Gérard Prensier

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.

Global impact of mature biofilm lifestyle on Escherichia coli K‐12 gene expression

The formation of biofilm results in a major lifestyle switch that is thought to affect the expression of multiple genes and operons. We used DNA arrays to study the global effect of biofilm formation on gene expression in mature Escherichia coli K‐12 biofilm. We show that, when biofilm is compared with the exponential growth phase, 1.9% of the genes showed a consistent up‐ or downregulation by a factor greater than two, and that 10% of the E. coli genome is significantly differentially expressed. The functions of the genes induced in these conditions correspond to stress response as well as energy production, envelope biogenesis and unknown functions. We provide evidence that the expression of stress envelope response genes, such as the psp operon or elements of the cpx and rpoE pathways, is a general feature of E. coli mature biofilms. We also compared biofilm with the stationary growth phase and showed that the biofilm lifestyle, although sharing similarities with the stationary growth phase, triggers the expression of specific sets of genes. Using gene disruption of 54 of the most biofilm‐induced genes followed by a detailed phenotypic study, we validated the biological relevance of our analysis and showed that 20 of these genes are required for the formation of mature biofilm. This group includes 11 genes of previously unknown function. These results constitute a comprehensive analysis of the global transcriptional response triggered in mature E. coli biofilms and provide insights into its physiological signature.

Lipid Remodeling of Murine Epididymosomes and Spermatozoa During Epididymal Maturation

Abstract We have isolated vesicular structures from mouse epididymal fluid, referred to as epididymosomes. Epididymosomes have a roughly spherical aspect and a bilayer membrane, and they are heterogeneous in size and content. They originate from the epididymal epithelium, notably from the caput region, and are emitted in the epididymal lumen by way of apocrine secretion. We characterized their membranous lipid profiles in caput and cauda epididymidal fluid samples and found that epididymosomes were particularly rich in sphingomyelin (SM) and arachidonic acid. The proportion of SM increased markedly during epididymal transit and represented half the total phospholipids in cauda epididymidal epididymosomes. The cholesterol:phospholipid ratio increased from 0.26 in the caput to 0.48 in the cauda epididymidis. Measures of epididymosomal membrane anisotropy revealed that epididymosomes became more rigid during epididymal transit, in agreement with their lipid composition. In addition, we have characterized the membrane lipid pattern of murine epididymal spermatozoa during their maturation. Here, we have shown that mouse epididymal spermatozoa were distinguished by high percentages of SM and polyunsaturated membranous fatty acids (PUFAs), principally represented by arachidonic, docosapentanoic, and docosahexanoic acids. Both SM and PUFA increased throughout the epididymal tract. In particular, we observed a threefold rise in the ratio of docosapentanoic acid. Epididymal spermatozoa had a constant cholesterol:phospholipid ratio (average, 0.30) during epididymal transit. These data suggest that in contrast with epididymosomes, spermatozoal membranes seem to become more fluid during epididymal maturation.

Changes in the Daphnia magna midgut upon ingestion of copper oxide nanoparticles: a transmission electron microscopy study.

