Search

Expression of p73, a p53 family member regulating cell growth and apoptosis, is maintained at low levels in mammalian cells, and cellular activation of p73 is usually controlled at the protein level. However, the precise molecular mechanisms by which p73 stability is regulated are unclear. During the search for interacting molecules with the COOH-terminal proline-rich region of p73, we identified a novel NEDD4-related protein (termed as NEDL2) which contains a C2 domain at its NH(2)-terminus, two WW domains, and a HECT domain at its COOH-terminus. As expected, NEDL2 catalyzed the ubiquitination of bacterial cellular proteins in vitro. Reciprocal co-immunoprecipitation experiments and in vitro pull-down assays revealed that NEDL2 bound to p73, which carries two putative PY motifs. p73 was efficiently ubiquitinated but stabilized in a NEDL2-dependent manner. Accordingly, p73 decayed at faster rates in the absence of NEDL2 than in its presence. Consistent with the NEDL2-mediated stabilization of p73, NEDL2 enhanced the p73-dependent transcriptional activation. Thus, our results suggest that NEDL2 activates the function of p73 by increasing its stability.

High pressure induces a cellular stress response. This response was investigated in lactic acid bacteria on the level of their proteome and cellular organization, as well as in changes in the bacterial cell membrane. It is evident that the cytoplasmic membrane is a sensitive target and mediator of pressure induced stress response and lethal effects. A working model was established for high pressure response and injury of cellular systems distinguishing direct (primary) from indirect (secondary) pressure effects: The most pressure sensitive targets are affected directly and instantly during the ramp above 50 MPa and appear to be macromolecules requiring association to function in cellular organization and the bacterial membrane. At higher pressures, typically present during pressure holding times, pressure mediated indirect intracellular changes, e. g. loss of ions or drop in pH, are synergic stress factors prone to co-determine high pressure stress response and cell death.

Strain DSK1 is a novel moderately barophilic bacterium isolated from the Japan Trench at a depth of 6,356 m. Phylogenetic analysis based on 16S ribosomal DNA sequences showed that strain DSK1 represents a separate lineage with the Shewanella barophiles branch and is closely related to Moritella marina. Comparisons of the temperature and pressure range for growth and some biochemical characteristics indicate that strain DSK1 differs from M. marina and Shewanella barophilic species. Furthermore, strain DSK1 displays a low level of DNA similarity to the Moritella and Shewanella type strains; however this isolate characteristically produces DHA (22:6) as a membrane fatty acids, and the fatty acid profile of this strain is similar to that of M. marina. Because of these differences, strain DSK1 appears to represent a novel deep-sea Moritella species. The name Moritella japonica is proposed. The type strain is JCM 10249.

Deep-sea bacteria have unique systems for gene and protein expression controlled by hydrostatic pressure. One of the sigma factors, sigma54, was found to play an important role in pressure-regulated transcription in a deep-sea piezophilic bacterium, Shewanella violacea. A glutamine synthetase gene (glnA) has been targeted as a model for the pressure-regulated promoter to investigate transcriptional regulation by the sigma54 factor. Recognition sites for sigma54 and sigma70 factors were observed at an upstream region of the glnA, and NtrC-binding sites were also identified at the same region. Primer extension analyses revealed that the transcription initiation sites of both promoters were determined and that transcription from the sigma54 site was regulated by elevated pressure. The sigma54 promoter is known to be activated by a two-component signal transduction system, the NtrB-NtrC phosphorylation relay. Our results suggested that this system might be regulated by deep-sea conditions and that the gene expression controlled by the sigma54 promoter was actually regulated by pressure. We propose a possible model of the molecular mechanisms for pressure-regulated transcription.

