An Integrative Database of ß-Lactamase Enzymes: Sequences, Structures, Functions, and Phylogenetic Trees.

ß-Lactamase enzymes have attracted substential medical attention from researchers and clinicians because of their clinical, ecological, and evolutionary interest. Here, we present a comprehensive online database of ß-lactamase enzymes. The current database is manually curated and incorporates the primary amino acid sequences, closest structural information in an external structure database (the Protein Data Bank [PDB]) and the functional profiles and phylogenetic trees of the four molecular classes (A, B, C, and D) of ß-lactamases. The functional profiles are presented according to the MICs and kinetic parameters that make them more useful for the investigators. Here, a total of 1,147 ß-lactam resistance genes are analyzed and described in the database. The database is implemented in MySQL and the related website is developed with Zend Framework 2 on an Apache server, supporting all major web browsers. Users can easily retrieve and visualize biologically important information using a set of efficient queries from a graphical interface. This database is freely accessible at http://ifr48.timone.univ-mrs.fr/beta-lactamase/public/.

Parsimony-based test for identifying changes in evolutionary trends for quantitative characters: implications for the origin of the amniotic egg.

The origin of the amniotic egg was a major event in vertebrate evolution and is thought to have contributed to the spectacular evolutionary radiation of amniotes. We test one of the most popular scenarios proposed by Carroll in 1970 to explain the origin of the amniotic egg using a novel method based on an asymmetric version of linear parsimony (aka Wagner parsimony) for identifying the most parsimonious split of a tree into two parts between which the evolution of the character is allowed to differ. The new method evaluates the cost of splitting a phylogenetic tree at a given node as the integral, over all pairs of asymmetry parameters, of the most parsimonious costs that can be achieved by using the first parameter on the subtree pending from this node and the second parameter elsewhere. By testing all the nodes, we then obtain the most parsimonious split of a tree with regard to the character values at its tips. Among the nine trees and two characters tested, our method yields a total of 517 parsimonious trend changes in Permo-Carboniferous stegocephalians, a single one of which occurs in a part of the tree (among stem-amniotes) where Carroll's scenario predicts that there should have been distinct changes in body size evolutionary trends. This refutes the scenario because the amniote stem does not appear to have elevated rates of evolutionary trend shifts. Our nodal body size estimates offer less discriminating power, but they likewise fail to find strong support for Carroll's scenario.

Genomic evidence for ameiotic evolution in the bdelloid rotifer Adineta vaga.

Loss of sexual reproduction is considered an evolutionary dead end for metazoans, but bdelloid rotifers challenge this view as they appear to have persisted asexually for millions of years. Neither male sex organs nor meiosis have ever been observed in these microscopic animals: oocytes are formed through mitotic divisions, with no reduction of chromosome number and no indication of chromosome pairing. However, current evidence does not exclude that they may engage in sex on rare, cryptic occasions. Here we report the genome of a bdelloid rotifer, Adineta vaga (Davis, 1873), and show that its structure is incompatible with conventional meiosis. At gene scale, the genome of A. vaga is tetraploid and comprises both anciently duplicated segments and less divergent allelic regions. However, in contrast to sexual species, the allelic regions are rearranged and sometimes even found on the same chromosome. Such structure does not allow meiotic pairing; instead, we find abundant evidence of gene conversion, which may limit the accumulation of deleterious mutations in the absence of meiosis. Gene families involved in resistance to oxidation, carbohydrate metabolism and defence against transposons are significantly expanded, which may explain why transposable elements cover only 3% of the assembled sequence. Furthermore, 8% of the genes are likely to be of non-metazoan origin and were probably acquired horizontally. This apparent convergence between bdelloids and prokaryotes sheds new light on the evolutionary significance of sex.

Full-length title: NRPPUR database search and in vitro analysis identify an NRPS-PKS biosynthetic gene cluster with a potential antibiotic effect.

