A stress protein is induced in the deep-sea barophilic hyperthermophile Thermococcus barophilus when grown under atmospheric pressure

The whole-cell protein inventory of the deep-sea barophilic hyperthermophile Thermococcus barophilus was examined by one-dimensional SDS gradient gel electrophoresis when grown under different pressure conditions at 85°C (T _opt). One protein (P60) with a molecular mass of approximately 60 kDa was prominent at low pressures (0.3 MPa hydrostatic pressure and 0.1 MPa atmospheric pressure) but not at deep-sea pressures (10, 30, and 40 MPa). About 17 amino acids were sequenced from the N-terminal end of the protein. Sequence homology analysis in the GenBank database showed that P60 most closely resembled heat-shock proteins in some sulfur-metabolizing Archaea. A high degree of amino acid identity (81%–93%) to thermosome subunits in Thermococcales strains was found. Another protein (P35) with molecular mass of approximately 35.5 kDa was induced at 40 MPa hydrostatic pressure but not under low-pressure conditions. No amino acid sequence homology was found for this protein when the 40 amino acids from the N-terminal end were compared with homologous regions of proteins from databases. A PTk diagram was generated for T. barophilus. The results suggest that P _habitat is about 35 MPa, which corresponds to the in situ pressure where the strain was obtained. Received: May 14, 1999 / Accepted: July 30, 1999     

Synchronous effects of temperature, hydrostatic pressure, and salinity on growth, phospholipid profiles, and protein patterns of four Halomonas species isolated from deep-sea hydrothermal-vent and sea surface environments

Four strains of euryhaline bacteria belonging to the genus Halomonas were tested for their response to a range of temperatures (2, 13, and 30 degrees C), hydrostatic pressures (0.1, 7.5, 15, 25, 35, 45, and 55 MPa), and salinities (4, 11, and 17% total salts). The isolates were psychrotolerant, halophilic to moderately halophilic, and piezotolerant, growing fastest at 30 degrees C, 0.1 MPa, and 4% total salts. Little or no growth occurred at the highest hydrostatic pressures tested, an effect that was more pronounced with decreasing temperatures. Growth curves suggested that the Halomonas strains tested would grow well in cool to warm hydrothermal-vent and associated subseafloor habitats, but poorly or not at all under cold deep-sea conditions. The intermediate salinity tested enhanced growth under certain high-hydrostatic-pressure and low-temperature conditions, highlighting a synergistic effect on growth for these combined stresses. Phospholipid profiles obtained at 30 degrees C indicated that hydrostatic pressure exerted the dominant control on the degree of lipid saturation, although elevated salinity slightly mitigated the increased degree of lipid unsaturation caused by increased hydrostatic pressure. Profiles of cytosolic and membrane proteins of Halomonas axialensis and H. hydrothermalis performed at 30 degrees C under various salinities and hydrostatic pressure conditions indicated several hydrostatic pressure and salinity effects, including proteins whose expression was induced by either an elevated salinity or hydrostatic pressure, but not by a combination of the two. The interplay between salinity and hydrostatic pressure on microbial growth and physiology suggests that adaptations to hydrostatic pressure and possibly other stresses may partially explain the euryhaline phenotype of members of the genus Halomonas living in deep-sea environments.     

Synchronous Effects of Temperature, Hydrostatic Pressure, and Salinity on Growth, Phospholipid Profiles, and Protein Patterns of Four Halomonas Species Isolated from Deep-Sea Hydrothermal- Vent and Sea Surface Environments

