Microbial Communities

Several components are frequently distinguished among deep-sea vent microbial communities: free-living and surface-bound bacteria in the warm and cold areas, invertebrate symbionts, thermophilic (and hyperthermophilic) organisms.

13.2.1 Free Living and Attached Bacteria in Warm and Cold Vent Areas

When approaching an active vent side with a manned-submersible, it is very common to observe bacterial mats on the sea bottom that can sometimes reach a thickness of several centimetres. The bacteria constituting these mats showed various morphotypes and metabolic types as well [19], but sulphur-oxidising bacteria showing filamentous forms (Beggiatoa, Thiotrix) seem to be dominant within these communities. In cold and warm seawater, at a short distance from the vents, bacterial densities are similar to those found in coastal marine waters, and range from 6x104 to 109 cells/ml. Autotrophic organisms may account for 10 to 2.2x106 cells/ml, the highest concentrations being unusual in common oceanic waters. However, measurements of autotrophic activities showed that the biomass production of free-living autotrophic bacteria was too low to sustain the invertebrate biomass present at the vent sites [23].

From the surface-bound and free-living communities, a variety of metabolic types (autotrophs, heterotrophs, sulphur-oxidisers, methane-oxidisers, nitrifyers, denitrifyers, sulphate reducers, etc.) has been reported [30]. Specific to the vent sites are the autotrophic and heterotrophic sulphur-oxidisers that gain their energy from aerobic oxidation of reduced sulphur compounds and particularly hydrogen sulphide, which is quite abundant in the vent fluids [14]. To a smaller extent, manganese-oxidising bacteria have also been reported [13] from the vent environment and the plumes. It must be noted that mesophilic halotolerant (16 % to 27 % NaCl) bacteria have been found in different vent plume samples collected at the Juan de Fuca Ridge, where they may form up to 28 % of the total microbial community, as estimated by means of MPN (most probable number method) viable counts [18]. Several isolates have been assigned to the genus Halomonas and Marinobacter, but they have not been fully identified. These organisms may have originated from sub sea-floor brine environments.

A few species have been isolated in pure culture and fully characterised. They are listed in Table 13.2. These strains do not reflect the real biodiversity of free-living mesophiles, but were obtained from experiments dedicated to autotrophy and sulphur oxidation, artificial surface colonisation or specific enrichment and screening for exopolysaccharide production [33].

Table 13.2 Mesophilic bacterial species isolated from deep-sea hydrothermal vents.

Genus

Species

Metabolism

Temperature range

References

Thiomicrospira

Crunogena

sulphur-oxidiser

mesophilic

38

Thibacillus

Hydrothermalis

sulphur-oxidiser

mesophilic

39

Hyphomicrobium

Hirschiana

heterotroph

mesophilic

4G

Hyphomicrobium

Jannaschiana

heterotroph

mesophilic

4G

Alteromonas

Macleodii/jijiensis

heterotroph

mesophilic

41

Alteromonas

Infernus

heterotroph

mesophilic

42

Vibrio

Diabolicus

heterotroph

mesophilic

43

13.2.2 Invertebrate Symbionts

The discovery of abundant animal communities (unknown species of tube worms, bivalves, polychaetes, shrimps, etc.) in the close vicinity of warm (10-30°C) water emissions was totally unexpected. How can we explain such animal (primary consumers) concentrations when the total darkness prevents photosynthetic primary production? Tissues of tube worms (Vestimentifera) and bivalves were examined with SEM (scanning electron microscope) and TEM (transmission electron microscope), and it was shown that they contained abundant bacterial cells. Further biochemical analyses indicated that these endosymbiotic bacteria were able to gain their energy from aerobic oxidation of hydrogen sulphide, and to fix carbon dioxide into organic matter [7].

