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.     

Different Pressure Resistance of Lactate Dehydrogenases from Hagfish is Dependent on Habitat Depth and Caused by Tetrameric Structure Dissociation

The effects of high hydrostatic pressure on lactate dehydrogenase (LDH) activities from two species of hagfish were examined. LDH from Eptatretus okinoseanus, a deep-sea species, retained 67% of the original activity even at 100 MPa. LDH activity from Eptatretus burgeri, a shallow-sea species, was completely lost at 50 MPa but recovered to the original value at 0.1 MPa. The tetrameric structure of LDH-A₄ from E. okinoseanus did not change at 50 MPa. In contrast, almost all LDH tetramers from E. burgeri dissociated to dimers and monomers at 50 MPa but reverted to tetramers at 0.1 MPa. These results show that the dissociation of tetramers caused the inactivation of E. burgeri LDH. The difference depends on the number 6 and 10 amino acids. The mechanism of the slight, gradual inactivation of E. okinoseanus LDH at high pressure differs and is probably due to the metamorphosis of its inner structures.     

Purification and characterization of novel extracellular endopolygalacturonases from a deep-sea yeast, Cryptococcus sp. N6, isolated from the Japan Trench

Two novel endo-type polygalacturonases (PGase), with molecular weights of 36 kDa and 40 kDa (named p36 and p40, respectively), were purified from the supernatant of the culture medium of a deep-sea yeast, strain N6, isolated from the Japan Trench. The N-terminal 20 amino acids of p36 and p40 were identical, and the sequence homology was 47.4% in comparison with the PGase of Fusarium moniliforme. A treatment of p40 with glycopeptidase F reduced the molecular weight to 36 kDa, suggesting that p40 possessed N-acetylglucosamines on its asparagine residues and p40 might be matured by glycosylation of p36.     

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     

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     

Purification and identification of the violaxanthin deepoxidase as a 43 kDa protein

Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH₄)₂SO₄ fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54–60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific activity obtained was 256 mol g^–1 s^–1 protein, which is at least 10-fold higher than reported earlier. We estimate that there is 1 VDE molecule per 20–100 electron transport chains. The 43 kDa protein was N-terminally sequenced, after protection of cysteine residues with -mercaptoethanol and iodoacetamid, and a unique sequence of 20 amino acids was obtained. The amino acid composition of the protein revealed a high abundance of charged and polar amino acids and remarkably, 11 cysteine residues. Two other proteins (39.5 kDa and 40 kDa) copurifying with VDE were also N-terminally sequenced. The N-terminal part of the 39.5 kDa protein showed complete sequence identity both with the N-terminal part of cyt b ₆ and an internal sequence of polyphenol oxidase.Abbreviations DMSO dimethylsulfoxid - HIC hydrophobic interaction chromatography - MGDG monogalactosyl diacylglycerol - VDE violaxanthin deepoxidase A preliminary report of these results was presented at the Xth Int. Congress on Photosynthesis, Montpellier, France, 1995.     

Metabolic selectivity and growth of<Emphasis Type="Italic"> Clostridium thermocellum</Emphasis> in continuous culture under elevated hydrostatic pressure

The continuous culture of Clostridium thermocellum, a thermophilic bacterium capable of producing ethanol from cellulosic material, is demonstrated at elevated hydrostatic pressure (7.0 MPa, 17.3 MPa) and compared with cultures at atmospheric pressure. A commercial limitation of ethanol production by C. thermocellum is low ethanol yield due to the formation of organic acids (acetate, lactate). At elevated hydrostatic pressure, ethanol:acetate (E/A) ratios increased >10² relative to atmospheric pressure. Cell growth was inhibited by approximately 40% and 60% for incubations at 7.0 MPa and 17.3 MPa, respectively, relative to continuous culture at atmospheric pressure. A decrease in the theoretical maximum growth yield and an increase in the maintenance coefficient indicated that more cellobiose and ATP are channeled towards maintaining cellular function in pressurized cultures. Shifts in product selectivity toward ethanol are consistent with previous observations of hydrostatic pressure effects in batch cultures. The results are partially attributed to the increasing concentration of dissolved product gases (H₂, CO₂) with increasing pressure; and they highlight the utility of continuous culture experiments for the quantification of the complex role of dissolved gas and pressure effects on metabolic activity.     

