Psychrophiles and polar regions

Most reviews of microbial life in cold environments begin with a lament of how little is known about the psychrophilic (cold-loving) inhabitants or their specific adaptations to the cold. This situation is changing, as research becomes better focused by new molecular genetic (and other) approaches, by awareness of accelerated environmental change in polar regions, and by strong interest in the habitability of frozen environments elsewhere in the solar system. This review highlights recent discoveries in molecular adaptation, biodiversity and microbial dynamics in the cold, along with the concept of eutectophiles, organisms living at the critical interface inherent to the phase change of water to ice.     

Bacterial diversity in the snow over Tibetan Plateau Glaciers

Bacterial diversity and cell abundance in the snow of the four glaciers (Guoqu, Zadang, East Rongbuk and Palong No. 4) located in different climatic zones of the Tibetan Plateau were investigated through culture-independent molecular analysis of 16S rRNA gene clone library and flow cytometry approaches. Cell abundance ranged from 0.68 × 10³ to 720 × 10³ cells mL⁻¹, with higher values in the northern glaciers than in the southern ones. Bacterial diversity was unexpectedly high in the snow habitats of the world’s highest plateau, with 15 common genera distributed widely among the glaciers. The bacterial diversity in the snow at different glaciers was related to the surrounding environments. The Guoqu Glacier, to the north near the desert zone and with the lowest temperature, preserved more bacteria closely related to a cold environment and soil than the other glaciers. However, in the Palong No. 4 Glacier located in the south warm region around vegetation, most bacteria were phylogenetically related to plant-associated bacteria.     

低温微生物及其酶类的研究概况

广泛分布在地球寒冷生境 ,如南北两极、高山、深海以及冰川中的低温微生物 ,不但为研究低温生态系统、生命起源与进化以及生物适冷机制提供了丰富的材料 ,同时在生物工程方面也具有潜在的巨大开发价值。国内外越来越多的科研人员对低温微生物及其产物的研究表现出了浓厚的兴趣。关于细胞膜和低温酶的研究 ,是目前微生物适冷机制研究中的 2个热点。就低温微生物的研究现状和适冷机制以及低温酶类的研究进行了综述。     

Antifreeze proteins in polar sea ice diatoms: diversity and gene expression in the genus Fragilariopsis

Fragilariopsis is a dominating psychrophilic diatom genus in polar sea ice. The two species Fragilariopsis cylindrus and Fragilariopsis curta are able to grow and divide below freezing temperature of sea water and above average sea water salinity. Here we show that antifreeze proteins (AFPs), involved in cold adaptation in several psychrophilic organisms, are widespread in the two polar species. The presence of AFP genes (afps) as a multigene family indicated the importance of this group of genes for the genus Fragilariopsis, possibly contributing to its success in sea ice. Protein phylogeny showed the potential mobility of afps, which appear to have crossed kingdom and domain borders, occurring in Bacteria, diatoms, crustaceans and fungi. Our results revealed a broad distribution of AFPs not only in polar organisms but also in taxa apparently not related to cold environments, suggesting that these proteins may be multifunctional. The relevance of AFPs to Fragilariopsis was also shown by gene expression analysis. Under stress conditions typical for sea ice, with subzero temperatures and high salinities, F. cylindrus and F. curta strongly expressed selected afps. An E/G point mutation in the Fragilariopsis AFPs may play a role in gene expression activity and protein function.     

Cold adaptation of a mesophilic subtilisin-like protease by laboratory evolution

Enzymes isolated from organisms native to cold environments generally exhibit higher catalytic efficiency at low temperatures and greater thermosensitivity than their mesophilic counterparts. In an effort to understand the evolutionary process and the molecular basis of cold adaptation, we have used directed evolution to convert a mesophilic subtilisin-like protease from Bacillus sphaericus, SSII, into its psychrophilic counterpart. A single round of random mutagenesis followed by recombination of improved variants yielded a mutant, P3C9, with a catalytic rate constant (k(cat)) at 10 degrees C 6.6 times and a catalytic efficiency (k(cat)/K(M)) 9.6 times that of wild type. Its half-life at 70 degrees C is 3.3 times less than wild type. Although there is a trend toward decreasing stability during the progression from mesophile to psychrophile, there is not a strict correlation between decreasing stability and increasing low temperature activity. A first generation mutant with a >2-fold increase in k(cat) is actually more stable than wild type. This suggests that the ultimate decrease in stability may be due to random drift rather than a physical incompatibility between low temperature activity and high temperature stability. SSII shares 77. 4% identity with the naturally psychrophilic protease subtilisin S41. Although SSII and S41 differ at 85 positions, four amino acid substitutions were sufficient to generate an SSII whose low temperature activity is greater than that of S41. That none of the four are found in S41 indicates that there are multiple routes to cold adaptation.     