BACKGROUND: Growing concern about the emergence of antibiotic resistance is compelling the pharmaceutical industry to search for new antimicrobial agents. The availability of genome sequences has enabled the development of computational mining as an important tool in the discovery of natural products with antibiotic effect. RESULTS: NRPPUR (Non-Ribosomal Peptide and Polyketide Urmite) is a new bioinformatic tool that was created to detect polyketides and non-ribosomal peptide gene clusters (PKS and NRPS) in bacterial genomes using the rpsBlast program. The NRPPUR database was constructed locally by assembling all 3505 available sequences of NRPS-PKS that have been identified by in silico approaches to date, with 164 Biosynthetic Gene Clusters (BGCs) derived from the published literature that have demonstrated antimicrobial activity in vitro. The in silico analysis of 49 intestinal human bacterial genomes using the NRPPUR made it possible to identify 91 BGCs including 89 clusters that had never previously been described. On average, intestinal human bacterial genomes devote nearly 0.8% (±1.4% s.d.) of their genome to NRPS/PKS biosynthesis, with Bacillus vallismortis, Streptomyces massiliensis and Bacillus subtilis genomes apportioning 8.4, 3.6 and 3.15% of their genomes, respectively. When using the cross-streak method, S. massiliensis displayed antibacterial activity against many Gram-positive and negative bacteria including methicillin-resistant Staphylococcus aureus (MRSA). CONCLUSIONS: NRPPUR has proven to be a very useful tool for the primary in silico selection of species with potential antimicrobial activity and human microbiota could be the future source of new antimicrobial discoveries. Further exploration of this and other ecological niches, coupled with high-throughput antibacterial activity screening should be envisaged.

A multispecies approach reveals hot spots and cold spots of diversity and connectivity in invertebrate species with contrasting dispersal modes.

Genetic diversity is crucial for species' maintenance and persistence, yet is often overlooked in conservation studies. Species diversity is more often reported due to practical constraints, but it is unknown if these measures of diversity are correlated. In marine invertebrates, adults are often sessile or sedentary and populations exchange genes via dispersal of gametes and larvae. Species with a larval period are expected to have more connected populations than those without larval dispersal. We assessed the relationship between measures of species and genetic diversity, and between dispersal ability and connectivity. We compiled data on genetic patterns and life history traits in nine species across five phyla. Sampling sites spanned 600 km in the northwest Mediterranean Sea and focused on a 50-km area near Marseilles, France. Comparative population genetic approaches yielded three main results. (i) Species without larvae showed higher levels of genetic structure than species with free-living larvae, but the role of larval type (lecithotrophic or planktotrophic) was negligible. (ii) A narrow area around Marseilles, subject to offshore advection, limited genetic connectivity in most species. (iii) We identified sites with significant positive contributions to overall genetic diversity across all species, corresponding with areas near low human population densities. In contrast, high levels of human activity corresponded with a negative contribution to overall genetic diversity. Genetic diversity within species was positively and significantly linearly related to local species diversity. Our study suggests that local contribution to overall genetic diversity should be taken into account for future conservation strategies.

The genome of the white-rot fungus Pycnoporus cinnabarinus: a basidiomycete model with a versatile arsenal for lignocellulosic biomass breakdown.

