Claire Geslin

The manganese and iron superoxide dismutases protect Escherichia coli from heavy metal toxicity.

Superoxide dismutases (SODs) are vital components that defend against oxidative stress through decomposition of superoxide radical. Escherichia coli contains two highly homologous SODs, a manganese- and an iron-containing enzyme (Mn-SOD and Fe-SOD, respectively). In contrast, a single Mn-SOD is present in Bacillus subtilis. In E. coli, the absence of SODs was found to be associated with an increased sensitivity to cadmium, nickel and cobalt ions. Mutants lacking either sodA or sodB exhibited metal resistance to levels comparable to that of the wild-type strain. Although sod-deficient mutant cells were more resistant to zinc than their wild-type counterpart, no differences between the strains were observed in the presence of copper. In B. subtilis, the sodA mutation had no effect on cadmium and copper resistance. These results suggest that intracellular generation of superoxide by cadmium, nickel and cobalt is toxic in E. coli. They support the participation of sod genes in its protection against metal stress.

Molecular diversity of new Thermococcales isolates from a single area of hydrothermal deep-sea vents as revealed by randomly amplified polymorphic DNA fingerprinting and 16S rRNA gene sequence analysis

ABSTRACT Members of the Thermococcales are anaerobic Archaea belonging to the kingdom Euryarchaea that are studied in many laboratories as model organisms for hyperthermophiles. We describe here a molecular analysis of 86 new Thermococcales isolates collected from six different chimneys of a single hydrothermal field located in the 13°N 104°W segment of the East Pacific ridge at a depth of 2,330 m. These isolates were sorted by randomly amplified polymorphic DNA (RAPD) fingerprinting into nine groups, and nine unique RAPD profiles were obtained. One RAPD group corresponds to new isolates of Thermococcus hydrothermalis, whereas all other groups and isolates with unique profiles are different from the 22 reference strains included in this study. Analysis of 16S rRNA gene sequences of representatives of each RAPD group and unique profiles showed that one group corresponds to Pyrococcus strains, whereas all the other isolates are Thermococcus strains. We estimated that our collection may contain at least 11 new species. These putative species, isolated from a single area of hydrothermal deep-sea vents, are dispersed in the 16S rRNA tree among the reference strains previously isolated from diverse hot environments (terrestrial, shallow water, hydrothermal vents) located around the world, suggesting that there is a high degree of dispersal of Thermococcales. About one-half of our isolates contain extrachromosomal elements that could be used to search for novel replication proteins and to develop genetic tools for hyperthermophiles.

TPV1, the first virus isolated from the hyperthermophilic genus Thermococcus

We describe a novel virus, TPV1 (Thermococcus prieurii virus 1), which was discovered in a hyperthermophilic euryarchaeote isolated from a deep-sea hydrothermal chimney sample collected at a depth of 2700 m at the East Pacific Rise. TPV1 is the first virus isolated and characterized from the hyperthermophilic euryarchaeal genus Thermococcus. TPV1 particles have a lemon-shaped morphology (140 nm × 80 nm) similar to the structures previously reported for Fuselloviruses and for the unclassified virus-like particle PAV1 (Pyrococcus abyssi virus 1). The infection with TPV1 does not cause host lysis and viral replication can be induced by UV irradiation. TPV1 contains a double-stranded circular DNA of 21.5 kb, which is also present in high copy number in a free form in the host cell. The TPV1 genome encompasses 28 predicted genes; the protein sequences encoded in 16 of these genes show no significant similarity to proteins in public databases. Proteins predicted to be involved in genome replication were identified as well as transcriptional regulators. TPV1 encodes also a predicted integrase of the tyrosine recombinase family. The only two genes that are homologous between TPV1 and PAV1 are TPV1-22 and TPV1-23, which encode proteins containing a concanavalin A-like lectin/glucanase domain that might be involved in virus-host recognition.

MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms

BackgroundThe production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms.ResultsWe present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented.ConclusionsThe objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation.

Observation of virus-like particles in high temperature enrichment cultures from deep-sea hydrothermal vents

A systematic search was carried out on samples collected in various geographically distant hydrothermal sites located on the East Pacific Rise (EPR 9 degrees N and 13 degrees N) and Mid-Atlantic Ridge (MAR 36 degrees N and 37 degrees N) to investigate the diversity of virus-like particles (VLPs) from deep-sea vents. Eighty-nine positive enrichment cultures were obtained from one hundred and one crude samples at 85 degrees C. VLPs were detected by electron microscopy in fifteen different enrichments. Among the different morphotypes observed, the lemon-shaped type prevailed but rods and novel pleomorphic morphologies were also observed. Several observations strongly suggested that host strains of the novel VLPs belong to the hyperthermophilic euryarchaeal order Thermococcales.

Living Side by Side with a Virus: Characterization of Two Novel Plasmids from Thermococcus prieurii, a Host for the Spindle-Shaped Virus TPV1

ABSTRACT Microbial cells often serve as an evolutionary battlefield for different types of mobile genetic elements, such as viruses and plasmids. Here, we describe the isolation and characterization of two new archaeal plasmids which share the host with the spindle-shaped Thermococcus prieurii virus 1 (TPV1). The two plasmids, pTP1 and pTP2, were isolated from the hyperthermophilic archaeon Thermococcus prieurii (phylum Euryarchaeota), a resident of a deep-sea hydrothermal vent located at the East Pacific Rise at 2,700-m depth (7�25′24 S, 107�47′66 W). pTP1 (3.1 kb) and pTP2 (2.0 kb) are among the smallest known plasmids of hyperthermophilic archaea, and both are predicted to replicate via the rolling-circle mechanism. The two plasmids and the virus TPV1 do not have a single gene in common and stably propagate in infected cells without any apparent antagonistic effect on each other. The compatibility of the three genetic elements and the high copy number of pTP1 and pTP2 plasmids (50 copies/cell) might be useful for developing new genetic tools for studying hyperthermophilic euryarchaea and their viruses.

