Piezophilic Bacteria Research Articles

Adaptations to high pressure of Nautilia sp. strain PV-1, a piezophilic Campylobacterium (aka Epsilonproteobacterium) isolated from a deep-sea hydrothermal vent.

Physiological and gene expression studies of deep-sea bacteria under pressure conditions similar to those experienced in their natural habitat are critical for understanding growth kinetics and metabolic adaptations to in situ conditions. The Campylobacterium (aka Epsilonproteobacterium) Nautilia sp. strain PV-1 was isolated from hydrothermal fluids released from an active deep-sea hydrothermal vent at 9° N on the East Pacific Rise. Strain PV-1 is a piezophilic, moderately thermophilic, chemolithoautotrophic anaerobe that conserves energy by coupling the oxidation of hydrogen to the reduction of nitrate or elemental sulfur. Using a high-pressure-high temperature continuous culture system, we established that strain PV-1 has the shortest generation time of all known piezophilic bacteria and we investigated its protein expression pattern in response to different hydrostatic pressure regimes. Proteogenomic analyses of strain PV-1 grown at 20 and 5MPa showed that pressure adaptation is not restricted to stress response or homeoviscous adaptation but extends to enzymes involved in central metabolic pathways. Protein synthesis, motility, transport, and energy metabolism are all affected by pressure, although to different extents. In strain PV-1, low-pressure conditions induce the synthesis of phage-related proteins and an overexpression of enzymes involved in carbon fixation.

Distinctive gene and protein characteristics of extremely piezophilic Colwellia

BackgroundThe deep ocean is characterized by low temperatures, high hydrostatic pressures, and low concentrations of organic matter. While these conditions likely select for distinct genomic characteristics within prokaryotes, the attributes facilitating adaptation to the deep ocean are relatively unexplored. In this study, we compared the genomes of seven strains within the genus Colwellia, including some of the most piezophilic microbes known, to identify genomic features that enable life in the deep sea.ResultsSignificant differences were found to exist between piezophilic and non-piezophilic strains of Colwellia. Piezophilic Colwellia have a more basic and hydrophobic proteome. The piezophilic abyssal and hadal isolates have more genes involved in replication/recombination/repair, cell wall/membrane biogenesis, and cell motility. The characteristics of respiration, pilus generation, and membrane fluidity adjustment vary between the strains, with operons for a nuo dehydrogenase and a tad pilus only present in the piezophiles. In contrast, the piezosensitive members are unique in having the capacity for dissimilatory nitrite and TMAO reduction. A number of genes exist only within deep-sea adapted species, such as those encoding d-alanine-d-alanine ligase for peptidoglycan formation, alanine dehydrogenase for NADH/NAD+ homeostasis, and a SAM methyltransferase for tRNA modification. Many of these piezophile-specific genes are in variable regions of the genome near genomic islands, transposases, and toxin-antitoxin systems.ConclusionsWe identified a number of adaptations that may facilitate deep-sea radiation in members of the genus Colwellia, as well as in other piezophilic bacteria. An enrichment in more basic and hydrophobic amino acids could help piezophiles stabilize and limit water intrusion into proteins as a result of high pressure. Variations in genes associated with the membrane, including those involved in unsaturated fatty acid production and respiration, indicate that membrane-based adaptations are critical for coping with high pressure. The presence of many piezophile-specific genes near genomic islands highlights that adaptation to the deep ocean may be facilitated by horizontal gene transfer through transposases or other mobile elements. Some of these genes are amenable to further study in genetically tractable piezophilic and piezotolerant deep-sea microorganisms.

Transcriptomic Analysis Reveals Common Adaptation Mechanisms Under Different Stresses for Moderately Piezophilic Bacteria.