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     

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     

Purification and Characterization of a DnaK-like and a GroEL-like Protein from Porphyromonas gingivalis

Heat-shock proteins of Porphyromonas gingivalis were demonstrated and two of them were purified and further characterized. The amplified de novo synthesis of two different proteins, with apparent molecular weights of 75 kDa and 68 kDa, was observed by autofluorography when a P. gingivalis culture incubated in a ¹⁴C-labeled amino acid mixture was shifted from 37°C to 44°C. Both proteins possessed ATP-binding abilities and were purified to almost homogeneity employing affinity chromatography on ATP-agarose followed by preparative SDS-PAGE. Purified 75 kDa and 68 kDa proteins had isoelectric points of 4.4 and 4.6, respectively. They were shown to be immunoreactive with commercial anti-DnaK and anti-GroEL polyclonal antibodies, respectively. Immunoblotting analysis of whole cells using antiserum raised against each purified protein from P. gingivalis, confirmed elevated synthesis of both proteins during thermal shock. A GroEL protein reacted strongly with antiserum against the 68 kDa protein. However, a DnaK protein reacted weakly with antiserum to the 75 kDa protein. Analysis of the N-terminal amino acid sequence of the DnaK-like protein (75 kDa) showed a high degree of homology with those of the HSP70 family including both prokaryotic and eukaryotic cells. The N-terminal amino acid analysis of the GroEL-like protein (68 kDa) indicated that it was identical to those of cloned GroEL homologues from P. gingivalis.     

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.     

New knowledge about the PHA-locus and P(3HB) granule-associated proteins in Chromatium vinosum

Summary The isolation of poly(3-hydroxybutyric acid) granules of Chromatium vinosum D was re-examined. Beside the PHA synthase and a 17 kDa protein, a 14 kDa protein was identified as predominant granule-associated protein. The M _r as well as the N-terminal amino acid sequence exhibited identity to ORF5_Cv, which is located within the pha-locus of C. vinosum. In addition, sequence alignements revealed new information about ORF4_Cv, which is also located within the pha-locus, and about the 17 kDa protein, which exhibited homology to heat shock proteins recently detected in Escherichia coli.     

Cloning and Sequence Analysis of the hemB Gene of Rhodothermus marinus

A Rhodothermus marinus gene, hemB, coding for 5-aminolevulinic acid (ALA) dehydratase (ALAD) has been cloned and sequenced. The reading frame of the hemB gene is 1020 base pairs encoding a protein of 340 amino acids with a calculated molecular mass of 37.4 kDa. The amino acid sequence shows homology with eubacterial and eukaryotic ALA dehydratases. A putative metal-binding site of the protein shows strongest homology with corresponding sites from plant ALA dehydratases that require Mg²⁺ for activity. It differs with respect to only one amino acid out of 20 from a corresponding site in pea ALAD. Received: 1 March 1999 / Accepted: 7 April 1999     

Characterization and gene cloning of a cold-active cellulase from a deep-sea psychrotrophic bacterium Pseudoalteromonas sp. DY3

The celX gene encoding an extracellular cold-active cellulase was isolated from a psychrotrophic bacterium, which was isolated from deep-sea sediment and identified as a Pseudoalteromonas species. It encoded a protein consisting of 492 amino acids with a calculated molecular mass of 52.7 kDa. The CelX consisted of an N-terminal catalytic domain belonging to glycoside hydrolase family 5 and a C-terminal cellulose-binding domain belonging to carbohydrate-binding module family 5. The long linker sequence connecting both domains was composed of 105 residues. The optimal temperature for cellulase activity of CelX was 40°C. The enzyme was most active at pH 6–7 and showed better resistance to alkaline condition. The zymogram activity analysis indicated that the CelX consisted of single enzyme component. The cellobiose was main hydrolysate of CelX.     

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.     

Novel Psychropiezophilic Oceanospirillales Species Profundimonas piezophila gen. nov., sp. nov., Isolated from the Deep-Sea Environment of the Puerto Rico Trench

The diversity of deep-sea high-pressure-adapted (piezophilic) microbes in isolated monoculture remains low. In this study, a novel obligately psychropiezophilic bacterium was isolated from seawater collected from the Puerto Rico Trench at a depth of ∼6,000 m. This isolate, designated YC-1, grew best in a nutrient-rich marine medium, with an optimal growth hydrostatic pressure of 50 MPa (range, 20 to 70 MPa) at 8°C. Under these conditions, the maximum growth rate was extremely slow, 0.017 h⁻¹, and the maximum yield was 3.51 × 10⁷ cells ml⁻¹. Cell size and shape changed with pressure, shifting from 4.0 to 5.0 μm in length and 0.5 to 0.8 μm in width at 60 MPa to 0.8- to 1.0-μm diameter coccoid cells under 20 MPa, the minimal pressure required for growth. YC-1 is a Gram-negative, facultatively anaerobic heterotroph. Its predominant cellular fatty acids are the monounsaturated fatty acids (MUFAs) C_16:1 and C_18:1. Unlike many other psychropiezophiles, YC-1 does not synthesize any polyunsaturated fatty acids (PUFAs). Phylogenetic analysis placed YC-1 within the family of Oceanospirillaceae, closely related to the uncultured symbiont of the deep-sea whale bone-eating worms of the genus Osedax. In common with some other members of the Oceanospirillales, including those enriched during the Deepwater Horizon oil spill, YC-1 is capable of hydrocarbon utilization. On the basis of its characteristics, YC-1 appears to represent both a new genus and a new species, which we name Profundimonas piezophila gen. nov., sp. nov.