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.     

Psychrophily is associated with differential energy partitioning, photosystem stoichiometry and polypeptide phosphorylation in Chlamydomonas raudensis

Chlamydomonas raudensis UWO 241 and SAG 49.72 represent the psychrophilic and mesophilic strains of this green algal species. This novel discovery was exploited to assess the role of psychrophily in photoacclimation to growth temperature and growth irradiance. At their optimal growth temperatures of 8 °C and 28 °C respectively, UWO 241 and SAG 49.72 maintained comparable photostasis, that is energy balance, as measured by PSII excitation pressure. Although UWO 241 exhibited higher excitation pressure, measured as 1-qL, at all growth light intensities, the relative changes in 1-qL were similar to that of SAG 49.72 in response to growth light. In response to suboptimal temperatures and increased growth irradiance, SAG 49.72 favoured energy partitioning of excess excitation energy through inducible, down regulatory processes (Φ_NPQ) associated with the xanthophyll cycle and antenna quenching, while UWO 241 favoured xanthophyll cycle-independent energy partitioning through constitutive processes involved in energy dissipation (Φ_NO). In contrast to SAG 49.72, an elevation in growth temperature induced an increase in PSI/PSII stoichiometry in UWO 241. Furthermore, SAG 49.72 showed typical threonine-phosphorylation of LHCII, whereas UWO 241 exhibited phosphorylation of polypeptides of comparable molecular mass to PSI reaction centres but the absence of LHCII phosphorylation. Thus, although both strains maintain an energy balance irrespective of their differences in optimal growth temperatures, the mechanisms used to maintain photostasis were distinct. We conclude that psychrophily in C. raudensis is complex and appears to involve differential energy partitioning, photosystem stoichiometry and polypeptide phosphorylation.     

Resolving the phylogenetic relationship between Chlamydomonas sp. UWO 241 and Chlamydomonas raudensis sag 49.72 (Chlorophyceae) with nuclear and plastid DNA sequences

The Antarctic psychrophilic green alga Chlamy‐domonas sp. UWO 241 is an emerging model for studying microbial adaptation to polar environments. However, little is known about its evolutionary history and its phylogenetic relationship with other chlamydomonadalean algae is equivocal. Here, we attempt to clarify the phylogenetic position of UWO 241, specifically with respect to Chlamydomonas rau‐densis SAG 49.72. Contrary to a previous report, we show that UWO 241 is a distinct species from SAG 49.72. Our phylogenetic analyses of nuclear and plastid DNA sequences reveal that UWO 241 represents a unique lineage within the Moewusinia clade (sensu Nakada) of the Chlamydomonadales (Chlorophyceae, Chlorophyta), closely affiliated to the marine species Chlamydomonas parkeae SAG 24.89.     

IDENTIFICATION OF A PSYCHROPHILIC GREEN ALGA FROM LAKE BONNEY ANTARCTICA: CHLAMYDOMONAS RAUDENSIS ETTL. (UWO 241) CHLOROPHYCEAE1

An unusual psychrophilic green alga was isolated from the deepest portion of the photic zone (<0.1% of incident PAR) at a depth of 17 m in the permanently ice‐covered lake, Lake Bonney, Antarctica. Here we identify and report the first detailed morphological and molecular examination of this Antarctic green alga, which we refer to as strain UWO 241. To determine the taxonomic identity, UWO 241 was examined using LM and TEM and partial sequences of the small subunit (SSU), internal transcribed spacer (ITS) 1 and ITS2 regions (including the 5.8S) of the ribosomal operon. These data were compared with those of previously described taxa. We identified UWO 241 as a strain of Chlamydomonas raudensis Ettl (SAG 49.72). Chlamydomonas raudensis is closely related to C. noctigama Korshikov (UTEX 2289) as well as foraminifer symbionts such as C. hedleyi Lee, Crockett, Hagen et Stone (ATCC 50216). In addition, its morphology, pigment complement, and phototactic response to temperature are reported. Chlamydomonas raudensis (UWO 241) contains relatively high levels of lutein and low chl a/b ratios (1.6±0.15), and the phototactic response was temperature dependent. The Antarctic isolate (UWO 241) included the typical photosynthetic pigments found in all chl a/b containing green algae. It possesses a small eyespot and, interestingly, was positively phototactic only at higher nonpermissive growth temperatures. Comparison of SSU and ITS rDNA sequences confirms the identification of the strain UWO 241 as C. raudensis Ettl and contradicts the previous designation as C. subcaudata Wille.     