Four strains of euryhaline bacteria belonging to the genus Halomonas were tested for their response to a range of temperatures (2, 13, and 30°C), hydrostatic pressures (0.1, 7.5, 15, 25, 35, 45, and 55 MPa), and salinities (4, 11, and 17% total salts). The isolates were psychrotolerant, halophilic to moderately halophilic, and piezotolerant, growing fastest at 30°C, 0.1 MPa, and 4% total salts. Little or no growth occurred at the highest hydrostatic pressures tested, an effect that was more pronounced with decreasing temperatures. Growth curves suggested that the Halomonas strains tested would grow well in cool to warm hydrothermal-vent and associated subseafloor habitats, but poorly or not at all under cold deep-sea conditions. The intermediate salinity tested enhanced growth under certain high-hydrostatic-pressure and low-temperature conditions, highlighting a synergistic effect on growth for these combined stresses. Phospholipid profiles obtained at 30°C indicated that hydrostatic pressure exerted the dominant control on the degree of lipid saturation, although elevated salinity slightly mitigated the increased degree of lipid unsaturation caused by increased hydrostatic pressure. Profiles of cytosolic and membrane proteins of Halomonas axialensis and H. hydrothermalis performed at 30°C under various salinities and hydrostatic pressure conditions indicated several hydrostatic pressure and salinity effects, including proteins whose expression was induced by either an elevated salinity or hydrostatic pressure, but not by a combination of the two. The interplay between salinity and hydrostatic pressure on microbial growth and physiology suggests that adaptations to hydrostatic pressure and possibly other stresses may partially explain the euryhaline phenotype of members of the genus Halomonas living in deep-sea environments.     

Purification of a ccb-type quinol oxidase specifically induced in a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F

We investigated for the first time the respiratory chain system of a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F. A membrane-bound ccb-type quinol oxidase, from cells grown at 60 MPa pressure, was purified to an electrophoretically homogeneous state. The purified enzyme complex consisted of four kinds of subunits with molecular masses of 98, 66, 18.5, and 15 kDa, and it contained 0.96 mol of protoheme and 1.95 mol of covalently bound heme c per mol of enzyme. Only protoheme in the enzyme reacted with CO and CN⁻, and the catalytic activity of the enzyme was 50% inhibited by 4 μM CN⁻. The isoelectric point of the native enzyme complex was determined to be 5.0. This enzyme was specifically induced only under conditions of elevated hydrostatic pressure, and high levels were expressed in cells grown at 60 MPa. The membranes isolated from cells grown at atmospheric pressure (0.1 MPa) exhibited high levels of both cytochrome c oxidase and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPDH2)-oxidase activity. These results suggest the presence of two kinds of respiratory chains regulated in response to pressure in the deep-sea bacterium DB-172F. Received: November 25, 1997 / Accepted: December 25, 1997     

Differential scanning calorimetry study on the inner membrane lipids prepared from barotolerant Pseudomonas sp. BT1

We investigated the properties of membrane lipids of barotolerant Pseudomonas sp. BT1 by differential scanning calorimetry and spectrophotometry using a system equipped with a hydrostatic pressure controller. In the case of cells grown under high pressure, an endothermic peak appeared under high-pressure measurement conditions. However, in the case of cells grown at 0.1 MPa, such a peak was not observed. It was also observed on spectrophotometry that the membrane lipids from cells grown at 30 MPa had stable properties in comparison with those grown at 0.1 MPa various hydrostatic pressures and temperatures.     

Solute accumulation in the deep-sea bacterium Photobacterium profundum

The identity and amounts of intracellular solutes in the deep-sea bacterium Photobacterium profundum strain SS9 were studied using nuclear magnetic resonance techniques. P. profundum strain SS9, a moderate piezophile which grows optimally at 20-30 MPa primarily accumulated glutamate and betaine, with lesser amounts of alanine, beta-hydroxybutyrate (beta-HB) and oligomers composed of the beta-HB units when grown at 0.1 MPa to early stationary phase. When grown at the optimal pressure, the cells preferentially increased intracellular concentrations of beta-HB and beta-HB oligomers, while the amino acid pools remained relatively constant. Since the organic solutes increased with increasing external NaCl in the medium, they are functioning as osmolytes. The beta-HB molecules represent a novel class of osmolytes, termed 'piezolytes,' whose cellular levels responded to hydrostatic pressure as well as osmotic pressure. Factors such as cell growth stage and temperature were also examined for their effect on the solute distribution in these cells.     

