Genomic Species Are Ecological Species as Revealed by Comparative Genomics in Agrobacterium tumefaciens

The definition of bacterial species is based on genomic similarities, giving rise to the operational concept of genomic species, but the reasons of the occurrence of differentiated genomic species remain largely unknown. We used the Agrobacterium tumefaciens species complex and particularly the genomic species presently called genomovar G8, which includes the sequenced strain C58, to test the hypothesis of genomic species having specific ecological adaptations possibly involved in the speciation process. We analyzed the gene repertoire specific to G8 to identify potential adaptive genes. By hybridizing 25 strains of A. tumefaciens on DNA microarrays spanning the C58 genome, we highlighted the presence and absence of genes homologous to C58 in the taxon. We found 196 genes specific to genomovar G8 that were mostly clustered into seven genomic islands on the C58 genome—one on the circular chromosome and six on the linear chromosome—suggesting higher plasticity and a major adaptive role of the latter. Clusters encoded putative functional units, four of which had been verified experimentally. The combination of G8-specific functions defines a hypothetical species primary niche for G8 related to commensal interaction with a host plant. This supports that the G8 ancestor was able to exploit a new ecological niche, maybe initiating ecological isolation and thus speciation. Searching genomic data for synapomorphic traits is a powerful way to describe bacterial species. This procedure allowed us to find such phenotypic traits specific to genomovar G8 and thus propose a Latin binomial, Agrobacterium fabrum, for this bona fide genomic species

Démasquage des gènes spécifiques d'une espèce génomique du complexeAgrobacterium tumefaciens par AFLP et multicapteur à ADN

National audienceUnmasking species specific genes inspecies G8 by AFLP and microarray. In the frame of studies undertaken to associate a biological concept to the present genomic definition of the bacterial species, a study was done to find species specific genes common to all members of a model species, the genomic species G8 of the species complex Agrobacterium tumefaciens. The study was firstly based upon the identification of the genomic origin of AFLP markers of the species by comparing experimental results to in silico simulations of AFLP of the complete genome sequence of the G8 reference strain C58. Due to size determination uncertainties and possible close co-migration of predicted fragments, only a little number of amplified AFLP markers could be localized without ambiguities in the genome of C58. Nevertheless, the study delivered a catalog of the genes common to all G8 members. Along to standard house-keeping genes, 25% of the G8 core genome also consisted of hypothetical non-conserved or "ORFan" genes. ORFans, which encode unknown functions, are good candidate to encode species specific functions and thus to allow for further studies about the ecological specialization of the species. The study also revealed that AFLP experimental results were influenced by epigenetic determinants that could have artefactually reduced the number of species specific genes delivered in the study, but not the confidence about those readily found common. The still unknown epigenetic phenomenon is thought to prevent the digestion of certain parts of the genomic DNA by endonucleases required for AFLP. It is likely the cause of the significantly higher number of genes experimentally found common to all species members around the centre of the linear chromosome, while the epigenetic phenomenon would have hampered digestions and then delivered artefactual not-common genes in the chromosome lateral branches.The second approach was based on a microarray constructed against the whole genome of C58. A comparative genome hybridization study conducted with members of all the A. tumefaciens complex allowed us to determine genes specific of the genomic species G8. Most of these genes are ORFans and knowing their functions requires additionnal studies. Nevertheless, the study showed that the species specific genes as well as other accessory genes were much more abundant on the linear than on the circular chromosome. The linear chromosome of A. tumefaciens therefore looks to be the preferred genome place for genetic innovations.Dans le contexte de la recherche générale sur le déterminisme génétique des espèces bactériennes, des gènes spécifiques communs à tous les membres d'une espèce modèle ont été recherchés chez l'espèce génomique G8 du complexe Agrobacterium tumefaciens. L'étude était basée sur l'identification de l'origine génomique des fragments AFLP marqueurs de l'espèce réalisée par simulation bioinformatique de l'AFLP à partir de la séquence du génome de la souche de référence C58. L'analyse a permis de dresser un catalogue de gènes communs à tous les membres de l'espèce. L'étude montre cependant que les résultats de l'AFLP expérimentale dépendent aussi de caractères épigénétiques qui en réduisent l'efficacité. Ce phénomène, dont le déterminisme n'est pas connu, ne gêne cependant pas l'identification des gènes communs de l'espèce mais a pu en réduire considérablement leur nombre. L'utilisation d'un multicapteur à ADN (ou biopuce) basé sur le génome complet de C58 avec des représentants de toutes les espèces du taxon a permis d'établir le catalogue de tous les gènes vraiment spécifiques à l'espèce G8 d'A. tumefaciens. Parmi ceux-ci, il y avait beaucoup de gènes non-conservés (i.e. sans orthologues connus dans les bases de données). Ces gènes orphelins qui codent a priori des fonctions inconnues et originales sont de bons candidats pour déterminer la niche écologique spécifique de l'espèce. L'étude montre en outre que le chromosome linéaire est le lieu privilégié des innovations génétiques dans ce taxon