BACKGROUND: Saprophytic filamentous fungi are ubiquitous micro-organisms that play an essential role in photosynthetic carbon recycling. The wood-decayer Pycnoporus cinnabarinus is a model fungus for the study of plant cell wall decomposition and is used for a number of applications in green and white biotechnology. RESULTS: The 33.6 megabase genome of P. cinnabarinus was sequenced and assembled, and the 10,442 predicted genes were functionally annotated using a phylogenomic procedure. In-depth analyses were carried out for the numerous enzyme families involved in lignocellulosic biomass breakdown, for protein secretion and glycosylation pathways, and for mating type. The P. cinnabarinus genome sequence revealed a consistent repertoire of genes shared with wood-decaying basidiomycetes. P. cinnabarinus is thus fully equipped with the classical families involved in cellulose and hemicellulose degradation, whereas its pectinolytic repertoire appears relatively limited. In addition, P. cinnabarinus possesses a complete versatile enzymatic arsenal for lignin breakdown. We identified several genes encoding members of the three ligninolytic peroxidase types, namely lignin peroxidase, manganese peroxidase and versatile peroxidase. Comparative genome analyses were performed in fungi displaying different nutritional strategies (white-rot and brown-rot modes of decay). P. cinnabarinus presents a typical distribution of all the specific families found in the white-rot life style. Growth profiling of P. cinnabarinus was performed on 35 carbon sources including simple and complex substrates to study substrate utilization and preferences. P. cinnabarinus grew faster on crude plant substrates than on pure, mono- or polysaccharide substrates. Finally, proteomic analyses were conducted from liquid and solid-state fermentation to analyze the composition of the secretomes corresponding to growth on different substrates. The distribution of lignocellulolytic enzymes in the secretomes was strongly dependent on growth conditions, especially for lytic polysaccharide mono-oxygenases. CONCLUSIONS: With its available genome sequence, P. cinnabarinus is now an outstanding model system for the study of the enzyme machinery involved in the degradation or transformation of lignocellulosic biomass.

Amphioxus FGF signaling predicts the acquisition of vertebrate morphological traits.

FGF signaling is one of the few cell-cell signaling pathways conserved among all metazoans. The diversity of FGF gene content among different phyla suggests that evolution of FGF signaling may have participated in generating the current variety of animal forms. Vertebrates possess the greatest number of FGF genes, the functional evolution of which may have been implicated in the acquisition of vertebrate-specific morphological traits. In this study, we have investigated the roles of the FGF signal during embryogenesis of the cephalochordate amphioxus, the best proxy for the chordate ancestor. We first isolate the full FGF gene complement and determine the evolutionary relationships between amphioxus and vertebrate FGFs via phylogenetic and synteny conservation analysis. Using pharmacological treatments, we inhibit the FGF signaling pathway in amphioxus embryos in different time windows. Our results show that the requirement for FGF signaling during gastrulation is a conserved character among chordates, whereas this signal is not necessary for neural induction in amphioxus, in contrast to what is known in vertebrates. We also show that FGF signal, acting through the MAPK pathway, is necessary for the formation of the most anterior somites in amphioxus, whereas more posterior somite formation is not FGF-dependent. This result leads us to propose that modification of the FGF signal function in the anterior paraxial mesoderm in an amphioxus-like vertebrate ancestor might have contributed to the loss of segmentation in the preotic paraxial mesoderm of the vertebrate head.

An automated approach for the identification of horizontal gene transfers from complete genomes reveals the rhizome of Rickettsiales.

BACKGROUND: Horizontal gene transfer (HGT) is considered to be a major force driving the evolutionary history of prokaryotes. HGT is widespread in prokaryotes, contributing to the genomic repertoire of prokaryotic organisms, and is particularly apparent in Rickettsiales genomes. Gene gains from both distantly and closely related organisms play crucial roles in the evolution of bacterial genomes. In this work, we focus on genes transferred from distantly related species into Rickettsiales species. RESULTS: We developed an automated approach for the detection of HGT from other organisms (excluding alphaproteobacteria) into Rickettsiales genomes. Our systematic approach consisted of several specialized features including the application of a parsimony method for inferring phyletic patterns followed by blast filter, automated phylogenetic reconstruction and the application of patterns for HGT detection. We identified 42 instances of HGT in 31 complete Rickettsiales genomes, of which 38 were previously unidentified instances of HGT from Anaplasma, Wolbachia, Candidatus Pelagibacter ubique and Rickettsia genomes. Additionally, putative cases with no phylogenetic support were assigned gene ontology terms. Overall, these transfers could be characterized as "rhizome-like". CONCLUSIONS: Our analysis provides a comprehensive, systematic approach for the automated detection of HGTs from several complete proteome sequences that can be applied to detect instances of HGT within other genomes of interest.

