[Show abstract][Hide abstract] ABSTRACT: 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 (HPt) 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.
PLoS ONE 09/2014; 9(9):e106831. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacteria of the genus Photobacterium thrive worldwide in oceans and show substantially varied lifestyles, including free-living, commensal, pathogenic, symbiotic, and piezophilic. Here, we present the genome sequence of a luminous, piezophilic Photobacterium phosphoreum strain, ANT-2200, isolated from a water column at 2,200 m depth in the Mediterranean Sea. It is the first genomic sequence of the P. phosphoreum group. An analysis of the sequence provides insight into the adaptation of bacteria to the deep-sea habitat.
[Show abstract][Hide abstract] ABSTRACT: Magnetotactic bacteria (MTB) are capable of synthesizing intracellular organelles, the magnetosomes, that are membrane-bounded magnetite or greigite crystals arranged in chains. Although MTB are widely spread in various ecosystems, few axenic cultures are available, and only freshwater Magnetospirillum spp. have been genetically analysed. Here, we present the complete genome sequence of a marine magnetotactic spirillum, Magnetospira sp. QH-2. The high number of repeats and transposable elements account for the differences in QH-2 genome structure compared with other relatives. Gene cluster synteny and gene correlation analyses indicate that the insertion of the magnetosome island in the QH-2 genome occurred after divergence between freshwater and marine magnetospirilla. The presence of a sodium-quinone reductase, sodium transporters and other functional genes are evidence of the adaptive evolution of Magnetospira sp. QH-2 to the marine ecosystem. Genes well conserved among freshwater magnetospirilla for nitrogen fixation and assimilatory nitrate respiration are absent from the QH-2 genome. Unlike freshwater Magnetospirillum spp., marine Magnetospira sp. QH-2 neither has TonB and TonB-dependent receptors nor does it grow on trace amounts of iron. Taken together, our results show a distinct, adaptive evolution of Magnetospira sp. QH-2 to marine sediments in comparison with its closely related freshwater counterparts.
[Show abstract][Hide abstract] ABSTRACT: 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 °C (optimum 65 °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 C15:0, C16:0, C17:0 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 °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.
Antonie van Leeuwenhoek 06/2013; · 2.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel obligately anaerobic, non-spore-forming, rod-shaped mesophilic, halophilic bacterium staining Gram-negative, was isolated from sediments of Guaymas basin. The strain, designated Ra1766G1T, grew at 20-40 °C (optimum 30-35 °C) and at pH 6.0-8.0 (optimum pH 6.5-7.5). It required 0.5%-7.5% NaCl (optimum 2%-3%) for growth. Sulfate, thiosulfate, elemental sulfur, sulfite, fumarate, nitrate and nitrite were not used as terminal electron acceptors. Strain Ra1766G1T used cellobiose, glucose, mannose, maltose, arabinose, raffinose, galactose, ribose, saccharose, pyruvate and xylose as electron donors. The main fermentation product from glucose metabolism was acetate. The predominant cellular fatty acids were anteiso-C15:0, iso-C15:0, anteiso DMA-C15:0 and C16:0. The main polar lipids consisted of diphosphatiglycerol, phosphatidylglycerol, glycolipids and phospholipids.The G+C content of the genomic DNA was 31.2 mol%. The closest phylogenetic relatives of Ra1766G1T were Natranaerovirga pectinivoraT (92.4% similarity), Natranaerovirga hydrolytica (90.2% similarity) and Defluviitalea saccharophilaT (88.9% similarity). On the basis of phylogenetic inference and phenotypic properties, strain Ra1766G1T (= DSM 24848T = JCMT= 16313) is proposed as the type strain of a novel species of a novel genus, Vallitalea guaymasensis gen. nov., sp. nov.
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 02/2013; · 2.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AM13 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Molecular Biology 10/2012; · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The pks genomic island of Escherichia coli encodes polyketide (PK) and nonribosomal peptide (NRP) synthases that allow assembly of a putative hybrid PK-NRP compound named colibactin that induces DNA double-strand breaks in eukaryotic cells. The pks-encoded machinery harbors an atypical essential protein, ClbP. ClbP crystal structure and mutagenesis experiments revealed a serine-active site and original structural features compatible with peptidase activity, which was detected by biochemical assays. Ten ClbP homologs were identified in silico in NRP genomic islands of closely and distantly related bacterial species. All tested ClbP homologs were able to complement a clbP-deficient E. coli mutant. ClbP is therefore a prototype of a new subfamily of extracytoplasmic peptidases probably involved in the maturation of NRP compounds. Such peptidases will be powerful tools for the manipulation of NRP biosynthetic pathways.
