A barophilic response by two hyperthermophilic, hydrothermal vent Archaea: An upward shift in the optimal temperature and acceleration of growth rate at supra-optimal temperatures by elevated pressure

Abstract The influence of elevated hydrostatic pressure on the growth rates of two hyperthermophilic Archaea isolated from hydrothermal vent environments (strains ES1 and ES4) was investigated over their entire temperature range for growth. Thermococcus celer , a shallow marine hyperthermophile was included in the study for comparative purposes. For one strain (ES4), the pressure at the site of collection (22 MPa) caused an upward shift in the optimal growth temperature of about 6°C compared to growth at 1 MPa. Although the optimal temperature for ES1 was unaffected by 22 MPa, elevated pressure stimulated the growth rate at supra-optimal temperatures. The temperature range for growth for both organisms was extended upward 2°C at 22 MPa pressure. For both strains 22 MPa had little effect on growth rates at sub-optimal temperatures. Growth was observed at pressures as high as 89 MPa for ES1 and 67 MPa for ES4, but with these higher pressures the temperature range for growth was narrowed, and the optimal temperature was shifted downward. Growth of Thermococcus celer was slightly stimulated by 22 MPa at its reported optimal temperature of 88°C, but was inhibited by higher pressure.     

Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.     

Shell nacre ultrastructure and depressurisation dissolution in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

This study describes the micro-morphological features of the shell nacre in the vent mytilid Bathymodiolus azoricus collected along a bathymetric gradient of deep-sea hydrothermal vents of the mid-Atlantic ridge (MAR). Pressure-dependent crystallisation patterns were detected in animals subjected to post-capture hydrostatic simulations. We provide evidence for the following: (1) shell micro morphology in B. azoricus is similar to that of several vent and cold-seep species, but the prismatic shell layers may vary among bathymodiolids; (2) nacre micro-morphology of mussels from three vent sites of the MAR did not differ significantly; minor differences do not appear to be related to hydrostatic pressure, but rather to calcium ion availability; (3) decompression stress may cause drop off in pH of the pallial fluid that damages nascent crystals, and in a more advanced phase, the aragonite tablets as well as the continuous layer of mature nacre; and (4) adverse effects of decompression on calcium salt deposition in shells was diminished by re-pressurisation of specimens. The implications of the putative influence of hydrostatic pressure on biomineralisation processes in molluscs are discussed. An erratum to this article can be found at     

Hydrostatic pressure is like high temperature and oxidative stress in the damage it causes to yeast

Abstract A comparison of barotolerance, thermotolerance and oxygen tolerance was made under different physiological conditions, such as heat shocked and recovered state, different growth phases and changes of physiological conditions by mutations. The three kinds of tolerance showed similar features under different physiological conditions. We suggest that the damage caused by hydrostatic pressure may be essentially the same as that due to high temperature and oxidative stress in yeast.     

Induction of barotolerance by heat shock treatment in yeast

In Saccharomyces cerevisiae, heat shock treatment provides protection against subsequent hydrostatic pressure damage. Such an induced hydrostatic pressure resistance (barotolerance) closely resembles the thermotolerance similarly induced by heat shock treatment. The parallel induction of barotolerance and thermotolerance by heat shock suggests that hydrostatic pressure and high temperature effects in yeast may be tightly linked physiologically.     

Synthetic reconstruction of extreme high hydrostatic pressure resistance in Escherichia coli

Although high hydrostatic pressure (HHP) is an interesting parameter to be applied in bioprocessing, its potential is currently limited by the lack of bacterial chassis capable of surviving and maintaining homeostasis under pressure. While several efforts have been made to genetically engineer microorganisms able to grow at sublethal pressures, there is little information for designing backgrounds that survive more extreme pressures. In this investigation, we analyzed the genome of an extreme HHP-resistant mutant of E. coli MG1655 (designated as DVL1), from which we identified four mutations (in the cra, cyaA, aceA and rpoD loci) causally linked to increased HHP resistance. Analysing the functional effect of these mutations we found that the coupled effect of downregulation of cAMP/CRP, Cra and the glyoxylate shunt activity, together with the upregulation of RpoH and RpoS activity, could mechanistically explain the increased HHP resistance of the mutant. Using combinations of three mutations, we could synthetically engineer E. coli strains able to comfortably survive pressures of 600–800 MPa, which could serve as genetic backgrounds for HHP-based biotechnological applications.     

