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International audience; Pseudothermotoga elfii strain DSM9442 and P. elfii subsp. lettingae strain DSM14385 are hyperthermophilic bacteria. P. elfii DSM9442 is a piezophile and was isolated from a depth of over 1600 m in an oil-producing well in Africa. P. elfii subsp. lettingae is piezotolerant and was isolated from a thermophilic bioreactor fed with methanol as the sole carbon and energy source. In this study, we analyzed both strains at the genomic and transcriptomic levels, paying particular attention to changes in response to pressure increases. Transcriptomic analyses revealed common traits of adaptation to increasing hydrostatic pressure in both strains, namely, variations in transport membrane or carbohydrate metabolism, as well as species-specific adaptations such as variations in amino acid metabolism and transport for the deep P. elfii DSM9442 strain. Notably, this work highlights the central role played by the amino acid aspartate as a key intermediate of the pressure adaptation mechanisms in the deep strain P. elfii DSM9442. Our comparative genomic and transcriptomic analysis revealed a gene cluster involved in lipid metabolism that is specific to the deep strain and that was differentially expressed at high hydrostatic pressures and might, thus, be a good candidate for a piezophilic gene marker in Pseudothermotogales.

Microorganisms living in deep-oil reservoirs face extreme conditions of elevated temperature and hydrostatic pressure. Within these microbial communities, members of the order Thermotogales are predominant. Among them, the genus Pseudothermotoga is widespread in oilfield-produced waters. The growth and cell phenotypes under hydrostatic pressures ranging from 0.1 to 50 MPa of two strains from the same species originating from subsurface, Pseudothermotoga elfii DSM9442 isolated from a deep African oil-producing well, and surface, P. elfii subsp. lettingae isolated from a thermophilic sulfate-reducing bioreactor, environments are reported for the first time. The data support evidence for the piezophilic nature of P. elfii DSM9442, with an optimal hydrostatic pressure for growth of 20 MPa and an upper limit of 40 MPa, and the piezotolerance of P. elfii subsp. lettingae with growth occurring up to 20 MPa only. Under the experimental conditions, both strains produce mostly acetate and propionate as volatile fatty acids with slight variations with respect to the hydrostatic pressure for P. elfii DSM9442. The data show that the metabolism of P. elfii DSM9442 is optimized when grown at 20 MPa, in agreement with its piezophilic nature. Both Pseudothermotoga strains form chained cells when the hydrostatic pressure increases, especially P. elfii DSM9442 for which 44% of cells is chained when grown at 40 MPa. The viability of the chained cells increases with the increase in the hydrostatic pressure, indicating that chain formation is a protective mechanism for P. elfii DSM9442.

Hot oil reservoirs harbor diverse microbial communities, with many of them inhabiting thermophilic or hyperthermophilic fermentative Thermotogae species. A new Thermotoga sp. strain TFO was isolated from an Californian offshore oil reservoir which is phylogenetically related to thermophilic species T. petrophila RKU-1T and T. naphthophila RKU-10T, isolated from the Kubiki oil reservoir in Japan. The average nucleotide identity and DNA–DNA hybridization measures provide evidence that the novel strain TFO is closely related to T. naphthophila RKU-10T, T. petrophila RKU-1T and can not be differentiated at the species level. In the light of these results, the reclassification of T. naphthophila RKU-10 and strain TFO as heterotypic synonyms of T. petrophila is proposed. A pangenomic survey of closely related species revealed 55 TFO strain-specific proteins, many of which being linked to glycosyltransferases and mobile genetic elements such as recombinases, transposases and prophage, which can contribute to genome evolution and plasticity, promoting bacterial diversification and adaptation to environmental changes. The discovery of a TFO-specific transport system dctPQM, encoding a tripartite ATP-independent periplasmic transporter (TRAP), has to be highlighted. The presence of this TRAP system assumes that it could assist in anaerobic n-alkane degradation by addition of fumarate dicarboxylic acid, suggesting a niche-specific gene pool which correlates with the oil reservoir that T. petrophila TFO inhabits. Finally, T. naphthophila RKU-10, T. petrophila RKU-1T, T. petrophila TFO form a distinct phylogenetic lineage with different geographic origins, share the same type of ecological niche including the burial history of fields. Theses findings might support the indigenous character of this species in oil reservoirs.