Isolation of oligotrophic yeasts from supraglacial environments of different altitude on the Gulkana Glacier (Alaska)

Psychrophilic yeasts have been isolated from supra- and subglacial ice at many sites worldwide. To understand the ecology of psychrophilic yeasts on glaciers, we focused on their adaptation to wide range of nutrient concentrations and their distribution with altitude on the Gulkana Glacier in Alaska. We found various culturable psychrophilic yeasts on the ice surfaces of the glacier, and 11 species were isolated with incubation at 4?°C in four different dilutions of agar medium. Some of our isolated species (Rhodotorula psychrophenolica, Rhodotorula aff. psychrophenolica, Rhodotorula glacialis, and Basidiomycota sp. 1) can grow on the low dissolved organic matter (DOC) concentrations medium (7.6?mg?L(-1) ) which is close to the typical level of supraglacial melt water, suggesting that these species can inhabit in any supraglacial meltwater. Otherwise, most of other species were isolated only from higher DOC concentration medium (183?mg?L(-1) -18.3?g?L(-1) ), suggesting that these are inhabitant around the cryoconite, because DOC concentrations in melted surface-ice contained cryoconite is much higher than in melted water. Similarity of altitudinal distribution between culturable yeast and algal biomass suggests that the ecological role played by the cold-adapted yeasts is as organic matter decomposers and nutrient cyclers in glacier ecosystem.     

Some like it cold: understanding the survival strategies of psychrophiles

Much of the Earth's surface, both marine and terrestrial, is either periodically or permanently cold. Although habitats that are largely or continuously frozen are generally considered to be inhospitable to life, psychrophilic organisms have managed to survive in these environments. This is attributed to their innate adaptive capacity to cope with cold and its associated stresses. Here, we review the various environmental, physiological and molecular adaptations that psychrophilic microorganisms use to thrive under adverse conditions. We also discuss the impact of modern “omic” technologies in developing an improved understanding of these adaptations, highlighting recent work in this growing field.     

Comparative void-volume analysis of psychrophilic and mesophilic enzymes: Structural bioinformatics of psychrophilic enzymes reveals sources of core flexibility

Background

Psychrophiles, cold-adapted organisms, have adapted to live at low temperatures by using a variety of mechanisms. Their enzymes are active at cold temperatures by being structurally more flexible than mesophilic enzymes. Even though, there are some indications of the possible structural mechanisms by which psychrophilic enzymes are catalytic active at cold temperatures, there is not a generalized structural property common to all psychrophilic enzymes.

Results

We examine twenty homologous enzyme pairs from psychrophiles and mesophiles to investigate flexibility as a key characteristic for cold adaptation. B-factors in protein X-ray structures are one way to measure flexibility. Comparing psychrophilic to mesophilic protein B-factors reveals that psychrophilic enzymes are more flexible in 5-turn and strand secondary structures. Enzyme cavities, identified using CASTp at various probe sizes, indicate that psychrophilic enzymes have larger average cavity sizes at probe radii of 1.4-1.5 Å, sufficient for water molecules. Furthermore, amino acid side chains lining these cavities show an increased frequency of acidic groups in psychrophilic enzymes.

Conclusions

These findings suggest that embedded water molecules may play a significant role in cavity flexibility, and therefore, overall protein flexibility. Thus, our results point to the important role enzyme flexibility plays in adaptation to cold environments.