Piezophiles, by the commonly accepted definition, grow faster under high hydrostatic pressure (HHP) than under ambient pressure and are believed to exist only in pressurized environments where life has adapted to HHP during evolution. However, recent findings suggest that piezophiles have developed a common adaptation strategy to cope with multiple types of stresses including HHP. These results raise a question on the ecological niches of piezophiles: are piezophiles restricted to habitats with HHP? In this study, we observed that the bacterial strains Sporosarcina psychrophila DSM 6497 and Lysinibacillus sphaericus LMG 22257, which were isolated from surface environments and then transferred under ambient pressure for half a century, possess moderately piezophilic characteristics with optimal growth pressures of 7 and 20MPa, respectively. Their tolerance to HHP was further enhanced by MgCl2 supplementation under the highest tested pressure of 50MPa. Transcriptomic analysis was performed to compare gene expression with and without MgCl2 supplementation under 50MPa for S. psychrophila DSM 6497. Among 4390 genes or transcripts obtained, 915 differentially expressed genes (DEGs) were identified. These DEGs are primarily associated with the antioxidant defense system, intracellular compatible solute accumulation, and membrane lipid biosynthesis, which have been reported to be essential for cells to cope with HHP. These findings indicate no in situ pressure barrier for piezophile isolation, and cells may adopt a common adaptation strategy to cope with different stresses.

Revisiting the genus Photobacterium: taxonomy, ecology and pathogenesis.

The genus Photobacterium, one of the eight genera included in the family Vibrionaceae, contains 27 species with valid names and it has received attention because of the bioluminescence and pathogenesis mechanisms that some of its species exhibit. However, the taxonomy and phylogeny of this genus are not completely elucidated; for example, P. logei and P. fischeri are now considered members of the genus Aliivibrio, and previously were included in the genus Vibrio. In addition, P. damselae subsp. piscicida was formed as a new combination for former Vibrio damsela and Pasteurella piscicida. Moreover, P. damselae subsp. damselae is an earlier heterotypic synonym of P. histaminum. To avoid these incovenences draft and complete genomic sequences of members of Photobacterium are increasingly becoming available and their use is now routine for many research laboratories to address diverse goals: species delineation with overall genomic indexes, phylogenetic analyses, comparative genomics, and phenotypic inference. The habitats and isolation source of the Photobacterium species include seawater, sea sediments, saline lake waters, and a variety of marine organisms with which the photobacteria establish different relationships, from symbiosis to pathogenic interactions. Several species of this genus contain bioluminescent strains in symbiosis with marine fish and cephalopods; in addition, other species enhance its growth at pressures above 1 atmosphere, by means of several high-pressure adaptation mechanisms and for this, they may be considered as piezophilic (former barophilic) bacteria. Until now, only P. jeanii, P. rosenbergii, P. sanctipauli, and the two subspecies of P. damselae have been reported as responsible agents of several pathologies on animal hosts, such as corals, sponges, fish and homeothermic animals. In this review we have revised and updated the taxonomy, ecology and pathogenicity of several members of this genus. [Int Microbiol 20(1): 1-10 (2017)].

Predominance of Viable Spore-Forming Piezophilic Bacteria in High-Pressure Enrichment Cultures from ~1.5 to 2.4 km-Deep Coal-Bearing Sediments below the Ocean Floor.

Phylogenetically diverse microorganisms have been observed in marine subsurface sediments down to ~2.5 km below the seafloor (kmbsf). However, very little is known about the pressure-adapted and/or pressure-loving microorganisms, the so called piezophiles, in the deep subseafloor biosphere, despite that pressure directly affects microbial physiology, metabolism, and biogeochemical processes of carbon and other elements in situ. In this study, we studied taxonomic compositions of microbial communities in high-pressure incubated sediment, obtained during the Integrated Ocean Drilling Program (IODP) Expedition 337 off the Shimokita Peninsula, Japan. Analysis of 16S rRNA gene-tagged sequences showed that members of spore-forming bacteria within Firmicutes and Actinobacteria were predominantly detected in all enrichment cultures from ~1.5 to 2.4 km-deep sediment samples, followed by members of Proteobacteria, Acidobacteria, and Bacteroidetes according to the sequence frequency. To further study the physiology of the deep subseafloor sedimentary piezophilic bacteria, we isolated and characterized two bacterial strains, 19R1-5 and 29R7-12, from 1.9 and 2.4 km-deep sediment samples, respectively. The isolates were both low G+C content, gram-positive, endospore-forming and facultative anaerobic piezophilic bacteria, closely related to Virgibacillus pantothenticus and Bacillus subtilis within the phylum Firmicutes, respectively. The optimal pressure and temperature conditions for growth were 20 MPa and 42°C for strain 19R1-5, and 10 MPa and 43°C for strain 29R7-12. Bacterial (endo)spores were observed in both the enrichment and pure cultures examined, suggesting that these piezophilic members were derived from microbial communities buried in the ~20 million-year-old coal-bearing sediments after the long-term survival as spores and that the deep biosphere may host more abundant gram-positive spore-forming bacteria and their spores than hitherto recognized.