Deep-sea bacteria have unique systems for the gene and protein expression controlled by hydrostatic pressure. One of the σ factors, σ 54 , was found to play an important role on the pressure-regulated transcription in a deep-sea piezophilic bacterium, Shewanella violacea . A glutamine synthetase gene ( glnA ) has been targeted as a model for the pressure-regulated promoter to investigate the transcriptional regulation by the σ 54 factor. Recognition sites for σ 54 and σ 70 factors were observed at an upstream region of the glnA and also NtrC-binding sites were identified at this same region. Primer extension analyses revealed that the transcription initiation sites of both promoters were determined and that the transcription from the σ 54 site was regulated by elevated pressure. The σ 54 promoter is known to be activated by a two components signal transduction system, NtrB-NtrC phospholylated relay. Our results suggested that this system might be regulated by deep-sea conditions and that the gene expression controlled by the σ 54 promoter was actually regulated by pressure. We proposed a possible model of the molecular mechanisms for pressure-regulated transcription.

The elongation of Escherichia coli cells, in response to high hydrostatic pressure growth, is a unique property of this bacterium. Our high pressure observation microscope system (HPOMS) has provided direct observation of living E. coli elongated cells under high pressure. To study elongation mechanisms of E. coli under high pressure, the formation of FtsZ ring which was an early event of cell division was examined by immuno-fluorescence microscopy (IFM), and the chromosomal DNA was also detected by DAPI staining. The FtsZ-ring formation and the condensed nucleoid structure was observed in the filamentous cells when the cells were fixed after releasing the pressure. Surprisingly, no FtsZ ring was observed in whole filamentous cells, when the cells were fixed without decompression. Also the chromosomal DNA was segregated but not condensed at the same condition. These results suggested that the cell division of E. coli was inhibited by high hydrostatic pressure at early stage in the cell cycle before the FtsZ-ring formation during cultivation, however, cells could grow continuously without septum formation, chromosomal DNA condensation.

Technological advancements in the recovery and cultivation of deep-sea microorganisms have resulted in the isolation of novel groups of bacteria which are adapted to high pressure. From these collections a number of new species have been identified, including members of the genera Shewanella, Moritella, Photobacterium and Colwellia Some of these isolates have been the subjects of investigations into pressure effects on gene, protein and fatty acid regulation and mechanisms of baro (piezo) adaptation or sensing. Cytochrome bd assembly and function appears to be critical to pressure adaptation in at least one Shewanella species, and the ToxR/S proteins are required for pressure-responsive regulation in a Photobacterium strain.

We have isolated the genes for quinol oxidase from a deep-sea piezophilic bacterium, Shewanella violacea. Analysis of the deduced amino acid sequences of the cyo subunits showed that this oxidase has high similarity to Escherichia coli bo-type quinol oxidase. Northern blot analysis showed that these genes are expressed at a high level when the bacterium is grown at elevated pressure. Upstream in the cyo-operon, a sigma54-binding motif and an octamer sequence unit were found, suggesting that these elements may play a role in regulation of expression of the cyo-operon in response to changes in pressure.

The genus Shewanella is one of the typical deep-sea bacterial genera. Two isolated deep-sea Shewanella species, Shewanella benthica and Shewanella violacea, were found to be able to grow better under high hydrostatic pressure conditions than at atmospheric pressure. These species are not only piezophilic (barophilic), but also psychrophilic. Many psychrophilic and psychrotolerant Shewanella species have been isolated and characterized from cold environments, such as seawater in Antarctica or the North Sea. Some of these cold-adapted Shewanella were shown to be piezotolerant, meaning that growth occurs in a high-pressure habitat. In this review, we propose that two major sub-genus branches of the genus Shewanella should be recognized taxonomically, one group characterized as high-pressure cold-adapted species that produce substantial amounts of eicosapentaenoic acid, and the other group characterized as mesophilic pressure-sensitive species.

The rpoA gene encoding the alpha subunit of RNA polymerase from the deep-sea piezophilic bacterium Shewanella violacea DSS12 was cloned and sequenced. The rpoA gene was found to encode a polypeptide consisting of 329 amino acids with a molecular mass of 36238 Da. S. violacea alpha protein was expressed in a ts Escherichia coli mutant, to confirm whether the rpoA gene is functional. It complemented this mutation, indicating a chimeric RNA polymerase is assembled at the non-permissive temperature. Recombinant alpha protein was overexpressed using an expression plasmid harboring the rpoA gene and purified to near homogeneity. Primer extension analysis revealed that two transcriptional initiation sites are recognized by sigma(70) RNA polymerase. It also indicated that pressure response (piezoresponse) in the alpha operon occurred at the transcriptional level, suggesting some positive regulators may interact with the transcriptional apparatus and regulate the expression of the operon at different pressure conditions.

