootstrapping phylogenies inferred from rearrangement data.

BackgroundLarge-scale sequencing of genomes has enabled the inference of phylogenies based on the evolution of genomic architecture, under such events as rearrangements, duplications, and losses. Many evolutionary models and associated algorithms have been designed over the last few years and have found use in comparative genomics and phylogenetic inference. However, the assessment of phylogenies built from such data has not been properly addressed to date. The standard method used in sequence-based phylogenetic inference is the bootstrap, but it relies on a large number of homologous characters that can be resampled; yet in the case of rearrangements, the entire genome is a single character. Alternatives such as the jackknife suffer from the same problem, while likelihood tests cannot be applied in the absence of well established probabilistic models.ResultsWe present a new approach to the assessment of distance-based phylogenetic inference from whole-genome data; our approach combines features of the jackknife and the bootstrap and remains nonparametric. For each feature of our method, we give an equivalent feature in the sequence-based framework; we also present the results of extensive experimental testing, in both sequence-based and genome-based frameworks. Through the feature-by-feature comparison and the experimental results, we show that our bootstrapping approach is on par with the classic phylogenetic bootstrap used in sequence-based reconstruction, and we establish the clear superiority of the classic bootstrap for sequence data and of our corresponding new approach for rearrangement data over proposed variants. Finally, we test our approach on a small dataset of mammalian genomes, verifying that the support values match current thinking about the respective branches.ConclusionsOur method is the first to provide a standard of assessment to match that of the classic phylogenetic bootstrap for aligned sequences. Its support values follow a similar scale and its receiver-operating characteristics are nearly identical, indicating that it provides similar levels of sensitivity and specificity. Thus our assessment method makes it possible to conduct phylogenetic analyses on whole genomes with the same degree of confidence as for analyses on aligned sequences. Extensions to search-based inference methods such as maximum parsimony and maximum likelihood are possible, but remain to be thoroughly tested.

Computational and Experimental Analysis of Redundancy in the Central Metabolism of Geobacter sulfurreducens

Previous model-based analysis of the metabolic network of Geobacter sulfurreducens suggested the existence of several redundant pathways. Here, we identified eight sets of redundant pathways that included redundancy for the assimilation of acetate, and for the conversion of pyruvate into acetyl-CoA. These equivalent pathways and two other sub-optimal pathways were studied using 5 single-gene deletion mutants in those pathways for the evaluation of the predictive capacity of the model. The growth phenotypes of these mutants were studied under 12 different conditions of electron donor and acceptor availability. The comparison of the model predictions with the resulting experimental phenotypes indicated that pyruvate ferredoxin oxidoreductase is the only activity able to convert pyruvate into acetyl-CoA. However, the results and the modeling showed that the two acetate activation pathways present are not only active, but needed due to the additional role of the acetyl-CoA transferase in the TCA cycle, probably reflecting the adaptation of these bacteria to acetate utilization. In other cases, the data reconciliation suggested additional capacity constraints that were confirmed with biochemical assays. The results demonstrate the need to experimentally verify the activity of key enzymes when developing in silico models of microbial physiology based on sequence-based reconstruction of metabolic networks.

A generic approach to identify Transcription Factor-specific operator motifs; Inferences for LacI-family mediated regulation in Lactobacillus plantarum WCFS1

BackgroundA key problem in the sequence-based reconstruction of regulatory networks in bacteria is the lack of specificity in operator predictions. The problem is especially prominent in the identification of transcription factor (TF) specific binding sites. More in particular, homologous TFs are abundant and, as they are structurally very similar, it proves difficult to distinguish the related operators by automated means. This also holds for the LacI-family, a family of TFs that is well-studied and has many members that fulfill crucial roles in the control of carbohydrate catabolism in bacteria including catabolite repression. To overcome the specificity problem, a comprehensive footprinting approach was formulated to identify TF-specific operator motifs and was applied to the LacI-family of TFs in the model gram positive organism, Lactobacillus plantarum WCFS1. The main premise behind the approach is that only orthologous sequences that share orthologous genomic context will share equivalent regulatory sites.ResultsWhen the approach was applied to the 12 LacI-family TFs of the model species, a specific operator motif was identified for each of them. With the TF-specific operator motifs, potential binding sites were found on the genome and putative minimal regulons could be defined. Moreover, specific inducers could in most cases be linked to the TFs through phylogeny, thereby unveiling the biological role of these regulons. The operator predictions indicated that the LacI-family TFs can be separated into two subfamilies with clearly distinct operator motifs. They also established that the operator related to the 'global' regulator CcpA is not inherently distinct from that of other LacI-family members, only more degenerate. Analysis of the chromosomal position of the identified putative binding sites confirmed that the LacI-family TFs are mostly auto-regulatory and relate mainly to carbohydrate uptake and catabolism.ConclusionOur approach to identify specific operator motifs for different TF-family members is specific and in essence generic. The data infer that, although the specific operator motifs can be used to identify minimal regulons, experimental knowledge on TF activity especially is essential to determine complete regulons as well as to estimate the overlap between TF affinities.