Journal of Biological Chemistry 07/2011; 286(41):35562-70. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Magnetotactic bacteria (MTB) mineralize nanosized magnetite or greigite crystals within cells and thus play an important role in the biogeochemical process. Despite decades of research, knowledge of MTB distribution and ecology, notably in areas subjected to oil industry activities, is still limited. In the present study, we investigated the presence of MTB in the Gulf of Fos, French Mediterranean coast, which is subjected to intensive oil industry activities. Microcosms containing sediments/water (1:2, v/v) from several sampling sites were monitored over several weeks. The presence of MTB was revealed in five of eight sites. Diverse and numerous MTB were revealed particularly from one site (named CAR), whilst temporal variations of a homogenous magnetotactic cocci population was shown within the LAV site microcosm over a 4-month period. Phylogenetic analysis revealed that they belonged to Alphaproteobacteria, and a novel genus from the LAV site was evidenced. Among the physicochemical parameters measured, a correlation was shown between the variation of MTB abundance in microcosms and the redox state of sulphur compounds.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 01/2011; 6(8):e23594. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel sulfate-reducing bacterium, designated C1TLV30(T), 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 C1TLV30(T) 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 C1TLV30(T) used as energy sources: lactate, fumarate, formate, malate, pyruvate and ethanol. The end products from lactate oxidation were acetate, H(2)S and CO(2) 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 C1TLV30(T) 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 C1TLV30(T) 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 C1TLV30(T) ( = DSM 21447(T) = JCM 1548(T)) is proposed to be assigned to a novel species of the genus Desulfovibrio, Desulfovibrio piezophilus sp. nov.
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 02/2010; 61(Pt 11):2706-2711. · 2.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report a multiplex real-time polymerase chain reaction method for detecting enterohemorrhagic Escherichia coli (EHEC) from strains or stool specimens. This assay detected the virulence genes stx1, stx2, and eae, without the use of probes. The method, which was validated on a collection of 143 EHEC strains, is simple, rapid, cost-effective, and sensitive.
[Show abstract][Hide abstract] ABSTRACT: The widespread magnetotactic bacteria have the peculiar capacity of navigation along the geomagnetic field. Despite their ubiquitous distribution, only few axenic cultures have been obtained worldwide. In this study, we reported the first axenic culture of magnetotactic bacteria isolated from the Mediterranean Sea. This magneto-ovoid strain MO-1 grew in chemically defined O(2) gradient minimal media at the oxic-anoxic transition zone. It is phylogenetically related to Magnetococcus sp. MC-1 but might represent a novel genus of Proteobacteria. Pulsed-field gel electrophoresis analysis indicated that the genome size of the MO-1 strain is 5 ± 0.5 Mb, with four rRNA operons. Each cell synthesizes about 17 magnetosomes within a single chain, two phosphorous-oxygen-rich globules and one to seven lipid storage granules. The magnetosomes chain seems to divide in the centre during cell division giving rise to two daughter cells with an approximately equal number of magnetosomes. The MO-1 cell possesses two bundles of seven individual flagella that were enveloped in a unique sheath. They swam towards the north pole with a velocity up to 300 μm per second with frequent change from right-hand to left-hand helical trajectory. Using a magneto-spectrophotometry assay we showed that MO-1 flagella were powered by both proton-motive force and sodium ion gradient, which is a rare feature among bacteria.
[Show abstract][Hide abstract] ABSTRACT: beta-Lactamases represent the major resistance mechanism of gram-negative bacteria against beta-lactam antibiotics. The amino acid sequences of these proteins vary widely, but all are located in the periplasm of bacteria. In this study, we investigated the translocation mechanism of representative beta-lactamases in an Escherichia coli model. N-terminal signal sequence analyses, antibiotic activity assay, and direct measurement of translocation of a green fluorescent protein (GFP) reporter fused to beta-lactamases revealed that most were exported via the Sec pathway. However, the Stenotrophomonas maltophilia L2 beta-lactamase was exported via the E. coli Tat translocase, while the S. maltophilia L1 beta-lactamase was Sec dependent. These results show the possible Tat-dependent translocation of beta-lactamases in the E. coli model system. In addition, the mutation of the cytoskeleton-encoding gene mreB, which may be involved in the spatial organization of penicillin-binding proteins, decreased the MIC of beta-lactams for beta-lactamase-producing E. coli. These findings provide new knowledge about beta-lactamase translocation, a putative new target for addressing beta-lactamase-mediated resistance.