Oocyte-somatic cells interactions, lessons from evolution.

BACKGROUND: Despite the known importance of somatic cells for oocyte developmental competence acquisition, the overall mechanisms underlying the acquisition of full developmental competence are far from being understood, especially in non-mammalian species. The present work aimed at identifying key molecular signals from somatic origin that would be shared by vertebrates. RESULTS: Using a parallel transcriptomic analysis in 4 vertebrate species - a teleost fish, an amphibian, and two mammals - at similar key steps of developmental competence acquisition, we identified a large number of species-specific differentially expressed genes and a surprisingly high number of orthologous genes exhibiting similar expression profiles in the 3 tetrapods and in the 4 vertebrates. Among the evolutionary conserved players participating in developmental competence acquisition are genes involved in key processes such as cellular energy metabolism, cell-to-cell communications, and meiosis control. In addition, we report many novel molecular actors from somatic origin that have never been studied in the vertebrate ovary. Interestingly, a significant number of these new players actively participate in Drosophila oogenesis. CONCLUSIONS: Our study provides a comprehensive overview of evolutionary-conserved mechanisms from somatic origin participating in oocyte developmental competence acquisition in 4 vertebrates. Together our results indicate that despite major differences in ovarian follicular structure, some of the key players from somatic origin involved in oocyte developmental competence acquisition would be shared, not only by vertebrates, but also by metazoans. The conservation of these mechanisms during vertebrate evolution further emphasizes the important contribution of the somatic compartment to oocyte quality and paves the way for future investigations aiming at better understanding what makes a good egg.

CASSIOPE: an expert system for conserved regions searches.

BACKGROUND: Understanding genome evolution provides insight into biological mechanisms. For many years comparative genomics and analysis of conserved chromosomal regions have helped to unravel the mechanisms involved in genome evolution and their implications for the study of biological systems. Detection of conserved regions (descending from a common ancestor) not only helps clarify genome evolution but also makes it possible to identify quantitative trait loci (QTLs) and investigate gene function.The identification and comparison of conserved regions on a genome scale is computationally intensive, making process automation essential. Three key requirements are necessary: consideration of phylogeny to identify orthologs between multiple species, frequent updating of the annotation and panel of compared genomes and computation of statistical tests to assess the significance of identified conserved gene clusters. RESULTS: We developed a modular system superimposed on a multi-agent framework, called CASSIOPE (Clever Agent System for Synteny Inheritance and Other Phenomena in Evolution). CASSIOPE automatically identifies statistically significant conserved regions between multiple genomes based on automated phylogenies and statistical testing. Conserved regions were searched for in 19 species and 1,561 hits were found. To our knowledge, CASSIOPE is the first system to date that integrates evolutionary biology-based concepts and fulfills all three key requirements stated above. All results are available at http://194.57.197.245/cassiopeWeb/displayCluster?clusterId=1 CONCLUSION: CASSIOPE makes it possible to study conserved regions from a chosen query genetic region and to infer conserved gene clusters based on phylogenies and statistical tests assessing the significance of these conserved regions.Source code is freely available, please contact: Pierre.pontarotti@univ-provence.fr.

The functional convergence of antibiotic resistance in ß-lactamases is not conferred by a simple convergent substitution of amino acid.