Azacycloundecenes, which are key intermediates in the synthesis of derivatives of the marine alkaloid manzamine C, are conveniently prepared using ring-closing olefin metathesis (RCM).

The ntrBC genes coding for the bacterial signal-transducing protein NtrB and the bacterial enhancer-binding protein NtrC of deep-sea piezophilic Shewanella violacea were cloned and their nucleotide sequences were analyzed. The conserved regions of NtrB and those of NtrC are well conserved in the case of the ntrBC products of S. violacea.

The effects of micronutrients on growth of Thermococcus guaymasensis and Thermococcus aggregans in a starchcontaining medium were investigated. A trace minerals solution, a vitamins solution and calcium chloride were omitted from the medium or added in different amounts. The growth rates of both species were not affected over a significant range of concentrations of these compounds, but appreciable inhibition of growth was observed after the addition of elemental sulfur to the medium. T. guaymasensis exhibited a significant tolerance to high amounts of trace element and vitamin solutions but growth was inhibited by the omission of these compounds from the medium. Moreover, both amylolytic and pullulytic activities increased in the presence of 6-fold higher amounts of trace element and vitamin solutions, compared to the concentrations used in the usual medium. In T. aggregans, both enzymatic activities were enhanced in the presence of either increased (4-fold) amounts of trace element and vitamin solutions, or after the addition of elemental sulfur to the medium. Furthermore, larger activities of starch-hydrolysing enzymes were detected with a 10-fold higher concentration of calcium chloride, compared to the usual medium, in the absence of trace element and vitamin solutions. When both Thermococcus species were tested for the tolerance to specific cations and oxyanions, T. guaymasensis exhibited higher tolerance compared to T. aggregans, the former strain being capable to grow in the presence of 6 mM Ni2+, 4 mM Cu2+, 1.5 mM SeO42-, and 1.5 mM MoO42-. The content of total cell proteins followed the pattern of starch-hydrolysing enzymes and an over-expression of proteins in the range of 35, 50 and 70 kDa was observed.

We have recently reported that a sigma(54)-like factor recognizes a DNA element, designated as region A, upstream of a pressure-regulated operon in piezophilic Shewanella violacea strain DSS12 (Nakasone et al., FEMS Microbiology Lett. 176 (1999) 351-356). In this study, we isolated and characterized the rpoN gene of this piezophilic bacterium. The rpoN gene was found to encode a putative protein consisting of 492 amino acid residues with a predicted molecular mass of 55359 Da. Significant homology was evident comparing the rpoN sequence of S. violacea with that of Escherichia coli (62.8% identity), Vibrio anguillarum (61.7% identity) and Pseudomonas putida (57.0% identity). The DNA-binding domain at the C-terminus of sigma(54) is well conserved in the case of the S. violacea rpoN gene product and the helix-turn-helix motif and the RpoN box are also present. In addition, the conserved glutamine-rich domain is present at the N-terminus. sigma(54) in S. violacea was expressed at a relatively constant level under various growth conditions as determined by both primer extension and Western blotting analyses. By means of a recombinant plasmid, a hexahistidine-tagged derivative of the sigma(54) from strain DSS12 was overexpressed in Escherichia coli and purified to near homogeneity. An electrophoretic mobility shift assay demonstrated that the purified sigma(54) protein specifically recognizes region A in the above-mentioned pressure-regulated operon.

