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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 β‐lactamases, a common mechanism of enzymatic resistance that inactivates a widely used class of antibiotics. We first obtained β‐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 β‐lactamase functional activity. We found that the functional activity of antibiotic resistance mediated by β‐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.

International audience; L'oeuf amniotique est un caractère-clé des amniotes. Son apparition fut un événement majeur qui contribua probablement au succès spectaculaire des amniotes dans les environnements terrestres, dont ils dominent largement la plupart des habitats. Leur groupe-frère, les lissamphibiens, s'est nettement moins diversifié et représente une biomasse bien plus faible. Nous testons une des hypothèses les plus populaires concernant l'origine de l'oeuf amniotique, hypothèse émise par Carroll (1970). Elle stipule que des amniotes-souches auraient initialement pondu un œuf anamniotique sur la terre ferme (comme le font aujourd'hui certains Plethodontidae). L'absence initiale des annexes extra-embryonnaires qui caractérisent l'oeuf amniotique contemporain aurait limité les échanges gazeux, limitant la taille de l'oeuf à moins de 1 cm de diamètre, et celle des adultes, à moins de 10 cm de longueur museau-cloaque. Ceci implique une diminution de taille chez certains amniotes-souches, car la plupart des stégocéphales permo-carbonifères mesuraient plus de 10 cm. Après l'apparition de l'oeuf amniotique (pourvu des annexes), la taille de l'oeuf et des adultes aurait augmenté.Bien que le registre fossile n'ait pas livré les œufs pertinents, il est possible de tester les prédictions de l'hypothèse de Carroll sur l'évolution de la taille corporelle. L'un d'entre nous avait effectué une première série de tests en 2004 (Laurin, 2004), à l'aide de la parcimonie des moindres carrés et des contrastes phylogénétiques indépendants. Une des prévisions de Carroll s'était vérifiée (il semble bien y avoir eu une légère tendance à l'augmentation de taille chez les premiers amniotes), mais pas les autres.Nous testons à nouveau l'hypothèse de Carroll à l'aide du même jeu de données (taille corporelle, âge géologique et phylogénie de 107 taxons permo-carbonifères) mais en utilisant des méthodes plus appropriées : la parcimonie linéaire asymétrique (Didier, 2017), permettant de mieux estimer la taille des ancêtres hypothétiques en présence de tendance évolutive, et une nouvelles méthode utilisant cette parcimonie pour identifier les régions de l'arbre où les tendances évolutives changent. En effet, l'hypothèse de Carroll suggère deux changements de tendances au sein des amniotes-souches ; une diminution de taille suivant le début de la ponte en milieu terrestre, puis une augmentation de taille suite à l'apparition de l'oeuf amniotique. Nos résultats (Didier et al., 2019) ne soutiennent pas l'hypothèse de Carroll. La plupart des estimations de taille des ancêtres sur la souche des amniotes suggèrent une longueur museau-cloaque supérieure à 10 cm ; seules des estimations pour certains nœuds sont inférieures à ce seuil, et uniquement si on postule la présence d'une tendance évolutive très forte vers une augmentation de taille corporelle. De plus, la souche des amniotes ne semble pas être une partie de l'arbre montrant un taux plus élevé que la moyenne de changements de tendances ; au contraire, un seul changement de tendance, sur 517 changements possibles, se situe dans cette partie de l'arbre. Nous ne pouvons cependant pas rejeter catégoriquement l'hypothèse de Carroll car cette partie de l'arbre est peu représentée dans le registre fossile, selon notre phylogénie préférée (mais elle l'est nettement mieux selon l'alternative plus ancienne, plus populaire, mais à notre avis moins bien soutenue). Une meilleure compréhension de l'origine des amniote attendra donc la découverte de fossiles supplémentaires.RéférencesCarroll R.L. (1970). Quantitative aspects of the amphibian-reptilian transition. Forma et Functio, 3 : 165-178.Didier G. (2017). Time-dependent-asymmetric-linear-parsimonious ancestral state reconstruction. Bulletin of mathematical biology, 79(10) : 2334-2355.Didier G., Chabrol O. & Laurin M. (2019). Parsimony-based test for identifying changes in evolutionary trends for quantitative characters: implications for the origin of the amniotic egg. Cladistics, doi:10.1111/cla.12371.Laurin M. (2004). The evolution of body size, Cope's rule and the origin of amniotes. Syst. Biol., 53(4) : 594-622.

[YG: Invited Keynote Lecture]; International audience

Figure S1. A Venn diagram of PKS, NRPS, and hybrid gene-cluster numbers in NRPUR database. The gene-cluster numbers of the total, bacteria, eukarya and archaea are shown in black, red, green, and grey, respectively. Table S1. The 164 NRPS-PKS BGC sequences identified from the literature search resulting from experimental data with validated antibacterial activity. Table S2. Summary of NRPS and PKS gene clusters found in the genomes of bacteria from the gut. Figure S2. Distribution of NRPS, PKS, and hybrid gene clusters in bacteria from the gut a) in all studied bacteria, b) in the studied phyla (PDF 313 kb)

International audience; 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.

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.

International audience; 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.

24 p.-8 fig.-4 tab.-- Levasseur, Anthony et al., [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., This work was funded by the Commission of the European Communities through the BIORENEW project (NMP2-CT-2006-026456 “White Biotechnology for added-value products from re newable plant polymers: Design of tailor-made biocatalysts and new industrial bioprocesses”) and the PEROXICATS project (KBBE-2010-4-265397 “Novel and more robust fungal peroxidases as industrial biocatalysts.”), and by the Spanish Ministerio de Economía y Competitividad (MINECO) through the HIPOP project (BIO2011-26694, “Screening and engineering of new high-redox-potential peroxidases”).

International audience; 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 recombi-nation, 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.