Effects of hydrostatic pressure on yeasts isolated from deep-sea hydrothermal vents

Hydrostatic pressure plays a significant role in the distribution of life in the biosphere. Knowledge of deep-sea piezotolerant and (hyper)piezophilic bacteria and archaea diversity has been well documented, along with their specific adaptations to cope with high hydrostatic pressure (HHP). Recent investigations of deep-sea microbial community compositions have shown unexpected micro-eukaryotic communities, mainly dominated by fungi. Molecular methods such as next-generation sequencing have been used for SSU rRNA gene sequencing to reveal fungal taxa. Currently, a difficult but fascinating challenge for marine mycologists is to create deep-sea marine fungus culture collections and assess their ability to cope with pressure. Indeed, although there is no universal genetic marker for piezoresistance, physiological analyses provide concrete relevant data for estimating their adaptations and understanding the role of fungal communities in the abyss. The present study investigated morphological and physiological responses of fungi to HHP using a collection of deep-sea yeasts as a model. The aim was to determine whether deep-sea yeasts were able to tolerate different HHP and if they were metabolically active. Here we report an unexpected taxonomic-based dichotomic response to pressure with piezosensitve ascomycetes and piezotolerant basidiomycetes, and distinct morphological switches triggered by pressure for certain strains.

Alterations in Membrane Phospholipid Fatty Acids of Gram‐Positive Piezotolerant Bacterium Sporosarcina sp. DSK25 in Response to Growth Pressure

Pressure is an important thermodynamic property of the ocean and the deep biosphere that affects microbial physiology and biochemistry. Here, we report on our investigation of the response of Gram-positive piezotolerant bacterium Sporosarcina sp. DSK25 to hydrostatic pressure. Strain DSK25 responded in an adaptive manner to upshifts of growth pressure and showed systematic changes in phospholipid fatty acids. As the pressure increased from 0.1 to 10 MPa (Megapascal), unsaturated fatty acids in DSK25 increased from 21.7 to 31.1% of total fatty acids, while the level of iso- and anteiso-branched fatty acids remained unchanged. At higher pressures (30, 50, and 60 MPa), the amount of unsaturated fatty acids decreased, and that of anteiso-branched fatty acids increased from 34.4 to 49.9% at the expense of iso-branched fatty acids. For the first time, two polyunsaturated fatty acids (PUFA), 18:2n-6 and 18:2n-x, with the latter having much higher abundance than the former, were identified in DSK25. The concentration of the PUFA increased with growth pressure. These results indicate the involvement of unsaturated and methyl-branched fatty acids in the modulation of bacteria membrane fluidity and function over environmentally relevant parameter (pressure). Piezotolerant bacterium Sporosarcina sp. DSK25 appears to utilize two regulatory mechanisms for adaptation to high pressure, a rapid-responding mechanism on transient scale, expressed as increased biosynthesis of monounsaturated fatty acids, and a long-term adaptation mechanism in increased synthesis of anteiso-branched and polyunsaturated fatty acids. Our results further suggest that Gram-positive piezophilic bacteria respond differently than Gram-negative bacteria in adaptation to high pressure.

Draft Genome Sequence of the Deep-Sea Bacterium Shewanella benthica Strain KT99

We report the draft genome sequence of the obligately piezophilic Shewanella benthica strain KT99 isolated from the abyssal South Pacific Ocean. Strain KT99 is the first piezophilic isolate from the Tonga-Kermadec trench, and its genome provides many clues on high-pressure adaptation and the evolution of deep-sea piezophilic bacteria.