Cuticle expansion during feeding in the tick Amblyomma hebraeum (Acari: Ixodidae): The role of hydrostatic pressure

Female Amblyomma hebraeum ticks (Acari: Ixodidae) increase their weight ∼10-fold during a ‘slow phase of engorgement’ (7–9 days), and a further 10-fold during the ‘rapid phase’ (12–24 h). During the rapid phase, the cuticle thins by half, with a plastic (permanent) deformation of greater than 40% in two orthogonal directions. A stress of 2.5 MPa or higher is required to achieve this degree of deformation (Flynn and Kaufman, 2015). Using a dimensional analysis of the tick body and applying the Laplace equation, we calculated that the tick must achieve high internal hydrostatic pressures in order to engorge fully: greater than 55 kPa at a fed:unfed mass ratio of ∼20:1, when cuticle thinning commences (Flynn and Kaufman, 2011). In this study we used a telemetric pressure transducer system to measure the internal hydrostatic pressure of ticks during feeding. Sustained periods of irregular high frequency (>20 Hz) pulsatile bursts of high pressure (>55 kPa) were observed in two ticks: they had been cannulated just prior to the rapid phase of engorgement, and given access to a host rabbit for completion of the feeding cycle. The pattern of periods of high pressure generation varied over the feeding cycle and between the two specimens. We believe that these pressures exceed those reported so far for any other animal.     

Induction of cell-mediated immunity against B16-BL6 melanoma in mice vaccinated with cells modified by hydrostatic pressure and chemical crosslinking

In the preceding paper we have demonstrated an increase in presentation of both major histocompatibility complex antigens (MHC) and a tumor-associated antigen of the weakly immunogenic B16 melanoma by a straight-forward technique. The method consists in modulating the tumor cell membrane by hydrostatic pressure and simultaneous chemical crosslinking of the cell-surface proteins. In B16-BL6 melanoma, the induced antigenic modulation was found to persist for over 48 h, which permitted the evaluation of the ability of modified B16-BL6 cells to induce immunity against unmodified B16-BL6 cells. In the present study, we have shown that a significant systemic immunity was induced only in mice that were immunized with modified B16-BL6 melanoma cells, whereas immunization with unmodified B16-BL6 cells had only a marginal effect when compared to the results in control sham-immunized mice. The induced immunity was specific since a single immunization affected the growth of B16-BL6 tumors but had no effect on MCA 106, an antigenically unrelated tumor. The addition of interleukin-2 to the immunization regimen had no effect on the antitumor responses induced by the modified B16-BL6 cells. The cell-mediated immunity conferred by immunization with treated B16-BL6 cells was confirmed in experiments in vitro where splenocytes from immunized mice could be sensitized to proliferate by the presence of B16-BL6 cells. In addition, the altered antigenicity of these melanoma cells appeared to correlate with their increased susceptibility to specific effectors. Thus,⁵¹Cr-labeled B16-BL6 target cells, modified by pressure and crosslinking, in comparison to control labeled target cells, were lysed in much greater numbers by effectors such as lymphokine-activated killer cells and allogeneic cytotoxic lymphocytes (anti-H-2^b), while such cells remained resistant to lysis by natural killer cells. Our findings indicate that the physical and chemical modifications of the tumor cells that are described here may be considered as a simple yet effective method for the preparation of tumor vaccines, which could be applied in tumor-bearing hosts.     

Increased projection of MHC and tumor antigens in murine B16-BL6 melanoma induced by hydrostatic pressure and chemical crosslinking

The B16-BL6 melanoma, like most spontaneously arising tumors, is poorly immunogenic and expresses low levels of major histocompatibility complex (MHC) antigens. Treatment of cells of this tumor in vitro by hydrostatic pressure in the presence of adenosine 2,3-dialdehyde (oxAdo), a membrane-impermeant crosslinker, caused elevated projection of MHC and a specific tumor antigen as demonstrated by flow-cytometric analysis. Maximum projection of both the MHC and the tumor antigens could be reached by application of 1200 atm for 15 min in the presence of 20 mM oxAdo. It is not yet clear whether this passive increase in availability of antigens on the cell surface originated from a dormant pool of antigens in the plasma membrane or from pressure-induced fusion of antigen-rich intracellular organelles (e.g. the endoplasmic reticulum). The immunogenic properties of the antigen-enriched B16-BL6 cells are described in the following paper.     

