Aurélien Barré

Life on arginine for Mycoplasma hominis: clues from its minimal genome and comparison with other human urogenital mycoplasmas.

Mycoplasma hominis is an opportunistic human mycoplasma. Two other pathogenic human species, M. genitalium and Ureaplasma parvum, reside within the same natural niche as M. hominis: the urogenital tract. These three species have overlapping, but distinct, pathogenic roles. They have minimal genomes and, thus, reduced metabolic capabilities characterized by distinct energy-generating pathways. Analysis of the M. hominis PG21 genome sequence revealed that it is the second smallest genome among self-replicating free living organisms (665,445 bp, 537 coding sequences (CDSs)). Five clusters of genes were predicted to have undergone horizontal gene transfer (HGT) between M. hominis and the phylogenetically distant U. parvum species. We reconstructed M. hominis metabolic pathways from the predicted genes, with particular emphasis on energy-generating pathways. The Embden–Meyerhoff–Parnas pathway was incomplete, with a single enzyme absent. We identified the three proteins constituting the arginine dihydrolase pathway. This pathway was found essential to promote growth in vivo. The predicted presence of dimethylarginine dimethylaminohydrolase suggested that arginine catabolism is more complex than initially described. This enzyme may have been acquired by HGT from non-mollicute bacteria. Comparison of the three minimal mollicute genomes showed that 247 CDSs were common to all three genomes, whereas 220 CDSs were specific to M. hominis, 172 CDSs were specific to M. genitalium, and 280 CDSs were specific to U. parvum. Within these species-specific genes, two major sets of genes could be identified: one including genes involved in various energy-generating pathways, depending on the energy source used (glucose, urea, or arginine) and another involved in cytadherence and virulence. Therefore, a minimal mycoplasma cell, not including cytadherence and virulence-related genes, could be envisaged containing a core genome (247 genes), plus a set of genes required for providing energy. For M. hominis, this set would include 247+9 genes, resulting in a theoretical minimal genome of 256 genes.

MolliGen, a database dedicated to the comparative genomics of Mollicutes

Bacteria belonging to the class Mollicutes were among the first ones to be selected for complete genome sequencing because of the minimal size of their genomes and their pathogenicity for humans and a broad range of animals and plants. At this time six genome sequences have been publicly released (Mycoplasma genitalium, Mycoplasma pneumoniae, Ureaplasma urealyticum-parvum, Mycoplasma pulmonis, Mycoplasma penetrans and Mycoplasma gallisepticum) and as the number of available mollicute genomes increases, comparative genomics analysis within this model group of organisms becomes more and more instructive. However, such an analysis is difficult to carry out without a suitable platform gathering not only the original annotations but also relevant information available in public databases or obtained by applying common bioinformatics methods. With the aim of solving these difficulties, we have developed a web-accessible database named MolliGen (http://cbi.labri.fr/outils/molligen/). After selecting a set of genomes the user can launch various types of search based on annotation, position on the chromosomes or sequence similarity. In addition, relationships of putative orthology have been precomputed to allow differential genome queries. The results are presented in table format with multiple links to public databases and to bioinformatic analyses such as multiple alignments or BLAST search. Specific tools were also developed for the graphical visualization of the results, including a multi- genome browser for displaying dynamic pictures with clickable objects and for viewing relationships of precomputed similarity. MolliGen is designed to integrate all the complete genomes of mollicutes as they become available.

Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak

BackgroundThe Fagaceae family comprises about 1,000 woody species worldwide. About half belong to the Quercus family. These oaks are often a source of raw material for biomass wood and fiber. Pedunculate and sessile oaks, are among the most important deciduous forest tree species in Europe. Despite their ecological and economical importance, very few genomic resources have yet been generated for these species. Here, we describe the development of an EST catalogue that will support ecosystem genomics studies, where geneticists, ecophysiologists, molecular biologists and ecologists join their efforts for understanding, monitoring and predicting functional genetic diversity.ResultsWe generated 145,827 sequence reads from 20 cDNA libraries using the Sanger method. Unexploitable chromatograms and quality checking lead us to eliminate 19,941 sequences. Finally a total of 125,925 ESTs were retained from 111,361 cDNA clones. Pyrosequencing was also conducted for 14 libraries, generating 1,948,579 reads, from which 370,566 sequences (19.0%) were eliminated, resulting in 1,578,192 sequences. Following clustering and assembly using TGICL pipeline, 1,704,117 EST sequences collapsed into 69,154 tentative contigs and 153,517 singletons, providing 222,671 non-redundant sequences (including alternative transcripts). We also assembled the sequences using MIRA and PartiGene software and compared the three unigene sets. Gene ontology annotation was then assigned to 29,303 unigene elements. Blast search against the SWISS-PROT database revealed putative homologs for 32,810 (14.7%) unigene elements, but more extensive search with Pfam, Refseq_protein, Refseq_RNA and eight gene indices revealed homology for 67.4% of them. The EST catalogue was examined for putative homologs of candidate genes involved in bud phenology, cuticle formation, phenylpropanoids biosynthesis and cell wall formation. Our results suggest a good coverage of genes involved in these traits. Comparative orthologous sequences (COS) with other plant gene models were identified and allow to unravel the oak paleo-history. Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 52,834 SSRs and 36,411 SNPs. All of these are available through the Oak Contig Browser http://genotoul-contigbrowser.toulouse.inra.fr:9092/Quercus_robur/index.html.ConclusionsThis genomic resource provides a unique tool to discover genes of interest, study the oak transcriptome, and develop new markers to investigate functional diversity in natural populations.

Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro- and micro-events that are shaping mycoplasma diversity

BackgroundWhile the genomic era is accumulating a tremendous amount of data, the question of how genomics can describe a bacterial species remains to be fully addressed. The recent sequencing of the genome of the Mycoplasma agalactiae type strain has challenged our general view on mycoplasmas by suggesting that these simple bacteria are able to exchange significant amount of genetic material via horizontal gene transfer. Yet, events that are shaping mycoplasma genomes and that are underlining diversity within this species have to be fully evaluated. For this purpose, we compared two strains that are representative of the genetic spectrum encountered in this species: the type strain PG2 which genome is already available and a field strain, 5632, which was fully sequenced and annotated in this study.ResultsThe two genomes differ by ca. 130 kbp with that of 5632 being the largest (1006 kbp). The make up of this additional genetic material mainly corresponds (i) to mobile genetic elements and (ii) to expanded repertoire of gene families that encode putative surface proteins and display features of highly-variable systems. More specifically, three entire copies of a previously described integrative conjugative element are found in 5632 that accounts for ca. 80 kbp. Other mobile genetic elements, found in 5632 but not in PG2, are the more classical insertion sequences which are related to those found in two other ruminant pathogens, M. bovis and M. mycoides subsp. mycoides SC. In 5632, repertoires of gene families encoding surface proteins are larger due to gene duplication. Comparative proteomic analyses of the two strains indicate that the additional coding capacity of 5632 affects the overall architecture of the surface and suggests the occurrence of new phase variable systems based on single nucleotide polymorphisms.ConclusionOverall, comparative analyses of two M. agalactiae strains revealed a very dynamic genome which structure has been shaped by gene flow among ruminant mycoplasmas and expansion-reduction of gene repertoires encoding surface proteins, the expression of which is driven by localized genetic micro-events.

Molecular and phenotypic profiling from the base to the crown in maritime pine wood‐forming tissue

Environmental, developmental and genetic factors affect variation in wood properties at the chemical, anatomical and physical levels. Here, the phenotypic variation observed along the tree stem was explored and the hypothesis tested that this variation could be the result of the differential expression of genes/proteins during wood formation. Differentiating xylem samples of maritime pine (Pinus pinaster) were collected from the top (crown wood, CW) to the bottom (base wood, BW) of adult trees. These samples were characterized by Fourier transform infrared spectroscopy (FTIR) and analytical pyrolysis. Two main groups of samples, corresponding to CW and BW, could be distinguished from cell wall chemical composition. A genomic approach, combining large-scale production of expressed sequence tags (ESTs), gene expression profiling and quantitative proteomics analysis, allowed identification of 262 unigenes (out of 3512) and 231 proteins (out of 1372 spots) that were differentially expressed along the stem. A good relationship was found between functional categories from transcriptomic and proteomic data. A good fit between the molecular mechanisms involved in CW-BW formation and these two types of wood phenotypic differences was also observed. This work provides a list of candidate genes for wood properties that will be tested in forward genetics.

Predicting the Minimal Translation Apparatus: Lessons from the Reductive Evolution of Mollicutes

Mollicutes is a class of parasitic bacteria that have evolved from a common Firmicutes ancestor mostly by massive genome reduction. With genomes under 1 Mbp in size, most Mollicutes species retain the capacity to replicate and grow autonomously. The major goal of this work was to identify the minimal set of proteins that can sustain ribosome biogenesis and translation of the genetic code in these bacteria. Using the experimentally validated genes from the model bacteria Escherichia coli and Bacillus subtilis as input, genes encoding proteins of the core translation machinery were predicted in 39 distinct Mollicutes species, 33 of which are culturable. The set of 260 input genes encodes proteins involved in ribosome biogenesis, tRNA maturation and aminoacylation, as well as proteins cofactors required for mRNA translation and RNA decay. A core set of 104 of these proteins is found in all species analyzed. Genes encoding proteins involved in post-translational modifications of ribosomal proteins and translation cofactors, post-transcriptional modifications of t+rRNA, in ribosome assembly and RNA degradation are the most frequently lost. As expected, genes coding for aminoacyl-tRNA synthetases, ribosomal proteins and initiation, elongation and termination factors are the most persistent (i.e. conserved in a majority of genomes). Enzymes introducing nucleotides modifications in the anticodon loop of tRNA, in helix 44 of 16S rRNA and in helices 69 and 80 of 23S rRNA, all essential for decoding and facilitating peptidyl transfer, are maintained in all species. Reconstruction of genome evolution in Mollicutes revealed that, beside many gene losses, occasional gains by horizontal gene transfer also occurred. This analysis not only showed that slightly different solutions for preserving a functional, albeit minimal, protein synthetizing machinery have emerged in these successive rounds of reductive evolution but also has broad implications in guiding the reconstruction of a minimal cell by synthetic biology approaches.