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.     

Psychrophily is associated with differential energy partitioning, photosystem stoichiometry and polypeptide phosphorylation in Chlamydomonas raudensis

Chlamydomonas raudensis UWO 241 and SAG 49.72 represent the psychrophilic and mesophilic strains of this green algal species. This novel discovery was exploited to assess the role of psychrophily in photoacclimation to growth temperature and growth irradiance. At their optimal growth temperatures of 8 degrees C and 28 degrees C respectively, UWO 241 and SAG 49.72 maintained comparable photostasis, that is energy balance, as measured by PSII excitation pressure. Although UWO 241 exhibited higher excitation pressure, measured as 1-qL, at all growth light intensities, the relative changes in 1-qL were similar to that of SAG 49.72 in response to growth light. In response to suboptimal temperatures and increased growth irradiance, SAG 49.72 favoured energy partitioning of excess excitation energy through inducible, down regulatory processes (Phi(NPQ)) associated with the xanthophyll cycle and antenna quenching, while UWO 241 favoured xanthophyll cycle-independent energy partitioning through constitutive processes involved in energy dissipation (Phi(NO)). In contrast to SAG 49.72, an elevation in growth temperature induced an increase in PSI/PSII stoichiometry in UWO 241. Furthermore, SAG 49.72 showed typical threonine-phosphorylation of LHCII, whereas UWO 241 exhibited phosphorylation of polypeptides of comparable molecular mass to PSI reaction centres but the absence of LHCII phosphorylation. Thus, although both strains maintain an energy balance irrespective of their differences in optimal growth temperatures, the mechanisms used to maintain photostasis were distinct. We conclude that psychrophily in C. raudensis is complex and appears to involve differential energy partitioning, photosystem stoichiometry and polypeptide phosphorylation.     

The Antarctic psychrophiles Chlamydomonas spp. UWO241 and ICE-MDV exhibit differential restructuring of photosystem I in response to iron

Chlamydomonas sp. UWO241 is a psychrophilic alga isolated from the deep photic zone of a perennially ice-covered Antarctic lake (east lobe Lake Bonney, ELB). Past studies have shown that C. sp. UWO241 exhibits constitutive downregulation of photosystem I (PSI) and high rates of PSI-associated cyclic electron flow (CEF). Iron levels in ELB are in the nanomolar range leading us to hypothesize that the unusual PSI phenotype of C. sp. UWO241 could be a response to chronic Fe-deficiency. We studied the impact of Fe availability in C. sp. UWO241, a mesophile, C. reinhardtii SAG11-32c, as well as a psychrophile isolated from the shallow photic zone of ELB, Chlamydomonas sp. ICE-MDV. Under Fe-deficiency, PsaA abundance and levels of photooxidizable P700 (ΔA₈₂₀/A₈₂₀) were reduced in both psychrophiles relative to the mesophile. Upon increasing Fe, C. sp. ICE-MDV and C. reinhardtii exhibited restoration of PSI function, while C. sp. UWO241 exhibited only moderate changes in PSI activity and lacked almost all LHCI proteins. Relative to Fe-excess conditions (200 µM Fe²⁺), C. sp. UWO241 grown in 18 µM Fe²⁺ exhibited downregulation of light harvesting and photosystem core proteins, as well as upregulation of a bestrophin-like anion channel protein and two CEF-associated proteins (NdsS, PGL1). Key enzymes of starch synthesis and shikimate biosynthesis were also upregulated. We conclude that in response to variable Fe availability, the psychrophile C. sp. UWO241 exhibits physiological plasticity which includes restructuring of the photochemical apparatus, increased PSI-associated CEF, and shifts in downstream carbon metabolism toward storage carbon and secondary stress metabolites.