Several studies have been published recently on high-pressure tolerance mechanisms in the yeast Saccharomyces cerevisiae , focusing on the role of intracellular trehalose and Hspl04 proteins. Abe and Horikoshi have been analyzing the physiological effects of non-lethal levels of hydrostatic pressure, below 100 MPa, in yeast, and they showed that elevated pressure promotes the acidification of vacuoles. The acidification that occurs under elevated pressure is ascribed to hydration and ionization of carbon dioxide generated through ethanol fermentation, which leads to accumulation of large number of protons in the cytoplasm. Recently, the term “piezophilic” was proposed to replace “barophilic” as the prefixes “baro” and “piezo,” derived from Greek, mean “weight” and “pressure,” respectively (Yayanos, 1995). Thus, the word piezophilic may be more suitable than barophilic to indicate bacteria that grow better at high pressure than at atmospheric pressure. This chapter opted to use the term “piezophilic bacteria,” which means high pressure loving bacteria, and focuses on the taxonomy of deep-sea piezophiles and the features of their gene expression and respiratory systems related to high pressure adaptation.

There has been a renewal of interest in the survival strategies employed by deep-sea, high-pressure-adapted (piezophilic) microorganisms as well as in the effects of high pressure on mesophilic, 1-atmosphere-pressure-adapted microorganisms. This is partly the result of a greater appreciation of the adaptations of microorganisms to life in extreme environments and partly the result of the development of new techniques for examining physiological and molecular processes as a function of pressure.

Background: Although a number of studies have investigated the induction of oral tolerance to several proteins, relatively little is known about the induction of oral tolerance to β-lactoglobulin, one of the major antigenic proteins in milk. Objective: We investigated the influence of the timing of the initial β-lactoglobulin exposure on oral tolerance induction and examined some characteristics of the tolerogenic immune response. Methods: BALB/c mice were given β-lactoglobulin prenatally or from the third or fifth postnatal week, bred for 17 weeks, and compared with unexposed control mice. Specific plasma anti-β-lactoglobulin antibodies (total IgG, IgG subclasses, IgM, and IgE), antigen-specific splenocyte responses, frequencies of antibody-producing cells, and cytokine production by splenocytes, intestinal mucosal lymphocytes, and Peyer’s patches were analyzed. Results: Differences were observed among the 4 groups of mice in changes of plasma anti-β-lactoglobulin antibody titers, antigen-specific T-cell proliferation, and frequencies of antibody-producing splenocytes, intestinal mucosal lymphocytes, and Peyer’s patch cells after the first exposure to β-lactoglobulin. The onset and duration of the immunologic responses were found to be dependent on the timing of antigen exposure. Prenatal exposure to antigen facilitated the induction of oral tolerance to β-lactoglobulin, whereas delayed antigen exposure retarded tolerance. The induction of oral tolerance was associated with increased IL-4 and/or IL-10 production and decreased IL-12 production. Conclusion: Our results suggest that the timing of initial antigen exposure greatly influences the induction of oral tolerance to β-lactoglobulin and that altered secretion of regulatory cytokines may be responsible for the differences in antibody production and oral tolerance induction. (J Allergy Clin Immunol 1999;104:870-8.)

The Japan Trench land slope at a depth of 6,400 m is the deepest cold-seep environment with Calyptogena communities. Sediment samples from inside and beside the Calyptogena communities were collected, and the microbial diversity in the sediment samples was studied by molecular phylogenetic techniques. From DNA extracted directly from the sediment samples, 16S rDNAs were amplified by the polymerase chain reaction method. The sequences of the amplified 16S rDNAs selected by restriction fragment length polymorphism analysis were determined and compared with sequences in DNA databases. The results showed that 33 different bacterial 16S rDNA sequences from the two samples analyzed fell into similar phylogenetic categories, the alpha-, gamma-, delta-, and varepsilon-subdivisions of Proteobacteria, Cytophaga, and gram-positive bacteria; some of the 16S rDNA sequences were common to both samples. delta- and varepsilon-Proteobacteria-related sequences were abundant in both sediments. These sequences are mostly related to sulfate-reducing or sulfur-reducing bacteria and epibionts, respectively. Eight different archaeal 16S rDNA sequences were cloned from the sediments. The majority of the archaeal 16S rDNA sequences clustered in Crenarchaeota and showed high similarities to marine group I archaeal rDNA. A Methanococcoides burtonii-related sequence obtained from the sediment clustered in the Euryarchaeota indicating that M. burtonii-related strains in the area of Calyptogena communities may contribute to production of methane in this environment. From these results, we propose a possible model of sulfur circulation within the microbial community and that of Calyptogena clams in the cold-seep environment.