Displaced Conformational Equilibrium in a Piezophilic Cytochrome P450

Cytochromes P450 from piezophilic bacteria living at extreme pressures provide a powerful tool for exploration of the substrate‐dependent conformational transitions in the enzyme, which involve large changes in hydration and are thus pressure‐sensitive. Cytochrome P450 of a deep‐sea bacteria Photobacterium profundum SS9 was cloned and expressed in E. coli. Purified P450‐SS9 was found to be predominantly in the high‐spin state. The enzyme was found to bind various substrates including fatty acids and their derivatives. The spin equilibrium could be displaced by substrate binding to either high‐spin (HS) or low‐spin (LS), depending on the structure. In contrast to all cytochromes P450 studied up to date, hydrostatic pressure failed to displace the spin equilibrium completely to the LS state in the substrate‐free P450‐SS9. Similar behavior is observed in HS‐complexes, whereas the spin equilibrium in the LS‐complexes becomes pressure‐sensitive. We also probed the accessibility of the heme pocket with H2O2–dependent heme depletion. The rate of this process, which was very slow in the substrate‐free enzyme and in HS‐complexes, is dramatically increased in LS‐complexes. These findings provide evidence of large‐scale changes in the openness and degree of hydration of the active site that are involved in the substrate‐dependent spin transitions in the enzyme. (Supported by NIH grant GM054995)

Procedure for isolation of the plastic degrading piezophilic bacteria from deep-sea environments

A procedure for the isolation of aliphatic polyester-degrading bacteria from deep-sea environments was developed using three step high-pressure cultivation. First, the sediments collected from the deep-sea floor, the Kurile and Japan Trenches at a depth of 5000-7000 m, were cultivated with poly e-caprolactone (PCL) films under low-temperature (4°C) and high-pressure (50MPa) conditions. Secondly, high-pressure continuous cultivation using the deep-sea baro-piezophile and thermophile isolation and cultivation (DEEPBATH) system was performed three times to accumulate piezophilic bacteria. Finally, piezophilic PCL degrading bacteria were isolated using the pressure bag method involving media with PCL granules. Using this procedure, we succeeded in isolating several piezophilic aliphatic polyester-degrading bacteria from the sediment samples of deep-trenches.

Cloning and characterization of dihydrofolate reductases from deep-sea bacteria

Enzymes from organisms living in deep-sea are thought to have characteristic pressure-adaptation mechanisms in structure and function. To better understand these mechanisms in dihydrofolate reductase (DHFR), an essential enzyme in living cells, we cloned, overexpressed and purified four new DHFRs from the deep-sea bacteria Shewanella violacea (svDHFR), Photobacterium profundum (ppDHFR), Moritella yayanosii (myDHFR) and Moritella japonica (mjDHFR), and compared their structure and function with those of Escherichia coli DHFR (ecDHFR). These deep-sea DHFRs showed 33-56% primary structure identity to ecDHFR while far-ultraviolet circular dichroism and fluorescence spectra suggested that their secondary and tertiary structures were not largely different. The optimal temperature and pH for deep-sea DHFRs activity were lower than those of ecDHFR and different from each other. Deep-sea DHFRs kinetic parameters K(m) and k(cat) were larger than those of ecDHFR, resulting in 1.5-2.8-fold increase of k(cat)/K(m) except for mjDHFR which had a 28-fold decrease. The enzyme activity of ppDHFR and mjDHFR (moderate piezophilic bacteria) as well as ecDHFR decreased as pressure increased, while svDHFR and myDHFR (piezophilic bacteria) showed a significant tolerance to pressure. These results suggest that DHFRs from deep-sea bacteria possess specific enzymatic properties adapted to their life under high pressure.

Psychromonas hadalis sp. nov., a novel piezophilic bacterium isolated from the bottom of the Japan Trench

An obligately piezophilic bacterium was isolated from sediment collected from the bottom of the Japan Trench at a depth of 7542 m. The isolated strain, designated K41G(T), was closely affiliated with members of the genus Psychromonas on the basis of 16S rRNA gene sequence analysis. Levels of DNA-DNA relatedness between strain K41G(T) and Psychromonas reference strains were significantly lower than that accepted as the phylogenetic definition of a species. The optimal temperature and pressure for growth of strain K41G(T) were 6 degrees C and 60 MPa, respectively. The DNA G+C content was 39.1 mol%. Whole-cell fatty acids consisted of significant amounts of unsaturated fatty acids C(16 : 1) (37 %) and C(14 : 1) (17 %), saturated fatty acid C(16 : 0) (31 %) and polyunsaturated fatty acid C(22 : 6) (8 %). Based on the taxonomic differences observed, strain K41G(T) is considered to represent a novel obligately piezophilic Psychromonas species. The name Psychromonas hadalis (type strain, K41G(T)=JCM 11830(T)=ATCC BAA-638(T)) is proposed. This is the second species of obligately piezophilic bacteria to be proposed in the genus Psychromonas.

