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.

Complete Genome Sequence of the Deep-Sea Bacterium Psychromonas Strain CNPT3.

Members of the genus Psychromonas are commonly found in polar and deep-sea environments. Here we present the genome of Psychromonas strain CNPT3. Historically, it was the first bacterium shown to piezoregulate the composition of its membrane lipids and to have a higher growth rate at 57 megapascals (MPa) than at 0.1 MPa.

Universal primers and PCR of gut contents to study marine invertebrate diets.

Determining the diets of marine invertebrates by gut content analysis is problematic. Many consumed organisms become unrecognizable once partly digested, while those with hard remains (e.g. diatom skeletons) may bias the analysis. Here, we adapt DNA-based methods similar to those used for microbial diversity surveys as a novel approach to study the diets of macrophagous (the deep-sea amphipods Scopelocheirus schellenbergi and Eurythenes gryllus) and microphagous (the bivalve Lucinoma aequizonata) feeders in the deep sea. Polymerase chain reaction (PCR) in conjunction with 'universal' primers amplified portions of the mitochondrial cytochrome c oxidase I (COI) gene for animals ingested by S. schellenbergi and E. gryllus and the 18S rRNA gene for lesser eukaryotes ingested by L. aequizonata. Amplified sequences were combined with sequences from GenBank to construct phylogenetic trees of ingested organisms. Our analyses indicate that S. schellenbergi, E. gryllus and L. aequizonata diets are considerably more diverse than previously thought, casting new light on the foraging strategies of these species. Finally, we discuss the strengths and weaknesses of this technique and its potential applicability to diet analyses of other invertebrates.

Evolutionary relationships of cultivated psychrophilic and barophilic deep-sea bacteria.

Evolutionary relationships of cultivated barophilic bacteria were determined. All psychrophilic and barophilic isolates were affiliated with one of five genera of the gamma subdivision of the class Proteobacteria ((gamma)-Proteobacteria): Shewanella, Photobacterium, Colwellia, Moritella, and a new group containing strain CNPT3. The data indicate that the barophilic phenotype has evolved independently in different (gamma)-Proteobacteria genera.

Microbiology to 10,500 meters in the deep sea.

Microorganisms in the deep sea live at high pressures, low and high temperatures, and in darkness. These parameters and their food supply govern their lives. The study of these creatures gives us an opportunity to see how life processes work at some of the highest temperatures and pressures of the biosphere. Cultured bacterial isolates can grow to over 100 MPa at 2 degrees C and to over 40 MPa at over 100 degrees C. These cultures comprise the foundation for the study of the molecular biology and biotechnology of these isolates. The PTk diagram shows how temperature and pressure affect the growth rate of a bacterium and helps in the search for relationships among bacteria from habitats differing in temperature and pressure.

Ultrastructural changes in an obligately barophilic marine bacterium after decompression.

The bacterial isolate MT-41 from 10,476 m, nearly the greatest ocean depth, is obligately barophilic. The purpose of this study was to describe the morphological changes in MT-41 due to nearly isothermal decompression followed by incubation at atmospheric pressure. Two cultures were grown at 103.5 MPa and 2 degrees C and then decompressed to atmospheric pressure (0.101 MPa). One of the cultures was fixed just before decompression. The other culture, kept at 0 degrees C, was sampled immediately and four more times over 168 h. The number of CFU (assayed at 103.5 MPa and 2 degrees C) declined with incubation time at atmospheric pressure. Decompression itself did not lead to immediate morphological changes. The ultrastructure, however, was altered with increasing time at atmospheric pressure. The first aberrations were intracellular vesicles and membrane fragments in the medium. After these changes were plasmolysis, cell lysis, the formation of extracellular vesicles, and the formation of ghost cells. Intact cells in the longest incubation at atmospheric pressure had the normal cytoplasmic granularity suggestive of ribosomes but had few and poorly stained fibrils in the bacterial nucleoids. From the practical standpoint, samples of hadal deep-sea regions need to be fixed either in situ or shortly after arrival at the sea surface even when recovered in insulated sampling gear. This should prevent drastic structural degradation of sampled cells, thus allowing both accurate estimates of deep-sea benthic standing stock and realistic morphological descriptions.