Antimicrobial Agents and Chemotherapy 12/2008; 53(1):242-8. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: BES-1 is an atypical extended-spectrum β-lactamase (ESBL), which only shares 47% identity with CTX-M enzymes, its most closely related ESBL. CTX-Ms and BES-1 hydrolyze preferentially Cefotaxime among third-generation cephalosporins. However, BES-1 differs from CTX-Ms by significant activity against Ceftazidime. A comparative structural study of BES-1 was undertaken to investigate the structural features involved in its peculiar properties. Methods: The crystal structure of BES-1 was determined at 1.5 Å resolution and compared with that of CTX-M-9. Molecular docking was then performed in BES-1 catalytic pocket with benzylpenicillin and third-generation cephalosporins. Results: BES-1 general structure was close to that of other class A β-lactamases, especially a conserved disposition of the catalytic residues (Ser70, Ser130, Glu166). However, BES-1 presented an enlargement of the entrance of its active site compared with CTX-M-9. This enlargement was mainly due to the original shape of the loop harboring the residues 101 to 106. This loop shifted 1.2 to 2 Å compared to that of CTX-M-9. Consequently, the residues 167 to 179 of the Ω loop, which is known to impair the accommodation of third-generation cephalosporins, also shifted 0.6 to 1.5 Å. Finally, the residue Arg220 established hydrogen bonds with residues Thr237. This last interaction led to a conformation of Thr237 hydroxyl group, which favors the accommodation of the carboxylic group of cephalosporins. Conclusion: The crystal structure of ESBL BES-1 exhibits a major enlargement of the binding site because of an original positioning of residues 101 to 106 and the association of the hydrogen-bound residues Thr237 and Arg 220, which favor the accommodation of third generation cephalosporins.
Infectious Diseases Society of America 2008 Annual Meeting; 10/2008
[Show abstract][Hide abstract] ABSTRACT: Background: Of the four classes of β-lactamases, class A enzymes are those most frequently encountered in clinical isolates. Oxyimino β-lactams escape the hydrolytic activity of most class A β-lactamases. However, their widespread use has led to the emergence of the class A extended-spectrum β-lactamases (ESBLs), especially the CTX-M-type enzymes, which have become highly prevalent since the late 90’s. Unlike the TEM and SHV class A β-lactamases, there have been few structural studies of the CTX-M family. In addition, the first and last steps of the catalytic reaction have never been investigated in class A β-lactamases. In this work, we determined the crystal structure of CTX-M-9 in complexes with b-lactams and hydrolyzed b-lactams to capture the enzyme at these steps in the catalytic reaction. Methods: An acylation-defective mutant of CTX-M-9 was constructed at position 70 by site-directed mutagenesis experiment. The mutant was crystallized and soaked with intact and hydrolyzed β-lactams. The structures of the corresponding complexes were then determined by X-ray diffraction. Results and conclusion: The structure of CTX-M-9 was obtained at high resolution in complex with β-lactams representing the enzyme before the acylation transition state and just before the expulsion of the hydrolyzed product. It emerged from these structures: (i) the role of Arg-276 in the capture of β-lactams in the solvent, (ii) a shift of the β3 strand and a high density of hydrogen bonds which allow the accommodation of third-generation β-lactams and (iii) a molecular rearrangement of β-lactams during hydrolysis which induces auto-expulsion of the hydrolyzed β-lactams.
Infectious Diseases Society of America 2008 Annual Meeting; 10/2008
[Show abstract][Hide abstract] ABSTRACT: Background: β-lactamases are a family of diverse periplasmic proteins that have a great ability to evolve. This poses a threat in the emergence of novel antibiotic resistance pathogens, especially in Escherichia coli. β-lactamases must be exported into the periplasm of Gram-negative bacteria to induce β-lactam resistance. In this study, we investigated the translocation mechanism of several β-lactamases from diverse origins in Escherichia coli. Methods: Signal sequences of β-lactamases were analyzed in silico with the TATFIND 1.2 and TatP programs. The native forms and fusions to GFP of six representative β-lactamases (TEM-1, CTX-M-14, BlaC, L1, L2, and AmpC) were then investigated in wildtype E. coli and isogenic mutants defective for the export systems Tat, Sec or the cytoskeleton encoding gene mreB. The export was monitored by the determination of MIC, cellular fractionations, enzymatic assays, western-blot and under a confocal fluorescent microscope. Results: N-terminal signal sequence analyses, antibiotic activity assay and direct measurement of translocation of GFP reporter fused to β-lactamases revealed that most were exported via the Sec-pathway. However, L2 β-lactamase was exported via the Tat translocase. The mutation of the cytoskeleton encoding gene mreB also weakly altered the translocation of β-lactamases. Conclusion: These results show for the first time the possibility of Tat-dependent antibiotic resistance in E. coli.