Vestimentifera

Riftia pachyptila is the most spectacular tube worm from the eastern Pacific vent environments. It lives within a tube made of chitin and proteins from which a plume of red gills appears, and the biggest individuals can reach a height of 2 metres. This organism does not have a digestive tract (no mouth or anus) but a specific organ called the trophosome which is full sulphur-oxidising bacteria [6]. Nutrients required for bacterial life, such as carbon dioxide as a carbon source, hydrogen sulphide as an energy source and molecular oxygen as an electron acceptor, are collected from the environment by the worm and provided to the bacteria through blood transportation. Oxygen and hydrogen sulphide are transported towards the trophosome endosymbionts on two distinct sites of a blood pigment, which is rather similar to haemoglobin [16].

Molluscs

Vent molluscs (bivalves and gastropods) also harbour symbionts. The large bivalve Calyptogena magnifica, which lives in several eastern Pacific vent sites, has a digestive tract that is probably not functional. The symbionts are sulphur-oxidisers and are located in specific cells within the gill filaments. Mussels such as Bathymodiolus are present with different species in all vent sites explored so far. They have a functional digestive tract but they still have symbionts within their gill filaments [25].

Fig. 13.2 Invertebrates from deep-sea hydrothermal vents with endo- and ecto-symbionts. Upper left: Bathymodiolus azorensis and Bythograeid crab (Segonzacia) in Menez Gwen (850 m depth); upper right: swarms of Rimicaris exoculata on chimney walls in the Rainbow hydrothermal vent field near the Azores Islands (2300 m depth); lower left: Alvinella pompejana and Paralvinella grasslei at 13°N on the East Pacific Rise (2630 m depth); lower right: Riftiapachyptila (Siboglinidae) at 13°N on the East Pacific Rise (2630 m depth). Courtesy of Ifremer/Atos, Ifremer/Phare and Ifremer/Hydronaut.

Fig. 13.2 Invertebrates from deep-sea hydrothermal vents with endo- and ecto-symbionts. Upper left: Bathymodiolus azorensis and Bythograeid crab (Segonzacia) in Menez Gwen (850 m depth); upper right: swarms of Rimicaris exoculata on chimney walls in the Rainbow hydrothermal vent field near the Azores Islands (2300 m depth); lower left: Alvinella pompejana and Paralvinella grasslei at 13°N on the East Pacific Rise (2630 m depth); lower right: Riftiapachyptila (Siboglinidae) at 13°N on the East Pacific Rise (2630 m depth). Courtesy of Ifremer/Atos, Ifremer/Phare and Ifremer/Hydronaut.

It has been reported that particularly by Bathymodiolus species possess methanotrophic symbionts [8], and dual symbiosis (methane-oxidising bacteria and sulphur-oxidising bacteria within the same host) have been described for certain mid-Atlantic Ridge mussel populations. Despite many attempts, no symbiont has been cultured in a laboratory so far. Their physiology has been deduced from detection of enzymes involved in sulphide oxidation and autotrophic carbon dioxide fixation, or by analysing their rRNA (5S, 16S) sequences which demonstrated that they belong to the gamma subdivision of Proteobacteria [12] (see phylogenetic tree, Fig. 12.1 in Chap. 12).

In the western Pacific, several species of gastropods inhabit active vent sites. Although less studied, these snails also harbour autotrophic bacteria within their gill filaments.

An important question for these symbionts is the problem of transmission along their life cycles. Molecular techniques based on amplification (PCR) of genes encoding for 16S rRNA, and in situ hybridisation showed that a direct (vertical) transmission exists for the bivalve Calyptogena magnifica through their oocytes [4]. However, in the case of the tube worm Riftia, symbionts probably come from the environment and infest the worm after its metamorphosis.

Polychaetous Annelids

While Bivalve and Vestimentifera live in moderate temperature areas, other invertebrates thrive close to smoker walls and hence are exposed to elevated temperatures. This is the case for the polychaetous annelid Alvinella pompejana (Fig. 13.2), whose one individual has even been photographed [9] while twisted around a temperature probe at 100°C! These animals build the tube in which they live onto the outer walls of active smokers from the eastern Pacific. The temperature of their environment is most probably in the range 40-60°C. Alvinella is probably one of the most thermotolerant metazoans. These animals harbour a very dense epibiotic microbial community on their integuments, and some epibionts are inserted at very precise locations, as confirmed by structural modification of the worm's cuticule. SEM observations [7] showed various morphotypes (filaments, rods, cocci, twisted and stalked forms), and some of them have been successfully cultured in the laboratory, showing their rather high metabolic diversity [30].