DNA has been extracted on site from the brines of the final crystallizing pond of an alkaline saltern at Lake Magadi, Kenya. Amplification of 16S rRNA genes followed by cloning, sequencing, and phylogenetic analysis has re- vealed the presence of two distinct new archaeal lineages. The majority of cloned sequences showed greater than 95% identity to each other, but only 88%-90% similarity to any cultivated haloalkaliphilic Archaea, and form a distinct cluster within the known Haloarchaea. Two cloned genes showed close similarity to each other but only 76% similar- ity to any known archaeal sequence, and therefore repre- sent a distinct phylotype only distantly related to the euryarchaeotal branch of the Archaea.

A novel coccoid-shaped, hyperthermophilic, anaerobic archaeon, strain RG-20, was isolated from a deep-sea hydrothermal vent fluid sample taken at 1394-m depth at the Mid-Okinawa Trough (27 degrees 32.7'N, 126 degrees 58.5'E). Cells of this isolate occur singly or in pairs and are about 0.8 to 2 microm in diameter. Growth was observed at temperatures between 50 degrees and 93 degrees C, with an optimum at 85 degrees C. The pH range for growth is 5.0-9.0, with an optimum around 7.0. Strain RG-20 requires 1%-4% of NaCl for growth, and cell lysis occurs at concentrations below 1%. The newly isolated strain grows preferentially in the presence of elemental sulfur on proteinaceous substrates such as yeast extract, peptone, or tryptone, and no growth was observed on carbohydrates, carboxylic acids, alcohols, or lipids. This microorganism is resistant to streptomycin, chloramphenicol, ampicillin, and kanamycin at concentrations up to 150 microg/ ml, but is susceptible to rifampicin. Analysis of the hydrolyzed core lipids by thin-layer chromatography (TLC) revealed the presence of archaeol and caldarchaeol. The mol% G+C content of the DNA is 55.8. Partial sequencing of the 16S rDNA indicates that strain RG-20 belongs to the genus Thermococcus. Considering these data and on the basis of the results from DNA-DNA hybridization studies, we propose that this strain should be classified as a new species named Thermococcus siculi (si'cu.li. L. gen. n. siculi, of the deep-sea [siculum, deep-sea in literature of Ovid], referring to the location of the sample site, a deep-sea hydrothermal vent). The type strain is isolate RG-20 (DSM No. 12349).

We have isolated two strains of extremely barophilic bacteria from sediment collected from the world's deepest ocean floor in the Mariana Trench, Challenger Deep, at a depth of 10 898 m [Kato C, Li L, Nogi Y, Nakamura Y, Tamaoka J, Horikoshi K (1998) Appl Environ Microbiol 64:1510-1513]. One strain, DB21MT-2, was identified as a strain of Shewanella benthica, and the other strain, DB21MT-5, is closely affiliated with members of the genus Moritella on the basis of 16S rDNA sequence analysis. The hybridization values for DNA-DNA relatedness between DB21MT-5 and the Moritella reference strains were significantly lower than that accepted as the phylogenetic definition of a species. Based on this and other taxonomic differences, strain DB21MT-5 appears to represent a novel obligately barophilic deep-sea Moritella species. The name Moritella yayanosii (JCM 10263) is proposed. This is the first proposed species of obligately barophilic bacteria of the genus Moritella.

Defining the diversity and distribution of natural microbial communities from sea sediments has been a long-standing challenge in the field of microbial ecology and evolution. It is generally estimated that about 95% of the organic matter produced photosynthetically in the surface of waters recycled in the upper 100-300 m (Jannasch and Taylor 1984). Only about 1% of photosynthetically produced organic carbon reaches the deep-sea floor. Therefore, the major nutritional characteristics of the deep-sea as a habitat for organisms are relatively low input of organic carbon and its consumption. The availability of nutrients, being geographically highly variable, and the elevated pressures characterized the deep sea sediments as potentially unique bacterial environments.

