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ABSTRACT: We developed an electrical modulation method for attachment and detachment of microorganisms. Living microorganisms suspended in non-nutritive media such as PBS(-) and artificial seawater were attracted by and selectively attached to indium tin oxide (ITO)/glass electrode regions to which a negative potential was applied. The microorganisms suspended in LB medium and glucose solution were not attracted to the ITO electrode. Dead microorganisms were not attracted to the ITO electrode. The living microorganisms were retrieved after detachment from the ITO electrode by application of a high-frequency triangular wave potential. When we applied this method to separate microorganisms from deep-sea sediment, bacteria belonging to 19 phyla and 23 classes were collected without undesirable high molecular weight contaminants such as humic acids. At the phylum and class level, respectively, 95 and 87 % of the phylotypes among electrically retrieved bacteria were common to the gene clones from the direct sediment DNA extraction. This technique is a novel useful method to prepare bacterial cells in a single population or a community for metagenomic analyses.
Marine Biotechnology 02/2013; · 3.43 Impact Factor
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ABSTRACT: To understand the pressure-adaptation mechanism of deep-sea enzymes, we studied the effects of pressure on the enzyme activity and structural stability of dihydrofolate reductase (DHFR) of the deep-sea bacterium Moritella profunda (mpDHFR) in comparison with those of Escherichia coli (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50MPa whereas ecDHFR was monotonically inactivated by pressure, suggesting inherent pressure-adaptation mechanisms in mpDHFR. The secondary structure of apo-mpDHFR was stable up to 80°C, as revealed by circular dichroism spectra. The free energy changes due to pressure and urea unfolding of apo-mpDHFR, determined by fluorescence spectroscopy, were smaller than those of ecDHFR, indicating the unstable structure of mpDHFR against pressure and urea despite the three-dimensional crystal structures of both DHFRs being almost the same. The respective volume changes due to pressure and urea unfolding were -45 and -53ml/mol at 25°C for mpDHFR, which were smaller (less negative) than the corresponding values of -77 and -85ml/mol for ecDHFR. These volume changes can be ascribed to the difference in internal cavity and surface hydration of each DHFR. From these results, we assume that the native structure of mpDHFR is loosely packed and highly hydrated compared with that of ecDHFR in solution.
Biochimica et Biophysica Acta 03/2012; 1824(3):511-9. · 4.66 Impact Factor
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ABSTRACT: Shewanella violacea DSS12 is a psychrophilic piezophile that optimally grows at 30MPa. It contains a substantial amount of eicosapentaenoic acid (EPA) in the membrane. Despite evidence linking increased fatty acid unsaturation and bacterial growth under high pressure, little is known of how the physicochemical properties of the membrane are modulated by unsaturated fatty acids in vivo. By means of the newly developed system performing time-resolved fluorescence anisotropy measurement under high pressure (HP-TRFAM), we demonstrate that the membrane of S. violacea is highly ordered at 0.1MPa and 10°C with the order parameter S of 0.9, and the rotational diffusion coefficient D(w) of 5.4μs(-1) for 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatriene in the membrane. Deletion of pfaA encoding the omega-3 polyunsaturated fatty acid synthase caused disorder of the membrane and enhanced the rotational motion of acyl chains, in concert with a 2-fold increase in the palmitoleic acid level. While the wild-type membrane was unperturbed over a wide range of pressures with respect to relatively small effects of pressure on S and D(w), the ΔpfaA membrane was disturbed judging from the degree of increased S and decreased D(w). These results suggest that EPA prevents the membrane from becoming hyperfluid and maintains membrane stability against significant changes in pressure. Our results counter the generally accepted concept that greater fluidity is a membrane characteristic of microorganisms that inhabit cold, high-pressure environments. We suggest that retaining a certain level of membrane physical properties under high pressure is more important than conferring membrane fluidity alone.