However, the dominant morphotypes that consist of filaments that are visible to the naked eye again escaped culturing. Analysis of their 16S rRNA revealed that they belong to the epsilon subdivision of Proteobacteria, a phylogenetic lineage whose cultured known members are microaerophilic or anaerobic and metabolise sulphur compounds [5]. (See Fig. 12.1 in Chap. 12 for the phylognetic tree.)

Crustaceans

According to their geographic location, vent invertebrate communities are characterised by specific assemblages (tube worms and Alvinellid worms in the eastern Pacific, Gastropods in the western Pacific, for instance). Atlantic vents are unique because of their spectacular clouds of shrimps swarming around active smokers. These shrimps belong to several genera, and the genus Rimicaris is one of the most abundant and most studied. Mouth parts of these shrimps are enlarged and covered by epibiotic filamentous and rod-shaped bacteria. Using molecular techniques, it was demonstrated that these ectosymbionts also belong to the epsilon subdivision of Proteobacteria [28]. They are not dependent on their host because they have been also found on sulphide rocks, even with no shrimps around. The trophic role of the epibiotic bacteria has not been clearly demonstrated for either annelid or crustacean symbiosis.

13.2.3 Thermophiles and Hyperthermophiles

Among the physical parameters influencing life, temperature is probably the most studied. For centuries, heat has been used (and is still being used) for bacterial decontamination and sterilisation. The first thermophilic organism (living optimally above 60°C) was discovered in the late 1960s. Thermus aquaticus was isolated by T. Brock [3] from a hot spring in Yellowstone National Park (USA). More than 30 years later, the enzyme DNA polymerase from Thermus aquaticus made DNA amplification possible through the polymerase chain reaction (PCR) with applications in basic biology, medical sciences, food microbiology and even criminology. In 1977, T. Brock isolated Sulfolobus acidocaldarius, the first hyperthermophilic organism (the optimal temperature for growth is above 80°C), a member of the third domain of life discovered the same year by C. Woese: the Archaea [34].

Novel Hydrothermal Vent in the Bacteria Domain

The occurrence of hot fluids venting at temperatures up to 350°C certainly influenced the search for hyperthermophilic organisms, with the aim of discovering organisms growing at above 100°C (the temperature of boiling water under atmospheric pressure). However, enrichment cultures of hydrothermal chimney samples at temperatures below 60°C allowed isolation of several organisms [1] from the Bacteria domain (Table 13.3). The first microorganisms isolated were strict anaerobes of the genus Thermotoga and Thermosipho fermenting organic matter. Moreover, among anaerobes, an autotrophic organism, Desulfurobacterium thermolithotrophum, which utilises hydrogen as the electron donor and thiosulphate as the electron acceptor, was described. Later, aerobic thermophiles were also described for the genera Thermus and Bacillus. These organisms were not the first found because the thermal gradient is very steep: only a few decimetres may be separating the hot anaerobic zone from the cold oxygenated deep water, and the border line between anaerobiosis and aerobiosis is at about 30°C. However, certain hydrothermal chimneys have a porosity that allows cold water intake and results in an internal circulation of warm water, slightly oxygenated at temperatures around 70°C. Further, relative genera such as Oceanothermus, Vulcanithermus, Caldithrix, Caloranaerobacter were isolated and described. Thermophilic sulphate-reducers such as Thermodesulfator indiensis, or iron-reducers such as Deferribacter abyssi have also been described recently.

In order to improve the knowledge about the diversity of vent Prokaryotes, molecular approaches (PCR amplification of 16S rRNA genes) were also used. For the Bacteria domain, this approach revealed the importance of the epsilon subgroup of Proteobacteria. Furthermore, culture methods allowed isolation of novel members from this lineage, belonging to the genera Nautilia, Caminibacter or Sulfurospirillum. These organisms are anaerobes or microaerophilic, and may use nitrate and/or sulphur as electron acceptors.