Isolation of a gene regulated by hydrostatic pressure in a deep-sea bacterium.

Barophilic bacteria inhabit the deep oceans, and the specific functional modifications and regulatory mechanisms which govern adaptation to hydrostatic pressure are beginning to be understood. For example, the rate of production of several proteins by some hydrothermal vent archaebacteria and the degree of saturation of membrane lipids in other deep-sea bacteria have been found to change as a result of cultivation at high pressure. We report here the cloning of gene, ompH, which encodes a major pressure-inducible protein of strain SS9, a gram-negative eubacterium isolated from a depth of 2.5 kilometres in the Sulu Sea. Messenger RNA encoded by ompH is expressed when cells are grown at 280 atm but not at 1 atm, indicating that transcription of the ompH gene is controlled by hydrostatic pressure. The function of the OmpH protein in adaptation to high pressure and the use of the ompH gene in studying how bacteria sense and respond to pressure is discussed.

Properties of the glucose transport system in some deep-sea bacteria.

Many deep-sea bacteria are specifically adapted to flourish under the high hydrostatic pressures which exist in their natural environment. For better understanding of the physiology and biochemistry of these microorganisms, properties of the glucose transport systems in two barophilic isolates (PE-36, CNPT-3) and one psychrophilic marine bacterium (Vibrio marinus MP1) were studied. These bacteria use a phosphoenol-pyruvate:sugar phosphotransferase system (PTS) for glucose transport, similar to that found in many members of the Vibrionaceae and Enterobacteriaceae. The system was highly specific for glucose and its nonmetabolizable analog, methyl alpha-glucoside (a-MG), and exhibited little affinity for other sugars tested. The temperature optimum for glucose phosphorylation in vitro was approximately 20 degrees C. Membrane-bound PTS components of deep-sea bacteria were capable of enzymatically cross-reacting with the soluble PTS enzymes of Salmonella typhimurium, indicating functional similarities between the PTS systems of these organisms. In CNPT-3 and V. marinus, increased pressure had an inhibitory effect on a-MG uptake, to the greatest extent in V. marinus. Relative to atmospheric pressure, increased pressure stimulated sugar uptake in the barophilic isolate PE-36 considerably. Increased hydrostatic pressure inhibited in vitro phosphoenolpyruvate-dependent a-MG phosphorylation catalyzed by crude extracts of V. marinus and PE-36 but enhanced this activity in crude extracts of the barophile CNPT-3. Both of the pressure-adapted barophilic bacteria were capable of a-MG uptake at higher pressures than was the nonbarophilic psychrophile, V. marinus.

Evolutional and ecological implications of the properties of deep-sea barophilic bacteria.

The rate of reproduction of deep-sea bacteria from six different capture depths between 1957 and 10,476 meters was studied as a function of temperature and pressure. The results showed the following: the true deep-sea bacteria of different depths have several characteristics, presumably evolutionally derived, distinguishing them from each other and from bacteria of atmospheric-pressure environments; pressure plays a significant role in determining the distribution of oceanic life; and pressure-adapted bacteria are easily recovered from and ubiquitous in the deep ocean. Organisms evolving in habitats of different temperatures and pressures need to be studied to understand the physical limits of life, the distribution of life within the earth and its oceans, the role of organisms in organic diagenesis and petroleum formation, and the possible existence of life on and within other planets.

Biochemical function and ecological significance of novel bacterial lipids in deep-sea procaryotes.

