Microbial adaptations to the psychrosphere/piezosphere

Low temperature and high pressure deep-sea environments occupy the largest fraction of the biosphere. Nevertheless, the molecular adaptations that enable life to exist under these conditions remain poorly understood. This article will provide an overview of the current picture on high pressure adaptation in cold oceanic environments, with an emphasis on genetic experiments performed on Photobacterium profundum. Thus far genes which have been found or implicated as important for pressure-sensing or pressure-adaptation include genes required for fatty acid unsaturation, the membrane protein genes toxR and rseC and the DNA recombination gene recD. Many deep-sea bacteria possess genes for the production of omega-3 polyunsaturated fatty acids. These could be of biotechnological significance since these fatty acids reduce the risk of cardiovascular disease and certain cancers and are useful as dietary supplements.     

Monounsaturated but Not Polyunsaturated Fatty Acids Are Required for Growth of the Deep-Sea Bacterium Photobacterium profundum SS9 at High Pressure and Low Temperature

There is considerable evidence correlating the production of increased proportions of membrane unsaturated fatty acids (UFAs) with bacterial growth at low temperatures or high pressures. In order to assess the importance of UFAs to microbial growth under these conditions, the effects of conditions altering UFA levels in the psychrotolerant piezophilic deep-sea bacterium Photobacterium profundum SS9 were investigated. The fatty acids produced by P. profundum SS9 grown at various temperatures and pressures were characterized, and differences in fatty acid composition as a function of phase growth, and between inner and outer membranes, were noted. P. profundum SS9 was found to exhibit enhanced proportions of both monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids when grown at a decreased temperature or elevated pressure. Treatment of cells with cerulenin inhibited MUFA but not PUFA synthesis and led to a decreased growth rate and yield at low temperature and high pressure. In addition, oleic acid-auxotrophic mutants were isolated. One of these mutants, strain EA3, was deficient in the production of MUFAs and was both low-temperature sensitive and high-pressure sensitive in the absence of exogenous 18:1 fatty acid. Another mutant, strain EA2, produced little MUFA but elevated levels of the PUFA species eicosapentaenoic acid (EPA; 20:5n-3). This mutant grew slowly but was not low-temperature sensitive or high-pressure sensitive. Finally, reverse genetics was employed to construct a mutant unable to produce EPA. This mutant, strain EA10, was also not low-temperature sensitive or high-pressure sensitive. The significance of these results to the understanding of the role of UFAs in growth under low-temperature or high-pressure conditions is discussed.     

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.     

Monounsaturated but not polyunsaturated fatty acids are required for growth of the deep-sea bacterium Photobacterium profundum SS9 at high pressure and low temperature

There is considerable evidence correlating the production of increased proportions of membrane unsaturated fatty acids (UFAs) with bacterial growth at low temperatures or high pressures. In order to assess the importance of UFAs to microbial growth under these conditions, the effects of conditions altering UFA levels in the psychrotolerant piezophilic deep-sea bacterium Photobacterium profundum SS9 were investigated. The fatty acids produced by P. profundum SS9 grown at various temperatures and pressures were characterized, and differences in fatty acid composition as a function of phase growth, and between inner and outer membranes, were noted. P. profundum SS9 was found to exhibit enhanced proportions of both monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids when grown at a decreased temperature or elevated pressure. Treatment of cells with cerulenin inhibited MUFA but not PUFA synthesis and led to a decreased growth rate and yield at low temperature and high pressure. In addition, oleic acid-auxotrophic mutants were isolated. One of these mutants, strain EA3, was deficient in the production of MUFAs and was both low-temperature sensitive and high-pressure sensitive in the absence of exogenous 18:1 fatty acid. Another mutant, strain EA2, produced little MUFA but elevated levels of the PUFA species eicosapentaenoic acid (EPA; 20:5n-3). This mutant grew slowly but was not low-temperature sensitive or high-pressure sensitive. Finally, reverse genetics was employed to construct a mutant unable to produce EPA. This mutant, strain EA10, was also not low-temperature sensitive or high-pressure sensitive. The significance of these results to the understanding of the role of UFAs in growth under low-temperature or high-pressure conditions is discussed.     