Novel Hydrothermal Vent in the Archaea Domain

Two lineages are distinguished for Archaea: Euryarchaeota and Crenarchaeota. The first lineage gathers organisms from various metabolic types (Table 13.4). The most frequently isolated lineages from deep-sea hydrothermal vents are Thermococcales, with the genera Pyrococcus and Thermococcus, for a total of about 15 abyssal species [1]. All are hyperthermophilic, grow optimally above 80°C, ferment organic compounds and particularly peptides. Most of them require elemental sulphur or a sulphur-containing amino-acid (cystein) in the culture medium, and produce hydrogen sulphide. The dominance of Thermococcales in collections of hydrothermal microorganisms does not necessarily correspond to the natural situation. Although the number of species described certainly corresponds to a real diversity, it must be noticed that these organisms are relatively easy to grow in the laboratory. These organisms are strict anaerobes, but may resist oxygen exposure if this occurs at low temperatures (< 20°C), or under organic carbon starvation [26]. All these observations could explain why they apparently dominate in enrichment cultures and consequently strain collections. Recently, a third Thermococcales genus, Paleococcus ferrophilus was described. This organism requires ferrous iron as the electron acceptor in the absence of elemental sulphur.

Among the Euryarchaeota, two sulphate-reducing species were described within the genus Archaeoglobus. Close to these sulphate-reducers, the genus Geoglobus, which is able to use ferric iron as electron acceptor was recently isolated. Methanogens (producing methane from hydrogen and carbon dioxide) belonging to the genera Methanococcus, renamed Methanocoaldococcus, Methanothermococcus and Methanopyrus were currently isolated. For several years, Methanopyrus kandleri remained the most thermophilic organism on Earth, growing optimally at 106°C, with a maximum temperature for growth at 110°C.

Crenarchaeota constitute the second main lineage for Archaea (a third lineage, Korarchaeota, is only known from 16S rRNA sequences). All are hyperthermophilic organisms metabolising sulphur or sulphur containing compounds. Several species have been isolated from deep-sea hydrothermal vents. They are strictly anaerobic, but show various metabolic types. They belong to the genera Desulfurococcus, Staphylococcus, Ignicoccus, Pyrodictium and Pyrolobus. The last genus includes the species Pyrolobus fumarii, which grows optimally at 110°C, with a maximum at 113°C. This is the most thermophilic organism presently known on Earth. Moreover, it can survive 2 hours exposure in an autoclave (120°C).

An additionnal lineage has been proposed within the Archaea with the discovery of Nanoarchaeum equitans, a small sized-organism (400 nm) living in very close association with Ignicoccus cells.

Comparison of thermophilic and hyperthermophilic genera isolated from deep-sea hydrothermal vents and other hot environments shows rather few differences. Acidophilic hyperthermophiles such as Sulfolobus have not been reported from deep-sea vents. Although hydrothermal fluids are acidic, they are quickly diluted into neutral (pH = 8) seawater. Consequently, a hot (less than 113°C) and acidic (pH < 5-6) biotope has not been reported yet at a vent site. Certain species such as Staphylothermus marinus have been reported from shallow and deep hot springs, and probably have a ubiquitous distribution. For most of these hyperthermophiles, a novelty was found at the species level as confimed by the numerous species called "profundus", "abyssi" or "hydrothermalis". However, several genera, particularly for the thermophilic Archaea recently described, seem to be only present in deep-sea vents (Pyrolobus, Paleococcus, Desulfurobacterium, Nautilia, Caminibacter, etc.). However, it is not firmly proven that they only thrive in deep environments, and perhaps shallow vent species are still waiting to be detected.

Acidithiobacillus Ferrooxidans

Fig. 13.3 Novel micro-organisms from deep-sea hydrothermal vents. Upper left: Pyrolobus fumarii, the most thermophilic organism known on Earth (reprinted from Blöchl et al. [37] with permission from Springer-Verlag), size 2.9 |m; upper right: Pyrococcus abyssi; lower: Desulfurobacterium thermolithotrophum.