     

Preliminary Study on Effects of Glacial Retreat on the Dominant Glacial Snow Bacteria in Laohugou Glacier No. 12

One impact of climate change is the rapid shrinking of glaciers, resulting in microorganisms deposited into glacial snow or ice being exposed to new environments such as glacier foreland. A pyrosequencing analysis based on the bacterial 16S rRNA gene showed that bacterial diversity was the highest in proglacial soil, followed by that of glacial snow in ablation zone, then by that of glacial snow in the accumulation area, finally by that of glacial snow in glacier terminus, with the combination of Chao1, ACE, Shannon and Simpson analysis. Eighteen phyla were detected from the 7 samples, but mainly composed of Proteobacteria, Actinobacteria and Bacteroidetes. Flavobacterium, Massilia, Pedobacter, Polaromonas were more abundant in glacial snow samples than in glacial soil sample. Massilia was rarely reported in other environments, implying the necessity for its conservation under scenarios of glacier and snowpack loss induced by climate change.     

Bacterial community structure on two alpine debris-covered glaciers and biogeography of Polaromonas phylotypes

High-elevation cold environments are considered ideal places to test hypotheses about mechanisms of bacterial colonization and succession, and about bacterial biogeography. Debris-covered glaciers (glaciers whose ablation area is mainly covered by a continuous layer of rock debris fallen from the surrounding mountains) have never been investigated in this respect so far. We used the Illumina technology to analyse the V5 and V6 hypervariable regions of the bacterial 16S rRNA gene amplified from 38 samples collected in July and September 2009 at different distances from the terminus on two debris-covered glaciers (Miage and Belvedere—Italian Alps). Heterotrophic taxa-dominated communities and bacterial community structure changed according to ice ablation rate, organic carbon content of the debris and distance from the glacier terminus. Bacterial communities therefore change during downwards debris transport, and organic carbon of these recently exposed substrates is probably provided more by allochthonous deposition of organic matter than by primary production by autotrophic organisms. We also investigated whether phylotypes of the genus Polaromonas, which is ubiquitous in cold environments, do present a biogeographical distribution by analysing the sequences retrieved in this study together with others available in the literature. We found that the genetic distance among phylotypes increased with geographic distance; however, more focused analyses using discrete distance classes revealed that both sequences collected at sites <100 km and at sites 9400–13 500 km to each other were more similar than those collected at other distance classes. Evidences of biogeographic distribution of Polaromonas phylotypes were therefore contrasting.     

Cold-adapted archaea

Many archaea are extremophiles. They thrive at high temperatures, at high pressure and in concentrated acidic environments. Nevertheless, the largest proportion and greatest diversity of archaea exist in cold environments. Most of the Earth's biosphere is cold, and archaea represent a significant fraction of the biomass. Although psychrophilic archaea have long been the neglected majority, the study of these microorganisms is beginning to come of age. This review casts a spotlight on the ecology, adaptation biology and unique science that is being realized from studies on cold-adapted archaea.     

Isolation and characterization of marine psychrophilic phage-host systems from Arctic sea ice

Phage-host systems from extreme cold environments have rarely been surveyed. This study is concerned with the isolation and characterization of three different phage-host systems from Arctic sea ice and melt pond samples collected north-west of Svalbard (Arctic). On the basis of 16S rDNA sequences, the three bacterial phage hosts exhibited the greatest similarity to the species Shewanella frigidimarina (96.0%), Flavobacterium hibernum (94.0%), and Colwellia psychrerythraea (98.4%), respectively. The host bacteria are psychrophilic with good growth at 0°C, resulting in a rapid formation of visible colonies at this temperature. The phages showed an even more pronounced adaptation to cold temperatures than the bacteria, with growth maxima below 14°C and good plaque formation at 0°C. Transmission electron microscopy (TEM) examinations revealed that the bacteriophages belonged to the tailed, double-stranded DNA phage families Siphoviridae and Myoviridae. All three phages were host-specific.Communicated by K. Horikoshi     