Biochimica et Biophysica Acta 03/2012; 1818(3):574-83. · 4.66 Impact Factor
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ABSTRACT: For efficient oxygen supply to pressurized culture, we developed a method using a highly pressurized membrane reactor with an air-saturated medium circulation system. The new method increased the cell growth of aerobic yeast approximately 20 folds larger than that in the case of using a conventional method.
Journal of Bioscience and Bioengineering 02/2012; 113(2):220-3. · 1.79 Impact Factor
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ABSTRACT: The influence of hydrostatic pressure on microbial sulfate reduction (SR) was studied using sediments obtained at cold seep sites from 5500 to 6200 m water depth of the Japan Trench. Sediment samples were stored under anoxic conditions for 17 months in slurries at 4°C and at in situ pressure (50 MPa), at atmospheric pressure (0.1 MPa), or under methanic conditions with a methane partial pressure of 0.2 MPa. Samples without methane amendment stored at in situ pressure retained higher levels of sulfate reducing activity than samples stored at 0.1 MPa. Piezophilic SR showed distinct substrate specificity after hydrogen and acetate addition. SR activity in samples stored under methanic conditions was one order of magnitude higher than in non-amended samples. Methanic samples stored under low hydrostatic pressure exhibited no increased SR activity at high pressure even with the amendment of methane. These new insights into the effects of pressure on substrate specific sulfate reducing activity in anaerobic environmental samples indicate that hydrostatic pressure must be considered to be a relevant parameter in ecological studies of anaerobic deep-sea microbial processes and long-term storage of environmental samples.
Frontiers in microbiology. 01/2012; 3:253.
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ABSTRACT: Cytochrome c₅ of pressure-sensitive Shewanella livingstonensis (SL cytc₅) exhibits lower thermal stability than a highly homologous counterpart of pressure-tolerant Shewanella violacea. This stability difference is due to an enthalpic effect that can be attributed to the amino acid residue at position 50 (Leu or Lys). These cytc₅ proteins are appropriate materials for understanding the protein stability mechanism.
Bioscience Biotechnology and Biochemistry 09/2011; 75(9):1859-61. · 1.28 Impact Factor
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ABSTRACT: We studied the quantum interference of electrons in the Bi (p(x), p(y)) orbital-derived j = 1/2 spin-split surface states at Bi/Ag(111)√3 × √3 surfaces of 10 monolayer thick Ag(111) films on Si(111) substrates. Surface electron standing waves were observed clearly at the energy (E) below the intersection of the two spin-split downward dispersing parabola bands (E(x)). The E dependence of the standing wave pattern reveals the dispersion as the average of the two spin-split surface bands due to the interference between |(k + Δ), ↑> and |-(k - Δ), ↑> [or (|(k - Δ), ↓>) and |-(k + Δ), ↓>] states. In contrast, it was impossible to deduce the dispersion from the standing wave pattern at E ≥ E(x) because the surface electron cannot find its backscattered state with the same spin polarization.
Physical Review Letters 07/2011; 107(2):027204. · 7.37 Impact Factor
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ABSTRACT: The facultative piezophile Shewanella violacea DSS12 is known to alter its respiratory components under the influence of hydrostatic pressure during growth, suggesting that it has a respiratory system that functions in adaptation to high pressure. We investigated the pressure- and temperature-dependencies of the respiratory terminal oxidase activity of the membrane of S. violacea relative to non-piezophilic Shewanella species. We observed that the activity in the membrane of S. violacea was more resistant to high pressure than those of non-piezophilic Shewanella even though DSS12 was cultured under atmospheric pressure. On the other hand, the temperature dependency of this activity was almost the same for all of the tested strain regardless of optimal growth temperature. Both high pressure and low temperature are expected to lower protein flexibility, causing a decrease in enzyme activity, but the results of this study suggest that the mechanism maintaining enzyme activity under high hydrostatic pressure is different from that at low temperature. Additionally, the responses of the activity to the pressure- and temperature-changes were independent of membrane lipid composition. Therefore, the piezotolerance of the respiratory terminal oxidases of S. violacea is perhaps dependent on the properties of the protein itself and not on the lipid composition of the membrane. Our observations suggest that S. violacea constitutively express piezotolerant respiratory terminal oxidases that serve adaptation to the deep-sea environment.
