Isolation and properties of barophilic and barotolerant bacteria from deep-sea mud samples

Several barophilic and barotolerant bacteria were isolated from deep-sea mud samples of Suruga Bay (2485 m depth), the Ryukyu Trench (5110 m depth), and the Japan Trench (land-side 6356 m, and sea-side 6269 m depth, respectivelys. The barophilic bacteria, strains DB5501, DB6101, DB6705 and DB6906, were albe to grow better under high hydrostatic pressures than under atmospheric pressure (0.1 megapascals; MPa). The optimal growth pressures for the barophilic bacteria were approximately 50 MPa at 10°C. The barotolerant strains DSK1 and DSS12 were determined to be psychrophilic, and had optimal growth temperatures of 10°C and 8°C, respectively. The degree of barophily and barotolerance was shown to be very dependent on temperature. For example, at 4°C the barophilic strains were indistinguishable from barotolerant bacteria, whereas at 15°C the barotolerant strains behaved more like the barophilic strains. Based on sequence analysis of 16S ribosomal DNA, all of the strains included in this study belong to the gamma subgroup of the Proteobacteria. Phylogenetic relations between the isolated strains and the known gamma subgroup bacteria suggested that the isolated strains belong to a new sub-branch of this group.     

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°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.     

New Method for Isolating Barophiles from Intestinal Contents of Deep-Sea Fishes Retrieved from the Abyssal Zone

We devised a new method (the dorayaki method) using marine agar under in situ pressures to isolate barophilic bacteria from the intestinal contents of three deep-sea fishes (two Coryphaenoides yaquinae samples and one Ilyophis sp. sample) retrieved from depths of 4,700 to 6,100 m in the Northwest Pacific Ocean. All 10 strains isolated from one sample (C. yaquinae) were obligately barophilic. One of the 10 strains did not grow at atmospheric pressure and 103.4 MPa but did grow well between 20.7 and 82.7 MPa, with optimal growth at 41.4 MPa. This method is useful for isolating psychrophilic and barophilic deep-sea bacteria.     

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°C at atmospheric pressure. Inactivation in samples from warmed cell suspensions was measured as the loss of colonyforming ability (CFA) at 10°C and 587 bars. At atmospheric pressure, there was a slow loss of CFA even at 10°C. The loss of CFA was rapid above 20°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.     

Population Sizes and Growth Pressure Responses of Intestinal Microfloras of Deep-Sea Fish Retrieved from the Abyssal Zone

The intestinal floras of seven deep-sea fish retrieved at depths of from 3,200 to 5,900 m were examined for population sizes and growth responses to pressure. Large populations of culturable bacteria, ranging from 1.1 x 10(sup6) to 3.6 x 10(sup8) cells per ml of contents, were detected when samples were incubated at conditions characteristic of those of the deep sea. Culturable cell counts at in situ pressures were greater than those at atmospheric pressure in all samples. Most of the strains isolated by the spread-plating method at atmospheric pressure later proved barophilic. Barophilic bacteria were the predominant inhabitants of the abyssal fish intestines.     

Stimulation du potentiel de dégradation dans les eaux marines profondes par les bactéries barotolérantes colonisant les fèces du plancton migrateur

At a depth of 1 150 m in the northwest Mediterranean, amino acid uptake and bacterial biomass production rates increased, in a 12-day time interval, 5- and 30-fold, respectively. Simultaneously, bacterial response to pressure changes evolved from barophilic to barotolerant. During the same period, scanning electron microscope observations and in situ observations using Underwater Video Profiler showed a 2-fold increase in particle concentrations, mainly due to faecal pellet production by zooplankton. This input, nutrient rich and largely colonized with bacteria unaffected by pressure variations since they originated from organisms that regularly migrate up and down through the water column, could produce such microbial activity peaking in the deep water masses.     