     

Microbial Degradation of Organic Pollutants in Soil in a Cold Climate

Cold-adapted microorganisms are potentially interesting for use in environmental biotechnology applications since a large part of the biosphere has low temperatures during at least parts of the year. Many studies have shown that both oil-contaminated and uncontaminated soils in the Arctic, the Antarctic and the Alps contain microbes that can degrade different hydrocarbons deriving from oils. A few studies have also been conducted on degradation of herbicides in soils at low temperatures. Furthermore, phenols and some polychlorinated biphenyl (PCB) congeners have proved to be degradable at low temperatures, using microorganisms isolated from sediments or soils. Additions of nitrogen and phosphorous to polluted soils have been shown to enhance the degradation of hydrocarbons in many cases. Bioaugmentation with hydrocarbon degrading cold-adapted microorganisms has given varying results. The inoculated microorganisms have probably been out-competed by the indigenous microorganisms in some cases. Different ways to increase the efficiency of microbial degradation of organic pollutants in soil in a cold climate is discussed.     

Remarkably low temperature optima for extracellular enzyme activity from Arctic bacteria and sea ice

Extracellular degradative enzymes released by psychrophilic marine bacteria (growing optimally at or below 15°C and maximally at 20°C) typically express activity optima at temperatures well above the upper growth limit of the producing strain. In the present study, we investigated whether or not near-zero Arctic environments contain extracellular enzymes with activity optimized to temperatures lower than previously reported. By applying fluorescently tagged substrate analogues to measure leucine-aminopeptidase and chitobiase activity, the occurrence of extracellular enzymatic activity (EEA) with remarkably low temperature optima (15°C) was documented in sea-ice samples. An extremely psychrophilic bacterial isolate, strain 34H, yielded an extract of cell-free protease with activity optimized at 20°C, the lowest optimum yet reported for cell-free EEA from a pure culture. The use of zymogram gels revealed the presence of three proteolytic bands (between 37 and 45 kDa) in the extract and the release of the greatest quantities of the proteases when the strain was grown at −1°C, suggesting a bacterial strategy for counteracting the effects of very cold temperatures on the catalytic efficiency of released enzymes. The detection of unusually cold-adapted EEA in environmental samples has ramifications not only to polar ecosystems and carbon cycling but also to protein evolution, biotechnology and bioremediation.     

Bacterial Adaptation to Low Temperature: Implications for Cold-Inducible Genes

Escherichia coli and later found to be a cold-shock response common to many bacterial species. CspA of 7.4 kD, a major cold-shock protein in E. coli, has been shown to share structural similarity with a class of eukaryotic Y box proteins which have RNA-binding domains. Transient synthesis of CspA upon cold shock is mediated by increased stabilization of the mRNA at low temperatures. The proposed role of some cold-shock proteins including CspA in the bacterial adaptation to low temperatures is to function as a RNA chaperone in the regulation of translation. Some enzymes of psychrotrophic or psychrophilic bacteria exhibit unique features of a cold-adapted enzyme, high catalytic activity at a low temperature and rapid inactivation at a moderate temperature. A monomeric isocitrate dehydrogenase isozyme (IDH-II) of a psychrophilic bacterium, Vibrio sp. strain ABE-1, is a typical cold-adapted enzyme. In addition, this enzyme is induced at low temperatures. Low temperature-dependent expression of icdll encoding IDH-II is controlled by two different cis-elements located at the untranslated upstream region of the gene, one is a silencer and the other is essential for the low temperature response. The physiological role of IDH-II is evaluated by transforming E. coli with icdll. The growth rate of the E. coli transformants at low temperatures is dependent on the level of expressed IDH-II activity. Received 11 January 1999/ Accepted in revised form 6 April 1999     

Photosynthetic Carbon Incorporation and Turnover in Antarctic Cryptoendolithic Microbial Communities: Are They the Slowest-Growing Communities on Earth?