AliasServer: a web server to handle multiple aliases used to refer to proteins

UNLABELLED AliasServer provides services that facilitate the assembly of data or datasets that make use of different identifiers for refering to the same protein. This resource relies on a database which contains, for a given organism, a non-redundant list of protein sequences associated with a set of aliases. AVAILABILITY AliasServer is available as an interactive Web server at http://cbi.labri.fr/outils/alias/ and as a web service using a SOAP interface. The complete tool, including sources and data, is available for local installations upon request. SUPPLEMENTARY INFORMATION Technical documentation is available at http://cbi.labri.fr/outils/alias/asdoc.pdf

Proteomic plasticity of two Eucalyptus genotypes under contrasted water regimes in the field.

Water deficit affects tree growth and limits wood production. In an attempt to identify the molecular triggers of adaptation mechanisms to water deficit in Eucalyptus, we investigated protein expression patterns of two ecophysiologically contrasted Eucalyptus genotypes. They were grown in the field in either natural conditions or irrigated for 7 weeks during the dry season in the Republic of Congo. At the phenotypic level, genotype (G), treatment (T) and/or G × T interaction effects were observed for above- and below-ground biomass-related traits. At the molecular level, changes in protein abundance were recorded in leaves (acidic pH 4-7, and basic pH 7-11, proteomes) and stems (acidic proteome) using two-dimensional gel electrophoresis (2-DE). One third of the detected protein spots displayed significant G, T and/or G × T effects, and 158 of them were identified by tandem mass spectrometry (LC-MS/MS) analysis. Thus, several proteins whose molecular plasticity was genetically controlled (i.e. G × T effect) were revealed, highlighting adaptive mechanisms to water deficit specific to each genotype, namely cell wall modification, cell detoxification and osmoregulation. Transcript abundances corresponding to G × T proteins were also investigated by quantitative RT-PCR. These proteins represent relevant targets to improve drought resistance in this ecologically and economically important forest tree genus.

Occurrence, plasticity, and evolution of the vpma gene family, a genetic system devoted to high-frequency surface variation in Mycoplasma agalactiae.

Mycoplasma agalactiae, an important pathogen of small ruminants, exhibits a very versatile surface architecture by switching multiple, related lipoproteins (Vpmas) on and off. In the type strain, PG2, Vpma phase variation is generated by a cluster of six vpma genes that undergo frequent DNA rearrangements via site-specific recombination. To further comprehend the degree of diversity that can be generated at the M. agalactiae surface, the vpma gene repertoire of a field strain, 5632, was analyzed and shown to contain an extended repertoire of 23 vpma genes distributed between two loci located 250 kbp apart. Loci I and II include 16 and 7 vpma genes, respectively, with all vpma genes of locus II being duplicated at locus I. Several Vpmas displayed a chimeric structure suggestive of homologous recombination, and a global proteomic analysis further indicated that at least 13 of the 16 Vpmas can be expressed by the 5632 strain. Because a single promoter is present in each vpma locus, concomitant Vpma expression can occur in a strain with duplicated loci. Consequently, the number of possible surface combinations is much higher for strain 5632 than for the type strain. Finally, our data suggested that insertion sequences are likely to be involved in 5632 vpma locus duplication at a remote chromosomal position. The role of such mobile genetic elements in chromosomal shuffling of genes encoding major surface components may have important evolutionary and epidemiological consequences for pathogens, such as mycoplasmas, that have a reduced genome and no cell wall.

Genome‐wide association links candidate genes to resistance to Plum Pox Virus in apricot (Prunus armeniaca)

In fruit tree species, many important traits have been characterized genetically by using single-family descent mapping in progenies segregating for the traits. However, most mapped loci have not been sufficiently resolved to the individual genes due to insufficient progeny sizes for high resolution mapping and the previous lack of whole-genome sequence resources of the study species. To address this problem for Plum Pox Virus (PPV) candidate resistance gene identification in Prunus species, we implemented a genome-wide association (GWA) approach in apricot. This study exploited the broad genetic diversity of the apricot (Prunus armeniaca) germplasm containing resistance to PPV, next-generation sequence-based genotyping, and the high-quality peach (Prunus persica) genome reference sequence for single nucleotide polymorphism (SNP) identification. The results of this GWA study validated previously reported PPV resistance quantitative trait loci (QTL) intervals, highlighted other potential resistance loci, and resolved each to a limited set of candidate genes for further study. This work substantiates the association genetics approach for resolution of QTL to candidate genes in apricot and suggests that this approach could simplify identification of other candidate genes for other marked trait intervals in this germplasm.