Species Composition and Barotolerance of Gut Microflora of Deep-Sea Benthic Macrofauna Collected at Various Depths in the Atlantic Ocean

The bacterial flora of marine animals collected at depths of 570 to 2,446 m was examined for population size and generic composition, and the barotolerant characteristics of selected bacterial isolates were determined. Total numbers of culturable, aerobic, heterotrophic bacteria were found to be low in animals collected at the greatest ocean depths sampled in this study. Vibrio spp. were predominant in 10 of 15 samples examined, and Photobacterium spp. and yeasts were the major components of the remainder. Pseudomonas, Achromobacter, and Flavobacterium spp. comprised minor components of the gut flora of deep-sea fish. Forty-six pure cultures isolated from samples of seven animals were tested for growth or viability after incubation for 1 week under pressures ranging from 100 to 750 atm. Strains of bacteria isolated from samples of fish intestine were more barotolerant than those from the stomach (P<0.01). When incubated at a pressure of 600 atm, viability of bacterial cultures originally isolated from fish caught at a depth of 570 m was significantly decreased in comparison with viability of cultures from animals caught at depths of 1,393 and 2,446 m (P<0.01). From results of this study, it is concluded that the gut microflora of animals that dwell in the deeper regions of the ocean are adapted to an increased hydrostatic pressure environment, that is, the gut microflora is less inhibited by elevated hydrostatic pressure with increasing depth from which the host animal was collected.     

A study of deep-sea natural microbial populations and barophilic pure cultures using a high-pressure chemostat

Continuous cultures in which a high-pressure chemostat was used were employed to study the growth responses of (i) deep-sea microbial populations with the naturally occurring carbon available in seawater and with limiting concentrations of supplemental organic substrates and (ii) pure cultures of copiotrophic barophilic and barotolerant deep-sea isolates in the presence of limiting carbon concentrations at various pressures, dilution rates, and temperatures. We found that the growth rates of natural populations could not be measured or were extremely low (e.g., a doubling time of 629 h), as determined from the difference between the dilution rate and the washout rate. A low concentration of supplemental carbon (0.33 mg/liter) resulted in positive growth responses in the natural population, which resulted in an increase in the number of cells and eventually a steady population of cells. We found that the growth responses to imposed growth pressure by barophilic and barotolerant pure-culture isolates that were previously isolated and characterized under high-nutrient-concentration conditions were maintained under the low-nutrient-concentration limiting conditions (0.33 to 3.33 mg of C per liter) characteristic of the deep-sea environment. Our results indicate that deep-sea microbes can respond to small changes in substrate availability. Also, barophilic microbes that are copiotrophic as determined by their isolation in the presence of high carbon concentrations and their preference for high carbon concentrations are versatile and are able to compete and grow as barophiles in the low-carbon-concentration oligotrophic deep-sea environment in which they normally exist.     

A Study of Deep-Sea Natural Microbial Populations and Barophilic Pure Cultures Using a High-Pressure Chemostat

Continuous cultures in which a high-pressure chemostat was used were employed to study the growth responses of (i) deep-sea microbial populations with the naturally occurring carbon available in seawater and with limiting concentrations of supplemental organic substrates and (ii) pure cultures of copiotrophic barophilic and barotolerant deep-sea isolates in the presence of limiting carbon concentrations at various pressures, dilution rates, and temperatures. We found that the growth rates of natural populations could not be measured or were extremely low (e.g., a doubling time of 629 h), as determined from the difference between the dilution rate and the washout rate. A low concentration of supplemental carbon (0.33 mg/liter) resulted in positive growth responses in the natural population, which resulted in an increase in the number of cells and eventually a steady population of cells. We found that the growth responses to imposed growth pressure by barophilic and barotolerant pure-culture isolates that were previously isolated and characterized under high-nutrient-concentration conditions were maintained under the low-nutrient-concentration limiting conditions (0.33 to 3.33 mg of C per liter) characteristic of the deep-sea environment. Our results indicate that deep-sea microbes can respond to small changes in substrate availability. Also, barophilic microbes that are copiotrophic as determined by their isolation in the presence of high carbon concentrations and their preference for high carbon concentrations are versatile and are able to compete and grow as barophiles in the low-carbon-concentration oligotrophic deep-sea environment in which they normally exist.     

Membrane lipids of a psychrophilic and barophilic deep-sea bacterium

In a psychrophilic and barophilic marine bacterial isolate of the genusAlteromonas, the ratio of total unsaturated versus saturated fatty acids in the membrane lipids increased when the organism was grown at increasing hydrostatic pressures and decreasing temperatures. This regulatory capacity, as well as the presence of relatively large amounts of 20:5 polyunsaturated fatty acid, appear to be functional in maintaining membrane fluidity within a range of pressures distinctly below and above the specific optimum and at typical deep sea temperatures.     

Enzymatic adaptation by bacteria under pressure.