Table 13.3 Novel Bacteria from deep-sea hydrothermal vents. (A: autotroph; H: heterotroph; F/S°: fermentative, elemental sulphur required; in the energy column, compounds on the left of "," are electron donors, compounds on the right of "/" are electron acceptors; |O2: microaerophilic; OC: organic carbon).

Table 13.3 Novel Bacteria from deep-sea hydrothermal vents. (A: autotroph; H: heterotroph; F/S°: fermentative, elemental sulphur required; in the energy column, compounds on the left of "," are electron donors, compounds on the right of "/" are electron acceptors; |O2: microaerophilic; OC: organic carbon).

Lineage

Genus

Species

Carbon source

Energy

Optimal T (°C)

Ref.

Aquificales

Persephonella

Marina

SA2-

70

44

Guayma-sensis

S2O32-

80

44

Hydrogeno-Phila

lO2 S2O32-

70

45

Desulfuro-bacteriales

Desulfuro-bacterium

Thermolitho-trophum

A

H2/S°

70

46

Crinifex

60-65

47

Thermo-togales

Marinitoga

Camini

H

F/S°

55

48

Piezzophila

H

F,S°

65

49

Thermotoga

sp.

H

F

80

50

Thermosipho

Melanensis

H

F/S°

70

51

Japonicus

H

F/S°

72

desulfuro-

desulfuro-

bacterium

Hydrogeno-Philum

A

H2/S°

75

53

Thermus/

Deino-

coccus

Thermus

sp.

H

OC/O2

70-80

philus

GY1211

H

OC/O2

75

55

Marinithermus

Hydro -thermalis

H

OC/O2

67

56

Vulcanithermus

57

Oceanithermus

Profundus

|O,

60

58

Deferri-bacteriales

Deferribacter

Desulfurican s abyssi

H

OC/S° NO3-, As

60-65

60

Table 13.3 (continued)

Firmicutes

Bacillus

sp.

H

o2/f

60-80

61

Caloranaero-bacter

azorensis

H

F

65

62

Caminicella

sporogenes

H

F

55-60

63

Proto-bacteria

Caminib acter

64

Nautilia

65

Sulfospirrilum

66

Thermodesul-fatator

67

Novel lineage

Caldithrix

abyssi

H/A

acetate, H2/NO3-F

65

62

Table 13.4 Novel Arachaea from deep-sea hydrothermal vents.

Lineage

Genus

Species

Carbon source

Energy

Optimal T (°C)

Ref.

Eury-archaeota

Methano-

Methano-

jannaschii

A

h2/co2

85

69

coccales

caldo-

infernus

A

h2/co„

85

70

coccus

vulcanius

A

h2/co„

80

71

Methano-

okinawensis

A

H2/CO2

60-65

72

thermo-coccus

Methano-

Methano-pyrus

kandleri

A

H2/CO2

98

73

pyrales

Thermoco-

Thermo-coccus

guaymensis

H

F,S°

88

74

ccales

aggregans

H

F,S°

88

74

barossi

H

F,S°

82

75

fumicolans

H

F,S°

85

76

hydrothermalis

H

F,S°

85

77

peptonophilus

H

F,S°

85-90

78

siculi

H

F,S°

85

79

chitonophagus

H

F,S°

85

80

barophilus

H

F,S°

85

81

atlanticus

H

F,S°

85

82

gammatolerans

H

F,S°

88

83

Pyrococcus

abyssi

H

F,S°

96

84

glycovorans

H

F,S°

95

85

horikoshii

H

F,S°

98

86

Paleococcus

ferrophilus

H

F,S° Fe+++ required

83

87

Table 13.4 (continued)

Archaeo-globales

Archaeo-globus

profundus

SO,2-

82

88

veneficus

SO32-

75-80

89

Geoglobus

ahangari

Fe

88

90

Cren-archaeota

Desulfuro-coccales

Ignicoccus

pacificus

A

h2/s°

90

91

Staphylo-thermus

marinus

H

F/S°

85-92

92

Pyrodictium

abyssi

H

F/S°

97

93

Pyrolobus

fumarii

S2O,2-

106

37

"Nano-archaeota"

Nano-archaeum

equitans

A?