Bacterial resistance to antibiotics is a serious medical and public health concern worldwide. Such resistance is conferred by a variety of mechanisms, but the extensive variability in levels of resistance across bacteria is a common finding. Understanding the underlying evolutionary processes governing this functional variation in antibiotic resistance is important as it may allow the development of appropriate strategies to improve treatment options for bacterial infections. The main objective of this study was to examine the functional evolution of ß-lactamases, a common mechanism of enzymatic resistance that inactivates a widely used class of antibiotics. We first obtained ß-lactamase protein sequences and minimal inhibitory concentration (MIC), a measure of antibiotic function, from previously published literature. We then used a molecular phylogenetic framework to examine the evolution of ß-lactamase functional activity. We found that the functional activity of antibiotic resistance mediated by ß-lactamase has evolved in a convergent manner within molecular classes, but is not associated with any single amino acid substitution. This suggests that the dynamics of convergent evolution in this system can vary between the functional and molecular (sequence) levels. Such disassociation may hamper bioinformatic approaches to antibiotic resistance determination and underscore the need for (less efficient but more effective) activity assays as an essential step in evaluating resistance in a given case.

Pithovirus sibericum, a new bona fide member of the "Fourth TRUC" club.

Nucleocytoplasmic large DNA viruses, or representatives of the proposed order Megavirales, include giant viruses of Acanthamoeba that were discovered over the last 12 years and are bona fide microbes. Phylogenies based on a few genes conserved amongst these megaviruses and shared by microbes classified as Eukarya, Bacteria, and Archaea, allowed for delineation of a fourth monophylogenetic group or "TRUC" (Things Resisting Uncompleted Classification) composed of the Megavirales representatives. A new Megavirales member named Pithovirus sibericum was isolated from a >30,000-year-old dated Siberian permafrost sample. This virion is as large as recently described pandoraviruses but has a genome that is approximately three to four times shorter. Our objective was to update the classification of P. sibericum as a new member of the "Fourth TRUC" club. Phylogenetic trees were constructed based on four conserved ancient genes and a phyletic analysis was concurrently conducted based on the presence/absence patterns of a set of informational genes from members of Megavirales, Bacteria, Archaea, and Eukarya. Phylogenetic analyses based on the four conserved genes revealed that P. sibericum is part of the fourth TRUC composed of Megavirales members, and is closely related to the families Marseilleviridae and Ascoviridae/Iridoviridae. Additionally, hierarchical clustering delineated four branches, and showed that P. sibericum is part of this fourth TRUC. Overall, phylogenetic and phyletic analyses using informational genes clearly indicate that P. sibericum is a new bona fide member of the "Fourth TRUC" club composed of representatives of Megavirales, alongside Bacteria, Archaea, and Eukarya.

Welcome to pandoraviruses at the 'Fourth TRUC' club.

Nucleocytoplasmic large DNA viruses, or representatives of the proposed order Megavirales, belong to families of giant viruses that infect a broad range of eukaryotic hosts. Megaviruses have been previously described to comprise a fourth monophylogenetic TRUC (things resisting uncompleted classification) together with cellular domains in the universal tree of life. Recently described pandoraviruses have large (1.9-2.5 MB) and highly divergent genomes. In the present study, we updated the classification of pandoraviruses and other reported giant viruses. Phylogenetic trees were constructed based on six informational genes. Hierarchical clustering was performed based on a set of informational genes from Megavirales members and cellular organisms. Homologous sequences were selected from cellular organisms using TimeTree software, comprising comprehensive, and representative sets of members from Bacteria, Archaea, and Eukarya. Phylogenetic analyses based on three conserved core genes clustered pandoraviruses with phycodnaviruses, exhibiting their close relatedness. Additionally, hierarchical clustering analyses based on informational genes grouped pandoraviruses with Megavirales members as a super group distinct from cellular organisms. Thus, the analyses based on core conserved genes revealed that pandoraviruses are new genuine members of the 'Fourth TRUC' club, encompassing distinct life forms compared with cellular organisms.

Mycobacteriophage-drived diversification of Mycobacterium abscessus.