Two strains of anaerobic, coccoid, saccharolytic, Gram-stain-negative bacteria were isolated from samples of anoxic hypersaline sediments of evaporation ponds in Tavira (Portugal) and Mallorca (Spain). Both isolates were moderately halophilic, neutrophilic and had a temperature optimum at 37 °C. The highest 16S rRNA gene sequence identity values were found with members of the genus Sedimentisphaera (84.9–88.2 %) within the order Sedimentisphaerales, class Phycisphaerae . The strain SM-Chi-D1T could be assigned to the family Sedimentisphaeraceae , while phylogenetic analyses based on 16S rRNA gene sequences and genomic data indicate that strain ST-NAGAB-D1T is both a member of a novel genus and a novel family. SM-Chi-D1T could be distinguished from other cultured members of the Sedimentisphaeraceae mainly by the stimulatory effect of sulfur on growth, lack of ethanol production during fermentation and several differences in the cellular fatty acids and polar lipids patterns. Main differential characteristics of ST-NAGAB-D1T were a polytrichous flagellation, the absence of branched chain fatty acids and presence of large proportions of the unsaturated cellular fatty acids C16 : 1 c9 and C18 : 1 c11. On the basis of genomic, chemotaxonomic, biochemical and physiological data, we propose the novel species and genera Anaerohalosphaera lusitana gen. nov., sp. nov., and Limihaloglobus sulfuriphilus gen. nov., sp. nov., represented by the type strains ST-NAGAB-D1T (=DSM 103484T=JCM 31926T=KCTC 15600T) and SM-Chi-D1T (=DSM 100118T=JCM 31927T=KCTC 15601T), respectively. In addition, we propose the novel family Anaerohalosphaeraceae fam. nov. to accommodate the genus Anaerohalosphaera.

International audience; Desulfovibrio piezophilus strain C1TLV30(T) is a mesophilic piezophilic sulfate-reducer isolated from Wood Falls at 1700 m depth in the Mediterranean Sea. In this study, we analysed the effect of the hydrostatic pressure on this deep-sea living bacterium at the physiologic and transcriptomic levels. Our results showed that lactate oxidation and energy metabolism were affected by the hydrostatic pressure. Especially, acetyl-CoA oxidation pathway and energy conservation through hydrogen and formate recycling would be more important when the hydrostatic pressure is above (26 MPa) than below (0.1 MPa) the optimal one (10 MPa). This work underlines also the role of the amino acid glutamate as a piezolyte for the Desulfovibrio genus. The transcriptomic analysis revealed 146 differentially expressed genes emphasizing energy production and conversion, amino acid transport and metabolism and cell motility and signal transduction mechanisms as hydrostatic pressure responding processes. This dataset allowed us to identify a sequence motif upstream of a subset of differentially expressed genes as putative pressure-dependent regulatory element.

International audience; In this study, we report for the first time the presence of magnetotactic bacteria (MTB) on the Northeastern Atlantic coast. Microscopy observations indicated a heterogeneous population of MTB morphotypes. The analysis of the 16S rDNA by pyrosequencing technology revealed four operational taxonomic sequence units affiliated within the Magnetococcales order, class Alphaproteobacteria. One of them was closely related to sequences of MTB from the Tunisian coast, central Mediterranean Sea. This work offers information on anew environmental context and on biogeography of MTB, highlights the putative impact that marine currents may have on MTB distribution on Earth, and underlines the role that pristine or polluted areas may play on the structure of the MTB communites.

International audience; RNA-seq was used to study the response of Desulfovibrio hydrothermalis, isolated from a deep-sea hydrothermal chimney on the East-Pacific Rise at a depth of 2,600 m, to various hydrostatic pressure growth conditions. The transcriptomic datasets obtained after growth at 26, 10 and 0.1 MPa identified only 65 differentially expressed genes that were distributed among four main categories: aromatic amino acid and glutamate metabolisms, energy metabolism, signal transduction, and unknown function. The gene expression patterns suggest that D. hydrothermalis uses at least three different adaptation mechanisms, according to a hydrostatic pressure threshold (HP t) that was estimated to be above 10 MPa. Both glutamate and energy metabolism were found to play crucial roles in these mechanisms. Quantitation of the glutamate levels in cells revealed its accumulation at high hydrostatic pressure, suggesting its role as a piezolyte. ATP measurements showed that the energy metabolism of this bacterium is optimized for deep-sea life conditions. This study provides new insights into the molecular mechanisms linked to hydrostatic pressure adaptation in sulfate-reducing bacteria.

