Microbial Detoxification of Sediments Underpins Persistence of Zostera marina Meadows.

Eelgrass meadows have attracted much attention not only for their ability to maintain marine ecosystems as feeding grounds for marine organisms but also for their potential to store atmospheric and dissolved CO2 as blue carbon. This study comprehensively evaluated the bacterial and chemical data obtained from eelgrass sediments of different scales along the Japanese coast to investigate the effect on the acclimatization of eelgrass. Regardless of the eelgrass habitat, approximately 1% Anaerolineales, Babeliales, Cytophagales, and Phycisphaerales was present in the bottom sediment. Sulfate-reducing bacteria (SRB) were present at 3.69% in eelgrass sediment compared to 1.70% in bare sediment. Sulfur-oxidizing bacteria (SOB) were present at 2.81% and 1.10% in the eelgrass and bare sediment, respectively. Bacterial composition analysis and linear discriminant analysis revealed that SOB detoxified H2S in the eelgrass meadows and that the larger-scale eelgrass meadows had a higher diversity of SOB. Our result indicated that there were regional differences in the system that detoxifies H2S in eelgrass meadows, either microbial oxidation mediated by SOB or O2 permeation via the physical diffusion of benthos. However, since bacterial flora and phylogenetic analyses cannot show bias and/or causality due to PCR, future kinetic studies on microbial metabolism are expected.

Distinctive gene and protein characteristics of extremely piezophilic Colwellia.

BACKGROUND: The 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. RESULTS: Significant 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. CONCLUSIONS: We 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.

Colwellia marinimaniae sp. nov., a hyperpiezophilic species isolated from an amphipod within the Challenger Deep, Mariana Trench.

An obligately piezophilic strain was isolated from an amphipod crustacean obtained in the Challenger Deep region of the Mariana Trench during the DEEPSEA CHALLENGE expedition. The strain, MTCD1T, grew at extremely high hydrostatic pressures, with a growth range of 80-140 MPa (optimum, 120 MPa) at 6 °C. Phylogenetic analyses based on the 16S rRNA gene sequence indicate that it is closely affiliated with the genus Colwellia. Comparative 16S rRNA gene sequence analyses revealed 95.7, 95.5 and 95.2 % similarity to Colwellia maris ABE-1T, Colwellia piezophila Y233GT and Colwellia psychrerythraea ATCC 27364T, respectively. The major cellular fatty acids were C16 : 1, C16 : 0 and C22 : 6 (docosahexaenoic acid), and the sole isoprenoid quinone produced was ubiqinone-8. DNA G+C content was 48.6 mol%. The strain was positive for oxidase and catalase activities. Based on the results from this study, strain MTCD1T is a novel Gram-negative species of the genus Colwellia, and the name Colwellia marinimaniae sp. nov. (type strain MTCD1T=ATCC TSD-5T=JCM 30270T) is proposed. It is the most piezophilic organism yet described.

Alicyclobacillus cellulosilyticus sp. nov., a thermophilic, cellulolytic bacterium isolated from steamed Japanese cedar chips from a lumbermill.

A thermophilic bacterium, strain Sueoka(T), was isolated from steamed Japanese cedar chips from a lumber mill in Gobo, Japan. The strain was able to grow on carboxymethyl cellulose at 60 °C, was Gram-stain-negative, and grew between 40.0 and 67.5 °C (optimum at 55 °C) and between pH 3.5 and 6.5 (optimum at pH 4.8). Comparative analysis of 16S rRNA gene sequences revealed 91.9 , 90.9 , and 90.8% similarity to Alicyclobacillus macrosporangiidus(T), Alicyclobacillus pomorum(T), and Alicyclobacillus acidocaldarius(T), respectively. The major quinone was MK-7 and the predominant cellular fatty acids were ω-cyclohexane C19 : 0 and ω-cyclohexane C17 : 0. The DNA G+C content was 60.8 mol%. Based on the results of this study, strain Sueoka(T) is a novel species of the genus Alicyclobacillus, and the namehttp://dx.doi.org/10.1601/nm.5071Alicyclobacillus cellulosilyticus sp. nov. (type strain Sueoka(T)  = JCM 18487(T)  = KCTC 33007(T)) is proposed.

Metagenome-Assembled Genome Sequence of Marine Rhizobiaceae sp. Strain MnEN-MB40S, Obtained from Manganese-Oxidizing Enrichment Culture.

Here, we report a new metagenome-assembled genome (MAG) from a marine Rhizobiaceae species. The MnEN-MB40S genome was assembled from a manganese-oxidizing enrichment culture metagenome. A 4.1-Mb MAG comprising 26 contigs, with a GC content of 60.0%, was obtained. This MAG contributes to the genomic information regarding the family Rhizobiaceae.