The Unique 16S rRNA Genes of Piezophiles Reflect both Phylogeny and Adaptation

In the ocean's most extreme depths, pressures of 70 to 110 megapascals prevent the growth of all but the most hyperpiezophilic (pressure-loving) organisms. The physiological adaptations required for growth under these conditions are considered to be substantial. Efforts to determine specific adaptations permitting growth at extreme pressures have thus far focused on relatively few gamma-proteobacteria, in part due to the technical difficulties of obtaining piezophilic bacteria in pure culture. Here, we present the molecular phylogenies of several new piezophiles of widely differing geographic origins. Included are results from an analysis of the first deep-trench bacterial isolates recovered from the southern hemisphere (9.9-km depth) and of the first gram-positive piezophilic strains. These new data allowed both phylogenetic and structural 16S rRNA comparisons among deep-ocean trench piezophiles and closely related strains not adapted to high pressure. Our results suggest that (i) the Circumpolar Deep Water acts as repository for hyperpiezophiles and drives their dissemination to deep trenches in the Pacific Ocean and (ii) the occurrence of elongated helices in the 16S rRNA genes increases with the extent of adaptation to growth at elevated pressure. These helix changes are believed to improve ribosome function under deep-sea conditions.

Fractionation of carbon isotopes in biosynthesis of fatty acids by a piezophilic bacterium Moritella japonica strain DSK1

We examined stable carbon isotope fractionation in biosynthesis of fatty acids of a piezophilic bacterium Moritella japonica strain DSK1. The bacterium was grown to stationary phase at pressures of 0.1, 10, 20, and 50 MPa in media prepared using sterile-filtered natural seawater supplied with glucose as the sole carbon source. Strain DSK1 synthesized typical bacterial fatty acids (C 14–19 saturated, monounsaturated, and cyclopropane fatty acids) as well as long-chain polyunsaturated fatty acids (PUFA) (20:6 ω3). Bacterial cell biomass and individual fatty acids exhibited consistent pressure-dependent carbon isotope fractionations relative to glucose. The observed Δδ FA-glucose (−1.0‰ to −11.9‰) at 0.1 MPa was comparable to or slightly higher than fractionations reported in surface bacteria. However, bulk biomass and fatty acids became more depleted in 13C with pressure. Average carbon isotope fractionation (Δδ FA-glucose) at high pressures was much higher than that for surface bacteria: −15.7‰, −15.3‰, and −18.3‰ at 10, 20, and 50 MPa, respectively. PUFA were more 13C depleted than saturated and monounsaturated fatty acids at all pressures. The observed isotope effects may be ascribed to the kinetics of enzymatic reactions that are affected by hydrostatic pressure and to biosynthetic pathways that are different for short-chain and long-chain fatty acids. A simple quantitative calculation suggests that in situ piezophilic bacterial contribution of polyunsaturated fatty acids to marine sediments is nearly two orders of magnitude higher than that of marine phytoplankton and that the carbon isotope imprint of piezophilic bacteria can override that of surface phytoplankton. Our results have important implications for marine biogeochemistry. Depleted fatty acids reported in marine sediments and the water column may be derived simply from piezophilic bacteria resynthesis of organic matter, not from bacterial utilization of a 13C-depleted carbon source (i.e., methane). The interpretation of carbon isotope signatures of marine lipids must be based on principles derived from piezophilic bacteria.