Effects of the piezo-tolerance of cultured deep-sea eel cells on survival rates, cell proliferation, and cytoskeletal structures

We investigated the pressure tolerance of deep-sea eel (Simenchelys parasiticus; habitat depth, 366–2,630 m) cells, conger eel (Conger myriaster) cells, and mouse 3T3-L1 cells. Although there were no living mouse 3T3-L1 and conger eel cells after 130 MPa (0.1 MPa = 1 bar) hydrostatic pressurization for 20 min, all deep-sea eel cells remained alive after being subjected to pressures up to 150 MPa for 20 min. Pressurization at 40 MPa for 20 min induced disruption of actin and tubulin filaments with profound cell-shape changes in the mouse and conger eel cells. In the deep-sea eel cells, microtubules and some actin filaments were disrupted after being subjected to hydrostatic pressure of 100 MPa and greater for 20 min. Conger eel cells were sensitive to pressure and did not grow at 10 MPa. Mouse 3T3-L1 cells grew faster under pressure of 5 MPa than at atmospheric pressure and stopped growing at 18 MPa. Deep-sea eel cells were capable of growth in pressures up to 25 MPa and stopped growing at 30 MPa. Deep-sea eel cells required 4 h at 20 MPa to finish the M phase, which was approximately fourfold the time required under atmospheric conditions.     

Effects of Hydrostatic Pressure on Growth and Luminescence of a Moderately-Piezophilic Luminous Bacteria Photobacterium phosphoreum ANT-2200

Bacterial bioluminescence is commonly found in the deep sea and depends on environmental conditions. Photobacterium phosphoreum ANT-2200 has been isolated from the NW Mediterranean Sea at 2200-m depth (in situ temperature of 13°C) close to the ANTARES neutrino telescope. The effects of hydrostatic pressure on its growth and luminescence have been investigated under controlled laboratory conditions, using a specifically developed high-pressure bioluminescence system. The growth rate and the maximum population density of the strain were determined at different temperatures (from 4 to 37°C) and pressures (from 0.1 to 40 MPa), using the logistic model to define these two growth parameters. Indeed, using the growth rate only, no optimal temperature and pressure could be determined. However, when both growth rate and maximum population density were jointly taken into account, a cross coefficient was calculated. By this way, the optimum growth conditions for P. phosphoreum ANT-2200 were found to be 30°C and, 10 MPa defining this strain as mesophile and moderately piezophile. Moreover, the ratio of unsaturated vs. saturated cellular fatty acids was found higher at 22 MPa, in agreement with previously described piezophile strains. P. phosphoreum ANT-2200 also appeared to respond to high pressure by forming cell aggregates. Its maximum population density was 1.2 times higher, with a similar growth rate, than at 0.1 MPa. Strain ANT-2200 grown at 22 MPa produced 3 times more bioluminescence. The proposed approach, mimicking, as close as possible, the in situ conditions, could help studying deep-sea bacterial bioluminescence and validating hypotheses concerning its role into the carbon cycle in the deep ocean.     

Iron reduction and mineralization of deep‐sea iron reducing bacterium Shewanella piezotolerans WP3 at elevated hydrostatic pressures

In this study, iron reduction and concomitant biomineralization of a deep‐sea iron reducing bacterium (IRB), Shewanella piezotolerans WP3, were systematically examined at different hydrostatic pressures (0.1, 5, 20, and 50 MPa). Our results indicate that bacterial iron reduction and induced biomineralization are influenced by hydrostatic pressure. Specifically, the iron reduction rate and extent consistently decreases with the increase in hydrostatic pressure. By extrapolation, the iron reduction rate should drop to zero by ~68 MPa, which suggests a possible shut‐off of enzymatic iron reduction of WP3 at this pressure. Nano‐sized superparamagnetic magnetite minerals are formed under all the experimental pressures; nevertheless, even as magnetite production decreases, the crystallinity and grain size of magnetite minerals increase at higher pressure. These results imply that IRB may play an important role in iron reduction, biomineralization, and biogeochemical cycling in deep‐sea environments.     