Draft Genome Sequence of Saccharomyces cerevisiae DJJ01, Isolated from Dojoji Temple in Gobo, Wakayama, Japan.

Saccharomyces cerevisiae strain DJJ01 was isolated from Dojoji Temple (Gobo, Wakayama, Japan) for development of local breweries. Here, we report the draft genome sequence of this strain to facilitate comparative genomic studies of yeast strains used for Japanese sake brewing.

Effect of hydrostatic pressure on the bilayer phase behavior of symmetric and asymmetric phospholipids with the same total chain length.

The bilayer phase transitions of palmitoylstearoyl-phosphatidylcholine (PSPC), diheptadecanoyl-PC (C17PC) and stearoylpalmitoyl-PC (SPPC) which have the same total carbon numbers in the two acyl chains were observed by differential scanning calorimetry and high-pressure optical method. As the temperature increased, these bilayers exhibited four phases of the subgel (Lc), lamellar gel (L beta'), ripple gel (P beta') and liquid crystal (L alpha), in turn. The Lc phase was observed only in the first heating scan after cold storage. The temperatures of the phase transitions were almost linearly elevated by applying pressure. The temperature-pressure phase diagrams and the thermodynamic quantities associated with the phase transitions were compared among the lipid bilayers. For all the bilayers studied, the pressure-induced interdigitated gel (L beta I) phase appeared above the critical interdigitation pressure (CIP) between the L beta' and P beta' phases. The CIPs for the PSPC, C17PC and SPPC bilayers were found to be 50.6, 79.1 and 93.0 MPa, respectively. Contribution of two acyl chains to thermodynamic properties for the phase transitions of asymmetric PSPC and SPPC bilayers was not even. The sn-2 acyl chain lengths of asymmetric PCs governed primarily the bilayer properties. The fluorescence spectra of Prodan in lipid bilayers showed the emission maxima characteristic of bilayer phases, which were dependent on the location of Prodan in the bilayers. Second derivative of fluorescent spectrum exhibited the original emission spectrum of Prodan to be composed of the distribution of Prodan into multiple locations in the lipid bilayer. The F''497/F''430 value, a ratio of second derivative of fluorescence intensity at 497 nm to that at 430 nm, is decisive evidence whether bilayer interdigitation will occur. With respect to the L beta'/L beta I phase transition in the SPPC bilayer, the emission maximum of Prodan exhibited the narrow-range red-shift from 441 to 449 nm, indicating that the L beta I phase in the SPPC bilayer has a less polar "pocket" formed by a space between uneven terminal methyl ends of the sn-1 and sn-2 chains, in which the Prodan molecule remains stably.

Thermotropic and barotropic phase transitions of N-methylated dipalmitoylphosphatidylethanolamine bilayers.

In order to understand the effect of polar head group modification on the thermotropic and barotropic phase behavior of phospholipid bilayer membranes, the phase transitions of dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidyl-N-methylethanolamine (DPMePE), dipalmitoylphosphatidyl-N,N-dimethylethanolamine (DPMe2PE) and dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were observed by differential scanning calorimetry and high-pressure optical methods. The temperatures of the so-called main transition from the gel (L(beta)) or ripple gel (P(beta)') phase to the liquid crystalline (L(alpha)) phase were almost linearly elevated by applying pressure. The slope of the temperature-pressure boundary, dT/dp, was in the range of 0.220-0.264 K MPa(-1) depending on the number of methyl groups in the head group of lipids. The main-transition temperatures of N-methylated DPPEs decreased with increasing size of head group by stepwise N-methylation. On the other hand, there was no significant difference in thermodynamic quantities of the main transition between the phospholipids. With respect to the transition from the subgel (L(c)) phase to the lamellar gel (L(beta) or L(beta)') phase, the transition temperatures were also elevated by applying pressure. In the case of DPPE bilayer the L(c)/L(beta) transition appeared at a pressure higher than 21.8 MPa. At a pressure below 21.8 MPa the L(c)/L(alpha) transition was observed at a temperature higher than the main-transition temperature. The main (L(beta)/L(alpha)) transition can be recognized as the transformation between metastable phases in the range from ambient pressure to 21.8 MPa. Polymorphism in the gel phase is characteristic of DPPC bilayer membrane unlike other lipid bilayers used in this study: the L(beta)', P(beta)' and pressure-induced interdigitated gel (L(beta)I) phases were observed only in the DPPC bilayer. Regarding the bilayers of DPPE, DPMePE and DPMe2PE, the interdigitation of acyl chain did not appear even at pressures as high as 200 MPa.

Effect of deuterium oxide on the thermodynamic quantities associated with phase transitions of phosphatidylcholine bilayer membranes.