This work is a follow-up of our previous paper (Heinlaan et al., 2008. Chemosphere 71, 1308-1316) where we showed about 50-fold higher acute toxicity of CuO nanoparticles (NPs) compared to bulk CuO to water flea Daphnia magna. In the current work transmission electron microscopy (TEM) was used to determine potential time-dependent changes in D. magna midgut epithelium ultrastructure upon exposure to CuO NPs compared to bulk CuO at their 48 h EC(50) levels: 4.0 and 175 mg CuO/L, respectively. Special attention was on potential internalization of CuO NPs by midgut epithelial cells. Ingestion of both CuO formulations by daphnids was evident already after 10 min of exposure. In the midgut lumen CuO NPs were dispersed whereas bulk CuO was clumped. By the 48th hour of exposure to CuO NPs (but not to equitoxic concentrations of bulk CuO) the following ultrastructural changes in midgut epithelium of daphnids were observed: protrusion of epithelial cells into the midgut lumen, presence of CuO NPs in circular structures analogous to membrane vesicles from holocrine secretion in the midgut lumen. Implicit internalization of CuO NPs via D. magna midgut epithelial cells was not evident however CuO NPs were no longer contained within the peritrophic membrane but located between the midgut epithelium microvilli. Interestingly, upon exposure to CuO NPs bacterial colonization of the midgut occurred. Ultrastructural changes in the midgut of D. magna upon exposure to CuO NPs but not to bulk CuO refer to its nanosize-related adverse effects. Time-dependent solubilisation of CuO NPs and bulk CuO in the test medium was quantified by recombinant Cu-sensor bacteria: by the 48th hour of exposure to bulk CuO, the concentration of solubilised copper ions was 0.05 ± 0.01 mg Cu/L that was comparable to the acute EC(50) value of Cu-ions to D. magna (48 h CuSO(4) EC(50) = 0.07 ± 0.01 mg Cu/L). However, in case of CuO NPs, the solubilised Cu-ions 0.01 ± 0.001 mg Cu/L, explained only part of the toxicity.

Thermotoga elfii sp. nov., a Novel Thermophilic Bacterium from an African Oil-producing Well

A thermophilic, glucose-fermenting, strictly anaerobic, rod-shaped bacterium, strain SEBR 6459T (T = type strain), was isolated from an African oil-producing well. This organism was identified as a member of the genus Thermotoga on the basis of the presence of the typical outer sheath-like structure (toga) and 16S rRNA signature sequences and its ability to grow on carbohydrates (glucose, arabinose, fructose, lactose, maltose, and xylose). Major differences in its 16S rRNA gene sequence, its lower optimum temperature for growth (66 degrees C), its sodium chloride range for growth (0 to 2.8%), its lack of lactate as an end product from glucose fermentation, and its peritrichous flagella indicate that strain SEBR 6459T is not similar to the three previously described Thermotoga species. Furthermore, this organism does not belong to any of the other genera related to the order Thermotogales that have been described. On the basis of these findings, we propose that this strain should be described as a new species, Thermotoga elfii. The type strain of T. elfii is SEBR 6459 (= DSM 9442).

Isolation and Characterization of Halothermothrix orenii gen. nov., sp. nov., a Halophilic, Thermophilic, Fermentative, Strictly Anaerobic Bacterium

The occurrence of thermophilic, halophilic anaerobic bacteria in the sediment of a Tunisian salted lake was tested in samples collected at 20-cm intervals down to a depth of 1.20 m. A long rod, present only in the 40- to 60-cm layer, was isolated at 60 degrees C in a medium containing 100 g of NaCl per liter and designated strain H168. This strain produced acetate, ethanol, H2, and CO2 from glucose metabolism. Fructose, xylose, ribose, cellobiose, and starch were also oxidized. The optimum temperature for growth was 60 degrees C. No growth was obtained at 42 or 70 degrees C. Strain H168 grew optimally in NaCl concentrations ranging from 50 to 100 g per liter, with the upper and lower limits of growth around 200 and 40 g per liter, respectively. The G+C ratio of the DNA was 39.6 mol%. Although halophilic, moderately thermophilic bacteria have been characterized among anaerobes, particularly within methanogens, strain H168 is the first true thermophilic (growing above 60 degrees C) halophilic anaerobic bacterium described so far. The phylogeny, physiology, morphology, lipid content, and high G+C content of strain H168 are sufficiently different from those of genera belonging to the family Haloanaerobiaceae to justify the definition of a new genus.