Pressure and temperature effects on growth and viability of the hyperthermophilic archaeon Thermococcus peptonophilus

We studied the effects of high temperatures and elevated hydrostatic pressures on the physiological behavior and viability of the extremely thermophilic deep-sea archaeon Thermococcus peptonophilus. Maximal growth rates were observed at 30 and 45 MPa although no significant increases in cell yields were detected. Growth at 60 MPa was slower. The optimal growth temperature shifted from 85° C at 30 MPa to 90–95° C at 45 MPa. Cell viability during the stationary phase was also enhanced under high pressure. A trend towards barophily at pressures greater than those encountered in situ at the sea floor was demonstrated at increasing growth temperatures. The viability of cells during starvation, at high temperature (90, 95° C), and at low temperature (10° C) was enhanced at 30 and 45 MPa as compared to atmospheric pressure. These results show that the extremely thermophilic archaeon T. peptonophilus is a barophile. Received: 21 October 1996 / Accepted: 5 February 1997     

Analysis of intracellular pH in the yeast Saccharomyces cerevisiae under elevated hydrostatic pressure: a study in baro- (piezo-) physiology

Hydrostatic pressure is a distinctive feature of deep-sea environments, and this thermodynamic parameter has potentially inhibitory effects on organisms adapted to living at atmospheric pressure. In the yeast Saccharomyces cerevisiae, hydrostatic pressure causes a delay in or cessation of growth. The vacuole is a large acidic organelle involved in degradation of cellular proteins or storage of ions and various metabolites. Vacuolar pH, as determined using the pH-sensitive fluorescent dye 6-carboxyfluorescein, was analyzed in a hydrostatic chamber with transparent windows under elevated hydrostatic pressure conditions. A pressure of 40–60 MPa transiently reduced the vacuolar pH by approximately 0.33. A vma3 mutant defective in vacuolar acidification showed no reduction of vacuolar pH after application of hydrostatic pressure, indicating that the transient acidification is mediated through the function of vacuolar H⁺-ATPase. The vacuolar acidification was observed only in the presence of fermentable sugars, and never observed in the presence of ethanol, glycerol, or 3-o-methyl-glucose as the carbon source. Analysis of a glycolysis-defective mutant suggested that glycolysis or CO₂ production is involved in the pressure-induced acidification. Hydration and ionization of CO₂ is facilitated by elevated hydrostatic pressure because a negative volume change (ΔV < 0) accompanies the chemical reaction. Moreover the glucose-induced cytoplasmic alkalization is inhibited by elevated hydrostatic pressure, probably because of inhibition of the plasma membrane H⁺-ATPase. Therefore, the cytoplasm tends to become acidic under elevated hydrostatic pressure conditions, and this could be crucial for cell survival. To maintain a favorable cytoplasmic pH, the yeast vacuoles may serve as proton sequestrants under hydrostatic pressure. We are investigating the physiological effects of hydrostatic pressure in the course of research in a new experimental field, baro- (piezo-) physiology. Received: January 22, 1998 / Accepted: February 16, 1998     

Vacuolar acidification in Saccharomyces cerevisiae induced by elevated hydrostatic pressure is transient and is mediated by vacuolar H+-ATPase

We analyzed the vacuolar acidification in response to elevated hydrostatic pressure in Saccharomyces cerevisiae. The vacuolar pH, defined using 6-carboxyfluorescein, was directly measured in a hyperbaric chamber with a transparent window under high hydrostatic pressure. The vacuole of strain X2180 became acidified at the onset of pressurization to an extent dependent on the magnitude of pressure applied. A pressure of 40–60 MPa transiently reduced the vacuolar pH by about 0.33 within 4 min. The transient acidification was observed in the presence of D-glucose, D-fructose, or D-mannose as a carbon source, but not 3-o-methyl-D-glucose, ethanol, or glycerol, suggesting that the generation of CO₂ was involved in the process. A vma3 mutant defective in vacuolar acidification showed no reduction of vacuolar pH when hydrostatic pressure was applied. This result indicates that the transient vacuolar acidification induced by elevated hydrostatic pressure is mediated through the function of the vacuolar H+-ATPase. Received: August 21, 1996 / Accepted: November 11, 1996     