?

90

94

The existence of very hot fluids certainly drove the search for hyperthermophiles into deep-sea hydrothermal vents. However, the upper temperature limit for life (113°C) reported for a deep vent organism (Pyrolobus fumarii) exceeds the maximum growth temperature of Methanopyrus kandleri, isolated also from a 100 m deep vent in Iceland, or of Pyrodictium occultum isolated from shallow vent at Vulcano Island (Sicily, Italy) only by 3°C.

Carbon, Energy Sources and Electron Acceptors

In the absence of light, deep-sea microorganisms gain their energy from oxido-reduction reactions. A variety of electron donors and acceptors has been found to be used by thermophiles and/or hyperthermophiles (Tables 13.3, 13.4). From these reactions, they obtain energy to fix carbon dioxide or to use organic compounds as carbon sources. Almost all metabolic types already known for chemotrophic Prokaryotes have been found and most of the biogeochemical cycles may function in the vent environment: carbon, sulphur, iron, etc. However, the nitrogen cycle does not seem to function at elevated temperatures. If nitrate and nitrite may be used as electron acceptors by several organisms, they apparently cannot be oxidised at elevated temperature, although aerobic nitrification was reported for moderate thermophiles. Anaerobic nitrication has been recently demonstrated for organisms of the Planctomycetales lineage [2], which utilise nitrite as the electron acceptor to oxidise ammonium. The search and discovery of such organisms in the hyperthermophilic range would definitely establish that an ecosystem can fully function at elevated temperatures.

13.2.4 Specific Adaptations Fluctuations of Environmental Conditions

At deep-sea hydrothermal vents, several parameters may fluctuate and affect the physiology of microorganisms. This is the case for oxygen and nutrient concentrations. Hyperthermophiles are mostly anaerobic and sensitive to exposure to oxygen. This situation may occur in situ as a result of a decrease of venting activity. Using P. abyssi as a model strain, it was shown that, although oxygen was toxic for this strain at growth temperature, P. abyssi was able to survive for several weeks at 4°C in the presence of oxygen. Similarly, this organism was not affected by starvation in a minimal medium (no organic carbon) for at least one month at 4°C, and only minimally at 95°C for several days [26]. Furthermore, cells were more resistant to oxygen under starvation conditions. These results may indicate that at least Pyrococcus, but probably also other vent organisms, are adapted to fluctuations occurring in vent environments, and may be disseminated from one vent field to another.

Hydrostatic Pressure

Among all physical parameters mentioned above, the hydrostatic pressure (of the water column) is the one which is characteristic of all deep vent sites. Responses to an elevated hydrostatic pressure have first been studied in microorganisms from cold deep environments [24, 29, 35]. Piezophilic and even obligate piezophilic organisms have been described, and their physiology and adaptations studied at the molecular level. However, much less is known for deep-sea vent thermophiles. One of the reasons is that during the collection of a black smoker sample, using a retaining-pressure collecting device is not so easy: a single very small metallic particle (frequent in the vent plume) may cause a leak in the sampler. For this reason, all the samples studied were decompressed when brought up to the surface. All enrichment cultures that lead to the isolation of organisms described above were carried out under atmospheric pressure, or a slightly hyperbaric pressure to avoid boiling of culture media for temperatures close to or above 100°C.