Bioscience Biotechnology and Biochemistry 05/2011; 75(5):919-24. · 1.28 Impact Factor
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ABSTRACT: Shewanella violacea DSS12, a deep-sea bacterium, produces eicosapentaenoic acid (EPA) as a component of membrane phospholipids. Although various isolates from the deep sea, such as Photobacterium profundum SS9, Colwellia psychrerythraea 34H and various Shewanella strains, produce EPA- or docosahexaenoic acid-containing phospholipids, the physiological role of these polyunsaturated fatty acids remains unclear. In this article, we illustrate the physiological importance of EPA for high-pressure adaptation in strain DSS12 with the help of an EPA-deficient mutant (DSS12(pfaA)). DSS12(pfaA) showed significant growth retardation at 30 MPa, but not at 0.1 MPa. We also found that DSS12(pfaA) grown at 30 MPa forms filamentous cells. When an EPA-containing phospholipid (sn-1-oleoly-sn-2-eicosapentaenoyl phosphatidylethanolamine) was supplemented, the growth retardation and the morphological defect of DSS12(pfaA) were suppressed, indicating that the externally added EPA-containing phospholipid compensated for the loss of endogenous EPA. In contrast, the addition of an oleic acid-containing phospholipid (sn-1,2-dioleoyl phosphatidylethanolamine) did not affect the growth and the morphology of the cells. Immunofluorescent microscopic analysis with anti-FtsZ antibody revealed a number of Z-rings and separated nucleoids in DSS12(pfaA) grown at 30 MPa. These results demonstrate the physiological importance of EPA for the later step of Z-ring formation of S. violacea DSS12 under high-pressure conditions.
Environmental Microbiology 04/2011; 13(8):2293-8. · 5.84 Impact Factor
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ABSTRACT: To examine whether dihydrofolate reductase (DHFR) from deep-sea bacteria has undergone molecular evolution to adapt to high-pressure environments, we cloned eight DHFRs from Shewanella species living in deep-sea and ambient atmospheric-pressure environments, and subsequently purified six proteins to compare their structures, stabilities, and functions. The DHFRs showed 74-90% identity in primary structure to DHFR from S. violacea, but only 55% identity to DHFR from Escherichia coli (ecDHFR). Far-ultraviolet circular dichroism and fluorescence spectra suggested that the secondary and tertiary structures of these DHFRs were similar. In addition, no significant differences were found in structural stability as monitored by urea-induced unfolding and the kinetic parameters, K(m) and k(cat); although the DHFRs from Shewanella species were less stable and more active (2- to 4-fold increases in k(cat)/K(m)) than ecDHFR. Interestingly, the pressure effects on enzyme activity revealed that DHFRs from ambient-atmospheric species are not necessarily incompatible with high pressure, and DHFRs from deep-sea species are not necessarily tolerant of high pressure. These results suggest that the DHFR molecule itself has not evolved to adapt to high-pressure environments, but rather, those Shewanella species with enzymes capable of retaining functional activity under high pressure migrated into the deep-sea.
