Eric Peyretaillade

Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi

Microsporidia are obligate intracellular parasites infesting many animal groups. Lacking mitochondria and peroxysomes, these unicellular eukaryotes were first considered a deeply branching protist lineage that diverged before the endosymbiotic event that led to mitochondria. The discovery of a gene for a mitochondrial-type chaperone combined with molecular phylogenetic data later implied that microsporidia are atypical fungi that lost mitochondria during evolution. Here we report the DNA sequences of the 11 chromosomes of the ∼2.9-megabase (Mb) genome of Encephalitozoon cuniculi (1,997 potential protein-coding genes). Genome compaction is reflected by reduced intergenic spacers and by the shortness of most putative proteins relative to their eukaryote orthologues. The strong host dependence is illustrated by the lack of genes for some biosynthetic pathways and for the tricarboxylic acid cycle. Phylogenetic analysis lends substantial credit to the fungal affiliation of microsporidia. Because the E. cuniculi genome contains genes related to some mitochondrial functions (for example, Fe–S cluster assembly), we hypothesize that microsporidia have retained a mitochondrion-derived organelle.

Complete Genome Sequence of Crohn's Disease-Associated Adherent-Invasive E. coli Strain LF82

BACKGROUND Ileal lesions of Crohns disease (CD) patients are abnormally colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells and macrophages. PRINCIPAL FINDINGS We report here the complete genome sequence of E. coli LF82, the reference strain of adherent-invasive E. coli associated with ileal Crohns disease. The LF82 genome of 4,881,487 bp total size contains a circular chromosome with a size of 4,773,108 bp and a plasmid of 108,379 bp. The analysis of predicted coding sequences (CDSs) within the LF82 flexible genome indicated that this genome is close to the avian pathogenic strain APEC_01, meningitis-associated strain S88 and urinary-isolated strain UTI89 with regards to flexible genome and single nucleotide polymorphisms in various virulence factors. Interestingly, we observed that strains LF82 and UTI89 adhered at a similar level to Intestine-407 cells and that like LF82, APEC_01 and UTI89 were highly invasive. However, A1EC strain LF82 had an intermediate killer phenotype compared to APEC-01 and UTI89 and the LF82 genome does not harbour most of specific virulence genes from ExPEC. LF82 genome has evolved from those of ExPEC B2 strains by the acquisition of Salmonella and Yersinia isolated or clustered genes or CDSs located on pLF82 plasmid and at various loci on the chromosome. CONCLUSION LF82 genome analysis indicated that a number of genes, gene clusters and pathoadaptative mutations which have been acquired may play a role in virulence of AIEC strain LF82.

Bacterial community changes during bioremediation of aliphatic hydrocarbon-contaminated soil

The microbial community response during the oxygen biostimulation process of aged oil-polluted soils is poorly documented and there is no reference for the long-term monitoring of the unsaturated zone. To assess the potential effect of air supply on hydrocarbon fate and microbial community structure, two treatments (0 and 0.056 mol h⁻¹ molar flow rate of oxygen) were performed in fixed bed reactors containing oil-polluted soil. Microbial activity was monitored continuously over 2 years throughout the oxygen biostimulation process. Microbial community structure before and after treatment for 12 and 24 months was determined using a dual rRNA/rRNA gene approach, allowing us to characterize bacteria that were presumably metabolically active and therefore responsible for the functionality of the community in this polluted soil. Clone library analysis revealed that the microbial community contained many rare phylotypes. These have never been observed in other studied ecosystems. The bacterial community shifted from Gammaproteobacteria to Actinobacteria during the treatment. Without aeration, the samples were dominated by a phylotype linked to the Streptomyces. Members belonging to eight dominant phylotypes were well adapted to the aeration process. Aeration stimulated an Actinobacteria phylotype that might be involved in restoring the ecosystem studied. Phylogenetic analyses suggested that this phylotype is a novel, deep-branching member of the Actinobacteria related to the well-studied genus Acidimicrobium.

Microsporidian invasion apparatus: identification of a novel polar tube protein and evidence for clustering of ptp1 and ptp2 genes in three Encephalitozoon species.

ABSTRACT Microsporidia are unicellular eukaryotes occuring as obligate intracellular parasites which produce resistant spores. A unique motile process is represented by the sudden extrusion of the sporal polar tube for initiating entry of the parasite into a new host cell. The complete sequence of an acidic proline-rich polar tube protein (renamed PTP1) has been previously reported for Encephalitozoon cuniculiand E. hellem. Our immunological investigations provided evidence for an additional PTP in E. cuniculi, termed PTP2. The corresponding gene was sequenced and then expressed inEscherichia coli. As expected, mouse antibodies raised against the recombinant protein reacted specifically with the polar tube. The singlecopy ptp1 and ptp2 genes ofE. cuniculi were tandemly arranged on chromosome VI. Polyadenylation of the mRNAs was demonstrated. Identification and sequencing of homologous genes in the two other human-infectingEncephalitozoon species (ptp2 in E. hellem and ptp1 and ptp2 in E. intestinalis) were facilitated by conserved gene clustering. PTP2 appears as a novel structural protein (30 kDa) with a basic lysine-rich core and an acidic tail. Unlike PTP1, this protein is devoid of large tandem repeats. The interspecies conservation of cysteine residues supports a major role of disulfide bridges in polar tube assembly. The two PTPs should serve as both molecular markers of spore differentiation and diagnostic tools.