Microbial diversity of deep-sea extremophiles--Piezophiles, Hyperthermophiles, and subsurface microorganisms]

Knowledge of our Planet's biosphere has increased tremendously during the last 10 to 20 years. In the field of Microbiology in particular, scientists have discovered novel "extremophiles", microorganisms capable of living in extreme environments such as highly acidic or alkaline conditions, at high salt concentration, with no oxygen, extreme temperatures (as low as -20 degrees C and as high as 300 degrees C), at high concentrations of heavy metals and in high pressure environments such as the deep-sea. It is apparent that microorganisms can exist in any extreme environment of the Earth, yet already scientists have started to look for life on other planets; the so-called "Exobiology" project. But as yet we have little knowledge of the deep-sea and subsurface biosphere of our own planet. We believe that we should elucidate the Biodiversity of Earth more thoroughly before exploring life on other planets, and these attempts would provide deeper insight into clarifying the existence of extraterrestrial life. We focused on two deep-sea extremophiles in this article; one is "Piezophiles", and another is "Hyperthermophiles". Piezophiles are typical microorganisms adapted to high-pressure and cold temperature environments, and located in deep-sea bottom. Otherwise, hyperthermophiles are living in high temperature environment, and located at around the hydrothermal vent systems in deep-sea. They are not typical deep-sea microorganisms, but they can grow well at high-pressure condition, just like piezophiles. Deming and Baross mentioned that most of the hyperthermophilic archaea isolated from deep-sea hydrothermal vents are able to grow under conditions of high temperature and pressure, and in most cases their optimal pressure for growth was greater than the environmental pressure they were isolated from. It is possible that originally their native environment may have been deeper than the sea floor and that there had to be a deeper biosphere. This implication suggests that the deep-sea hydrothermal vents are the windows to a deep subsurface biosphere. A vast array of chemoautotrophic deep-sea animal communities have been found to exist in cold seep environments, and most of these animals are common with those found in hydrothermal vent environments. Thus, it is possible to consider that the cold seeps are also one of slit windows to a deep subsurface biosphere. We conclude that the deep-sea extremophiles are very closely related into the unseen majority in subsurface biosphere, and the subsurface biosphere probably concerns to consider the "exobiology".     

Genome scanning for detecting adaptive genes along environmental gradients in the Japanese conifer,Cryptomeria japonica

Local adaptation is important in evolutionary processes and speciation. We used multiple tests to identify several candidate genes that may be involved in local adaptation from 1026 loci in 14 natural populations of Cryptomeria japonica, the most economically important forestry tree in Japan. We also studied the relationships between genotypes and environmental variables to obtain information on the selective pressures acting on individual populations. Outlier loci were mapped onto a linkage map, and the positions of loci associated with specific environmental variables are considered. The outlier loci were not randomly distributed on the linkage map; linkage group 11 was identified as a genomic island of divergence. Three loci in this region were also associated with environmental variables such as mean annual temperature, daily maximum temperature, maximum snow depth, and so on. Outlier loci identified with high significance levels will be essential for conservation purposes and for future work on molecular breeding.     

Environmental adaptation: genomic analysis of the piezotolerant and psychrotolerant deep-sea iron reducing bacterium Shewanella piezotolerans WP3

Shewanella species are widespread in various environments. Here, the genome sequence of Shewanella piezotolerans WP3, a piezotolerant and psychrotolerant iron reducing bacterium from deep-sea sediment was determined with related functional analysis to study its environmental adaptation mechanisms. The genome of WP3 consists of 5,396,476 base pairs (bp) with 4,944 open reading frames (ORFs). It possesses numerous genes or gene clusters which help it to cope with extreme living conditions such as genes for two sets of flagellum systems, structural RNA modification, eicosapentaenoic acid (EPA) biosynthesis and osmolyte transport and synthesis. And WP3 contains 55 open reading frames encoding putative c-type cytochromes which are substantial to its wide environmental adaptation ability. The mtr-omc gene cluster involved in the insoluble metal reduction in the Shewanella genus was identified and compared. The two sets of flagellum systems were found to be differentially regulated under low temperature and high pressure; the lateral flagellum system was found essential for its motility and living at low temperature.     

Genome sequence of Oceanobacillus iheyensis isolated from the Iheya Ridge and its unexpected adaptive capabilities to extreme environments

Oceanobacillus iheyensis HTE831 is an alkaliphilic and extremely halotolerant Bacillus-related species isolated from deep-sea sediment. We present here the complete genome sequence of HTE831 along with analyses of genes required for adaptation to highly alkaline and saline environments. The genome consists of 3.6 Mb, encoding many proteins potentially associated with roles in regulation of intracellular osmotic pressure and pH homeostasis. The candidate genes involved in alkaliphily were determined based on comparative analysis with three Bacillus species and two other Gram-positive species. Comparison with the genomes of other major Gram-positive bacterial species suggests that the backbone of the genus Bacillus is composed of approximately 350 genes. This second genome sequence of an alkaliphilic Bacillus-related species will be useful in understanding life in highly alkaline environments and microbial diversity within the ubiquitous bacilli.     

Piezotolerance of the respiratory terminal oxidase activity of the piezophilic Shewanella violacea DSS12 as compared with non-piezophilic Shewanella species

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.     