Extremophiles 12/2010; 15(2):165-75. · 2.94 Impact Factor
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Eiji Aono,
Tomoya Baba,
Takeshi Ara,
Tatsunari Nishi,
Tomoko Nakamichi,
Eiji Inamoto,
Hiromi Toyonaga,
Miki Hasegawa,
Yuki Takai,
Yoshiko Okumura,
Miki Baba,
Masaru Tomita, Chiaki Kato,
Taku Oshima,
Kaoru Nakasone,
Hirotada Mori
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ABSTRACT: Remineralization of organic matter in deep-sea sediments is important in oceanic biogeochemical cycles, and bacteria play a major role in this process. Shewanella violacea DSS12 is a psychrophilic and piezophilic gamma-proteobacterium that was isolated from the surface layer of deep sea sediment at a depth of 5110 m. Here, we report the complete genome sequence of S. violacea and comparative analysis with the genome of S. oneidensis MR-1, isolated from sediments of a freshwater lake. Unlike S. oneidensis, this deep-sea Shewanella possesses very few terminal reductases for anaerobic respiration and no c-type cytochromes or outer membrane proteins involved in respiratory Fe(iii) reduction, which is characteristic of most Shewanella species. Instead, the S. violacea genome contains more terminal oxidases for aerobic respiration and a much greater number of putative secreted proteases and polysaccharases, in particular, for hydrolysis of collagen, cellulose and chitin, than are encoded in S. oneidensis. Transporters and assimilatory reductases for nitrate and nitrite, and nitric oxide-detoxifying mechanisms (flavohemoglobin and flavorubredoxin) are found in S. violacea. Comparative analysis of the S. violacea genome revealed the respiratory adaptation of this bacterium to aerobiosis, leading to predominantly aerobic oxidation of organic matter in surface sediments, as well as its ability to efficiently use diverse organic matter and to assimilate inorganic nitrogen as a survival strategy in the nutrient-poor deep-sea floor.
Molecular BioSystems 07/2010; 6(7):1216-26. · 3.53 Impact Factor
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ABSTRACT: 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.
Journal of biochemistry 04/2010; 147(4):591-9. · 1.95 Impact Factor
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ABSTRACT: We obtained 19 individual DNA fragments encoding 2-deoxy-scyllo-inosose synthase involved in the biosynthesis of aminoglycoside antibiotics from deep-sea sediments of the Pacific Ocean. Compared with genes from land-based environmental DNA, they showed low diversity. Combined with our previous study concerning the discovery of other aminoglycoside-biosynthetic genes from the same deep-sea samples, we suggest the importance of exploration of multiple biosynthetic genes to determine the diversity of aminoglycoside producers. We found that the deep sea is a useful source for screening of these genes.
Bioscience Biotechnology and Biochemistry 01/2010; 74(5):1102-5. · 1.28 Impact Factor
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Physical Review B. 01/2010; 82(16):1-7.
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Chiaki Kato
Seikagaku. The Journal of Japanese Biochemical Society 12/2009; 81(12):1094-100. · 0.04 Impact Factor
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ABSTRACT: 3-isopropylmalate dehydrogenase (IPMDH)-encoding leuB genes were obtained from the obligate piezophile Shewanella benthica DB21MT-2 and non-piezophile Shewanella oneidensis MR-1. The genes were expressed in Escherichia coli and the proteins were purified using His-tag. The estimated kinetic parameters of these enzymes indicated that IPMDH of S. benthica DB21MT-2 is more tolerant of high pressure than that of S. oneidensis MR-1. Thus such an adaptation is one of the mechanisms bacteria utilize for survival at high pressures.
