Psychrophilic Diatom Research Articles

Ice nucleating properties of the sea ice diatom Fragilariopsis cylindrus and its exudates

Abstract. In this study, we investigated the ice nucleation activity of the Antarctic sea ice diatom Fragilariopsis cylindrus. Diatoms are the main primary producers of organic carbon in the Southern Ocean, and the Antarctic sea ice diatom F. cylindrus is one of the predominant species. This psychrophilic diatom is abundant in open waters and within sea ice. It has developed several mechanisms to cope with the extreme conditions of its environment, for example, the production of ice-binding proteins (IBPs) and extracellular polymeric substances known to alter the structure of ice. Here, we investigated the ice nucleation activity of F. cylindrus using a microfluidic device containing individual sub-nanolitre (∼90 µm) droplet samples. The experimental method and a newly implemented Poisson-statistics-based data evaluation procedure applicable to samples with low ice nucleating particle concentrations were validated by comparative ice nucleation experiments with well-investigated bacterial samples from Pseudomonas syringae (Snomax®). The experiments reveal an increase of up to 7.2 ∘C in the ice nucleation temperatures for seawater containing F. cylindrus diatoms when compared to pure seawater. Moreover, F. cylindrus fragments also show ice nucleation activity, while experiments with the F. cylindrus ice-binding protein (fcIBP) show no significant ice nucleation activity. A comparison with experimental results from other diatoms suggests a universal behaviour of polar sea ice diatoms, and we provide a diatom-mass-based parameterization of their ice nucleation activity for use in models.

Alanine to serine substitutions drive thermal adaptation in a psychrophilic diatom cytochrome c6

In this study, we investigate the thermodynamic mechanisms by which electron transfer proteins adapt to environmental temperature by directly comparing the redox properties and folding stability of a psychrophilic cytochrome c and a mesophilic homolog. Our model system consists of two cytochrome c6 proteins from diatoms: one adapted specifically to polar environments, the other adapted generally to surface ocean environments. Direct electrochemistry shows that the midpoint potential for the mesophilic homolog is slightly higher at all temperatures measured. Cytochrome c6 from the psychrophilic diatom unfolds with a melting temperature 10.4°C lower than the homologous mesophilic cytochrome c6. Changes in free energy upon unfolding are identical, within error, for the psychrophilic and mesophilic protein; however, the chemical unfolding transition of the psychrophilic cytochrome c6 is more cooperative than for the mesophilic cytochrome c6. Substituting alanine residues found in the mesophile with serine found in corresponding positions of the psychrophile demonstrates that burial of the polar serine both decreases the thermal stability and decreases the midpoint potential. The mutagenesis data, combined with differences in the m-value of chemical denaturation, suggest that differences in solvent accessibility of the hydrophobic core underlie the adaptation of cytochrome c6 to differing environmental temperature.

A Mutagenesis Study to Investigate the Role of Alanine to Serine Mutations in the Adaptation of a Diatom Cytochrome c6 to Cold Temperatures

Proteins in the cytochrome c family share a covalently bond heme cofactor and clear spectroscopic features in the visible wavelengths, making them ideal model systems to investigate protein folding. We have previously identified two homologous cytochrome c6 proteins, one from the psychrophilic diatom Fragilariopsis cylindrus and another from the mesophilic diatom Thalassiosira pseudonana. We found that, while the two proteins only differ by 11 amino acid positions (89% identity), the F. cylindrus cytochrome c6 melts at a temperature 6.4°C lower than the T. pseudonana homolog. We predict that differences in the solvent accessibility of the hydrophobic cores of the two proteins will account for the differences in their stability over temperature. Using an amino acid sequence alignment, we identified three candidate amino acid positions that contain a non-polar alanine residue in the mesophilic protein, but the hydrophilic residue serine in the psychrophilic protein. Therefore, we predicted that, if serine were substituted for alanine in the context of the amino acid sequence of the mesophilic cytochrome c6, the melting temperature of the resulting variant should more closely match the melting temperature of the psychrophilic protein. In this study, we test our hypothesis by evaluating the melting temperatures of single-site alanine to serine amino acid variants of the T. pseudonana protein. We used site-directed mutagenesis to generate A86S, A93S, and A126S variants of T. pseudonana cytochrome c6, and recombinantly expressed and purified the proteins. Comparison of the melting temperatures of the variants with the melting temperatures of wild-type T. pseudonana and F. cylindrus proteins will provide evidence for the role of hydrophilic residues in destabilizing the hydrophobic core to allow for greater flexibility at lower temperatures.