Ecotype Diversity and Conversion in Photobacterium profundum Strains

Photobacterium profundum is a cosmopolitan marine bacterium capable of growth at low temperature and high hydrostatic pressure. Multiple strains of P. profundum have been isolated from different depths of the ocean and display remarkable differences in their physiological responses to pressure. The genome sequence of the deep-sea piezopsychrophilic strain Photobacterium profundum SS9 has provided some clues regarding the genetic features required for growth in the deep sea. The sequenced genome of Photobacterium profundum strain 3TCK, a non-piezophilic strain isolated from a shallow-water environment, is now available and its analysis expands the identification of unique genomic features that correlate to environmental differences and define the Hutchinsonian niche of each strain. These differences range from variations in gene content to specific gene sequences under positive selection. Genome plasticity between Photobacterium bathytypes was investigated when strain 3TCK-specific genes involved in photorepair were introduced to SS9, demonstrating that horizontal gene transfer can provide a mechanism for rapid colonisation of new environments.     

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.     

Micro- and Macro-Geographic Scale Effect on the Molecular Imprint of Selection and Adaptation in Norway Spruce

Forest tree species of temperate and boreal regions have undergone a long history of demographic changes and evolutionary adaptations. The main objective of this study was to detect signals of selection in Norway spruce (Picea abies [L.] Karst), at different sampling-scales and to investigate, accounting for population structure, the effect of environment on species genetic diversity. A total of 384 single nucleotide polymorphisms (SNPs) representing 290 genes were genotyped at two geographic scales: across 12 populations distributed along two altitudinal-transects in the Alps (micro-geographic scale), and across 27 populations belonging to the range of Norway spruce in central and south-east Europe (macro-geographic scale). At the macrogeographic scale, principal component analysis combined with Bayesian clustering revealed three major clusters, corresponding to the main areas of southern spruce occurrence, i.e. the Alps, Carpathians, and Hercynia. The populations along the altitudinal transects were not differentiated. To assess the role of selection in structuring genetic variation, we applied a Bayesian and coalescent-based F _ST-outlier method and tested for correlations between allele frequencies and climatic variables using regression analyses. At the macro-geographic scale, the F _ST-outlier methods detected together 11 F _ST-outliers. Six outliers were detected when the same analyses were carried out taking into account the genetic structure. Regression analyses with population structure correction resulted in the identification of two (micro-geographic scale) and 38 SNPs (macro-geographic scale) significantly correlated with temperature and/or precipitation. Six of these loci overlapped with F _ST-outliers, among them two loci encoding an enzyme involved in riboflavin biosynthesis and a sucrose synthase. The results of this study indicate a strong relationship between genetic and environmental variation at both geographic scales. It also suggests that an integrative approach combining different outlier detection methods and population sampling at different geographic scales is useful to identify loci potentially involved in adaptation.     

Thermodynamic and functional characteristics of deep-sea enzymes revealed by pressure effects

Hydrostatic pressure analysis is an ideal approach for studying protein dynamics and hydration. The development of full ocean depth submersibles and high-pressure biological techniques allows us to investigate enzymes from deep-sea organisms at the molecular level. The aim of this review was to overview the thermodynamic and functional characteristics of deep-sea enzymes as revealed by pressure axis analysis after giving a brief introduction to the thermodynamic principles underlying the effects of pressure on the structural stability and function of enzymes.     

The Secret to Successful Deep-Sea Invasion: Does Low Temperature Hold the Key?

There is a general consensus that today’s deep-sea biodiversity has largely resulted from recurrent invasions and speciations occurring through homogenous waters during periods of the Phanerozoic eon. Migrations likely continue today, primarily via isothermal water columns, such as those typical of Polar Regions, but the necessary ecological and physiological adaptations behind them are poorly understood. In an evolutionary context, understanding the adaptations, which allow for colonisation to high-pressure environments, may enable us to predict future events. In this investigation, we examine pressure tolerance during development, in the shallow-water neogastropod Buccinum undatum using thermally acclimated egg masses from temperate and sub-polar regions across the species range. Fossil records indicate neogastropods to have a deep-water origin, suggesting shallow-water species may be likely candidates for re-emergence into the deep sea. Our results show population level differences in physiological thresholds, which indicate low temperature acclimation to increase pressure tolerance. These findings imply this species is capable of deep-sea penetration through isothermal water columns prevailing at high latitudes. This study gives new insight into the fundamentals behind past and future colonisation events. Such knowledge is instrumental to understand better how changes in climate envelopes affect the distribution and radiation of species along latitudinal as well as bathymetric temperature gradients.     

Barophiles: deep-sea microorganisms adapted to an extreme environment

The deep-sea environment is characterized by high pressure and low temperature but in the vicinity of hydrothermal vents regions of extremely high temperature exist. Deep-sea microorganisms have specially adapted features that enable them to live and grow in this extreme environment. Recent research on the physiology and molecular biology of deep-sea barophilic bacteria has identified pressure-regulated operons and shown that microbial growth is influenced by the relationship between temperature and pressure in the deep-sea environment.