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     

Role of nitric oxide in the response of Saccharomyces cerevisiae cells to heat shock and high hydrostatic pressure

Nitric oxide (NO) is a simple and unique molecule that has diverse functions in organisms, including intracellular and intercellular messenger. The influence of NO on cell growth of Saccharomyces cerevisiae and as a signal molecule in stress response was evaluated. Respiring cells were more sensitive to an increase in intracellular NO concentration than fermentatively growing cells. Low levels of NO demonstrated a cytoprotective effect during stress from heat-shock or high hydrostatic pressure. Induction of NO synthase was isoform-specific and dependent on the metabolic state of the cells and the stress response pathway. These results support the hypothesis that an increase in intracellular NO concentration leads to stress protection.     

Entrainment of the larval release rhythm of the crab Rhithropanopeus harrisii (Brachyura: Xanthidae) by cycles in hydrostatic pressure

This study investigated the entrainment of a larval release rhythm by determining whether a tidal cycle in hydrostatic pressure could entrain the circatidal rhythm in larval release by the crab Rhithropanopeus harrisii (Gould). Ovigerous females were collected from a non-tidal estuary. The time of larval release by individual crabs was monitored under constant conditions with a time-lapse video system. Crabs with mature embryos at the time of collection had a pronounced circadian rhythm in larval release with a free running period of 25.1 h. Crabs with immature embryos that were maintained under constant conditions from the time of collection until larval release retained a weak circadian rhythm. Other crabs with immature embryos were exposed to a tidal cycle in step changes in hydrostatic pressure equivalent to 1 m of water. This cycle entrained a circatidal rhythm in larval release. The free-running period was 12.1 h and larvae were released at the time of the transition from low to high pressure. Although past studies demonstrated that a tidal cycle in hydrostatic pressure could entrain activity rhythms in crustaceans, this is the first study to show that pressure can entrain a larval release rhythm.     

Intracellular localization of the 90 kDA heat shock protein (HSP90alpha) determined by expression of a EGFP-HSP90alpha-fusion protein in unstressed and heat stressed 3T3 cells

Heat shock protein 90 (Hsp90) is an abundant protein and essential for all eukaryotic cells. The expression of Hsp90 is further enhanced after exposure to stress factors, e.g. a heat shock. Many proteins interacting with Hsp90 as well as the various functions for Hsp90 have been described. In this study, an Hsp90alpha fusion protein along with the enhanced green fluorescence protein (EGFP) was expressed under the control of the human cytomegalovirus immediate early promoter. EGFP-Hsp90alpha was mainly localized in the cytoplasm, with only minor amounts inside the nuclei. No EGFP-Hsp90alpha could be detected inside the nucleoli. Following exposure to elevated temperatures, higher amounts of EGFP-Hsp90alpha are inside the nucleus, but not within the nucleoli. As the most remarkable finding under these conditions, an association of EGFP-Hsp90alpha with the nuclear membrane became visible.     

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     

The effects of hydrostatic pressure on the low-K m GTPase in brain membranes from two congeneric marine fishes

