Understanding the Evolutionary Relationships and Major Traits of \u3cem\u3eBacillus\u3c/em\u3e through Comparative Genomics

Background: The presence of Bacillus in very diverse environments reflects the versatile metabolic capabilities of a widely distributed genus. Traditional phylogenetic analysis based on limited gene sampling is not adequate for resolving the genus evolutionary relationships. By distinguishing between core and pan-genome, we determined the evolutionary and functional relationships of known Bacillus. Results: Our analysis is based upon twenty complete and draft Bacillus genomes, including a newly sequenced Bacillus isolate from an aquatic environment that we report for the first time here. Using a core genome, we were able to determine the phylogeny of known Bacilli, including aquatic strains whose position in the phylogenetic tree could not be unambiguously determined in the past. Using the pan-genome from the sequenced Bacillus, we identified functional differences, such as carbohydrate utilization and genes involved in signal transduction, which distinguished the taxonomic groups. We also assessed the genetic architecture of the defining traits of Bacillus, such as sporulation and competence, and showed that less than one third of the B. subtilis genes are conserved across other Bacilli. Most variation was shown to occur in genes that are needed to respond to environmental cues, suggesting that Bacilli have genetically specialized to allow for the occupation of diverse habitats and niches. Conclusions: The aquatic Bacilli are defined here for the first time as a group through the phylogenetic analysis of 814 genes that comprise the core genome. Our data distinguished between genomic components, especially core vs. pan-genome to provide insight into phylogeny and function that would otherwise be difficult to achieve. A phylogeny may mask the diversity of functions, which we tried to uncover in our approach. The diversity of sporulation and competence genes across the Bacilli was unexpected based on previous studies of the B. subtilis model alone. The challenge of uncovering the novelties and variations among genes of the non-subtilis groups still remains. This task will be best accomplished by directing efforts toward understanding phylogenetic groups with similar ecological niches

Groups without cultured representatives dominate eukaryotic picophytoplankton in the oligotrophic South East Pacific Ocean

Background: Photosynthetic picoeukaryotes (PPE) with a cell size less than 3 µm play a critical role in oceanic primary production. In recent years, the composition of marine picoeukaryote communities has been intensively investigated by molecular approaches, but their photosynthetic fraction remains poorly characterized. This is largely because the classical approach that relies on constructing 18S rRNA gene clone libraries from filtered seawater samples using universal eukaryotic primers is heavily biased toward heterotrophs, especially alveolates and stramenopiles, despite the fact that autotrophic cells in general outnumber heterotrophic ones in the euphotic zone. Methodology/Principal Findings: In order to better assess the composition of the eukaryotic picophytoplankton in the South East Pacific Ocean, encompassing the most oligotrophic oceanic regions on earth, we used a novel approach based on flow cytometry sorting followed by construction of 18S rRNA gene clone libraries. This strategy dramatically increased the recovery of sequences from putative autotrophic groups. The composition of the PPE community appeared highly variable both vertically down the water column and horizontally across the South East Pacific Ocean. In the central gyre, uncultivated lineages dominated: a recently discovered clade of Prasinophyceae (IX), clades of marine Chrysophyceae and Haptophyta, the latter division containing a potentially new class besides Prymnesiophyceae and Pavlophyceae. In contrast, on the edge of the gyre and in the coastal Chilean upwelling, groups with cultivated representatives (Prasinophyceae clade VII and Mamiellales) dominated. Conclusions/Significance: Our data demonstrate that a very large fraction of the eukaryotic picophytoplankton still escapes cultivation. The use of flow cytometry sorting should prove very useful to better characterize specific plankton populations by molecular approaches such as gene cloning or metagenomics, and also to obtain into culture strains representative of these novel groups

Inferring evolutionary histories of pathway regulation from transcriptional profiling data