Lipid content and fatty acid composition of Porosira glacialis and Attheya longicornis in response to carbon dioxide (CO2) aeration.

In the current study two novel psychrophilic diatoms Porosira glacialis and Attheya longicornis were tested for suitability to CO2 mitigation coupled with production of the physiologically requisite omega– 3 fatty acids. This study is in line with the worldwide conducted research aimed at applying biorefinery concept to heavy polluting industries. Since the production of algal high value compounds, i.e. essential fatty acids, relies on utilization of residual CO2 emissions coming from industry, the costs of such production maybe substantially reduced. Besides, the ecological benefits of the biorefinery concept being implemented are obvious, since CO2 is one of the major greenhouse gases. The current research has shown that one of the tested microalgal species, the diatom P. glacialis showed good tolerance to high (20–25%) levels of CO2 and maintained growth rates comparable to controls. The total lipid content in the CO2 aerated culture increased from 8.91 to 10.57% in cell dry mass. Additionally, the content of docosahexaenoic acid (DHA) increased from 3.90 to 5.75%, while the concentration of eicosapentaenoic acid (EPA) decreased from 26.59 to 23.66%. In contrast, A. longicornis did not demonstrate any significant increase in total lipid content. Besides, its growth was hampered by high levels of CO2 aeration.

Metabolic influence of psychrophilic diatoms on travertines at the Huanglong Natural Scenic District of China.

Diatoms are a highly diversified group of algae that are widely distributed in aquatic ecosystems, and various species have different nutrient and temperature requirements for optimal growth. Here, we describe unusual psychrophilic diatoms of Cymbella in a travertine deposition environment in southwestern China in winter season. Travertine surfaces are colonized by these psychrophilic diatoms, which form biofilms of extracellular polysaccharide substances (EPS) with active metabolic activities in extremely cold conditions. The travertine in Huanglong, is a typical single crystalline calcite with anisotropic lattice distortions of unit cell parameters along axes of a and c, and this structure is suggestive of some level of metabolic mediation on mineralization. Near-edge X-ray absorption fine structure spectroscopy (NEXAFS) results further confirmed the occurrence of biogenic distortion of the crystal lattice of travertine calcite. Overall, our results imply that the metabolic influence of psychrophilic diatoms may be particularly important for promoting formation and dissolution of travertine in extremely cold environments of Huanglong. The EPS of psychrophilic diatoms will protect travertine from HCO3− etching and provide template for forming travertine when water re-flowing, in warm season.

Gross and net production during the spring bloom along the Western Antarctic Peninsula.

This study explores some of the physiological mechanisms responsible for high productivity near the shelf in the Western Antarctic Peninsula despite a short growing season and cold temperature. We measured gross and net primary production at Palmer Station during the summer of 2012/2013 via three different techniques: incubation with H2 (18) O; incubation with (14) CO2 ; and in situ measurements of O2 /Ar and triple oxygen isotope. Additional laboratory experiments were performed with the psychrophilic diatom Fragilariopsis cylindrus. During the spring bloom, which accounted for more than half of the seasonal gross production at Palmer Station, the ratio of net-to-gross production reached a maximum greater than c. 60%, among the highest ever reported. The use of multiple techniques showed that these high ratios resulted from low heterotrophic respiration and very low daylight autotrophic respiration. Laboratory experiments revealed a similar ratio of net-to-gross O2 production in F. cylindrus and provided the first experimental evidence for an important level of cyclic electron flow (CEF) in this organism. The low ratio of community respiration to gross primary production observed during the bloom at Palmer Station may be characteristic of high latitude coastal ecosystems and partially supported by a very active CEF in psychrophilic phytoplankton.

Slow carboxylation of Rubisco constrains the rate of carbon fixation during Antarctic phytoplankton blooms.