Infectious Diseases Society of America 2008 Annual Meeting; 10/2008
[Show abstract][Hide abstract] ABSTRACT: Shiga toxin-producing Escherichia coli (STEC) has been associated with food-borne diseases ranging from uncomplicated diarrhea to hemolytic-uremic syndrome (HUS). While most outbreaks are associated with E. coli O157:H7, about half of the sporadic cases may be due to non-O157:H7 serotypes. To assess the pathogenicity of STEC isolated from dairy foods in France, 40 strains isolated from 1,130 raw-milk and cheese samples were compared with 15 STEC strains isolated from patients suffering from severe disease. The presence of genes encoding Shiga toxins (stx(1), stx(2), and variants), intimin (eae and variants), adhesins (bfp, efa1), enterohemolysin (ehxA), serine protease (espP), and catalase-peroxidase (katP) was determined by PCR and/or hybridization. Plasmid profiling, ribotyping, and pulsed-field gel electrophoresis (PFGE) were used to further compare the strains at the molecular level. A new stx(2) variant, stx(2-CH013), associated with an O91:H10 clinical isolate was identified. The presence of the stx(2), eae, and katP genes, together with a combination of several stx(2) variants, was clearly associated with human-pathogenic strains. In contrast, dairy food STEC strains were characterized by a predominance of stx(1), with a minority of isolates harboring eae, espP, and/or katP. These associations may help to differentiate less virulent STEC strains from those more likely to cause disease in humans. Only one dairy O5 isolate had a virulence gene panel identical to that of an HUS-associated strain. However, the ribotype and PFGE profiles were not identical. In conclusion, most STEC strains isolated from dairy products in France showed characteristics different from those of strains isolated from patients.
Applied and Environmental Microbiology 05/2008; 74(7):2118-28. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Magnetotactic bacteria are a diverse group of motile prokaryotes that are ubiquitous in aquatic habitats and cosmopolitan
in distribution. In this study, we collected magnetotactic bacteria from the Mediterranean Sea. A remarkable diversity of
morphotypes was observed, including multicellular types that seemed to differ from those previously found in North and South
America. Another interesting organism was one with magnetosomes arranged in a six-stranded bundle which occupied one third
of the cell width. The magnetosome bundle was evident even under optic microscopy. These cells were connected together and
swam as a linear entire unit. Magnetosomes did not always align up to form a straight linear chain. A chain composed of rectangle
magnetosomes bent at a position with an oval crystal. High resolution transmission electron microscopy analysis of the crystal
at the pivotal position suggested uncompleted formation of the crystal. This is the first report of Mediterranean magnetotactic
bacteria, which should be useful for studies of biogeochemical cycling and geohistory of the Mediterranean Sea.
Journal of Ocean University of China 10/2007; 6(4):355-359.
[Show abstract][Hide abstract] ABSTRACT: Shigella surface protein IcsA and its cytoplasmic derivatives are localized to the old pole of rod-shaped cells when expressed in Escherichia coli. In spherical mreB cells, IcsA is targeted to ectopic sites and close to one extremity of actin-like MamK filament. To gain insight into the properties of the sites containing polar material, we studied the IcsA localization in spherical cells. GFP was exported into the periplasm via the Tat pathway and used as a periplasmic space marker. GFP displayed zonal fluorescence in both mreB and rodA-pbpA spherical E. coli cells, indicating an uneven periplasmic space. Deconvolution images revealed that the cytoplasmic IcsA fused to mCherry was localized outside or at the edge of the GFP zones. These observations strongly suggest that polar material is restricted to the positions where the periplasm possesses particular structural or biochemical properties.
Biochemical and Biophysical Research Communications 03/2007; 353(2):493-500. · 2.28 Impact Factor