One of the outstanding challenges in comparative genomics is to interpret the evolutionary importance of regulatory variation between species. Rigorous molecular evolution-based methods to infer evidence for natural selection from expression data are at a premium in the field, and to date, phylogenetic approaches have not been well-suited to address the question in the small sets of taxa profiled in standard surveys of gene expression. We have developed a strategy to infer evolutionary histories from expression profiles by analyzing suites of genes of common function. In a manner conceptually similar to molecular evolution models in which the evolutionary rates of DNA sequence at multiple loci follow a gamma distribution, we modeled expression of the genes of an \emph{a priori}-defined pathway with rates drawn from an inverse gamma distribution. We then developed a fitting strategy to infer the parameters of this distribution from expression measurements, and to identify gene groups whose expression patterns were consistent with evolutionary constraint or rapid evolution in particular species. Simulations confirmed the power and accuracy of our inference method. As an experimental testbed for our approach, we generated and analyzed transcriptional profiles of four \emph{Saccharomyces} yeasts. The results revealed pathways with signatures of constrained and accelerated regulatory evolution in individual yeasts and across the phylogeny, highlighting the prevalence of pathway-level expression change during the divergence of yeast species. We anticipate that our pathway-based phylogenetic approach will be of broad utility in the search to understand the evolutionary relevance of regulatory change.Comment: 30 pages, 12 figures, 2 tables, contact authors for supplementary table

The nuclear receptors of Biomphalaria glabrata and Lottia gigantea: Implications for developing new model organisms

© 2015 Kaur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedNuclear receptors (NRs) are transcription regulators involved in an array of diverse physiological functions including key roles in endocrine and metabolic function. The aim of this study was to identify nuclear receptors in the fully sequenced genome of the gastropod snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni and compare these to known vertebrate NRs, with a view to assessing the snail's potential as a invertebrate model organism for endocrine function, both as a prospective new test organism and to elucidate the fundamental genetic and mechanistic causes of disease. For comparative purposes, the genome of a second gastropod, the owl limpet, Lottia gigantea was also investigated for nuclear receptors. Thirty-nine and thirty-three putative NRs were identified from the B. glabrata and L. gigantea genomes respectively, based on the presence of a conserved DNA-binding domain and/or ligand-binding domain. Nuclear receptor transcript expression was confirmed and sequences were subjected to a comparative phylogenetic analysis, which demonstrated that these molluscs have representatives of all the major NR subfamilies (1-6). Many of the identified NRs are conserved between vertebrates and invertebrates, however differences exist, most notably, the absence of receptors of Group 3C, which includes some of the vertebrate endocrine hormone targets. The mollusc genomes also contain NR homologues that are present in insects and nematodes but not in vertebrates, such as Group 1J (HR48/DAF12/HR96). The identification of many shared receptors between humans and molluscs indicates the potential for molluscs as model organisms; however the absence of several steroid hormone receptors indicates snail endocrine systems are fundamentally different.The National Centre for the Replacement, Refinement and Reduction of Animals in Research, Grant Ref:G0900802 to CSJ, LRN, SJ & EJR [www.nc3rs.org.uk]