Amino acid substitutions in malate dehydrogenases of piezophilic bacteria isolated from intestinal contents of deep-sea fishes retrieved from the abyssal zone

To examine the occurrence in other deep-sea bacteria of two amino acid substitutions (Ala-180 and His-229) in malate dehydrogenase (MDH) found previously in the deep-sea piezophilic Moritella sp. strain 2D2, we cloned and sequenced MDH genes of deep-sea piezophilic Moritella and Shewanella strains isolated from intestinal contents of deep-sea fishes, as well as other Moritella species from deep-sea water and sediments: M. marina, M. japonica, and M. yayanosii. The piezophilic Moritella strains had a Val residue or an Ala residue at position 180 and all the Moritella strains except for one had a His residue at position 229. However, four piezophilic-strain-specific substitutions at positions 103, 111, 229, and 283 were found to be completely conserved in the MDH of the intestinal Moritella strains of deep-sea fishes, indicating the substitutions may be habitat-specific. The piezophilic Shewanella strains had a Val residue and a Gln residue at positions 180 and 229, respectively. However, the MDHs of the Shewanella strains had five piezophilic-strain-specific substitutions at positions 61, 65, 107, 161, and 202. Therefore, the enzymatic strategies for responding to deep-sea high pressure environments of the MDHs between the genera Moritella and Shewanella are potentially different. Moreover, homology modeling shows these substitutions found in the MDHs of both genera except for position 229 in the subunit interface are located on the exposed region of the MDH molecules, indicating the substitutions may be related to the hydration state of the molecules.

Colwellia piezophila sp. nov., a novel piezophilic species from deep-sea sediments of the Japan Trench

Two strains of obligately piezophilic bacteria were isolated from sediment collected from the bottom surface of a small canyon on the seaward slope of the Japan Trench at a depth of 6278 m. The isolated strains, Y223GT and Y251E, are closely affiliated with members of the genus Colwellia on the basis of 16S rRNA gene sequence analysis. The G + C contents of both strains were about 39 mol%. DNA-DNA hybridization values between these strains and Colwellia reference strains were significantly lower than those accepted as the phylogenetic definition of a species. The novel strains are Gram-negative, polarly flagellated and facultatively anaerobic. The optimal pressure for growth was 60 MPa at both 4 and 10 degrees C; the most rapid growth rate was observed at 10 degrees C and 60 MPa. No growth occurred at 15 degrees C under any pressure studied. The major isoprenoid quinone is Q-8. The predominant cellular fatty acids are C16 : 0 and C16 : 1. Based on the taxonomic differences observed, the isolated strains appear to represent a novel obligately piezophilic Colwellia species. The name Colwellia piezophila sp. nov. (type strain Y223GT = JCM 11831T = ATCC BAA-637T) is proposed.

Biosynthesis and dietary uptake of polyunsaturated fatty acids by piezophilic bacteria

The biochemistry of piezophilic bacteria is unique in that piezophiles produce polyunsaturated fatty acids (PUFAs). A pertinent question is if piezophilic bacteria synthesize PUFA de novo, through dietary uptake, or both. This study was undertaken to examine the biosynthesis and cellular uptake of PUFAs by piezophilic bacteria. A moderately piezophilic ( Shewanella violacea DSS12) and two hyperpiezophilic bacteria ( S. benthica DB21MT-2 and Moritella yayanosii DB21MT-5) were grown under 50 MPa (megapascal) and 100 MPa, respectively, in media containing marine broth 2216 supplemented with arachidonic acid (AA, sodium salt) and/or antibiotic cerulenin. There was active uptake and cellular incorporation of AA in the hyperpiezophilic bacteria DB21MT-2 (14.7% of total fatty acids) and DB21MT-5 (1.4%), but no uptake was observed in DSS12. When cells were treated with cerulenin, all three strains incorporated AA into cell membranes (13–19%). The biosynthesis of monounsaturated fatty acids was significantly inhibited (10–37%) by the addition of cerulenin, whereas the concentrations of PUFAs increased by 2–4 times. These results suggest that piezophilic bacteria biosynthesize and/or incorporate dietary polyunsaturated fatty acids that are important for their growth and piezoadaptation. The significance of these findings is also discussed in the context of phenotypic classification of piezophiles.

Phospholipid FA of piezophilic bacteria from the deep sea.