Anthropization level of Lascaux Cave microbiome shown by regional‐scale comparisons of pristine and anthropized caves

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Identification of Genomic Species in Agrobacterium Biovar 1 by AFLP Genomic Markers

Biovar 1 of the genus Agrobacterium consists of at least nine genomic species that have not yet received accepted species names. However, rapid identification of these organisms in various biotopes is needed to elucidate crown gall epidemiology, as well as Agrobacterium ecology. For this purpose, the AFLP methodology provides rapid and unambiguous determination of the genomic species status of agrobacteria, as confirmed by additional DNA-DNA hybridizations. The AFLP method has been proven to be reliable and to eliminate the need for DNA-DNA hybridization. In addition, AFLP fragments common to all members of the three major genomic species of agrobacteria, genomic species G1 (reference strain, strain TT111), G4 (reference strain, strain B6, the type strain of Agrobacterium tumefaciens), and G8 (reference strain, strain C58), have been identified, and these fragments facilitate analysis and show the applicability of the method. The maximal infraspecies current genome mispairing (CGM) value found for the biovar 1 taxon is 10.8%, while the smallest CGM value found for pairs of genomic species is 15.2%. This emphasizes the gap in the distribution of genome divergence values upon which the genomic species definition is based. The three main genomic species of agrobacteria in biovar 1 displayed high infraspecies current genome mispairing values (9 to 9.7%). The common fragments of a genomic species are thus likely “species-specific” markers tagging the core genomes of the species

Single acquisition of protelomerase gave rise to speciation of a large and diverse clade within the Agrobacterium/Rhizobium supercluster characterized by the presence of a linear chromid

International audienceLinear chromosomes are atypical in bacteria and likely a secondary trait derived from ancestral circular molecules. Within the Rhizobiaceae family, whose genome contains at least two chromosomes, a particularity of Agrobacterium fabrum (formerly A. tumefaciens) secondary chromosome (chromid) is to be linear and hairpin-ended thanks to the TelA protelomerase. Linear topology and telA distributions within this bacterial family was screened by pulse field gel electrophoresis and PCR. In A. rubi, A. larrymoorei, Rhizobium skierniewicense, A. viscosum, Agrobacterium sp. NCPPB 1650, and every genomospecies of the biovar 1/A. tumefaciens species complex (including R. pusense, A. radiobacter, A. fabrum, R. nepotum plus seven other unnamed genomospecies), linear chromid topologies were retrieved concomitantly with telA presence, whereas the remote species A. vitis, Allorhizobium undicola, Rhizobium rhizogenes and Ensifer meliloti harbored a circular chromid as well as no telA gene. Moreover, the telA phylogeny is congruent with that of recA used as a marker gene of the Agrobacterium phylogeny. Collectively, these findings strongly suggest that single acquisition of telA by an ancestor was the founding event of a large and diverse clade characterized by the presence of a linear chromid. This clade, characterized by unusual genome architecture, appears to be a relevant candidate to serve as a basis for a possible redefinition of the controversial Agrobacterium genus. In this respect, investigating telA in sequenced genomes allows to both ascertain the place of concerned strains into Agrobacterium spp. and their actual assignation to species/genomospecies in this genus

Démasquage des gènes spécifiques d'une espèce génomique du complexeAgrobacterium tumefaciens par AFLP et multicapteur à ADN