The bilayer phase transitions of three kinds of phospholipids, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and dihexadecylphosphatidylcholine (DHPC), in deuterium oxide (D(2)O) and hydrogen oxide (H(2)O) were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The DSC measurements showed that the substitution of H(2)O by D(2)O affected the pretransition temperatures and the main-transition enthalpies of all PC bilayers. The temperature-pressure phase diagrams for these PC bilayer membranes in both solvents were constructed by use of the data of light-transmittance measurements. Regarding the main transition of all PC bilayer membranes, there was no appreciable difference between the transition temperatures in D(2)O and H(2)O under high pressure. On the other hand, the phase transitions among the gel phases including the pretransition were significantly affected by the solvent substitution. The thermodynamic quantities of phase transitions for the PC bilayer membranes were evaluated and the differences in thermodynamic properties by the water substitution were considered from the difference of interfacial-free energy per molecule in the bilayer in both solvents. It was proved that the substitution of H(2)O by D(2)O causes shrinkage of the molecular area of phospholipid at bilayer interface due to the difference in bond strength between deuterium and hydrogen bonds and produces the great influence on the bilayer phase with the smaller area. Further, the induction of bilayer interdigitation in D(2)O turned out to need higher pressures than in H(2)O.

Bilayer phase transitions of N-methylated dioleoylphosphatidylethanolamines under high pressure.

The bilayer phase transitions of four kinds of unsaturated phospholipids with different-sized polar head groups, dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidyl-N-methylethanolamine (DOMePE), dioleoylphosphatidyl-N,N-dimethylethanolamine (DOMe2PE) and dioleoylphosphatidylcholine (DOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light-transmittance. DSC thermogram and light-transmittance curve for each phospholipid vesicle solution exhibited only one phase transition under ambient pressure, respectively. The light-transmittance of DOPC solution at pressure higher than 234 MPa abruptly increased stepwise at two temperatures, which corresponds to the appearance of stable subgel and lamellar gel phases under high pressure in addition to the liquid crystal phase. The constructed temperature (T)-pressure (p) phase diagrams were compared among these phospholipids. The phase-transition temperatures of the phospholipids decreased stepwise by N-methylation of the head group. The slops of the T-p phase boundary (dT/dp) of DOPE, DOMePE and DOMe2PE bilayers (0.127-0.145 K MPa-1) were found to be close to that of the transition from the lamellar crystal (or subgel; Lc) phase to the liquid crystal (Lalpha) phase for DOPC bilayer (0.131 K MPa-1). On the other hand, the dT/dp value of the main transition from the lamellar gel (Lbeta) phase to the Lalpha phase for DOPC bilayer (0.233 K MPa-1) was significantly different from that of the Lc/Lalpha transition, hence both curves intersected with each other at 234 MPa. The thermodynamic quantities associated with the phase transition of DOPE, DOMePE and DOMe2PE bilayers had also similar values to those for the Lc/Lalpha transition of DOPC bilayer. Taking into account of the values of transition temperature, dT/dp and thermodynamic quantities compared with the corresponding results of saturated phospholipids, we identified the phase transitions observed in the DOPE, DOMePE and DOMe2PE bilayers as the transition from the Lc phase to the Lalpha phase although they have been regarded as the main transition in the previous studies. The Lbeta phase is probably unstable for DOPE, DOMePE and DOMe2PE bilayers at all pressures, it exists as a metastable phase at pressures below 234 MPa while as a stable phase at pressures above 234 MPa in DOPC bilayer. The difference in phase stability among the phospholipid bilayers is originated from that in hydration structure of the polar head groups.

Pressure-induced phase transitions of lipid bilayers observed by fluorescent probes Prodan and Laurdan.

The fluorescence spectra of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and 6-dodecanoyl-2-(dimethylamino)naphthalene (Laurdan) in bilayer membranes of 1,2-distearoylphosphatidylcholine (DSPC) were observed as a function of pressure at constant temperature. The emission spectra of Prodan and Laurdan varied with the pressure-induced states of bilayer membranes. The maximum emission wavelength (lambda(max)) of Prodan characteristic of the liquid crystalline (L(alpha)), lamellar gel (L(beta)') and pressure-induced interdigitated gel (L(beta)I) phases of the DSPC bilayer was 480, 440 and 500 nm, respectively. On the other hand, the lambda(max) of Laurdan characteristic of the L(alpha) and L(beta)' phases was 480 and 440 nm in a similar manner as Prodan probe. However, no change in the lambda(max) was observed in spite of the occurrence of the interdigitation of bilayer. Since the lambda(max) reflects the solvent property around the probe molecules, we could speculate about the location of fluorescent probe in the bilayer membranes. In the L(alpha) phase the same chromophore group of Prodan and Laurdan probes distributes around phosphate group of lipid (i.e., polar region). The transformation of bilayer into the L(beta)' phase causes the Prodan and Laurdan molecules to move into the glycerol backbone (i.e., less polar) region. In the ripple gel (P(beta)') phase, the emission spectrum of Prodan shows a broad peak at about 480 nm and a shoulder around 440 nm, which means that the Prodan molecules are widespread over the wide range from the glycerol backbone to the hydrophilic part of bilayer. The P(beta)'/L(beta)I phase transition causes the Prodan molecule to squeeze out from the glycerol backbone region and to move the hydrophilic region near the bilayer surface. Contrarily, the Laurdan molecule was not squeezed out from the glycerol backbone region because the long acyl chain of Laurdan serves as an anchor in the hydrophobic core of bilayer. The ratio of fluorescence intensity of Prodan at 480 nm to that at 440 nm, F(480)/F(440), is available to observation of bilayer phase transitions. The plot of F(480)/F(440) versus pressure seems to be useful for the recognition of bilayer phase transition, especially the bilayer interdigitation.