The Mariana Trench, Challenger Deep (11°22’N. 142°25’E), is the deepest ocean bottom in the world. Sediment samples have been obtained from this extreme environment by the unmanned submersible Kaiko (Kyo et al. 1995) in three dives(nos. 21, 22, and 23) at a depth of 11 000 m (Kato et al. 1997). It seems likely that these were the first sediment samples recovered from the world’s deepest point with no microbiological contamination. In 1960 Jacques Piccard and D. Walsh dived to the world’s deepest point aboard the manned submersible Trieste. They reported seeing several deep-sea animals and fishes there (Piccard and Dietz 1961). Unfortunately, that dive was not for scientific purposes, and no films were made or samples collected, so their story could not be confirmed scientifically.

A pressure-regulated operon has been cloned and sequenced from deep-sea barophilic Shewanella strains. To understand pressure-regulated mechanisms of gene expression, a regulatory element upstream of the pressure-regulated operon from Shewanella sp. strain DSS12 was studied. Regions A and B were classified by sequence analysis. A unique octamer motif, AAGGTAAG, was found to be repeated in tandem 13 times in region B. An electrophoretic mobility shift assay demonstrated that a σ54-like factor recognizes region A and other unknown factors recognize region B. Different shift patterns of the protein–DNA complexes were observed when extracts of cells cultured at 0.1 MPa or 50 MPa were incubated with a DNA probe specific for region B. These results indicate that the deep-sea strain DSS12 expresses different DNA-binding factors under different pressure conditions.

A novel, moderately barophilic bacterium was isolated from a sediment sample obtained from the Ryukyu Trench, at a depth of 5110 m. The isolate, designated strain DSJ4, is a Gram-negative rod capable of growth between 4 degrees C and 18 degrees C under atmospheric pressure, with optimum growth displayed at 10 degrees C, and capable of growth at pressures between 0.1 MPa and 70 MPa at 10 degrees C, with optimum growth displayed at 10 MPa. Strain DSJ4 is a moderately barophilic bacterium, and shows no significant change in growth at pressures up to 50 MPa. Phylogenetic analysis of the 16S rRNA sequence of strain DSJ4 places this strain within the Photobacterium subgroup of the family Vibrionaceae, closely related to the strain SS9 that was independently isolated from the Sulu Trough. The temperature and pressure ranges for growth, cellular fatty acid composition, and assorted physiological and biochemical characteristics indicate that these strains differ from other Photobacterium species. Furthermore, both SS9 and DSJ4 displayed a low level of DNA similarity to other Photobacterium type strains. Based on these differences, these strains are proposed to represent a new deep-sea-type species. The name Photobacterium profundum (JCM10084) is proposed.

8 páginas.-- 5 figuras.-- 2 tablas.-- 46 referencias.-- El autor González Grau, Juan Miguel pertenece actualmente al Instituto de Recursos Naturales y Agrobiología de Sevilla, A hyperthermophilic, anaerobic archaeon was isolated from hydrothermal fluid samples obtained at the Okinawa Trough vents in the NE Pacific Ocean, at a depth of 1395m. The strain is obligately heterotrophic, and utilizes complex proteinaceous media (peptone, tryptone, or yeast extract), or a 21-amino-acid mixture supplemented with vitamins, as growth substrates. Sulfur greatly enhances growth. The cells are irregular cocci with a tuft of flagella, growing optimally at 98 degrees C (maximum growth temperature 102 degrees C), but capable of prolonged survival at 105 degrees C. Optimum growth was at pH 7 (range 5-8) and NaCl concentration 2.4% (range 1%-5%). Tryptophan was required for growth, in contrast to the closely related strains Pyrococcus furiosus and P. abyssi. Thin sections of the cell, viewed by transmission electron microscopy, revealed a periplasmic space similar in appearance to the envelope of P. furiosus. The predominant cell membrane component was tetraether lipid, with minor amounts of diether lipids. Treatment of the cells by mild osmotic shock released an extract that contained a Zn(2+)-dependent alkaline phosphatase. Phylogenetic analysis of the sequences encoding 16S rRNA and glutamate dehydrogenase places the isolate with certainty within the genus Pyrococcus although there is relatively low DNA-DNA hybridization (< 63%) with described species of this genus. Based on the reported results, we propose a new species, to be named Pyrococcus horikoshii sp.nov.