High-latitude oceans are areas of high primary production despite temperatures that are often well below the thermal optima of enzymes, including the key Calvin Cycle enzyme, Ribulose 1,5 bisphosphate carboxylase oxygenase (Rubisco). We measured carbon fixation rates, protein content and Rubisco abundance and catalytic rates during an intense diatom bloom in the Western Antarctic Peninsula (WAP) and in laboratory cultures of a psychrophilic diatom (Fragilariopsis cylindrus). At -1°C, the Rubisco turnover rate, kcat (c) , was 0.4 C s(-1) per site and the half saturation constant for CO2 was 15 μM (vs c. 3 C s(-1) per site and 50 μM at 20°C). To achieve high carboxylation rates, psychrophilic diatoms increased Rubisco abundance to c. 8% of biomass (vs c. 0.6% at 20°C), along with their total protein content, resulting in a low carbon : nitrogen ratio of c. 5. In psychrophilic diatoms, Rubisco must be almost fully active and near CO2 saturation to achieve carbon fixation rates observed in the WAP. Correspondingly, total protein concentrations were close to the highest ever measured in phytoplankton and likely near the maximum possible. We hypothesize that this high protein concentration, like that of Rubisco, is necessitated by slow enzyme rates, and that carbon fixation rates in the WAP are near a theoretical maximum.

Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation.

Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis. Its main structural element is an irregular β-helix with six loops of 18 or more residues that lies alongside an α-helix. β-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the β-helical fold is the nonsequential arrangement of loops that places the N- and C termini inside the solenoid of β-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi.

Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53°C±0.53°C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34°C±0.25°C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.

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.

Dynamic response of the transcriptome of a psychrophilic diatom, Chaetoceros neogracile, to high irradiance

Large-scale RNA profiling revealed that high irradiance differentially regulated 577 out of 1,439 non-redundant genes of the Antarctic marine diatom Chaetoceros neogracile, represented on a custom cDNA chip, during 6 h of treatment. Among genes that were up- or down-regulated more than twofold within 30 min of treatment (310/1,439), about half displayed an acclimatory response during 6 h under high light. Expression of the remaining non-acclimatory genes also rapidly returned to initial levels within 30 min following a shift to low irradiance. High light altered expression of most of the photosynthesis genes (48/70), in contrast to genes in other functional categories. In addition, opposite response patterns were provoked in genes encoding fucoxanthin chlorophyll a/c binding protein (FCP), the main component of the diatom light-harvesting complex; high irradiance caused a decrease in expression of most FCP genes, but drove the rapid and specific up-regulation of ten others. C. neogracile responded very promptly to a change in light intensity by rapidly adjusting the transcript levels of FCP genes up-regulated by high light, and these dynamic adjustments coincided well with diatoxanthin (Dtx) levels formed by the xanthophyll cycle under the same conditions. The observation that the non-photochemical quenching (NPQ) capacity of this polar diatom was highly dependent on Dtx, which could bind to FCP and trigger NPQ, suggests that the up-regulated FCP gene products may participate in a photoprotective process as Dtx-binding proteins.

A new class of ice-binding proteins discovered in a salt-stress-induced cDNA library of the psychrophilic diatom Fragilariopsis cylindrus (Bacillariophyceae)

The psychrophilic pennate diatom Fragilariopsis cylindrus is a dominant polar diatom adapted to grow at extremely low temperatures and high salinities prevailing in the brine channels of sea ice. As a basis for deeper investigations of this physiological trait, we used an EST approach to find candidate genes involved in acclimation to salt stress in this diatom. From 2880 cDNA clones sequenced from the 5′ end, 1691 high-quality tentative unique sequences were assembled and analysed. Only 62% of these sequences have homologues in the genomes of two mesophilic diatoms, Thalassiosira pseudonana (centric) and Phaeodactylum tricornutum (pennate), which share 73–78% of their genes. Of the 1691 sequences, 44.2% could be functionally characterized by comparison with the Swiss-Prot and RefSeq databases. These include sequences encoding different ionic transporters and antiporters, reflecting the requirement to re-establish the ion homeostasis disturbed by exogenous salt stress. Furthermore, numerous genes encoding heat shock proteins (hsps), genes related to oxidative stress, and three key genes involved in the proline synthesis pathway, the most important organic osmolyte synthesized in F. cylindrus were identified. A major outcome of this analysis is the finding of four full-length ORFs showing significant similarities to ice-binding proteins (IBPs), which have been shown in a parallel study to be specific to sea ice diatoms, giving evidence of their ability to shape their habitat.