Enhanced thermotolerance by hydrostatic pressure in the deep-sea hyperthermophile Pyrococcus strain ES4

Abstract: The combined effect of hydrostatic pressure and heat shock on thermotolerance was examined in the deep-sea hyperthermophilic archaeon Pyrococcus strain ES4. Pressure equivalent to the depth of isolation (22 MPa) enhanced ES4's survival at super-optimal temperatures (101–108°C) relative to low pressure (3 MPa). Pressure also raised the temperature at which a putative heat-shock protein (98 kDa) accumulated. ES4 grown at 95°C and 3 MPa displayed immediate enhanced thermotolerance to 105°C after being shifted to 22 MPa. Cultures grown at 95°C and 22 MPa and then heat shocked at 105°C and 3 MPa retained enhanced thermotolerance after decompression. These results suggest that this deep-sea hyperthermophile has developed pressure-induced responses that include increased survival to hyperthermal conditions.     

Numerical simulation of the mechanics of a yeast cell under high hydrostatic pressure

The mechanical effects of the compression of a yeast cell (Saccharomyces cerevisiae) under high hydrostatic pressure used for the processing of food and food ingredients are modelled and simulated with the finite-element method. The cell model consists of a cell wall, cytoplasm a lipid filled vacuole and the nucleus. Material parameters have been taken from literature or have been derived from thermodynamic relationships of water and lipids under high hydrostatic pressure. The model has been validated for a pressure load up to 250 MPa. Comparison of the volume reduction to in situ experimental observations reveals very good agreement. Dimensional analysis of the governing equations shows that transient pressure application in a high-pressure food process does not enhance structural inactivation (mechanical damage), unless pressure oscillation frequencies of 700 MHz are applied. The deformation of the cell under pressure deviates strongly from isotropic volume reduction. Especially, organelle membranes exhibit large effective strain values. Hydrostatic stress conditions are preserved in the interior part of the cell. A pressure load of 400 MPa, which is critical upon disruption of cell organelle membranes, generates an effective strain up to 80%. In the cell wall, the stress state is heterogeneous. Von-Mises stress reaches the critical value upon failure of the cell wall of 70+/-4 MPa at a pressure load between 415 and 460 MPa.     

Physiological Responses to Stress Conditions and Barophilic Behavior of the Hyperthermophilic Vent Archaeon Pyrococcus abyssi

The physiology of the deep-sea hyperthermophilic, anaerobic vent archaeon Pyrococcus abyssi, originating from the Fiji Basin at a depth of 2,000 m, was studied under diverse conditions. The emphasis of these studies lay in the growth and survival of this archaeon under the different conditions present in the natural habitat. Incubation under in situ pressure (20 MPa) and at 40 MPa increased the maximal and minimal growth temperatures by 4(deg)C. In situ pressure enhanced survival at a lethal high temperature (106 to 112(deg)C) relative to that at low pressure (0.3 MPa). The whole-cell protein profile, analyzed by one-dimensional sodium dodecyl sulfate gel electrophoresis, did not change in cultures grown under low or high pressure at optimal and minimal growth temperatures, but several changes were observed at the maximal growth temperature under in situ pressure. The complex lipid pattern of P. abyssi grown under in situ and 0.1- to 0.5-MPa pressures at different temperatures was analyzed by thin-layer chromatography. The phospholipids became more complex at a low growth temperature at both pressures but their profiles were not superimposable; fewer differences were observed in the core lipids. The polar lipids were composed of only one phospholipid in cells grown under in situ pressure at high temperatures. Survival in the presence of oxygen and under starvation conditions was examined. Oxygen was toxic to P. abyssi at growth range temperature, but the strain survived for several weeks at 4(deg)C. The strain was not affected by starvation in a minimal medium for at least 1 month at 4(deg)C and only minimally affected at 95(deg)C for several days. Cells were more resistant to oxygen in starvation medium. A drastic change in protein profile, depending on incubation time, was observed in cells when starved at growth temperature.     