Many microorganisms from the deep-sea display high-pressure-adapted--also described as barophilic or piezophilic--growth characteristics. Phylogenetic studies have revealed that a large proportion of the barophilic bacteria currently in culture collections belong to a distinct subgroup of the genus Shewanella, referred to as the "barophile branch." Many of the basic properties of barophiles that enable their survival at extremes of pressure remain to be elucidated. However, several genes whose expression is regulated by pressure, or which appear to be critical to baroadaptation, have been uncovered. One such operon, whose presence appears to be restricted to the "barophile branch," has been identified in DNA samples obtained from sediments recovered in the deepest ocean trench. In the case of another set of pressure-regulated genes, regulatory elements required for pressure signaling have been uncovered. The nature and regulation of these genes is discussed.

We have obtained sediment samples from the world's deepest sea-bottom, the Mariana Trench challenger point at a depth of 10,898 m, using the new unmanned submersible Kaiko. DNA was extracted from the sediment, and DNA fragments encoding several prokaryotic ribosomal RNA small-subunit sequences and pressure-regulated gene clusters, typically identified in deep-sea adapted bacteria, were amplified by the polymerase chain reaction. From the sequencing results, at least two kinds of bacterial 16S rRNAs closely related to those of the genus Pseudomonas and deep-sea adapted marine bacteria, and archaeal 16S rRNAs related to that of a planktonic marine archaeon were identified. The sequences of the amplified pressure-regulated clusters were more similar to those of deep-sea barophilic bacteria than those of barotolerant bacteria. These results suggest that deep-sea adapted barophilic bacteria, planktonic marine archaea, and some of the world's most widespread bacteria (the genus Pseudomonas) coexist on the world's deepest sea-bottom.

7 páginas.-- 7 figuras.-- 34 referencias.-- El autor González Grau, Juan Miguel pertenece actualmente al Instituto de Recursos Naturales y Agrobiología de Sevilla, 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., F. Canganella was supported by a short-term fellowship from the Organization for Economical and Cooperative Development (Paris)

Escherichia coli strain JD518, a cydD-deficient mutant, displayed temperature-sensitive and pressure-sensitive growth. The defective cydD gene in this strain was complemented by open reading frame 3 (ORF3), previously identified in DNA from a barotolerant bacterium, strain DSS12, allowing growth of the cydD mutant under high temperature and high pressure conditions. Spectrophotometrical analysis indicated that the cytochrome bd complex which is assembled by the CydD protein was expressed in E. coli strain JD518 carrying the ORF3 gene at the same level as occurred in the wild-type strain. Our results indicate that the cydD gene functions are required for cell stability under the condition of high pressure stress in bacteria.

A pressure-regulated operon was cloned and sequenced from a barophilic bacterium strain DB6705. Gene expression by this promoter was regulated at the level of transcription by elevated pressure in the barophilic strain. The promoter sequence was similar to the promoter of ompH from Photobacterium sp. strain SS9.

4 páginas.-- 1 figuras.-- 1 tabla.-- 10 referencias.-- El autor González Grau, Juan Miguel pertenece actualmente al Instituto de Recursos Naturales y Agrobiología de Sevilla, The culturability of a strictly anaerobic, extremely thermophilic archaeon, Thermococcus peptonophilus (optimal growth temperature: 85° C), was studied during survival stages at various temperatures (98, 85, 70, and 4° C). Total cell number (determined by DAPI staining), active cells (rhodamine-stained cells), and culturable cells (using most-probable-number) were counted over time. The number of culturable cells decreased under each condition tested. The total number of cells significantly decreased only at temperatures close to the maximum for growth (98° C); at this temperature, the cells spontaneously lysed. Our results suggested that survival at 4° C in oxygenated waters might be a mechanism for the dispersion of extreme thermophiles in the ocean. In addition, we proved the existence of T. peptonophilus cells in several physiological states: culturable cells, active non-culturable cells, inactive non-culturable cells, and dead cells. Cell death was caused by cellular lysis.