Hence, the question of the effect of elevated hydrostatic pressure on deep-sea thermophiles is still open. Several microbiologists exposed deep-sea thermophilic organisms isolated under atmospheric pressure to elevated hydrostatic pressure, using pressurised bioreactors. Several responses were observed: some organisms were baro-sensitive, and showed a slower growth rate under pressure; others were barotolerant, and their growth was not affected by pressure. However, most of the species studied appeared to be barophiles, and their growth rates were enhanced by hydrostatic pressure. Moreover, their optimal growth temperatures increased by a few °C. For instance, for Pyrococcus abyssi, while the maximum growth temperature under atmospheric pressure is 102°C, it reached 105-106°C under elevated hydrostatic pressure. Similarly, the optimum growth temperature increased from 96 to 100°C. Similar observations reported by several authors were compiled and discussed by Deming and Baross [11], who noted that for all baro-hyperthermophiles, the pressure allowing optimal growth was always above the pressure existing at the capture depth (for an organism isolated from a sample collected at 2000 m depth, the optimum pressure for growth was 40 MPa). This point is very remarkable since for baro-psychrophiles the optimum pressure for growth was always below the pressure existing at the capture depth.

Another experimental approach consisted in isolating hyperthermophilic organisms from enrichment cultures (and subcultures) under elevated hydrostatic pressure. This method allowed isolation of a novel Archaea species (Thermococcus barophilus) and a novel Bacteria species (Marinitoga piezzophila). For both organisms, which are not real taxinomic novelties, but only novel species, growth rates were enhanced under hydrostatic pressure, but growth remained possible under atmospheric pressure. In the case of Marinitoga, cell divisions were very slow under atmospheric pressure and cell morphology was clearly modified. As observed for other microorganisms, the maximum growth temperature was increased, but the optimal growth temperature was not affected by pressure increases. Further information was obtained from a study of total proteins of T. barophilus cultured under elevated hydrostatic pressure or atmospheric pressure [27]. Under hydrostatic pressure, an unknown 35 kDa protein was expressed. Under atmospheric pressure, a 60 kDa protein was expressed, which corresponded to a stress protein already known for other hyperthermophilic Archaea. All these data confirm that this Thermococcus species is a true baro-thermophile.

Heavy Metals

Several heterotrophic strains (Acinetobacter, Alteromonas, Pseudomonas, Vibrio) have been isolated from alvinellid Polycheates living on the chimney walls, and appeared resistant (or multi-resistant) to heavy metals such as cadmium, zinc, silver, arsenate and particularly copper [21]. This feature confirmed the adaptation of at least a part of the worm's microflora to the elevated metal concentrations existing in the vent environment. About 20 % of the strains studied harboured one or several plasmids (up to five) of sizes ranging from 4.6 to 157 kb. The occurrence of plasmids with some similarities for strains belonging to different phyla could suggest the existence of genetic transfers in the vent environments. Further studies involving a thermophilic Bacillus from deep-sea vents showed that exposure to cadmium and copper induce the expression of proteins including a manganese superoxide dismutase. Experiments with E. coli demonstrated that superoxide dismutase is involved in the defence against oxidative stress mediated by heavy metals, but the possibility that superoxide dismutase might also reduce metal toxicity by some process of metal storage was not excluded.

Ionising Radiation

Several hyperthermophilic Archaea have been shown to resist ionising radiation, but not as much as Deinococcus radiodurans, the most radioresistant microorganism known so far. In the vent environment, it was reported that invertebrates living on black smoker were exposed to natural radiation one hundred times above that received by Man on Earth. For this reason, responses of the Archaeon Pyrococcus abyssi to ionising radiation have been studied. It was observed that this organism did not show any loss of viability until 2 kGy of y-irradiation. It was then established that P. abyssi did not have a DNA protection mechanism that could explain its radioresistance although the chromosomic DNA was fully restored, under optimal growth conditions, within 2 hours following gamma irradiation. Experimental data indicated that strategies used by P. abyssi included an uncoupling of DNA repair and DNA synthesis, and a prevention of accumulation of genetic mistakes by exporting damaged DNA.

In order to isolate more radioresistant hyperthermophiles, enrichment cultures under conditions designed for growth of Thermococcales were exposed to elevated doses of radiation (20 to 30 kGy), and then processed for strain isolation [22]. Three radioresistant strains were obtained, and the most radioresistant (about the same range as Deinococcus radiodurans) was fully described as a novel species: Thermococcus gammatolerans.

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