Diversity and genomics of Antarctic marine micro-organisms

Marine bacterioplanktons are thought to play a vital role in Southern Ocean ecology and ecosystem function, as they do in other ocean systems. However, our understanding of phylogenetic diversity, genome-enabled capabilities and specific adaptations to this persistently cold environment is limited. Bacterioplankton community composition shifts significantly over the annual cycle as sea ice melts and phytoplankton bloom. Microbial diversity in sea ice is better known than that of the plankton, where culture collections do not appear to represent organisms detected with molecular surveys. Broad phylogenetic groupings of Antarctic bacterioplankton such as the marine group I Crenarchaeota, alpha-Proteobacteria (Roseobacter-related and SAR-11 clusters), gamma-Proteobacteria (both cultivated and uncultivated groups) and Bacteriodetes-affiliated organisms in Southern Ocean waters are in common with other ocean systems. Antarctic SSU rRNA gene phylotypes are typically affiliated with other polar sequences. Some species such as Polaribacter irgensii and currently uncultivated gamma-Proteobacteria (Ant4D3 and Ant10A4) may flourish in Antarctic waters, though further studies are needed to address diversity on a larger scale. Insights from initial genomics studies on both cultivated organisms and genomes accessed through shotgun cloning of environmental samples suggest that there are many unique features of these organisms that facilitate survival in high-latitude, persistently cold environments.     

Ecology under lake ice

Winter conditions are rapidly changing in temperate ecosystems, particularly for those that experience periods of snow and ice cover. Relatively little is known of winter ecology in these systems, due to a historical research focus on summer ‘growing seasons’. We executed the first global quantitative synthesis on under‐ice lake ecology, including 36 abiotic and biotic variables from 42 research groups and 101 lakes, examining seasonal differences and connections as well as how seasonal differences vary with geophysical factors. Plankton were more abundant under ice than expected; mean winter values were 43.2% of summer values for chlorophyll a, 15.8% of summer phytoplankton biovolume and 25.3% of summer zooplankton density. Dissolved nitrogen concentrations were typically higher during winter, and these differences were exaggerated in smaller lakes. Lake size also influenced winter‐summer patterns for dissolved organic carbon (DOC), with higher winter DOC in smaller lakes. At coarse levels of taxonomic aggregation, phytoplankton and zooplankton community composition showed few systematic differences between seasons, although literature suggests that seasonal differences are frequently lake‐specific, species‐specific, or occur at the level of functional group. Within the subset of lakes that had longer time series, winter influenced the subsequent summer for some nutrient variables and zooplankton biomass.     

RUBISCO ADAPTATION TO LOW TEMPERATURES: A COMPARATIVE STUDY IN PSYCHROPHILIC AND MESOPHILIC UNICELLULAR ALGAE

Some properties of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) from two psychrophilic Chloromonas species have been investigated in relation to their adaptation to cold environments. Contrary to the situation usually encountered with psychrophilic enzymes, the carboxylase activity of both purified "cold" RUBISCO enzymes was lower at low temperatures than that found with the enzyme of the mesophilic alga Chlamydomonas reinhardtii Dangeard. Moreover, the apparent optimal temperature for RUBISCO carboxylase activity was similar for psychrophilic and mesophilic enzymes. Psychrophilic RUBISCOs, however, showed a greater thermosensitivity than the C. reinhardtii enzyme. Genes encoding small and large subunits of RUBISCO from one psychrophilic isolate were sequenced. Comparison of the deduced amino acid sequences to those of higher plants and green algae revealed the substitution of a very highly conserved residue (cysteine₂₄₇ → serine in the large subunit) that could be responsible, at least in part, for the increased thermosensitivity of the "cold" enzyme. Interestingly, the relative amount of RUBISCO subunits found in the psychrophilic isolates was about twice as high as the amount observed in C. reinhardtii and five other mesophilic algae. The high production of a key enzyme to counterbalance its poor catalytic efficiency at low temperature could constitute a novel type of adaptive mechanism to cold environments.     