Isolation and characterization of Thermus thermophilus Gy1211 from a deep-sea hydrothermal vent

We examined a single, non-spore-forming, aerobic, thermophilic strain that was isolated from a deep-sea hydrothermal vent in the Guaymas Basin at a depth of 2000 m and initially placed in a phenetic group with Thermus scotoductus (X-1). We identified this deep-sea isolate as a new strain belonging to Thermus thermophilus using several parameters. DNA–DNA hybridization under stringent conditions showed 74% similarity between the deep-sea isolate and T. thermophilus HB-8^T (T = type strain). Phenotypic characteristics, such as the utilization of carbon sources, hydrolysis of different compounds, and antibiotic sensitivity were identical in the two strains. The polar lipids composition showed that strain Gy1211 belonged to the genus Thermus. The fatty acids composition indicated that this strain was related to the marine T. thermophilus strain isolated from the Azores. The new isolate T. thermophilus strain Gy1211 grew optimally at 75°C, pH 8.0, and 2% NaCl. A hydrostatic pressure of 20 MPa, similar to the in situ hydrostatic pressure of the deep-sea vent from which the strain was isolated, had no effect on growth. Strain HB-8^T, however, showed slower growth under these conditions. Received: November 26, 1997 / Accepted: May 20, 1999     

Isolation and complete genome sequence of Halorientalis hydrocarbonoclasticus sp. nov., a hydrocarbon-degrading haloarchaeon

Bioremediation in hypersaline environments is particularly challenging since the microbes that tolerate such harsh environments and degrade pollutants are quite scarce. Haloarchaea, however, due to their inherent ability to grow at high salt concentrations, hold great promise for remediating the contaminated hypersaline sites. This study aimed to isolate and characterize novel haloarchaeal strains with potentials in hydrocarbon degradation. A haloarchaeal strain IM1011 was isolated from Changlu Tanggu saltern near Da Gang Oilfield in Tianjin (China) by enrichment culture in hypersaline medium containing hexadecane. It could degrade 57 ± 5.2% hexadecane (5 g/L) in the presence of 3.6 M NaCl at 37 °C within 24 days. To get further insights into the mechanisms of petroleum hydrocarbon degradation in haloarchaea, complete genome (3,778,989 bp) of IM1011 was sequenced. Phylogenetic analysis of 16S rRNA gene, RNA polymerase beta-subunit (rpoB’) gene and of the complete genome suggested IM1011 to be a new species in Halorientalis genus, and the name Halorientalis hydrocarbonoclasticus sp. nov., is proposed. Notably, with insights from the IM1011 genome sequence, the involvement of diverse alkane hydroxylase enzymes and an intact β-oxidation pathway in hexadecane biodegradation was predicted. This is the first hexadecane-degrading strain from Halorientalis genus, of which the genome sequence information would be helpful for further dissecting the hydrocarbon degradation by haloarchaea and for their application in bioremediation of oil-polluted hypersaline environments.

     

Expression of Vitreoscilla hemoglobin in Gordonia amarae enhances biosurfactant production

The gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb) was electroporated into Gordonia amarae, where it was stably maintained, and expressed at about 4 nmol VHb g⁻¹ of cells. The maximum cell mass (OD₆₀₀) of vgb-bearing G. amarae was greater than that of untransformed G. amarae for a variety of media and aeration conditions (2.8-fold under normal aeration and 3.4-fold under limited aeration in rich medium, and 3.5-fold under normal aeration and 3.2-fold under limited aeration in mineral salts medium). The maximum level of trehalose lipid from cultures grown in rich medium plus hexadecane was also increased for the recombinant strain, by 4.0-fold in broth and 1.8-fold in cells under normal aeration and 2.1-fold in broth and 1.4-fold in cells under limited aeration. Maximum overall biosurfactant production was also increased in the engineered strain, by 1.4-fold and 2.4-fold for limited and normal aeration, respectively. The engineered strain may be an improved source for producing purified biosurfactant or an aid to microorganisms bioremediating sparingly soluble contaminants in situ.