The fatty acid composition of the membrane lipids in 11 deep-sea bacterial isolates was determined. The fatty acids observed were typical of marine vibrios except for the presence of large amounts of long-chain polyunsaturated fatty acids (PUFAs). These long-chain PUFAs were previously thought to be absent in procaryotes, with the notable exception of a single marine Flexibacter sp. In three barophilic strains tested at 2 degrees C, there was a general increase in the relative amount of PUFAs as pressure was increased from a low growth pressure towards the optimal growth pressure. In Vibrio marinus MP-1, a psychrophilic strain, PUFAs were found to increase as a function of decreasing temperature at constant atmospheric pressure. These results suggest the involvement of PUFAs in the maintenance of optimal membrane fluidity and function over environmentally relevant temperatures and pressures. Furthermore, since these lipids are essential nutrients for higher taxa and are found in large amounts in the lipids of deep-sea vertebrates and invertebrates, an important, specific role for deep-sea bacteria in abyssal food webs is implicated.

Adaptation of the membrane lipids of a deep-sea bacterium to changes in hydrostatic pressure.

The fatty acid composition of the cell membrane of the barophilic marine bacterium CNPT3 was found to vary as a function of pressure. Greater amounts of unsaturated fatty acids were present in bacteria growing at higher pressures. The results suggest adaptations in the membrane lipids to environmentally relevant pressures. This response to pressure appears to be analogous to temperature-induced membrane adaptations observed in other organisms.

Spectrofluorometric determination of bacterial DNA base composition.

A spectrofluorometric technique for bacterial DNA base composition has been developed. This fast and simple technique requires two fluorescent dyes and a few inexpensive reagents. The data from this assay indicate that the guanine-cytosine content obtained was within acceptable statistical limits in comparison to commonly cited literature values. The spectrofluorometric technique is reliable and reproducible.

High-pressure-temperature gradient instrument: use for determining the temperature and pressure limits of bacterial growth.

A pressurized temperature gradient instrument allowed a synoptic determination of the effects of temperature and pressure on the reproduction of bacteria. The instrument consisted of eight pressure vessels housed parallel to each other in an insulated aluminum block in which a linear temperature gradient was supported. For a given experiment, eight pressures between 1 and 1,100 bars were chosen; the linear temperature gradient was established over an interval within -20 to 100 degrees C. Pure cultures and natural populations were studied in liquid or solid medium either in short (ca. 2-cm) culture tubes or in long (76.2-cm) glass capillaries. In the case of a pure culture, experiments with the pressurized temperature gradient instrument determined values of temperature and pressure that bounded its growth. Feasibility experiments with mixed populations of bacteria from water samples from a shallow depth of the sea showed that the instrument may be useful in identifying the extent to which a natural population is adapted to the temperatures and pressures at the locale of origin of the sample. Additional conceived uses of the instrument included synoptic determinations of cell functions other than reproduction and of biochemical activities.

Possible artefactual basis for apparent bacterial growth at 250 degrees C.

Baross and Deming reported that thermophilic marine bacteria isolated from the vicinity of a submarine hot-spring grow at temperatures up to at least 250 degrees C. They did not, however, conduct the appropriate control experiments to eliminate the possibilities of chemical artefacts or contamination. Here, in experiments using the same growth medium, the same temperature and pressure apparatus and the same sampling and analytical procedures, we report results nearly identical to theirs. We conclude that their evidence indicating bacterial growth at 250 degrees C may be due to artefacts produced in the medium and to contaminants introduced during sample processing.

Reproduction of Bacillus stearothermophilus as a Function of Temperature and Pressure.

The colony-forming ability and the rate of reproduction of Bacillus stearothermophilus were determined as a function of temperature and pressure. Colonies were formed between 39 and 70 degrees C at atmospheric pressure and between 54 and 67 degrees C at 45 MPa. Colonies did not form at 55.9 MPa. The rate of reproduction in broth cultures decreased with increasing pressure at all temperatures. The rate of reproduction diminished rapidly with pressure above 10.4 MPa. Therefore, increased hydrostatic pressure was not sufficient to enable B. stearothermophilus to function beyond the temperature limiting growth and reproduction at atmospheric pressure, and B. stearothermophilus should grow in naturally or artificially warmed regions of the deep sea, where the pressure is less than approximately 50 MPa, although growth rates would be low above 10 MPa.