National audienceUnmasking species specific genes inspecies G8 by AFLP and microarray. In the frame of studies undertaken to associate a biological concept to the present genomic definition of the bacterial species, a study was done to find species specific genes common to all members of a model species, the genomic species G8 of the species complex Agrobacterium tumefaciens. The study was firstly based upon the identification of the genomic origin of AFLP markers of the species by comparing experimental results to in silico simulations of AFLP of the complete genome sequence of the G8 reference strain C58. Due to size determination uncertainties and possible close co-migration of predicted fragments, only a little number of amplified AFLP markers could be localized without ambiguities in the genome of C58. Nevertheless, the study delivered a catalog of the genes common to all G8 members. Along to standard house-keeping genes, 25% of the G8 core genome also consisted of hypothetical non-conserved or "ORFan" genes. ORFans, which encode unknown functions, are good candidate to encode species specific functions and thus to allow for further studies about the ecological specialization of the species. The study also revealed that AFLP experimental results were influenced by epigenetic determinants that could have artefactually reduced the number of species specific genes delivered in the study, but not the confidence about those readily found common. The still unknown epigenetic phenomenon is thought to prevent the digestion of certain parts of the genomic DNA by endonucleases required for AFLP. It is likely the cause of the significantly higher number of genes experimentally found common to all species members around the centre of the linear chromosome, while the epigenetic phenomenon would have hampered digestions and then delivered artefactual not-common genes in the chromosome lateral branches.The second approach was based on a microarray constructed against the whole genome of C58. A comparative genome hybridization study conducted with members of all the A. tumefaciens complex allowed us to determine genes specific of the genomic species G8. Most of these genes are ORFans and knowing their functions requires additionnal studies. Nevertheless, the study showed that the species specific genes as well as other accessory genes were much more abundant on the linear than on the circular chromosome. The linear chromosome of A. tumefaciens therefore looks to be the preferred genome place for genetic innovations.Dans le contexte de la recherche générale sur le déterminisme génétique des espèces bactériennes, des gènes spécifiques communs à tous les membres d'une espèce modèle ont été recherchés chez l'espèce génomique G8 du complexe Agrobacterium tumefaciens. L'étude était basée sur l'identification de l'origine génomique des fragments AFLP marqueurs de l'espèce réalisée par simulation bioinformatique de l'AFLP à partir de la séquence du génome de la souche de référence C58. L'analyse a permis de dresser un catalogue de gènes communs à tous les membres de l'espèce. L'étude montre cependant que les résultats de l'AFLP expérimentale dépendent aussi de caractères épigénétiques qui en réduisent l'efficacité. Ce phénomène, dont le déterminisme n'est pas connu, ne gêne cependant pas l'identification des gènes communs de l'espèce mais a pu en réduire considérablement leur nombre. L'utilisation d'un multicapteur à ADN (ou biopuce) basé sur le génome complet de C58 avec des représentants de toutes les espèces du taxon a permis d'établir le catalogue de tous les gènes vraiment spécifiques à l'espèce G8 d'A. tumefaciens. Parmi ceux-ci, il y avait beaucoup de gènes non-conservés (i.e. sans orthologues connus dans les bases de données). Ces gènes orphelins qui codent a priori des fonctions inconnues et originales sont de bons candidats pour déterminer la niche écologique spécifique de l'espèce. L'étude montre en outre que le chromosome linéaire est le lieu privilégié des innovations génétiques dans ce taxon

Rapid and Efficient Identification of Agrobacterium Species by recA Allele Analysis

International audienceThe analysis of housekeeping recA gene sequences from 138 strains from 13 species or genomic species of Agrobacterium, nine being biovar 1 genomospecies, and the others Agrobacterium larrymoorei, Agrobacterium rubi, Agrobacterium sp. NCPPB 1650, and Agrobacterium vitis and one “former” Agrobacterium species, Rhizobium rhizogenes, led to the identification of 50 different recA alleles and to a clear delineation of the 14 species or genomospecies entirely consistent with that obtained by amplified fragment length polymorphism (AFLP) analysis. The relevance of a recA sequencing approach for epidemiological analyses was next assessed on agrobacterial Tunisian isolates. All Tunisian isolates were found to belong to the Agrobacterium tumefaciens/ biovar 1 species complex by both biochemical tests and rrs sequencing. recA sequence analysis further permitted their unambiguous assignment to A. tumefaciens genomospecies G4, G6, G7, and G8 in total agreement with the results of an AFLP-based analysis. At subspecific level, several Tunisian recA alleles were novel, indicating the power and accuracy of recA-based typing for studies of Agrobacterium spp

Identification de fonctions « spécifiques d'espèce » impliquées dans l'individualisation en écovar de l’espèce G8 du complexe Agrobacterium tumefaciens

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Identification de fonctions « spécifiques d'espèce » impliquées dans l'individualisation en écovar de l’espèce G8 du complexe Agrobacterium tumefaciens

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