To investigate, the effects of hydrostatic pressure on transmembrane signaling in cold-adapted marine fishes, we examined the high-affinity GTPase activity in two congeneric marine fishes, Sebastolobus alascanus and S. altivelis. In brain membranes there are two GTPase activities, one with a low K _m and one with a high K _m for GTP. The high-affinity GTPase activity, characteristic of the subunits of the guanine nucleotide binding protein pool, was stimulated by the A₁ adenosine receptor agonists N ⁶(R-phenylisopropyl)adenosine and N ⁶-cyclopentyladenosine, and the muscarinic cholinergic agonist carbamyl choline. Pertussis toxin-catalyzed ADP-ribosylation of the membranes for 2 h at 5°C prior to the GTPase assay decreased the basal GTPase activity 30–40% and abolished N ⁶ (R-phenylisopropyl)adenosine stimulation of GTP hydrolysis. Basal high-affinity hydrolysis of GTP, measured at 0.3 mol·1^-1GTP, was stimulated 22% in both species by 340 atm pressure. At 340 atm pressure, the apparent K _m of GTP is decreased approximately 10% in each of the species, and the V _max values are increased 11 and 15.9% in S. alascanus and S. altivelis, respectively. The apparent volume changes associated with the decreased K _m of GTP and the increased V _max ranged from-7.0 to-9.9 ml·mol^-1. Increased pressure markedly decreased the efficacy of N ⁶ (R-phenylisopropyl) adenosine, N ⁶-cylcopentyladenosine and carbamyl choline in stimulating GTPase activity. The effects of increased hydrostatic pressure on transmembrane signal transduction by the A₁ adenosine receptor-inhibitory guanine nucleotide binding protein-adenylyl cyclase system may stem, at least in part, from pressure-increased GTP hydrolysis and the concomitant termination of inhibitory signal transduction.Abbreviations [³H] DPCPX ³H cyclopentyl-1, 3-dipropylxanthine - AppNHp 5-adenylylimidodiphosphate - cpm counts per minute - CPA N ⁶-cyclopentyladenosine - EDTA ethylenediaminetetra acetic acid - EGTA ethyleneglycol-bis (-aminoethylether) N, N, N, N-totra-acctic acid - G protein guanine nucleotide binding protein - G_i inhibitory G protein - G_o other G protein, common in brain membranes - G_s stimulatory G protein - GTPase guanosine triphosphatase - K _i inhibition constant - K _m Michaelis constant - pK _a log of the dissociation constant - R-PIA N ⁶ (R-phenylisopropyl) adenosine - TRIS tris[hydroxymethyl]aminomethane - V_max maximal velocity - [-³²P]GTP [-³²P] guanosine 5-triphosphate (tetra (triethylammonium) salt)     

Ultrastructural effects of pressure stress to the nucleus in Saccharomyces cerevisiae: a study by immunoelectron microscopy using frozen thin sections

Abstract The effects of hydrostatic pressure on subcellular structures, particularly the nucleus, of Saccharomyces cerevisiae were investigated by immunoelectron microscopy. Cells were treated with hydrostatic pressure from 0.1 to 400 MPa for 10 min at room temperature. Frozen thin sections of the cells revealed that spindle pole bodies disappeared at 100 MPa. At 150 MPa, the deposition of gold panicles for anti α-tubulin was noticed in the nucleus, although the filamentous structure of microtubules was lost. At 200 MPa, fewer gold particles were scattered in the nucleus and the nuclear membrane in several portions was also observed to be open at 300 MPa. These results show that elements of the nuclear division apparatus were susceptible to pressure stress, particularly spindle pole bodies and microtubules. The damage to spindle pole bodies, microtubules, and nuclear membrane caused by pressure stress was followed by the inhibition of nuclear division. After the release of pressure, the spindle pole bodies and microtubules of pressurized cells at below 200 MPa regained their normal appearance at 24 h.     

The interaction of HspA1A with TLR2 and TLR4 in the response of neutrophils induced by ovarian cancer cells in vitro

Inducible heat shock protein (HspA1A) promotes tumor cell growth and survival. It also interacts with effector cells of the innate immune system and affects their activity. Recently, we showed that the direct contact of ovarian cancer cells, isolated from tumor specimens, with neutrophils intensified their biological functions. Our current experiments demonstrate that the activation of neutrophils, followed by an increased production of reactive oxygen species, by cancer cells involves the interaction of HspA1A from cancer cells with Toll-like receptors 2 and 4 expressed on the neutrophils’ surface. Our data may have a practical implication for targeted anticancer therapies based, among other factors, on the inhibition of HspA1A expression in the cancer cells.     

A comparison of circatidal rhythmicity and entrainment by hydrostatic pressure cycles in the rock goby, Gobius paganellus L. and the shanny, Lipophrys pholis (L.)

The intertidal teleosts Gobius paganellus and Lipophrys pholis show endogenous circatidal activity rhythms when recorded in constant conditions. Under these conditions, the rhythm of L. pholis is the more precise which may indicate stronger coupling between underlying circalunadian oscillators in this species. In G. pagunellus the inter-oscillator coupling may be weaker and this could enable a more subtle interpretation of tidal fluctuations than in L. pholis . The oscillators may, however, be fundamentally different in the two species; circalunadian in G. paganellus and circatidal in L. pholis .
When exposed to hydrostatic pressure cycles of tidal frequency both species responded pre- dominantly to increasing pressure, which suggests that in the wild they are likely to be most active on the rising tide. Hydrostatic pressure cycles are confirmed as a zeitgeber for both species by the successful entrainment of some individuals. The lack of entrainment of others impIies that additional zeitgebers are required for complete entrainment.