Long-Term Temperature Acclimation of Photosynthesis in Steady-State Cultures of the Polar Diatom Fragilariopsis cylindrus

Cultures of the obligate psychrophilic diatom Fragilariopsis cylindrus (Grunow) were grown for 4 months under steady-state conditions at -1 degrees C and +7 degrees C (50 micromol photons m(-2) s(-1)) prior to measurements in order to investigate long-term acclimation of photosynthesis to both temperatures. No differences in maximum intrinsic quantum yield of PS II (F(V)/F(M)) and relative electron transport rates could be detected at either temperature after 4 months of acclimation. Measurements of photosynthesis (relative electron transport rates) vs. irradiance (P vs. E curves) revealed similar values for relative light utilization efficiency (alpha = 0.57 at -1 degrees C, alpha = 0.60 at +7 degrees C) but higher values for irradiance levels at which photosynthesis saturates (E(K)) at -1 degrees C and, therefore, higher maximum photosynthesis (P(MAX) = 54 (relative units) at -1 degrees C, P(MAX) = 49 at +7 degrees C). Nonphotochemical quenching (NPQ) measurements at 385 mumol photons m(-2) s(-1) indicated higher (37%) NPQ for diatoms grown at -1 degrees C compared to +7 degrees C, which was possibly related to a 2-fold increase in the concentration of the pigment diatoxanthin and a 9-fold up-regulation of a gene encoding a fucoxanthin chlorophyll a,c-binding protein. Expression of the D1 protein encoding gene psbA was ca. 1.5-fold up-regulated at -1 degrees C, whereas expression levels of other genes from Photosystem II (psbC, psbU, psbO), as well as rbcL, the gene encoding the Rubisco large subunit were similar at both temperatures. However, a 2-fold up-regulation of a plastid glyceraldehyde-P dehydrogenase at -1 degrees C indicated enhanced Calvin cycle activity. This study revealed for the first time that a polar diatom could efficiently acclimate photosynthesis over a wide range of polar temperatures given enough time. Acclimation of photosynthesis at -1 degrees C was probably regulated similarly to high light acclimation.

DIMETHYLSULFONIOPROPIONATE PRODUCTION BY PSYCHROPHILIC DIATOM ISOLATES1

Dimethylsulfoniopropionate (DMSP) production by psychrophilic diatom strains, Chaetoceros sp., Navicula sp., and Nitzschia sp., were experimentally analyzed. All strains showed rapid growth (0.3–0.5 d−1) under cold culture conditions (1.7°C). DMSP concentrations (both as particulate, DMSPp, and dissolved, DMSPd) were negligible in cultures of Chaetoceros sp., whereas those for Navicula sp. and Nitzschia sp. increased toward the end of the 56‐day experiments. The ratio of DMSPp:chl a of the latter two species was approximately 5 in the early exponential growth phase and decreased slightly toward the early stationary phase. During the stationary phase, when chl a and fluorescence remained constant, the ratios in both species increased linearly by up to approximately 6.5 times the value at the start of the stationary phase. This growth‐related DMSP production by diatoms may result in the low concentrations of DMSPp observed in the early stage of diatom blooms under natural conditions.

PHOTOSYNTHESIS AND COLD ACCLIMATION: MOLECULAR EVIDENCE FROM A POLAR DIATOM1

The psychrophilic diatom Fragilariopsis cylindrus (Grunow) Krieger in Helmcke & Krieger was used to investigate photosynthesis and growth under freezing temperatures. Gene expression during a temperature shift from +5° C to −1.8° C was studied under 3 and 35 μmol photons·m−2·s−1 by using a macroarray. These measurements were paralleled by determination of fluorescence induction at PSII and pigment analysis. The shift to −1.8° C at 35 μmol photons·m−2·s−1 caused a marginal decrease of photosynthetic quantum yield (Fv/Fm) from 0.61 to 0.52 with fast recovery after 1 day. The ratio of chl c to chl a increased from 3.1 to 5.5, and the ratio of diatoxanthin to diadinoxanthin increased from 0.7 to 5.0. Genes encoding proteins of PSII (psbA, psbC) and for carbon fixation (rbcL) were down‐regulated, whereas genes encoding chaperons (hsp70) and genes for plastid protein synthesis and turnover (elongation factor EfTs, ribosomal protein rpS4, ftsH protease) were up‐regulated. In contrast, cold exposure at 3 μmol photons·m−2·s−1 induced a marginal increase in Fv/Fm from 0.61 to 0.63 and a strong increase in fucoxanthin concentrations from 0.04 up to 0.12 pg·cell−1. This was paralleled by up‐regulation of fcp genes. The ratio of chl c to chl a also increased from 3.1 to 4.2, as did the ratio of diatoxanthin to diadinoxanthin from 0.7 to 2.2. Down‐regulation of psbA, psbC, and rbcL could also be measured but not up‐regulation of hsp70, EfTs, rpS4, and the ftsH protease. The latter genes are probably necessary to avoid cold shock photoinhibition only at higher light intensities.