Directed evolution of ancestral and modern enzymes

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 18-10-2019Esta tesis tiene embargado el acceso al texto completo hasta el 18-04-2021Ancestral sequence reconstruction (ASR) and resurrection (i.e. functional expression in a heterologous host) allows enzymes with different properties to be disclosed while its combination with directed evolution may lead to the development of a new generation of biocatalysts. In this Doctoral Thesis we have explored the combination of such powerful methods using as blueprints two different enzyme systems, Rubisco and laccase. In the first chapter of this Thesis we reconstructed and resurrected (in Escherichia coli) Precambrian Rubisco nodes which were evolved in parallel with the extant Rubisco counterpart. An in vitro dual high-throughput screening (HTS) method was set out to identify thermostable and functional variants after- applying a palette of directed evolution strategies. The stronger tolerance to mutational loads, the improved expression and the different kinetic behavior were some of the traits that highlighted in the Precambrian enzyme. Particularly, the evolved ancestral Clone B2 stood out as a case study of this elusive protein due to its alternative performance in the classical equilibrium of Rubisco kinetic constants. In the second chapter we focused ASR and directed evolution on basidiomycete laccases. Firstly, ancestral nodes from the Paleozoic were reconstructed and resurrected in Saccharomyces cerevisiae. The resurrected enzymes showed a higher heterologous expression and a broader pH stability profile than the modern -laboratory evolved- counterpart. The most promising ancestral node was subjected to structure-guided evolution for the oxidation of β–diketones, an unusual type of redox mediators capable to initiate the polymerization of vinyl monomers. The final chapter of the Thesis deals with consensus design, a long-standing protein engineering method to increase stability without compromising activity. We applied an in-house consensus method to stabilize a laboratory evolved high-redox potential laccase. Multiple sequence alignments were carried out and computationally refined by applying relative entropy and mutual information thresholds. Through this approach, an ensemble of 20 consensus mutations were identified, 18 of which were consensus ancestral mutations. After analyzing potential epistasis by site directed recombination in vivo, the best mutant was characterized displaying dramatically improved thermostability, kinetic values and secretion levels.La reconstrucción y resurrección (i.e. expresión funcional en un hospedador heterólogo) de secuencias ancestrales permite obtener enzimas con diferentes propiedades que al ser combinadas con la evolución dirigida pueden dar lugar al desarrollo de una nueva generación de biocatalizadores. En la presente Tesis Doctoral hemos explorado la combinación de estos potentes métodos usando como modelos dos sistemas enzimáticos diferentes, Rubisco y lacasa. En el primer capítulo se reconstruyeron y resucitaron (en Escherichia coli) nodos de rubiscos Precámbricas con el fin de evolucionarlos en paralelo con una versión moderna de Rubisco. Se desarrolló un método de cribado dual in vitro para poder identificar variantes termoestables y funcionales tras aplicar varias estrategias de evolución dirigida. Las enzimas Precámbricas destacaron por una alta tolerancia a tasas mutagénicas, expresión funcional mejorada y valores cinéticos diferentes a los de las enzimas modernas. En particular, la rubisco ancestral evolucionada, clon B2, despuntó como caso de estudio de esta complicada enzima debido al comportamiento alternativo que muestra con respecto al equilibrio clásico de las constantes cinéticas de la Rubisco. En el segundo capítulo se llevo a cabo la resurrección y evolución dirigida de lacasas de basidiomicetos. En primer lugar se reconstruyeron y resucitaron en Saccharomyces cerevisiae nodos ancestrales del Paleozoico. Las enzimas ancestrales mostraron mayor nivel de expresión heteróloga así como un perfil de estabilidad a diferentes pHs más amplio que el de la versión –evolucionada en el laboratorio- moderna. El nodo ancestral más prometedor fue sometido a evolución estructuralmente guiada para la oxidación de β-dicetonas, un tipo de mediador redox poco usual capaz de iniciar la polimerización de monómeros de vinilo. El capítulo final de la Tesis trata sobre el diseño consenso, un método clásico de ingeniería de proteínas para aumentar la estabilidad sin afectar a la actividad. Se aplicó un método consenso propio para la estabilización de una lacasa de alto potencial redox evolucionada en el laboratorio. Se llevó a cabo un alineamiento de múltiples secuencias que fue refinado computacionalmente mediante el uso de los marcadores de entropía relativa e información mutua. Mediante este procedimiento se identificaron 20 mutaciones consenso, 18 de las cuales corresponden a mutaciones ancestrales-consenso. Se analizó la posible epistasia de estas mutaciones mediante recombinación dirigida in vivo y se caracterizó el mejor mutante que presentó mayores niveles de estabilidad, valores cinéticos y secreciónLa presente Tesis Doctoral se ha llevado a cabo gracias a la financiación recibida a través de una beca para la formación de personal investigador (FPI) del Ministerio de Economía y Competitividad (BES-2014-068887) dentro de los proyectos nacionales DEWRY (BIO2013-43407-R) y LIGNOLUTION (BIO2016-79106-R)

Molecular phylogeny of beetle associated diplogastrid nematodes suggests host switching rather than nematode-beetle coevolution