Methanocalculus halotolerans gen. nov., sp. nov., isolated from an oil-producing well

Two irregular coccoid methanogens designated SEBR 4845T and FR1T were isolated from an oilfield in Alsace, France. Strain SEBR 4845T (T = type strain) is a hydrogenotrophic halotolerant methanogen, which grows optimally at 5% NaCI (w/v) and tolerates up to 12% NaCI. It does not use methylated compounds and therefore cannot be ascribed to any of the known genera of the halophilic methylotrophic methanogens. It differs from hydrogenotrophic members of the orders Methanococcales and Methanomicrobia les in the NaCI growth range (0-12% NaCI), which is the widest reported to data for any hydrogenotrophic methanogen. 16S rRNA gene sequence analysis indicated that strain SEBR 4845T is a novel isolate for which a new genus is proposed, Methanocalculus halotolerans gen. nov., sp. nov. (= OCM470T) that might be indigenous to the oilfield ecosystem. Strain FR1T (=OCM 471) is a moderately halophilic methanogen which growths optimally at 10% NaCI and tolerates up to 20% NaCI. It grows on trimethylamine and methanol as carbon and energy sources. The G+C content of its DNA is 43 mol%. It is therefore phenotypically and genotypically related to members of the genus Methanohalophilus. This report provides evidence that methylotrophic and hydrogenotrophic, but not aceticlastic methanogens are present in a saline subsurface oilfield environment, as already observed in surface saline to hypersaline environments.

Bacillus thermoamylovorans sp. nov., a Moderately Thermophilic and Amylolytic Bacterium

A moderately thermophilic, facultatively anaerobic, amylolytic bacterium was isolated from palm wine, a tropical alcoholic beverage that was sampled in Senegal. The cells were gram positive, catalase positive, non-spore forming, rod shaped, and slightly motile with peritrichous flagella. The strain which we examined did not possess cytochrome and produced L-(+)-lactate, acetate, ethanol, and formate but not hydrogen during carbohydrate fermentation. Growth occurred at pH values ranging from 5.4 to 8.5, and optimum growth occurred at around pH 7.0. The optimum temperature for growth was around 50 degrees C, and the upper temperature limit for growth was 58 degrees C. The guanine-plus-cytosine content of the DNA was 38.8 +/- 0.2 mol%. A sequence analysis of the 16S rRNA gene revealed that the new organism is closely related phylogenetically to members of genus Bacillus. Despite the lack of spores, we propose that on the basis of phylogenetic characteristics, the new isolate should be classified as a new Bacillus species, Bacillus thermoamylovorans. The type strain is strain DKP (= Collection of Institut Pasteur CNCM I-1378).

Desulfohalobium retbaense gen. nov., sp. nov., a Halophilic Sulfate-Reducing Bacterium from Sediments of a Hypersaline Lake in Senegal

Sulfate-reducing bacterial strain HR100 was isolated from sediments of Retba Lake, a pink hypersaline lake in Senegal. The cells were motile, nonsporulating, and rod shaped with polar flagella and incompletely oxidized a limited range of substrates to acetate and CO2. Acetate and vitamins were required for growth and could be replaced by Biotrypcase or yeast extract. Sulfate, sulfite, thiosulfate, and elemental sulfur were used as electron acceptors and were reduced to H2S. Growth occurred at pH values ranging from 5.5 to 8.0. The optimum temperature for growth was 37 to 40°C. NaCl and MgCl2 were required for growth; the optimum NaCl concentration was near 10%. The guanine-plus-cytosine content of the DNA was 57.1 ± 0.2 mol%. On the basis of the morphological and physiological properties of this strain, we propose that it should be classified in a new genus, Desulfohalobium, which includes a single species, Desulfohalobium retbaense. The type strain is strain DSM 5692.

Hepatitis C virus budding at lipid droplet-associated ER membrane visualized by 3D electron microscopy

The mechanisms underlying hepatitis C virus (HCV) morphogenesis remain elusive, but lipid droplets have recently been shown to be important organelles for virus production. We investigated the interaction between HCV-like particles and lipid droplets by three-dimensional reconstructions of serial ultrathin electron microscopy sections of cells producing the HCV core protein. The budding of HCV-like particles was mostly initiated at membranes close to the lipid droplets rather than at membranes directly apposed to the lipid droplets. This may have important implications for our understanding of the complex relationship between HCV and lipids and may make easier to dissect out the HCV life cycle.