Death of a hadal deep-sea bacterium after decompression.

An obligately barophilic bacterium that was recovered from a depth of 10,476 meters in the Pacific Ocean slowly lost colony-forming ability (assayed at 101.3 megapascals and 2 degrees C) during incubation at atmospheric pressure and 0 degrees C.

Dependence of reproduction rate on pressure as a hallmark of deep-sea bacteria.

Strains of bacteria in axenic culture were isolated from samples of depths between 1,957 and 10,476 m of the Pacific Ocean. All of the bacteria from this range of depths were barophilic. The pressure at which the rate of reproduction was maximal was found to be correlated with the depth of origin of the isolates.

Thermal Inactivation of a Deep-Sea Barophilic Bacterium, Isolate CNPT-3.

The barophilic deep-sea bacterium, isolate CNPT-3, was inactivated by exposures to temperatures between 10 and 32 degrees C at atmospheric pressure. Inactivation in samples from warmed cell suspensions was measured as the loss of colonyforming ability (CFA) at 10 degrees C and 587 bars. At atmospheric pressure, there was a slow loss of CFA even at 10 degrees C. The loss of CFA was rapid above 20 degrees C and only slightly affected by high pressures. The first-order rate constants for thermal inactivation fit the Arrhenius equation with an activation energy of 43 kcal (ca. 179.9 kJ)/mol. Light microscopy and scanning transmission electron microscopy revealed morphological changes due to warming of the cells. The changes ensued the loss of CFA. The results supported the hypothesis from an earlier work that indigenous (autochthonous) deep-sea bacteria from cold deep seas are both barophilic and psychrophilic. If ultimately sustained, these characteristics may be useful in designing experiments to assess the relative importance of the autochthonous and allochthonous bacteria in the deep sea. The data were used to evaluate how barophilic bacteria may have been missed in many investigations because of warming of the cells during sample retrieval from the sea or during cultivation in the laboratory. The evaluation revealed the need for temperature and pressure data during retrieval of samples and cultivation in the laboratory. Most deep-ocean microbiology may be possible with thermally insulated equipment for retrieval from the sea and with high-pressure vessels for laboratory incubations.

Obligately barophilic bacterium from the Mariana trench.

An amphipod (Hirondellea gigas) was retrieved with decompression in an insulated trap from an ocean depth of 10,476 m. Bacterial isolates were obtained from the dead and cold animal by using silica gel medium incubated at 1000 bars (1 bar = 10(5) Pa) and 2 degrees C. The isolate designated MT41 was found to be obligately barophilic and did not grow at a pressure close to that of 380 bars found at average depths of the sea. The optimal generation time of about 25 hr was at 2 degrees C and 690 bars. The generation time at 2 degrees C and 1,035 bars, a pressure close to that at the depth of origin, was about 33 hr. Among the conclusions are: (i) pressure is an important determinant of zonation along the water column of the sea; (ii) some obligately barophilic bacteria survive decompressions; (iii) the pressure of optimal growth at 2 degrees C appears to be less than the pressure at the depth of origin and may be diagnostic for the depth of origin; (iv) rates of reproduction are slow yet significant and an order of magnitude greater than previously thought; and (v) much of deep-sea microbiology may have been done with spurious deep-sea organisms due to warming of samples.

Isolation of a deep-sea barophilic bacterium and some of its growth characteristics.

A bacterium, a spirillum, has been isolated from a deep-sea sample and has been found to grow optimally at about 500 bars and 2 degrees to 4 degrees C. These conditions are similar to those prevailing at the 5700-meter depth from which the sample was collected. The organism grows at these pressures and temperatures with a generation time of between 4 and 13 hours; at atmospheric pressure and 2 degrees to 4 degrees C, the generation time is about 3 to 4 days.