A study of enzymic adaptation under hydrostatic pressure by moderately barotolerant bacteria that can grow at pressure up to about 500 atm revealed that some adaptive processes are relatively insensitive to pressure, whereas others are sufficiently barosensitive to compromise survival capacity in situations requiring adaptation to new substrates under pressure. Examples of the former include adaptation of Escherichia coli to arabinose catabolism for growth and adaptation of Streptococcus faecalis to catabolism of lactose, ribose, or maltose. Examples of the latter include derepression of the lac operon in Escherichia coli and induction of penicillinase synthesis by Bacillus licheniformis. For both these barosensitive systems, pressure had little effect on enzyme levels in constitutive strains or in bacteria that had previously been induced at 1 atm. Moreover, it had no detectable effect on penicillinase secretion. However, pressures of 300 to 400 atm were found to reduce markedly rates and extents of enzyme synthesis by bacteria undergoing derepression or adaptation. This inhibitory effect of pressure was reflected in greater barosensitivity with extended lag and slower growth of initially unadapted Escherichia coli cells inoculated into minimal medium with lactose as sole source of carbon and fuel, and by major reductions in the minimal inhibitory concentrations of penicillin G for unadapted B. licheniformis cells inoculated into complex, antibiotic-containing media. Cyclic adenosine 5'-monophosphate did not reverse pressure inhibition of derepression of the lac operon, and catabolite repression was complete under pressure. However, derepression of the lac operon was more sensitive to pressure at low concentrations of inducer than at high concentrations. Apparent volume changes for derepression were 94 and 60 ml/mol at inducer concentrations of about 0.5 and 5 mM, respectively. Pressure was found not to be inhibitory for uptake of beta-galactosides; in fact, it was somewhat stimulatory. Therefore, results were interpreted in terms of inducer binding and subsequent conversion of an operator-inducer-repressor complex to inactive repressor and operator. Both reactions appeared to result in an increase in volume, the former more so than the latter. We found also that 200 atm was actually stimulatory for growth of Escherichia coli in minimal media, and the bacterium was in a sense barophilic.     

Positive Pressure Effect on Manganese Binding by Bacteria in Deep-Sea Hydrothermal Plumes

A positive pressure effect (1.4 to 3.3×) on the binding of Mn²⁺ by a natural population of bacteria in a deep-sea hydrothermal plume was discovered over the intermediate pressure range of 1 to 200 atm (1 to 200 bars; ca. 1.01 × 10² to 2.03 × 10⁴ kPa). The data suggest Mn²⁺ binding is functionally barophilic rather than simply barotolerant.     

A Pressurized Chemostat for the Study of Marine Barophilic and Oligotrophic Bacteria

A continuous culture system that allows bacteria to be grown in steady-state populations under pressures of up to 700 atm (71 MPa) was constructed and tested. With readily available or slightly modified high-pressure chromatography equipment, a continuous flow of sterile medium is pressurized and passed through a 500-ml nylon-coated titanium reactor at flow rates of 0.01 to 10 ml min(sup-1). The pressure in the reactor is controlled by a backpressure regulator with greater than 1% accuracy. In test experiments, a culture of a psychro- and barophilic marine isolate from a depth of 4,900 m (strain F1-A, identified as a Shewanella sp.) was grown at 1, 300, and 450 atm (0.1, 30.4, and 40.5 MPa) and dilution rates of 60 and 90% of the organism's maximum growth rate (determined at 1 atm) in the required complex medium at levels of 3.3 and 0.33 mg of dissolved organic carbon per liter in the reservoir. Growth limitation by carbon was assured by an appropriate C/N/P ratio of the medium. The data indicate that barophilic growth characteristics in steady-state cultures of this psychro- and barophilic deep-sea isolate were positively affected by a decreasing growth rate at the higher of two substrate concentrations in the reservoir. After a 10-fold lowering of the substrate concentration, the effect was reversed. Under these conditions, the cell viability increased significantly, especially at the higher of the two pressures tested. The basic design of the system can principally also be used for growth studies on hyperthermophilic bacteria and archaea.     

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°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.     