The main forms of terrestrial life in the cold, desolate Ross Desert of Antarctica are lichen-dominated or cyanobacterium-dominated cryptoendolithic (hidden in rock) microbial communities. Though microbial community biomass (as measured by extractable lipid phosphate) was well within the range of values determined for other microbial communities, community lipid carbon turnover times (calculated from community lipid biomass, rates of community photosynthetic carbon incorporation into lipids versus temperature, and the in situ temperature record) were among the longest on Earth (ca. 20,000 years). When the temperature is above freezing and moisture is present, moderate rates of photosynthesis can be measured. Lichen communities had a psychrophilic temperature response (maximal rate of 4.5 ng of C h^-1 m^-2 at 10°C) while cyanobacteria communities had maximal rates at 20 to 30°C (3 ng of C h^-1 m^-2). These extraordinarily slowly growing communities were not nutrient limited. No significant changes in photosynthetic metabolism were observed upon additions of 100 nM to 1 mM nitrate, ammonium, phosphate, and manganese. These simple, tenacious microbial communities demonstrate strategies of survival under conditions normally considered too extreme for life.     

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.

     

Molecular basis of cold adaptation

Cold-adapted, or psychrophilic, organisms are able to thrive at low temperatures in permanently cold environments, which in fact characterize the greatest proportion of our planet. Psychrophiles include both prokaryotic and eukaryotic organisms and thus represent a significant proportion of the living world. These organisms produce cold-evolved enzymes that are partially able to cope with the reduction in chemical reaction rates induced by low temperatures. As a rule, cold-active enzymes display a high catalytic efficiency, associated however, with a low thermal stability. In most cases, the adaptation to cold is achieved through a reduction in the activation energy that possibly originates from an increased flexibility of either a selected area or of the overall protein structure. This enhanced plasticity seems in turn to be induced by the weak thermal stability of psychrophilic enzymes. The adaptation strategies are beginning to be understood thanks to recent advances in the elucidation of the molecular characteristics of cold-adapted enzymes derived from X-ray crystallography, protein engineering and biophysical methods. Psychrophilic organisms and their enzymes have, in recent years, increasingly attracted the attention of the scientific community due to their peculiar properties that render them particularly useful in investigating the possible relationship existing between stability, flexibility and specific activity and as valuable tools for biotechnological purposes.     

Temperature adaptation of DNA ligases from psychrophilic organisms

DNA ligases operating at low temperatures have potential advantages for use in biotechnological applications. For this reason, we have characterized the temperature optima and thermal stabilities of three minimal Lig E-type ATP-dependent DNA ligase originating from Gram-negative obligate psychrophilic bacteria. The three ligases, denoted Vib-Lig, Psy-Lig, and Par-Lig, show a remarkable range of thermal stabilities and optima, with the first bearing all the hallmarks of a genuinely cold-adapted enzyme, while the latter two have activity and stability profiles more typical of mesophilic proteins. A comparative approach based on sequence comparison and homology modeling indicates that the cold-adapted features of Vib-Lig may be ascribed to differences in surface charge rather than increased local or global flexibility which is consistent with the contemporary emerging paradigm of the physical basis of cold adaptation of enzymes.

     

Low temperature-induced systems failure in Escherichia coli: insights from rescue by cold-adapted chaperones

The growth of Escherichia coli cells is impaired at temperatures below 21 degrees C and stops at 7.5 degrees C; however, growth of a transgenic strain producing the cold-adapted chaperones Cpn60 and Cpn10 from the psychrophilic bacterium Oleispira antarctica is good at low temperatures. The E. coli cpn(+) transgene offers a novel opportunity for examining the essential protein for cell viability at low temperatures. By screening a large-scale protein map (proteome) of cells of K-12 and its Cpn(+) transgene incubated at 4 degrees C, we identified 22 housekeeping proteins involved in systems failure of E. coli when confronted with low temperature. Through co-immunoprecipitation of Cpn60, Northern blot, and in vitro refolding, we systematically identified that protein-chaperone interactions are key determinants of their protein functions at low temperatures. Furthermore, chromosomal gene deletion experiments suggest that the mechanism of cold-induced systems failure in E. coli is cold-induced inactivation of the GroELS chaperonins and the resulting failure to refold cold-inactivated Dps, ClpB, DnaK and RpsB proteins. These findings: (1) indicate the potential importance of chaperones in cold sensitivity, cold adaptation and cold tolerance in cellular systems, and (2) suggest the identity of a few key cold-sensitive chaperone-interacting proteins that get inactivated and ultimately cause systems failure in E. coli cells at low temperatures.     