Ice encapsulation protects rather than disturbs the freezing lichen

Arctic and alpine terricolous lichens are adapted to harsh environments and are tolerant to extremely low temperatures when metabolically inactive. However, there are reports indicating that freezing can be lethal to metabolically active lichens. With a projected warmer and more unstable climate, winter precipitation at high latitudes will fall more frequently as rain, causing snowmelt and encapsulating terricolous lichens in ice or exposing them to large temperature fluctuations. Lichens are a major winter food source for reindeer in most parts of the circumpolar region. A laboratory experiment tested how three hydrated reindeer forage lichen species covered by snow, encapsulated in ice, or uncovered responded to storage at freezing temperatures and subsequent warming. Photosynthetic performance (maximal fluorescence of dark-adapted samples and net photosynthetic rates) was significantly lower in lichens not insulated by snow or ice, whereas there were few differences between the snow and ice treatments. It is suggested that snow and ice provide sufficiently moist environments to improve extracellular and reduce intracellular ice nucleation activity. Ice encapsulation, which is often lethal to vascular plants, did not have any negative effects on the studied lichens. The results indicate that complete snow and ice melt followed by refreezing can be detrimental to terricolous lichen ecosystems. Reduced lichen biomass will have a negative effect both on reindeer winter survival and the indigenous peoples who herd reindeer.     

Identity and physiology of a new psychrophilic eukaryotic green alga, <Emphasis Type="Italic">Chlorella</Emphasis> sp., strain BI,isolated from a transitory pond near Bratina Island,Antarctica

Permanently low temperature environments are one of the most abundant microbial habitats on earth. As in most ecosystems, photosynthetic organisms drive primary production in low temperature food webs. Many of these phototrophic microorganisms are psychrophilic; however, functioning of the photosynthetic processes of these enigmatic psychrophiles (the "photopsychrophiles") in cold environments is not well understood. Here we describe a new chlorophyte isolated from a low temperature pond, on the Ross Ice Shelf near Bratina Island, Antarctica. Phylogenetic and morphological analyses place this strain in the Chlorella clade, and we have named this new chlorophyte Chlorella BI. Chlorella BI is a psychrophilic species, exhibiting optimum temperature for growth at around 10 degrees C. However, psychrophily in the Antarctic Chlorella was not linked to high levels of membrane-associated poly-unsaturated fatty acids. Unlike the model Antarctic lake alga, Chlamydomonas raudensis UWO241, Chlorella BI has retained the ability for dynamic short term adjustment of light energy distribution between photosystem II (PS II) and photosystem I (PS I). In addition, Chlorella BI can grow under a variety of trophic modes, including heterotrophic growth in the dark. Thus, this newly isolated photopsychrophile has retained a higher versatility in response to environmental change than other well studied cold-adapted chlorophytes.     

Polymer hydrolysis in a cold climate

In this review we discuss the activity of an ecologically significant group of psychrophilic bacteria, which are involved in the hydrolysis of plant cell wall polymers. Until now these organisms have been largely overlooked, despite the key role they play in releasing organic carbon fixed by primary producers in permanently cold environments such as Antarctica. This review details a specific group of plant cell wall polymer-degrading enzymes known as β-glycanases. Studies on "cold" enzymes in general are in their infancy, but it has been shown that many exhibit structural and functional modifications that enable them to function at low temperature. β-Glycanases in particular are intriguing because their substrates (cellulose and xylan) are very refractile, which may indicate that their "cold" modifications are pronounced. In addition, mesophilic β-glycanases have been extensively studied and the current state of our knowledge is reviewed. This body of information can be exploited to enable meaningful comparative studies between mesophilic and psychrophilic β-glycanases. The aim of such investigations is to obtain a deeper insight into those structural and functional modifications that enable these enzymes to function at low temperature and to examine the evolutionary relationship between mesophilic and psychrophilic β-glycanases. Received: December 21, 1998 / Accepted: February 3, 1999     

A physical study on the thermal ice ridge in a closed deep lake: Lake Kuttara, Hokkaido, Japan

An ice-ridge line more than 2,000 m long was discovered in a closed, deep lake—Lake Kuttara, Hokkaido, Japan—in the ice-covered season of 1995. To clarify when and how the ice-ridge line built up, meteorology and ice and water temperatures were monitored, and the density, thermal conductivity, and thickness of the covered ice were measured from January through March 1995. The covered ice on 4 and 5 March 1995, which corresponded to the period with the thickest lake ice, consisted of snow ice 0.05- to 0.07-m thick and candle ice 0.18- to 0.21-m thick. Numerical simulations for the ice thickness and temperature were carried out using a one-dimensional, unsteady, three-layer model. The simulated results, which were reasonable to the observations, indicate that due to the ice contraction and expansion, the ice-ridge line started to construct on 2 February and was completed the next day.