Sulfur vesicles from Thermococcales: A possible role in sulfur detoxifying mechanisms

The euryarchaeon Thermococcus prieurii inhabits deep-sea hydrothermal vents, one of the most extreme environments on Earth, which is reduced and enriched with heavy metals. Transmission electron microscopy and cryo-electron microscopy imaging of T. prieurii revealed the production of a plethora of diverse membrane vesicles (MVs) (from 50 nm to 400 nm), as is the case for other Thermococcales. T. prieurii also produces particularly long nanopods/nanotubes, some of them containing more than 35 vesicles encased in a S-layer coat. Notably, cryo-electron microscopy of T. prieurii cells revealed the presence of numerous intracellular dark vesicles that bud from the host cells via interaction with the cytoplasmic membrane. These dark vesicles are exclusively found in conjunction with T. prieurii cells and never observed in the purified membrane vesicles preparations. Energy-Dispersive-X-Ray analyses revealed that these dark vesicles are filled with sulfur. Furthermore, the presence of these sulfur vesicles (SVs) is exclusively observed when elemental sulfur was added into the growth medium. In this report, we suggest that these atypical vesicles sequester the excess sulfur not used for growth, thus preventing the accumulation of toxic levels of sulfur in the hosts cytoplasm. These SVs transport elemental sulfur out of the cell where they are rapidly degraded. Intriguingly, closely related archaeal species, Thermococcus nautili and Thermococcus kodakaraensis, show some differences about the production of sulfur vesicles. Whereas T. kodakaraensis produces less sulfur vesicles than T. prieurii, T. nautili does not produce such sulfur vesicles, suggesting that Thermococcales species exhibit significant differences in their sulfur metabolic pathways.

Effect of variation of environmental conditions on the microbial communities of deep-sea vent chimneys, cultured in a bioreactor

Both cultivation and molecular techniques were used to investigate the microbial diversity and dynamic of a deep-sea vent chimney. The enrichment cultures performed in a gas-lift bioreactor were inoculated with a black smoker chimney sample collected on TAG site on the mid-Atlantic ridge. To mimic as close as possible environmental conditions, the cultures were performed in oligotrophic medium with nitrogen, hydrogen and carbon dioxide (N2/H2/CO2) gas sweeping. Also, the temperature was first settled at a temperature of 85°C and colloidal sulphur was added. Then, the temperature was lowered to 60°C and sulphur was omitted. Archaeal and bacterial diversity was studied in both culture and natural samples. Through 16S rRNA gene sequences analysis of the enrichment cultures microorganisms affiliated to Archeoglobales, Thermococcales were detected in both conditions while, Deferribacterales and Thermales were detected only at 65°C in the absence of sulphur. Single-stranded conformational polymorphism and quantitative PCR permit to study the microbial community dynamic during the two enrichment cultures. The effect of environmental changes (modification of culture conditions), i.e. temperature, medium composition, electron donors and acceptors availability were shown to affect the microbial community in culture, as this would happen in their environment. The effect of environmental changes, i.e. temperature and medium composition was shown to affect the microbial community in culture, as this could happen in their environment. The modification of culture conditions, such as temperature, organic matter concentration, electron donors and acceptors availability allowed to enrich different population of prokaryotes inhabiting hydrothermal chimneys.

11 Deep-sea Thermococcales and their Genetic Elements: Plasmids and Viruses

Publisher Summary The first, and now the most numerous, organisms isolated from deep-sea hydrothermal vents belong to the order Thermococcales, within the Euryarchaeota in the domain Archaea. They are in general neutrophilic, strictly anaerobic hyperthermophilic peptide and sugar fermenters, that can reduce elemental sulfur to hydrogen sulfide (Palaeococcus may use ferric iron as electron acceptor), and they are rather easy to handle and to grow in the laboratory, probably because they are oxygen-resistant, if exposure occurs at room temperature or below. Despite the existence of a huge collection of Thermococcales strains, very few plasmidic elements are reported so far in the hyperthermophilic euryarchaeotes. The first is a small (3.5 kb) cryptic plasmid found in a high copy number in the marine species ‘‘P. abyssi’’ strain GE5. This plasmid, pGT5, is a stable multicopy plasmid (25–30 copies per chromosome) and can be isolated from cultures at different growth phase and in the temperature range from 75 up to 105oC. Among the Thermococcales, the most important order of the phylum Euryarchaeota, only one virus is isolated and completely characterized to date. Screening of enrichment cultures of Thermococcales has revealed an unexpected diversity among VLPs (virus-like particles) in deep-sea hyperthermophilic environments.

A simple procedure to determine the infectivity and host range of viruses infecting anaerobic and hyperthermophilic microorganisms

Plaque assay is the method traditionally used to isolate and purify lytic viruses, to determine the viral titer and host range. Whereas most bacterioviruses are either temperate or lytic, the majority of known archeoviruses are not lytic (i.e. they are temperate or chronic). In view of the widespread occurrence of such viruses in extreme environments, we designed an original method, called the inverted spot test, to determine the host range and infectivity of viruses isolated from anaerobic hyperthermophilic and sulfur-reducing microorganisms. Here, we used this approach to prove for the first time the infectivity of Pyrococcus abyssi virus 1 (PAV1) and to confirm the host range of Thermococcus prieurii virus 1 (TPV1), the only two viruses isolated so far from any of the described marine hyperthermophilic archaea (Euryarchaeota phylum, Thermococcales order).