Enzymatic profiles of 11 barophilic bacteria under in situ conditions: evidence for pressure modulation of phenotype

Barophilic bacteria are microorganisms that grow preferentially (facultative barophiles) or exclusively (obligate barophiles) under elevated hydrostatic pressure. Barophilic bacteria have been isolated from a variety of deep-sea environments. Attempts to characterize these organisms have been hampered by a lack of appropriate methodologies. A colorimetric method for the detection of 19 constitutively expressed enzymes under in situ conditions of pressure and temperature has been devised, using a simple modification of the commercially available API ZYME enzyme assay kit. By using this method, enzyme profiles of 11 barophilic isolates, including an obligate barophile, were determined. Nine of the 10 facultatively barophilic isolates examined exhibited a change of phenotype in at least one enzyme reaction when tested at 1 atm (1 atm = 101.29 kPa), compared with results obtained under in situ pressure. The assay is simple and rapid and allows for direct determination of enzyme activity under conditions of high pressure and low temperature.     

Enzymatic profiles of 11 barophilic bacteria under in situ conditions: evidence for pressure modulation of phenotype.

Barophilic bacteria are microorganisms that grow preferentially (facultative barophiles) or exclusively (obligate barophiles) under elevated hydrostatic pressure. Barophilic bacteria have been isolated from a variety of deep-sea environments. Attempts to characterize these organisms have been hampered by a lack of appropriate methodologies. A colorimetric method for the detection of 19 constitutively expressed enzymes under in situ conditions of pressure and temperature has been devised, using a simple modification of the commercially available API ZYME enzyme assay kit. By using this method, enzyme profiles of 11 barophilic isolates, including an obligate barophile, were determined. Nine of the 10 facultatively barophilic isolates examined exhibited a change of phenotype in at least one enzyme reaction when tested at 1 atm (1 atm = 101.29 kPa), compared with results obtained under in situ pressure. The assay is simple and rapid and allows for direct determination of enzyme activity under conditions of high pressure and low temperature.     

Observations of Barophilic Microbial Activity in Samples of Sediment and Intercepted Particulates from the Demerara Abyssal Plain

To better understand the ecological significance of pressure effects on bacteria in the abyssobenthic boundary layer, experimental suspensions of sediments and sinking particulates were prepared from samples collected in boxcore and bottom-moored sediment traps at two stations (depth, 4,470 and 4,850m) in the Demerara abyssal plain off the coast of Brazil. Replicate samples were incubated shipboard at 3°C and at both atmospheric and deep-sea pressures (440 or 480 atm [4.46 × 10⁴ or 4.86 × 10⁴ kPa]) following the addition of [¹⁴C]glutamic acid (<10 μg liter⁻¹) or yeast extract (0.025%) and the antibiotic nalidixic acid (0.002%). In seven of the eight samples supplemented with isotope, a barophilic microbial response was detected, i.e., substrate incorporation and respiration were greater under in situ pressure than at 1 atm (101.3 kPa). In the remaining sample, prepared from a sediment trap warmed to 24°C before recovery, pressure was observed to inhibit substrate utilization. Total bacterial counts by epifluorescence microscopy decreased with depth in each sediment core, as did utilization of glutamic acid. Significant percentages of the total bacterial populations in cold sediment trap samples (but not the prewarmed one or any boxcore sample) were abnormally enlarged and orange fluorescing after incubation with yeast extract and nalidixic acid under deep-sea conditions. Results indicated that in the deep sea, barophilic bacteria play a predominant role in the turnover of naturally low levels of glutamic acid, and the potential for intense microbial activity upon nutrient enrichment is more likely to occur in association with recently settled particulates, especially fecal pellets, than in buried sediments.     

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.     

Undecompressed microbial populations from the deep sea.

Metabolic transformations of glutamate and Casamino Acids by natural microbial populations collected from deep waters (1,600 to 3,100 m) were studied in decompressed and undecompressed samples. Pressure-retaining sampling/incubation vessels and appropriate subsampling/incubation vessels and appropriate subsampling techniques permitted time course experiments. In all cases the metabolic activity in undecompressed samples was lower than it was when incubated at 1 atm. Surface water controls showed a reduced activity upon compression. The processes involving substrate incorporation into cell material were more pressure sensitive than was respiration. The low utilization of substrates, previously found by in situ incubations for up to 12 months, was confirmed and demonstrated to consist of an initial phase of activity, in the range of 5 to 60 times lower than the controls, followed by a stationary phase of virtually no substrate utilization. No barophilic growth response (higher rates at elevated pressure than at 1 atm) was recorded; all populations observed exhibition various degrees of barotolerance.     

Molecular analyses of the sediment of the 11000-m deep Mariana Trench

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 identifed in deep-sea adapted bacteria, were amplifed 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 identifed. The sequences of the amplifed 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. Received: October 10, 1996 / Accepted: March 3, 1997