New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, <i>Chloromonas</i> sp.

<div><p>Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, <i>Chloromonas</i> sp., was identified and characterized. The length of the <i>Chloromonas</i> sp. IBP (<i>ChloroIBP</i>) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the <i>ChloroIBP</i> cDNA was 34.0 kDa. Expression of the <i>ChloroIBP</i> gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of <i>ChloroIBP</i> was cloned and over-expressed in <i>Escherichia coli</i> to investigate the IBP’s biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. <i>In silico</i> structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of <i>ChloroIBP</i> showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP.</p></div

Phylogenetic tree of SSU (18S rDNA) sequences among microorganisms genetically-close to <i>Chloromonas</i> sp.

The sequences used for analysis were acquired from the NCBI database and aligned by the ClustalW algorithm [24]. The tree was generated by the maximum likelihood (ML) method. The probabilities from maximum parsimony and distance methods (left and right values, respectively) were obtained by bootstrap analysis of 5000 repetitions.</p

Pictures of <i>Chloromonas</i> sp., and single ice crystal shapes from the extracellular fraction of <i>Chloromonas</i> sp.

(A) light micrograph showing chloroplast and flagella; (b) electron micrographs showing ultrastructural features by longitudinal and cross-section views (Cp, Chloroplast; F, Flagella; G, Golgi complex; L, Lumen; Mt, Mitochondria; N, Nucleus; S, Starch (C and D) ice crystal shapes formed by ice-binding proteins secreted from Chloromonas sp showing hexagonal morphology.</p

In-gel mobility of ChloroIBP.

<p>(A) Extracellular recombinant Trx-ChloroIBP visualized by immunoblotting following SDS-PAGE analysis in a redox experiment. 1, Trx-ChloroIBP treated with β-mercaptoethanol; 2, Trx-ChloroIBP oxidized by ambient air; 3, Trx-ChloroIBP alkylated by iodoacetamide after treatment with β-mercaptoethanol. (B) Topological change of native ChloroIBP. Treatment with reagents was the same as for (A). (C) Location of native ChloroIBP secreted into culture media on a native polyacrylamide gel. (M, protein markers representing bovine serum albumin based on the protein structure of P.69 pertactin (66) and L-lactic dehydrogenase (140); 1, silver staining of extracellular proteins secreted from <i>Chloromonas</i> sp.; 2, Periodic-acid staining of extracellular proteins from <i>Chloromonas</i> sp.; 3, Immunoblot band detected by anti-ChloroIBP antibody to a blot of Lane 1. (D) Topological movement of native ChloroIBP separated on a native polyacrylamide gel after reduction (Re) or under oxidizing conditions (Oxi).</p

Genomic DNA structure of the ChloroIBP gene and prediction of secondary structure of ChloroIBP.

<p>(A) The genomic structure consisted of 11 introns (I) and 12 exons (E) presented by white and gray boxes, respectively. The signal peptide is coloured orange. Lengths of the introns and exons are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154056#pone.0154056.s010" target="_blank">S2 Table</a>. (B) Deduced amino acid sequence of ChloroIBP with the signal peptide sequence double-underlined. A box with black colour shows the peptide sequences for detection of ChloroIBP in western blot analysis. Potential T-X-T ice-binding motifs are displayed in a bold, red font. Cysteine residues are shown in bold letters. The red box and blue lines indicate α-helix and coils, respectively. Orange arrows indicate β-strands.</p

Northern blot analysis of thermal and freezing stresses.

<p>(A and C) Electrophoretic data for control RNAs. (B) Autoradiogram of transcriptional change in ChloroIBP mRNA levels with thermal conditions. N, normal cells; 0.5, 30-min incubation at 25°C; 1, 1 h-incubation at 25°C; 2, 2-h incubation at 25°C. (D) Autoradiogram of transcriptional change in ChloroIBP mRNA levels with freezing condition. N, normal cells; 1/4, 25% of medium occupied by ice slush; 1/2, 50% of medium occupied by ice slush; C.F., 100% of medium occupied by ice slush.</p

Localization and levels of ChloroIBP production according to freezing condition.

<p>(A) Top—SDS-PAGE analysis of extracellular proteins from frozen cultures (1); culture where one quarter of the volume is ice slush (2); and culture grown under normal conditions (3). Bottom—immunoblot of the gel transfer where ChoroIBP migrates. (B) SDS-PAGE and immunoblot analysis. Top—Coomassie blue staining of samples prepared at the cold-acclimated (0°C, shaking incubation for 3 days, lanes 1–4) and normal (4°C, lanes 5–8) conditions. M, Marker proteins; 1 and 5, Total crude extracts; 2 and 6, Intracellular soluble proteins; 3 and 7, Cell debris samples; 4 and 8, Extracellular proteins. Bottom—immunoblot of the gel transfer where ChoroIBP migrates.</p

Plot of thermal hysteresis activity as a function of protein concentration of wild-type and mutant Trx-ChloroIBP.

<p>Colours and symbols indicate the different ChloroIBP mutants. Values shown are averaged S.D. of three replicates of the measurement of TH. Insets show single ice crystal morphology obtained with each Trx-ChloroIBP at the highest protein concentration assayed. Scale bars indicate 100 μm.</p

Southern blotting of Chloromonas genomic DNA to analyze the gene family encoding ChloroIBP.

<p>Left: Ethidium bromide-stained agarose gel showing: M, 1 kb DNA ladder; B, <i>Bam</i>HI-digested <i>Chloromonas</i> genomic DNA; K, <i>Kpn</i>I-digested <i>Chloromonas</i> genomic DNA; Xb, <i>Xba</i>I-digested <i>Chloromonas</i> genomic DNA; U, Undigested nuclear DNA as a control. Right: Autoradiogram of the probed blot from the gel on the left.</p

Thermal hysteresis activity as a function of Trx-ChloroIBP concentration.

<p>Insets show the morphological changes of single ice crystals at different recombinant ChloroIBP concentrations. BSA solution (5 mg/ml) was used as a control. Scale bars indicate 100 μm.</p