<p>Abstract</p> <p>Background</p> <p>Nematodes are putatively the most species-rich animal phylum. They have various life styles and occur in a variety of habitats, ranging from free-living nematodes in aquatic or terrestrial environments to parasites of animals and plants. The rhabditid nematode <it>Caenorhabditis elegans </it>is one of the most important model organisms in modern biology. <it>Pristionchus pacificus </it>of the family of the Diplogastridae has been developed as a satellite model for comparison to <it>C. elegans</it>. The Diplogastridae, a monophyletic clade within the rhabditid nematodes, are frequently associated with beetles. How this beetle-association evolved and whether beetle-nematode coevolution occurred is still elusive. As a prerequisite to answering this question a robust phylogeny of beetle-associated Diplogastridae is needed.</p> <p>Results</p> <p>Sequences for the nuclear small subunit ribosomal RNA and for 12 ribosomal protein encoding nucleotide sequences were collected for 14 diplogastrid taxa yielding a dataset of 5996 bp of concatenated aligned sequences. A molecular phylogeny of beetle-associated diplogastrid nematodes was established by various algorithms. Robust subclades could be demonstrated embedded in a phylogenetic tree topology with short internal branches, indicating rapid ancestral divergences. Comparison of the diplogastrid phylogeny to a comprehensive beetle phylogeny revealed no major congruence and thus no evidence for a long-term coevolution.</p> <p>Conclusion</p> <p>Reconstruction of the phylogenetic history of beetle-associated Diplogastridae yields four distinct subclades, whose deep phylogenetic divergence, as indicated by short internal branch lengths, shows evidence for evolution by successions of ancient rapid radiation events. The stem species of the Diplogastridae existed at the same time period when the major radiations of the beetles occurred. Comparison of nematode and beetle phylogenies provides, however, no evidence for long-term coevolution of diplogastrid nematodes and their beetle hosts. Instead, frequent host switching is observed. The molecular phylogeny of the Diplogastridae provides a framework for further examinations of the evolution of these associations, for the study of interactions within the ecosystems, and for investigations of diplogastrid genome evolution.</p

Design and development of learning model for compression and processing of deoxyribonucleic acid genome sequence

Owing to the substantial volume of human genome sequence data files (from 30-200 GB exposed) Genomic data compression has received considerable traction and storage costs are one of the major problems faced by genomics laboratories. This involves a modern technology of data compression that reduces not only the storage but also the reliability of the operation. There were few attempts to solve this problem independently of both hardware and software. A systematic analysis of associations between genes provides techniques for the recognition of operative connections among genes and their respective yields, as well as understandings into essential biological events that are most important for knowing health and disease phenotypes. This research proposes a reliable and efficient deep learning system for learning embedded projections to combine gene interactions and gene expression in prediction comparison of deep embeddings to strong baselines. In this paper we preform data processing operations and predict gene function, along with gene ontology reconstruction and predict the gene interaction. The three major steps of genomic data compression are extraction of data, storage of data, and retrieval of the data. Hence, we propose a deep learning based on computational optimization techniques which will be efficient in all the three stages of data compression

Recovering complete and draft population genomes from metagenome datasets.

Assembly of metagenomic sequence data into microbial genomes is of fundamental value to improving our understanding of microbial ecology and metabolism by elucidating the functional potential of hard-to-culture microorganisms. Here, we provide a synthesis of available methods to bin metagenomic contigs into species-level groups and highlight how genetic diversity, sequencing depth, and coverage influence binning success. Despite the computational cost on application to deeply sequenced complex metagenomes (e.g., soil), covarying patterns of contig coverage across multiple datasets significantly improves the binning process. We also discuss and compare current genome validation methods and reveal how these methods tackle the problem of chimeric genome bins i.e., sequences from multiple species. Finally, we explore how population genome assembly can be used to uncover biogeographic trends and to characterize the effect of in situ functional constraints on the genome-wide evolution

Prevalence of SOS-mediated control of integron integrase expression as an adaptive trait of chromosomal and mobile integrons