An anaerobic thermophilic bacterium designated CA9F1 was isolated from a thermal spring in France. Strain CA9F1 was observed to grow at temperatures between 55 and 70 A degrees C (optimum 65 A degrees C) and at pH between 6.8 and 9.5 (optimum pH 7.4). Strain CA9F1 does not require salt for growth (0-10 g l(-1) NaCl), with an optimum at 1 g l(-1). The DNA G+C content was determined to be 38.5 mol% (Tm). The major cellular fatty acids identified were C-15:0, C-16:0, C-17:0 iso. Based on phenotypic, chemotaxonomic and genotypic properties, strain CA9F1 was identified as Thermovenabulum gondwanense and this species was studied in more detail. Strain CA9F1 is a Gram-positive bacterium which forms a complex and regular multilayered cell wall structure, here characterised as being due to the presence of an S-layer. The network covers the entire cell surface and forms a hexagonal structure resembling that observed for Deinococcus radiodurans. The main protein component of the S-layer possesses domains comparable to that of the S-layer protein of Halothermothrix orenii. The characteristics of the strain were compared to that of T. gondwanese R270(T) isolated from microbial mats thriving in the thermal waters of a Great Artesian Basin bore runoff channel at 66 A degrees C, in Australia. Significant differences were observed between CA9F1 and the type strain. One of the major physiological differences is the inability of CA9F1 to reduce Fe(III). An emended description of T. gondwanense is given.

Desulfovibrio hydrothermalis AM13 T is a piezophilic, mesophilic, hydrogenotrophic sulfate-reducing bacterium collected from a deep-sea hydrothermal chimney on the East Pacific Rise (2,600 m depth, 13°N). We report the genome sequence of this bacterium, which includes a 3,702,934-bp chromosome and a circular plasmid of 5,328 bp.

Desulfovibrio piezophilus strain C1TLV30(T) is a piezophilic anaerobe that was isolated from wood falls in the Mediterranean deep-sea. D. piezophilus represents a unique model for studying the adaptation of sulfate-reducing bacteria to hydrostatic pressure. Here, we report the 3.6 Mbp genome sequence of this piezophilic bacterium. An analysis of the genome revealed the presence of seven genomic islands as well as gene clusters that are most likely linked to life at a high hydrostatic pressure. Comparative genomics and differential proteomics identified the transport of solutes and amino acids as well as amino acid metabolism as major cellular processes for the adaptation of this bacterium to hydrostatic pressure. In addition, the proteome profiles showed that the abundance of key enzymes that are involved in sulfate reduction was dependent on hydrostatic pressure. A comparative analysis of orthologs from the non-piezophilic marine bacterium D. salexigens and D. piezophilus identified aspartic acid, glutamic acid, lysine, asparagine, serine and tyrosine as the amino acids preferentially replaced by arginine, histidine, alanine and threonine in the piezophilic strain. This work reveals the adaptation strategies developed by a sulfate reducer to a deep-sea lifestyle.

pks genomic island of Escherichia coli is involved in the synthesis of the non-ribosomal peptide-type genotoxin colibactin, which has been suggesting as affecting the host immune response and having an impact on cancer development. The pks-encoded enzyme ClbP is an atypical peptidase that contributes to the synthesis of colibactin. In this work, we identified key features of ClbP. Bacterial fractionation and Western-blot analysis revealed the docking of ClbP to the bacterial inner membrane via a C-terminal domain harboring three predicted transmembrane helices. Whereas only one helix was necessary for the location in the inner membrane, the complete sequence of the C-terminal domain was necessary for ClbP bioactivity. In addition, the N-terminal sequence of ClbP allowed the SRP/Sec/YidC- and MreB-dependent translocation of the enzymatic domain in the periplasmic compartment, a feature also essential for ClbP bioactivity. Finally, the comparison of ClbP structure with that of the paralogs FmtA-like and AmpC revealed at an extremity of the catalytic groove a negative electrostatic potential surface characteristic of ClbP. Site-directed mutagenesis experiments identified in this zone two aspartic residues that were important for ClbP bioactivity. Overall, these results suggest a model for precolibactin activation by ClbP and pave a way for the design of inhibitors targeting colibactin production.