低温微生物的冷适应机理及其应用

低温微生物广泛分布于极地、冰川、永久冻土和深海等寒冷环境,其冷适应能力是多种机理共同作用的结果,包括酶的低温催化活性、低温下膜流动性的保持、冷休克蛋白、抗冻蛋白以及抗冻保护剂等.低温微生物主要应用于催化低温发酵、表达热不稳定蛋白质、生产抗冻保护剂和冬季治理污水等领域.     

Chlamydomonas raudensis (UWO 241), Chlorophyceae,exhibits the capacity for rapid D1 repair in response to chronic photoinhibition at low temperature1

Maximum photosynthetic capacity indicates that the Antarctic psychrophile Chlamydomonas raudensis H. Ettl UWO 241 is photosynthetically adapted to low temperature. Despite this finding, C. raudensis UWO 241 exhibited greater sensitivity to low‐temperature photoinhibition of PSII than the mesophile Chlamydomonas reinhardtii P. A. Dang. However, in contrast with results for C. reinhardtii, the quantum requirement to induce 50% photoinhibition of PSII in C. raudensis UWO 241 (50 μmol photons) was comparable at either 8°C or 29°C. To our knowledge, this is the first report of a photoautotroph whose susceptibility to photoinhibition is temperature independent. In contrast, the capacity of the psychrophile to recover from photoinhibition of PSII was sensitive to temperature and inhibited at 29°C. The maximum rate of recovery from photoinhibition of the psychrophile at 8°C was comparable to the maximum rate of recovery of the mesophile at 29°C. We provide evidence that photoinhibition in C. raudensis UWO 241 is chronic rather than dynamic. The photoinhibition‐induced decrease in the D1 content in C. raudensis recovered within 30 min at 8°C. Both the recovery of the D1 content as well as the initial fast phase of the recovery of F_v/F_m at 8°C were inhibited by lincomycin, a chloroplast protein synthesis inhibitor. We conclude that the susceptibility of C. raudensis UWO 241 to low‐temperature photoinhibition reflects its adaptation to low growth irradiance, whereas the unusually rapid rate of recovery at low temperature exhibited by this psychrophile is due to a novel D1 repair cycle that is adapted to and is maximally operative at low temperature.     

Evidence for phenotypic plasticity in the Antarctic extremophile Chlamydomonas raudensis Ettl. UWO 241

Life in extreme environments poses unique challenges to photosynthetic organisms. The ability for an extremophilic green alga and its genetic and mesophilic equivalent to acclimate to changes in their environment was examined to determine the extent of their phenotypic plasticities. The Antarctic extremophile Chlamydomonas raudensis Ettl. UWO 241 (UWO) was isolated from an ice-covered lake in Antarctica, whereas its mesophilic counterpart C. raudensis Ettl. SAG 49.72 (SAG) was isolated from a meadow pool in the Czech Republic. The effects of changes in temperature and salinity on growth, morphology, and photochemistry were examined in the two strains. Differential acclimative responses were observed in UWO which include a wider salinity range for growth, and broader temperature- and salt-induced fluctuations in F(v)/F(m), relative to SAG. Furthermore, the redox state of the photosynthetic electron transport chain, measured as 1-q(P), was modulated in the extremophile whereas this was not observed in the mesophile. Interestingly, it is shown for the first time that SAG is similar to UWO in that it is unable to undergo state transitions. The different natural histories of these two strains exert different evolutionary pressures and, consequently, different abilities for acclimation, an important component of phenotypic plasticity. In contrast to SAG, UWO relied on a redox sensing and signalling system under the growth conditions used in this study. It is proposed that growth and adaptation of UWO under a stressful and extreme environment poises this extremophile for better success under changing environmental conditions.