Pierre Peyret

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.

Production and consumption of methane in freshwater lake ecosystems

The atmospheric concentration of methane (CH(4)), a major greenhouse gas, is mainly controlled by the activities of methane-producing (methanogens) and methane-consuming (methanotrophs) microorganisms. Freshwater lakes are identified as one of the main CH(4) sources, as it was estimated that they contribute to 6-16% of natural CH(4) emissions. It is therefore critical to better understanding the biogeochemical cycling of CH(4) in these ecosystems. In this paper, the effects of environmental factors on methanogenic and methanotrophic rates are reviewed and an inventory of the methanogens and methanotrophs at the genus/species level in freshwater lakes is given. We focus on the anaerobic oxidation of methane, which is a still poorly known process but increasingly reported in freshwater lakes.

Phylogenomic data support a seventh order of Methylotrophic methanogens and provide insights into the evolution of Methanogenesis.

Increasing evidence from sequence data from various environments, including the human gut, suggests the existence of a previously unknown putative seventh order of methanogens. The first genomic data from members of this lineage, Methanomassiliicoccus luminyensis and “Candidatus Methanomethylophilus alvus,” provide insights into its evolutionary history and metabolic features. Phylogenetic analysis of ribosomal proteins robustly indicates a monophyletic group independent of any previously known methanogenic order, which shares ancestry with the Marine Benthic Group D, the Marine Group II, the DHVE2 group, and the Thermoplasmatales. This phylogenetic position, along with the analysis of enzymes involved in core methanogenesis, strengthens a single ancient origin of methanogenesis in the Euryarchaeota and indicates further multiple independent losses of this metabolism in nonmethanogenic lineages than previously suggested. Genomic analysis revealed an unprecedented loss of the genes coding for the first six steps of methanogenesis from H2/CO2 and the oxidative part of methylotrophic methanogenesis, consistent with the fact that M. luminyensis and “Ca. M. alvus” are obligate H2-dependent methylotrophic methanogens. Genomic data also suggest that these methanogens may use a large panel of methylated compounds. Phylogenetic analysis including homologs retrieved from environmental samples indicates that methylotrophic methanogenesis (regardless of dependency on H2) is not restricted to gut representatives but may be an ancestral characteristic of the whole order, and possibly also of ancient origin in the Euryarchaeota. 16S rRNA and McrA trees show that this new order of methanogens is very diverse and occupies environments highly relevant for methane production, therefore representing a key lineage to fully understand the diversity and evolution of methanogenesis.

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.

GoArrays: highly dynamic and efficient microarray probe design

MOTIVATION The use of oligonucleotide microarray technology requires a very detailed attention to the design of specific probes spotted on the solid phase. These problems are far from being commonplace since they refer to complex physicochemical constraints. Whereas there are more and more publicly available programs for microarray oligonucleotide design, most of them use the same algorithm or criteria to design oligos, with only little variation. RESULTS We show that classical approaches used in oligo design software may be inefficient under certain experimental conditions, especially when dealing with complex target mixtures. Indeed, our biological model is a human obligate parasite, the microsporidia Encephalitozoon cuniculi. Targets that are extracted from biological samples are composed of a mixture of pathogen transcripts and host cell transcripts. We propose a new approach to design oligonucleotides which combines good specificity with a potentially high sensitivity. This approach is original in the biological point of view as well as in the algorithmic point of view. We also present an experimental validation of this new strategy by comparing results obtained with standard oligos and with our composite oligos. A specific E.cuniculi microarray will overcome the difficulty to discriminate the parasite mRNAs from the host cell mRNAs demonstrating the power of the microarray approach to elucidate the lifestyle of an intracellular pathogen using mix mRNAs.

Molecular Evidence for the Existence of Two Species of Marteilia in Europe

Abstract Marteilia refringens is one of the most significant pathogens of bivalve molluscs. Previous sequencing of the small subunit ribosomal RNA gene of M. refringens isolates derived from the infected mussels (Mytilus edulis and Mytilus galloprovinciallis) and the oyster (Ostrea edulis) in Europe did not reveal genetic polymorphisms despite indications from epizootiological data that distinct types may exist. We investigated the existence of polymorphisms in the internal transcribed spacer region of the ribosomal RNA genes. The sequences of this region proved to be clearly dimorphic among Marteilia from five sampling sites. The distribution of the two genetic types, named “O” and “M”, appeared to be linked to the host species, oysters and mussels, respectively. We therefore support the recognition of two species of Marteilia in Europe and propose that the “O” type corresponds to M. refringens and the “M” type to M. maurini.

Identification of microbial communities involved in the methane cycle of a freshwater meromictic lake

Lake Pavin is a meromictic crater lake located in the French Massif Central area. In this ecosystem, most methane (CH(4)) produced in high quantity in the anoxic bottom layers, and especially in sediments, is consumed in the water column, with only a small fraction of annual production reaching the atmosphere. This study assessed the diversity of methanogenic and methanotrophic populations along the water column and in sediments using PCR and reverse transcription-PCR-based approaches targeting functional genes, i.e. pmoA (α-subunit of the particulate methane monooxygenase) for methanotrophy and mcrA (α-subunit of the methyl-coenzyme M reductase) for methanogenesis as well as the phylogenetic 16S rRNA genes. Although methanogenesis rates were much higher in sediments, our results confirm that CH(4) production also occurs in the water column where methanogens were almost exclusively composed of hydrogenotrophic methanogens, whereas both hydrogenotrophs and acetotrophs were almost equivalent in the sediments. Sequence analysis of markers, pmoA and the 16S rRNA gene, suggested that Methylobacter may be an important group actively involved in CH(4) oxidation in the water column. Two main phylotypes were characterized, one of which could consume CH(4) under conditions where the oxygen amount is undetectable.

ON PROTEINS OF THE MICROSPORIDIAN INVASIVE APPARATUS : COMPLETE SEQUENCE OF A POLAR TUBE PROTEIN OF ENCEPHALITOZOON CUNICULI

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that can cause opportunistic infections in AIDS patients. Spore invasion of host cells involves extrusion of a polar tube. After immunocytochemical identification of several polar tube proteins (PTPs) in E. cuniculi, a major PTP was isolated from two‐dimensional gels and two peptide fragments were sequenced. The complete nucleotide sequence of the corresponding gene was obtained using a combination of PCR amplification and cloning techniques. The gene exists as a single copy per haploid genome and encodes an acidic proline‐rich protein, with a deduced molecular mass of 37 kDa, that contains four tandemly arranged 26‐amino‐acid repeats. An N‐terminal region of 22 residues represents a cleaved signal peptide, probably involved in the targeting of the PTP. No similarity with known proteins has been found. The protein was expressed in Escherichia coli, purified and injected into mice. The antisera reacted specifically with the polar tube in indirect immunofluorescence assays and electron microscope immunocytochemistry. Further identification of conserved and variable PTP structural motifs should be useful for diagnostic purposes and new therapeutic strategies.

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.