A novel sulfate-reducing bacterium, designated C1TLV30T, was isolated from wood falls at a depth of 1693 m in the Mediterranean Sea. Cells were motile vibrios (2–4×0.5 µm). Strain C1TLV30T grew at temperatures between 15 and 45 °C (optimum 30 °C) and at pH 5.4–8.6 (optimum 7.3). It required NaCl for growth (optimum at 25 g NaCl l−1) and tolerated up to 80 g NaCl l−1. Strain C1TLV30T used as energy sources: lactate, fumarate, formate, malate, pyruvate and ethanol. The end products from lactate oxidation were acetate, H2S and CO2 in the presence of sulfate as terminal electron acceptor. Besides sulfate, thiosulfate and sulfite were also used as terminal electron acceptors, but not elemental sulfur, fumarate, nitrate or nitrite. Strain C1TLV30T possessed desulfoviridin and was piezophilic, growing optimally at 10 MPa (range 0–30 MPa). The membrane lipid composition of this strain was examined to reveal an increase in fatty acid chain lengths at high hydrostatic pressures. The G+C content of the genomic DNA was 49.6 % and the genome size was estimated at 3.5±0.5 Mb. Phylogenetic analysis of the SSU rRNA gene sequence indicated that strain C1TLV30T was affiliated to the genus Desulfovibrio with Desulfovibrio profundus being its closest phylogenetic relative (similarity of 96.4 %). On the basis of SSU rRNA gene sequence comparisons and physiological characteristics, strain C1TLV30T ( = DSM 21447T = JCM 1548T) is proposed to be assigned to a novel species of the genus Desulfovibrio, Desulfovibrio piezophilus sp. nov.

Enterohemorrhagic Escherichia coli (EHEC) are food-borne pathogens that can cause serious infections ranging from diarrhea to hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). Translocation of Shiga-toxins (Stx) from the gut lumen to underlying tissues is a decisive step in the development of the infection, but the mechanisms involved remain unclear. Many bacterial pathogens target the follicle-associated epithelium, which overlies Peyer's patches (PPs), cross the intestinal barrier through M cells and are captured by mucosal macrophages. Here, translocation across M cells, as well as survival and proliferation of EHEC strains within THP-1 macrophages were investigated using EHEC O157:H7 reference strains, isogenic mutants, and 15 EHEC strains isolated from HC/HUS patients. We showed for the first time that E. coli O157:H7 strains are able to interact in vivo with murine PPs, to translocate ex vivo through murine ileal mucosa with PPs and across an in vitro human M cell model. EHEC strains are also able to survive and to produce Stx in macrophages, which induce cell apoptosis and Stx release. In conclusion, our results suggest that the uptake of EHEC by M cells and underlying macrophages in the PP may be a critical step in Stx translocation and release in vivo. A new model for EHEC infection in humans is proposed that could help in a fuller understanding of EHEC-associated diseases.

16S rDNAs amplified by PCR from 22 hyperthermophilic methanococci isolated from deep-sea hydrothermal vents were compared with those of the six type strains of the genus Methanococcus by RFLP analysis. Restriction fragments obtained with HaeIII enabled four of the type species to be distinguished. Restrictions with Hhal, BstUI and Mspl were necessary to differentiate Methanococcus jannaschii and Methanococcus fervens. The results indicate that the 16S rDNA PCR-RFLP method provides a rapid and reliable tool for the identification of newly isolated hyperthermophilic Methanococcus spp.

International audience; As part of our ongoing project of identification of actin-binding proteins implicated in the cell transition (flagellate to amoeboid/adherent) of Trichomonas vaginalis, we have characterized an alpha-actinin-related protein in this parasite. The protein (P100) has a molecular mass of 100 kDa and an isoelectric point of 5.5. A monoclonal antibody raised against this protein co-localizes with the actin network. P100 gene transcripts are co-expressed with actin throughout the cell cycle. Analysis of the deduced protein sequence reveals three domains: an N-terminal actin-binding region; a central region rich in alpha-helix; and a C-terminal domain with Ca(2+)-binding capacity. Whereas the N- and C-terminal regions are well-conserved as compared to other alpha-actinins, we observe in the central region an atypical distribution of residues in five repeats. The sequence of the repeats does not show any homology with the rod domain of the other alpha-actinins, except for the first repeat which shows some similarity. The four other repeats of T. vaginalis P100 appear to result from a duplication event which is not detectable in the other sequences.