Bioscience Biotechnology and Biochemistry 11/2009; 73(11):2541-3. · 1.28 Impact Factor
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ABSTRACT: To understand sulfur oxidation in thioauto-trophic deep-sea clam symbionts, we analyzed the recently reported genomes of two chemoautotrophic symbionts of Calyptogena okutanii (Candidatus Vesicomyosocius okutanii strain HA: Vok) and C. magnifica (Candidatus Ruthia magnifica strain Cm: Rma), and examined the sulfur oxidation gene expressions in the Vok by RT-PCR. Both symbionts have genes for sulfide-quinone oxidoreductase (sqr), dissimilatory sulfite reductase (dsr), reversible dissimilatory sulfite reductase (rdsr), sulfur-oxidizing multienzyme system (sox)(soxXYZA and soxB but lacking soxCD), adenosine phosphosulfate reductase (apr), and ATP sulfurylase (sat). While these genomes share 29 orthologous genes for sulfur oxidation implying that both symbionts possess the same sulfur oxidation pathway, Rma has a rhodanese-related sulfurtransferase putative gene (Rmag0316) that has no corresponding ortholog in Vok, and Vok has one unique dsrR (COSY0782). We propose that Calyptogena symbionts oxidize sulfide and thiosulfate, and that sulfur oxidation proceeds as follows. Sulfide is oxidized to sulfite by rdsr. Sulfite is oxidized to sulfate by apr and sat. Thiosulfate is oxidized to zero-valence sulfur by sox, which is then reduced to sulfide by dsr. In addition, thiosulfate may also be oxidized into sulfate by another component of sox. The result of the RT-PCR showed that genes (dsrA, dsrB, dsrC, aprA, aprB, sat, soxB, and sqr) encoding key enzymes catalyzing sulfur oxidation were all equally expressed in the Vok under three different environmental conditions (aerobic, semioxic, and aerobic under high pressure at 9 MPa), indicating that all sulfur oxidation pathways function simultaneously to support intracellular symbiotic life.
Extremophiles 10/2009; 13(6):895-903. · 2.94 Impact Factor
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ABSTRACT: Shewanella violacea DSS12 is facultative piezophile isolated from the deep-sea. The expression of cydDC genes (required for d-type cytochrome maturation) of the organism is regulated by hydrostatic pressure. In this study, we analyzed the nucleotide sequence upstream of cydDC in detail and found that there are putative binding sites for the NarL protein which is part of a two-component regulatory system also containing the sensor protein NarX. Furthermore, we identified the narQP genes (homologues of narXL) from S. violacea DSS12 and demonstrated the heterologous expression of narP in Escherichia coli. These results will be helpful in examining pressure regulation of gene expression in S. violacea at the molecular level.
07/2009; 19(3):308-312.
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ABSTRACT: The deep-sea denitrifier Pseudomonas sp. strain MT-1 has two distinct gene clusters encoding dissimilatory nitrate reductases, periplasmic nitrate reductase (Nap) and membrane-bound nitrate reductase (Nar). In order to investigate the physiological roles of these enzymes, we determined the nitrate reductase activity of the soluble and membrane fractions from MT-1 and the type strain of Pseudomonas stutzeri (closely related with MT-1) grown under various conditions. In MT-1, the activities of both fractions were highest when the cells were grown anaerobically in the presence of nitrate under atmospheric pressure. However, the activity of the soluble fraction decreased when the cells were grown under high pressure, whereas that of membrane fraction remained constant. Further, the activity of the soluble fraction decreased when the enzyme reaction was performed at low temperature, although that of membrane fraction was not similarly affected. Additionally, the results of RT-PCR showed that expression of the nar genes was strongly induced under high pressure. In contrast, P. stutzeri(T) showed no such response following a shift in growth pressure. These results suggest that MT-1 possesses a special mechanism for adaptation to the low-temperature and high-pressure environments of the deep sea, and that Nar is the main dissimilatory nitrate reductase in MT-1 in such environments.
Bioscience Biotechnology and Biochemistry 05/2009; 73(4):896-900. · 1.28 Impact Factor
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ABSTRACT: Two genes for alternative sigma factors, sigma(E2) and sigma(E3), classified in the extracytoplasmic function sigma family for RNA polymerases, were identified in the deep-sea piezophilic bacterium Shewanella violacea DSS12. Amino acid alignments revealed that the domains for transcriptional functions were comparatively conserved compared with Escherichia coli sigma(E) in both proteins. Core-binding analysis suggested that both proteins function as sigma factors.
Bioscience Biotechnology and Biochemistry 02/2009; 73(1):200-2. · 1.28 Impact Factor