The interactive effects of temperature and silicon limitation on the psychrophilic ice diatom Pseudonitszchia seriata

The interactive effects of temperature and silicon limitation on the psychrophilic ice diatom Pseudonitszchia seriata

Photosynthetic and Respiratory Rates of Two Psychrophilic Diatoms

The photosynthetic rates in two psychrophilic diatoms, Chaetoceros sp. strain K3-10 and Nitzschia sp. K3-3 for cells grown at 0 degrees C were 8 to 10 microliters O(2) evolved per milligram dry weight per hour, and 10-fold higher, about 80 for cells grown at 10 degrees C. The respiration rates followed the same pattern, with a value of around 1 microliter dark uptake per milligram dry weight per hour for both organisms grown at 0 degrees C, and 6 to 10 for cells grown at 10 degrees C. When cells grown at 0 degrees C were immediately shifted to 10 degrees C or cells grown at 10 degrees C were shifted to 0 degrees C, the respiratory rates quickly adapted to values characteristic of cells grown at the shift temperature. On the other hand, the light-saturated rate of O(2) evolution showed much less immediate adaptation, especially on the up shift, 0 degrees to 10 degrees C. The chlorophyll a content of 0 degrees C grown cells was about 0.5% of dry weight, in 10 degrees C grown cells 1.3% (strain K3-10) and 2.2% (strain K3-3). In addition to a diminished chlorophyll a content in 0 degrees C grown cells, there seemed proportionally (by absorbance and calculation) less c to a than in 10 degrees C grown cells. The relative fluorescence excitation spectra of 680-nm emission also showed a lower contribution by both chlorophyll c and fucoxanthin in 0 degrees C grown cells of Chaetoceros sp. strain K3-10 as compared to 10 degrees C grown cells. The data at hand suggest that in psychrophilic diatoms continuously growing at 0 degrees C there may be problems associated with synthesis of an effective accessory pigment system, and as a working hypothesis it is suggested this is related to restriction of synthesis of one or several accessory pigment proteins.

Sensitivity of two psychrophilic diatoms to crude oil and fuel oil

Two pure cultures of diatoms, a Nitzschia sp. and a Chaetoceros sp., were grown at 0°C or 10°C in the presence of two crude oils and two fuel oils. The petroleum samples were absorbed onto filter paper discs and added directly to the algal cultures. In all cases the organisms were much more sensitive to the crude oils or fuel oils when growing at 0°C than at 10°C. The rate of photosynthesis measured by an oxygen electrode for Nitzschia sp. grown and tested at 0°C was not affected during 3 hours' incubation with some four times the amount of a crude oil or fuel oil that blocked growth. The experiments lead to the cautious suggestion that psychrophilic algae will prove some fivefold more sensitive than mesophilic algae to petroleum pollution.

Isolation and Growth of Psychrophilic Diatoms from the Ice-edge in the Bering Sea

Five pure cultures of diatoms, Thalassiosira sp., Navicula sp., Nitzschia sp., and two closely related Chaetoceros spp., were isolated from enrichment cultures made from ice and water samples from the ice-edge in the Bering Sea. Four cultures were studied in detail. The isolates did not grow above 18 °C; optimum growth was from 10 to 14 °C. The Nitzschia sp. and Chaetoceros spp. grew reproducibly at 0 ± 0·2 °C, albeit with long generation times of 6 to 7 d. Generation times at 10 °C were 0·8 to 1·9 d. The long generation times at 0 °C appeared intrinsic; growth rates were not increased by addition of ammonia, complex organic hydrolysates or light and dark cycles. The elemental analysis and culture density of the algae were reasonable. For example, the elemental analysis of Nitzschia sp. grown at 0 °C was C, 34·51%; H, 5·00%; N, 5·12%; ash, 30·3%, very similar to the values for cells grown at 10 °C. Cell yields of 0·5 mg dry weight ml−1 were routinely achieved. These appear to be among the first pure cultures of psychrophilic diatoms and possibly of microalgae in general.