We have isolated several microorganisms that are adapted to living in the extremes of the deep-sea environment. They include barophilic bacteria, which are able to grow at high hydrostatic pressure, but that are unable to grow at atmospheric pressure, and organic-solvent-tolerant bacteria, which are able to grow in the presence of toxic organic solvents such as toluene or benzene. In this review, we describe how to isolate such extremophiles, and we outline the characteristics of several strains that have been recovered from the deep-sea environment.

We have isolated several microorganisms that are adapted to living in the extremes of the deep-sea environment characterized as high hydrostatic pressure. They include barophilic bacteria, which are able to grow at high hydrostatic pressure, but that are unable to grow at atmospheric pressure, and barotolerant bacteria, which are able to grow almost same ability in both high pressure and atmospheric pressure conditions. A new application field, high pressure biotechnology, will be developing from the deep-sea microbial research in the future.

High hydrostatic pressure affects gene expression in microorganisms and three types of responses have been recognized: 1) high pressure-inducible, 2) pressure tolerant (independent), and 3) high pressure-repressible. Our observations suggest that mainly types 1) and 2) occur in deep-sea bacteria adapted to high pressure conditions (barophilic and barotolerant bacteria), however, in addition, type 3) occurs in Escherichia coli adapted to atmospheric pressure conditions. Our results are consistent with the hypothesis that high pressure-inducible and pressure tolerant gene expression is an ancient evolutionary trait which is shared by many bacteria.

A new dihydrofolate reductase (svDHFR) was purified from a deep-sea bacterium, Shewanella violacea strain DSS12, isolated from the Ryukyu Trench (5110 m). In contrast with E. coli DHFR, the enzyme ...

Elevated hydrostatic pressure can influence gene and protein expression in both 1 atmosphere-adapted and high pressure-adapted microorganisms. Here we review experiments documenting these effects and describe their significance towards understanding the molecular bases of life in deep-sea high pressure environments.

Fluorescent protein-based indicators for intracellular environment conditions such as pH and ion concentrations are commonly used to study the status and dynamics of living cells. Despite being an important factor in many biological processes, pressure, however, is an exception. Development of the pressure sensitive fluorescent protein would blaze a new path to the future biology. Recently, we enhanced the pressure sensitivity of yellow fluorescent protein (YFP) by inserting several glycines into it. Our mutation enhanced the interaction between the chromophore and the solvent that is responsible for pressure changing. In response to changes in pressure, a spectrum shift and an intensity change of the fluorescence was observed, indicating applicability of the present YFP for measuring the intracellular pressure on a live specimen. By measuring the fluorescence of the mutant YFP, we succeeded in measuring the intracellular pressure changes that follow actin filament disruption in living cells. This study shows that our mutation enhanced unknown properties of YFP that may allow us to measure the intracellular pressure.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Recombinant plasmids with the chloramphenicol acetyltransferase (CAT) structural gene behind several kinds of promoters were tested for expression in Escherichia coli during growth at atmospheric pressure (0.1 MPa) and at high pressure (30 MPa). Expression of the CAT gene from the lac promoter was remarkably activated (approx. 78-fold) by high pressure in the absence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). The stimulation of the CAT activity by the lac promoter at high pressure did not simply result from an increased plasmid copy number, because the CAT activities from the other promoters and beta-lactamase activities were unaffected at high pressure.

In this study, observation of the reaction of H2O2 at the surface of stainless steel utilizing electrochemical impedance spectroscopy (EIS) directly in the high purity water at 288oC was performed to determine the corrosive condition and the corrosion behavior of the stainless steel in high temperature and high purity water containing H2O2. Obtained results were summarized as follows; 1) The EIS data were measured directly in high temperature water without electrolyte injection. 2) The conductivity of the high temperature water at 288oC calculated by Rsol was 8.3x10-6S/cm. 3) The Rp was 9.0x103ohm•cm2 for 100ppb of H2O2, and the estimated corrosion rate was about 30μm/year. 4) The reciprocal of the polarization resistance had a linear relationship to the H2O2 concentration. It indicated that the corrosion current would be determined by the cathodic reaction of H2O2. 5) The estimated DH2O2 was 1.5x10-4cm2/s.