Background: Integrons are found in hundreds of environmental bacterial species, but are mainly known as the agents responsible for the capture and spread of antibiotic-resistance determinants between Gram-negative pathogens. The SOS response is a regulatory network under control of the repressor protein LexA targeted at addressing DNA damage, thus promoting genetic variation in times of stress. We recently reported a direct link between the SOS response and the expression of integron integrases in Vibrio cholerae and a plasmid-borne class 1 mobile integron. SOS regulation enhances cassette swapping and capture in stressful conditions, while freezing the integron in steady environments. We conducted a systematic study of available integron integrase promoter sequences to analyze the extent of this relationship across the Bacteria domain. Results: Our results showed that LexA controls the expression of a large fraction of integron integrases by binding to Escherichia coli-like LexA binding sites. In addition, the results provide experimental validation of LexA control of the integrase gene for another Vibrio chromosomal integron and for a multiresistance plasmid harboring two integrons. There was a significant correlation between lack of LexA control and predicted inactivation of integrase genes, even though experimental evidence also indicates that LexA regulation may be lost to enhance expression of integron cassettes. Conclusions: Ancestral-state reconstruction on an integron integrase phylogeny led us to conclude that the ancestral integron was already regulated by LexA. The data also indicated that SOS regulation has been actively preserved in mobile integrons and large chromosomal integrons, suggesting that unregulated integrase activity is selected against. Nonetheless, additional adaptations have probably arisen to cope with unregulated integrase activity. Identifying them may be fundamental in deciphering the uneven distribution of integrons in the Bacteria domain

Phylogeny of the nematode genus Pristionchus and implications for biodiversity, biogeography and the evolution of hermaphroditism

<p>Abstract</p> <p>Background</p> <p>The nematode <it>Pristionchus pacificus </it>has originally been developed as a satellite organism for comparison to <it>Caenorhabditis elegans</it>. A 10X coverage of the whole genome of <it>P. pacificus </it>is available, making <it>P. pacificus </it>the first non-<it>Caenorhabditis </it>nematode with a fully sequenced genome. The macroevolutionary comparison between <it>P. pacificus </it>and <it>C. elegans </it>has been complemented by microevolutionary studies of closely related strains and species within the genus <it>Pristionchus</it>. In addition, new understanding of the biology of <it>Pristionchus </it>from field studies, demonstrating a close association with various scarab beetles and the Colorado potato beetle, supports consideration of this nematode in studies of ecosystems. In the course of field studies on four continents more than 1,200 isolates were established from 15,000 beetle specimens representing 18 <it>Pristionchus </it>species. Two remarkable features of the <it>Pristionchus </it>– beetle association are the high species specificity of the interaction and the interception of the beetle's sex communication system for host recognition by the nematodes, as suggested by chemotaxis studies. Evolutionary interpretations of differences in developmental, behavioral and ecological patterns require a phylogenetic framework of the genus <it>Pristionchus</it>.</p> <p>Results</p> <p>Here, we provide a robust phylogeny of all 18 available <it>Pristionchus </it>species based on a set of 27 ribosomal protein genes encompassing a total of 10,971 bp. The phylogenetic tree provides evidence for North American and European clades, which are embedded in a deeper clade that includes Asian species. It also indicates putative invasion events. Of the 18 <it>Pristionchus </it>species, 13 are gonochoristic and five are hermaphroditic. The phylogeny indicates that all hermaphroditic species have arisen independently within the genus <it>Pristionchus</it>.</p> <p>Conclusion</p> <p>Combined ribosomal protein cDNA data can provide the basis for reconstruction of a robust phylogenetic framework for microevolutionary and biogeographic analyses. An additional major implication of our studies is the use of <it>Pristionchus </it>for nematode biodiversity assessments. While some species are represented by more than 100 isolates, others were found less than four times. Such patterns were observed on all continents and in all phylogenetic clades indicating that species asymmetry is a widespread phenomenon, which can now be further investigated by molecular tools.</p