Effect of pressure on the Prodan fluorescence in bilayer membranes of phospholipids with varying acyl chain lengths.

The fluorescence spectra of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) were observed as a function of pressure for the bilayer membrane systems of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC). The wavelength of the emission maximum, lambdamax, was found to be 480, 430, and 500 nm for the liquid crystalline (Lalpha), ripple gel (P'beta), and pressure-induced interdigitated gel (LbetaI) phase, respectively. Since the lambdamax reflects the solvent property around the probe molecules, we could speculate on the location of the Prodan molecules in the bilayer membranes; in the Lalpha phase of the lipid bilayer, the Prodan molecules distribute around the phosphate of the lipids (i.e. the polar region). The Lalpha/P'beta phase transition caused the Prodan molecules to move into the less polar region near the glycerol backbone. The fluorescence intensity of the Prodan in the P'beta phase was dependent on the chain length of the lipids and on pressure; the shorter the chain length of the lipid, the stronger the fluorescence intensity of the Prodan. Moreover, for the DLPC bilayer membrane system, the fluorescence intensity at 430 nm increased with increasing pressure, indicating that the partition of Prodan into the DLPC bilayer membrane is promoted by applying pressure. In the case of the DPPC and DSPC bilayers, as the pressure increased further, the pressure-induced interdigitation caused the Prodan molecules to squeeze out of the glycerol backbone region and to move the hydrophilic region near the bilayer surface. The ratio of fluorescence intensity at 480 nm to that at 430 nm, F480/F430, showed a sharp change at the phase-transition pressure. In the case of the DPPC and DSPC bilayers, the values of F480/F430 showed an abrupt increase above a certain pressure higher than the Lalpha/P'beta transition pressure, which corresponds to the interdigitation from the P'beta to the LbetaI phase. The plot of F480/F430 versus pressure is available for recognition of the bilayer phase transitions, especially the bilayer interdigitation.

Pressure study on symmetric and asymmetric phospholipid bilayers: effect of vesicle size on Prodan fluorescence.

The bilayer phase behavior of symmetric and asymmetric phosphatidylcholines (PCs), 1,2-diheptadecanoyl-PC (C17PC), 1-palmitoyl-2-stearoyl-PC (PSPC), and 1-stearoyl-2-palmitoyl-PC (SPPC), with different vesicle sizes were investigated by a high-pressure fluorescence method using the polarity-sensitive fluorescent probe Prodan. The second derivative of fluorescence spectra for all the PCs of small-sized vesicle showed four minima characteristic of four membrane states on the spectra irrespective of the acyl-chain symmetry, whereas those of large-sized vesicle had one more minimum originating from the most hydrophilic site at the membrane surface. These findings indicate that Prodan molecules can distribute into multiple sites in the bilayer and move around the head-group region depending on the vesicle size. The behavior of the spectra in the SPPC bilayer suggested that the interdigitated gel phase had a less polar "pocket" formed by a space between uneven terminal methyl ends of the sn-1 and sn-2 chains. It turned out that the curvature of vesicles affects the distribution of the Prodan molecules in all phases, more particularly in the interdigitated gel phase.

Recovery of tobacco BY-2 cells after high hydrostatic pressure treatment.

The recovery of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells in Linsmaire and Skoog medium after treatment at high hydrostatic pressure was investigated using an Evans Blue staining method to discriminate live from dead cells. The survival of BY-2 cells just after the high-pressure treatment at 5 degrees C and 25 degrees C decreased abruptly at pressures higher than 50 MPa and 100 MPa, respectively. Furthermore, almost all of the BY-2 cells treated at 5 degrees C and 25 degrees C recovered pressures below 25 MPa and 75 MPa, respectively. However, no BY-2 cells recovered at pressures above 100 MPa at either temperature.