Phylogenetic analysis of the small subunit ribosomal RNA of Marteilia refringens validates the existence of phylum Paramyxea (Desportes and Perkins, 1990)

Abstract Marteilia refringens is recognized as one of the most significant pathogens of bivalve molluscs. The nucleotide sequence of the small subunit ribosomal RNA gene of Marteilia refringens is used to elucidate the phylogenetic position of the phylum Paramyxea. Genomic DNA was extracted from sporangia of Marteilia, purified from infected blue mussels, Mytilus edulis, and flat oysters, Ostrea edulis. The sequences obtained from Marteilia species purified from both oysters and mussels were identical. The sequence identity was confirmed by in situ hybridization using a DNA probe targeted to a variable region of the ribosomal DNA. The small subunit ribosomal RNA gene sequence of M. refringens is very different from all known sequences of eukaryotic organisms, including those of myxosporeans and haplosporeans. Therefore, the phylum Paramyxea should continue to be recognized as an independent eukaryotic phylum.

The Human Gut Chip "HuGChip'', an explorative phylogenetic microarray for determining gut microbiome diversity at family level

Evaluating the composition of the human gut microbiota greatly facilitates studies on its role in human pathophysiology, and is heavily reliant on culture-independent molecular methods. A microarray designated the Human Gut Chip (HuGChip) was developed to analyze and compare human gut microbiota samples. The PhylArray software was used to design specific and sensitive probes. The DNA chip was composed of 4,441 probes (2,442 specific and 1,919 explorative probes) targeting 66 bacterial families. A mock community composed of 16S rRNA gene sequences from intestinal species was used to define the threshold criteria to be used to analyze complex samples. This was then experimentally verified with three human faecal samples and results were compared (i) with pyrosequencing of the V4 hypervariable region of the 16S rRNA gene, (ii) metagenomic data, and (iii) qPCR analysis of three phyla. When compared at both the phylum and the family level, high Pearsons correlation coefficients were obtained between data from all methods. The HuGChip development and validation showed that it is not only able to assess the known human gut microbiota but could also detect unknown species with the explorative probes to reveal the large number of bacterial sequences not yet described in the human gut microbiota, overcoming the main inconvenience encountered when developing microarrays.

Genome Sequence of "Candidatus Methanomassiliicoccus intestinalis" Issoire-Mx1, a Third Thermoplasmatales-Related Methanogenic Archaeon from Human Feces

ABSTRACT “Candidatus Methanomassiliicoccus intestinalis” Issoire-Mx1 is a methanogenic archaeon found in the human gut and is a representative of the novel order of methanogens related to Thermoplasmatales. Its complete genome sequence is presented here.

Detecting unknown sequences with DNA microarrays: explorative probe design strategies.

Designing environmental DNA microarrays that can be used to survey the extreme diversity of microorganisms existing in nature, represents a stimulating challenge in the field of molecular ecology. Indeed, recent efforts in metagenomics have produced a substantial amount of sequence information from various ecosystems, and will continue to accumulate large amounts of sequence data given the qualitative and quantitative improvements in the next-generation sequencing methods. It is now possible to take advantage of these data to develop comprehensive microarrays by using explorative probe design strategies. Such strategies anticipate genetic variations and thus are able to detect known and unknown sequences in environmental samples. In this review, we provide a detailed overview of the probe design strategies currently available to construct both phylogenetic and functional DNA microarrays, with emphasis on those permitting the selection of such explorative probes. Furthermore, exploration of complex environments requires particular attention on probe sensitivity and specificity criteria. Finally, these innovative probe design approaches require exploiting newly available high-density microarray formats.

Gene Capture Coupled to High-Throughput Sequencing as a Strategy for Targeted Metagenome Exploration

Next-generation sequencing (NGS) allows faster acquisition of metagenomic data, but complete exploration of complex ecosystems is hindered by the extraordinary diversity of microorganisms. To reduce the environmental complexity, we created an innovative solution hybrid selection (SHS) method that is combined with NGS to characterize large DNA fragments harbouring biomarkers of interest. The quality of enrichment was evaluated after fragments containing the methyl coenzyme M reductase subunit A gene (mcrA), the biomarker of methanogenesis, were captured from a Methanosarcina strain and a metagenomic sample from a meromictic lake. The methanogen diversity was compared with direct metagenome and mcrA-based amplicon pyrosequencing strategies. The SHS approach resulted in the capture of DNA fragments up to 2.5 kb with an enrichment efficiency between 41 and 100%, depending on the sample complexity. Compared with direct metagenome and amplicons sequencing, SHS detected broader mcrA diversity, and it allowed efficient sampling of the rare biosphere and unknown sequences. In contrast to amplicon-based strategies, SHS is less biased and GC independent, and it recovered complete biomarker sequences in addition to conserved regions. Because this method can also isolate the regions flanking the target sequences, it could facilitate operon reconstructions.

Annotation of microsporidian genomes using transcriptional signals

High-quality annotation of microsporidian genomes is essential for understanding the biological processes that govern the development of these parasites. Here we present an improved structural annotation method using transcriptional DNA signals. We apply this method to re-annotate four previously annotated genomes, which allow us to detect annotation errors and identify a significant number of unpredicted genes. We then annotate the newly sequenced genome of Anncaliia algerae. A comparative genomic analysis of A. algerae permits the identification of not only microsporidian core genes, but also potentially highly expressed genes encoding membrane-associated proteins, which represent good candidates involved in the spore architecture, the invasion process and the microsporidian-host relationships. Furthermore, we find that the ten-fold variation in microsporidian genome sizes is not due to gene number, size or complexity, but instead stems from the presence of transposable elements. Such elements, along with kinase regulatory pathways and specific transporters, appear to be key factors in microsporidian adaptive processes.