BACKGROUND: Mycobacterium abscessus is an emerging opportunistic pathogen which diversity was acknowledged by the recent description of two subspecies accommodating M. abscessus, Mycobacterium bolletii and Mycobacterium massiliense isolates. RESULTS: Here, genome analysis found 1-8 prophage regions in 47/48 M. abscessus genomes ranging from small prophage-like elements to complete prophages. A total of 20,304 viral and phage proteins clustered into 853 orthologous groups. Phylogenomic and phylogenetic analyses based on prophage region homology found three main clusters corresponding to M. abscessus, M. bolletii and M. massiliense. Analysing 135 annotated Tape Measure Proteins found thirteen clusters and four singletons, suggesting that at least 17 mycobacteriophages had infected M. abscessus during its evolution. The evolutionary history of phages differed from that of their mycobacterial hosts. In particular, 33 phage-related proteins have been horizontally transferred within M. abscessus genomes. They comprise of an integrase, specific mycobacteriophage proteins, hypothetical proteins and DNA replication and metabolism proteins. Gene exchanges, loss and gains which occurred in M. abscessus genomes have been driven by several mycobacteriophages. CONCLUSIONS: This analysis of phage-mycobacterium co-evolution suggests that mycobacteriophages are playing a key-role in the on-going diversification of M. abscessus. REVIEWERS: This article was reviewed by Eric Bapteste, Patrick Forterre and Eugene Koonin.

To tree or not to tree? Genome-wide quantification of recombination and reticulate evolution during the diversification of strict intracellular bacteria.

It is well known that horizontal gene transfer (HGT) is a major force in the evolution of prokaryotes. During the adaptation of a bacterial population to a new ecological niche, and particularly for intracellular bacteria, selective pressures are shifted and ecological niches reduced, resulting in a lower rate of genetic connectivity. HGT and positive selection are therefore two important evolutionary forces in microbial pathogens that drive adaptation to new hosts. In this study, we use genomic distance analyses, phylogenomic networks, tree topology comparisons, and Bayesian inference methods to investigate to what extent HGT has occurred during the evolution of the genus Rickettsia, the effect of the use of different genomic regions in estimating reticulate evolution and HGT events, and the link of these to host range. We show that ecological specialization restricts recombination occurrence in Rickettsia, but other evolutionary processes and genome architecture are also important for the occurrence of HGT. We found that recombination, genomic rearrangements, and genome conservation all show evidence of network-like evolution at whole-genome scale. We show that reticulation occurred mainly, but not only, during the early Rickettsia radiation, and that core proteome genes of every major functional category have experienced reticulated evolution and possibly HGT. Overall, the evolution of Rickettsia bacteria has been tree-like, with evidence of HGT and reticulated evolution for around 10-25% of the core Rickettsia genome. We present evidence of extensive recombination/incomplete lineage sorting (ILS) during the radiation of the genus, probably linked with the emergence of intracellularity in a wide range of hosts.

FunGene-DB: a web-based tool for Polyporales strains authentication.

Polyporales are extensively studied wood-decaying fungi with applications in white and green biotechnologies and in medicinal chemistry. We developed an open-access, user-friendly, bioinformatics tool named FunGene-DB (http://www.fungene-db.org). The goal was to facilitate the molecular authentication of Polyporales strains and fruit-bodies, otherwise subjected to morphological studies. This tool includes a curated database that contains ITS1-5.8S-ITS2 rDNA genes screened through a semi-automated pipeline from the International Nucleotide Sequence Database (INSD), and the similarity search BLASTn program. Today, the web-accessible database compiles 2379 accepted sequences, among which 386 were selected as reference sequences (most often fully identified ITS sequences for which a voucher, strain or specimen, has been deposited in a public-access collection). The restriction of the database to one reference sequence per species (or per clade for species complex) allowed most often unequivocal analysis. We conclude that FunGene-DB is a promising tool for molecular authentication of Polyporales. It should be especially useful for scientists who are not expert mycologists but who need to check the identity of strains (e.g. for culture collections, for applied microbiology).