Phospholipid FA (PLFA) profiles were determined on four piezophilic bacteria from the deep sea: Moritella japonica DSK1, Shewanella violacea DSS12, S. benthica DB6705, and S. benthica DB21MT-2. The total concentrations of PLFA were higher in strains grown at low pressure (DSK1, 10 MPa, 27.0 mg/g dry wt cells; DSS12, 50 MPa, 24.0 mg/g), and lower in strains grown at high pressure (DB6705, 85 MPa, 1.9 mg/g; DB21MT-2, 100 MPa, 3.0 mg/g). The piezophilic bacteria were characterized by a high abundance of unsaturated FA (62-73% of total FA). In particular, PUFA were detected in all piezophiles examined. Moritella japonica DSK1 produced 22:6n-3 (DHA), whereas the three Shewanella strains produced 20:5n-3 (EPA) with trace amounts of DHA. The detection of low levels of the medium-chain-length PUFA 18:2n-6 and 18:3 (DSK1) and 20:2 (DB6705 and DB21MT-2) suggests that the biosynthesis of EPA and DHA may be regulated by the formation and desaturation of di- and tri-unsaturated FA.

Characterization of the Pressure-induced Intermediate and Unfolded State of Red-shifted Green Fluorescent Protein—A Static and Kinetic FTIR, UV/VIS and Fluorescence Spectroscopy Study

The green fluorescence proteins (GFP) are widely used as reporters in molecular and cell biology. For their use it in high-pressure microbiology and biotechnology studies, their structural properties, thermodynamic parameters and stability diagrams have to be known. We investigated the pressure stability of the red-shifted green fluorescent protein (rsGFP) using Fourier-transform infrared spectroscopy, fluorescence and UV/Vis spectroscopy. We found that rsGFP does not unfold up to ∼9 kbar at room temperature. Its unique three-dimensional structure is held responsible for the high-pressure stability. At higher temperatures, its secondary structure collapses below 9 kbar (e.g. the denaturation pressure at 58 °C is 7.8 kbar). The analysis of the IR data shows that the pressure-denatured state contains more disordered structures at the expense of a decrease of intramolecular β-sheets. As indicated by the large volume change of Δ V° u≈−250(±50) ml mol −1 at 58 °C, this highly cooperative transition can be interpreted as a collapse of the β-can structure of rsGFP. For comparison, the temperature-induced unfolding of rsGFP has also been studied. At high temperature (T m =78 ° C), the unfolding resulted in the formation of an aggregated state. Contrary to the pressure-induced unfolding, the temperature-induced unfolding and aggregation of GFP is irreversible. From the FT-IR data, a tentative p, T-stability diagram for the secondary structure collapse of GFP has been obtained. Furthermore, changes in fluorescence and absorptivity were found which are not correlated to the secondary structural changes. The fluorescence and UV/Vis data indicate smaller conformational changes in the chromophore region at much lower pressures (∼4 kbar) which are probably accompanied by the penetration of water into the β-can structure. In order to investigate also the kinetics of this initial step, pressure-jump relaxation experiments were carried out. The partial activation volumes observed indicate that the conformational changes in the chromophore region when passing the transition state are indeed rather small, thus leading to a comparably small volume change of −20 ml mol −1 only. The use of the chromophore absorption and fluorescence band of rsGFP in using GFP as reporter for gene expression and other microbiological studies under high pressure conditions is thus limited to pressures of about 4 kbar, which still exceeds the pressure range relevant for studies in vivo in micro-organisms, including piezophilic bacteria from deep-sea environments.

Psychromonas kaikoae sp. nov., a novel from the deepest piezophilic bacterium cold-seep sediments in the Japan Trench.

Two strains of obligately piezophilic bacteria were isolated from sediment collected from the deepest cold-seep environment with chemosynthesis-based animal communities within the Japan Trench, at a depth of 7434 m. The isolated strains, JT7301 and JT7304T, were closely affiliated with members of the genus Psychromonas on the basis of 16S rDNA sequence analysis. Hybridization values for DNA-DNA relatedness between these strains and the Psychromonas antarctica reference strain were significantly lower than that accepted as the phylogenetic definition of a species. The optimal temperature and pressure for growth of the isolates were 10 degrees C and 50 MPa and they produced both eicosapentaenoic acid (C20:5omega3) and docosahexaenoic acid (C22:6) in the membrane layer. Based on the taxonomic differences observed, the isolated strains appear to represent a novel obligately piezophilic Psychromonas species. The name Psychromonas kaikoae sp. nov. (type strain JT7304T = JCM 11054T = ATCC BAA-363T) is proposed. This is the first proposed obligately piezophilic species of the genus Psychromonas.