Shoji Hatano

Cryoprotective activities of group 3 late embryogenesis abundant proteins from Chlorella vulgaris C-27.

The nucleotide sequence of hiC12, isolated as a cDNA clone of hardening-induced Chlorella (hiC) genes, was identified. The clone encodes a late embryogenesis abundant (LEA) protein having six repeats of a 11-mer amino acid motif, although in a slightly imperfect form. To overexpress the hiC6 1) and hiC12 genes, their coding regions were PCR amplified and subcloned into a pGEX-1λT vector. The HIC6 and HIC12 proteins were expressed as GST fusion proteins in E. coli, then purified. The two HIC proteins were found to be effective in protecting a freeze-labile enzyme, LDH, against freeze-inactivation. On a molar concentration basis, they were about 3.1×106 times more effective in protecting LDH than sucrose and as effective as BSA. Cryoprotection tests with five kinds of chain-shortened polypeptides, synthesized based on the 11-mer amino acid motif of the HIC6 protein showed that the cryoprotective activity decreased with a decrease in the repeating units of the 11-mer motif. In fact, cryoprotective activities of three kinds of single 11-mer amino acids were very low even at high concentrations. All the results suggested that the sufficiently repeated 11-mer motif is required for the cryoprotective activities of Chlorella LEA proteins.

Introduction of the hiC6 Gene, which Encodes a Homologue of a Late Embryogenesis Abundant (LEA) Protein, Enhances Freezing Tolerance of Yeast

Summary The hiC6 gene of Chlorella vulgaris, sharing sequence similarity with a late embryogenesis abundant (lea) gene, was introduced into Saccharomyces cerevisiae. It was inserted on a multicopy plasmid under the transcriptional control of the yeast GAL 1 promoter. Expression of HIC6 protein was confirmed by immunochemical methods. Expression level of the protein was increased gradually with the induction-time by galactose. With maximum induction time, the freeze-tolerance of yeast transformed with hiC6 gene was approximately 3.3 times (from 20 % to 66 % survival rate) higher than that of the control yeast.

Two Low-temperature-inducible Chlorella Genes for Δ12 and ω-3 Fatty Acid Desaturase (FAD): Isolation of Δ12 and ω-3 fad cDNA Clones,…

In an attempt to clarify the involvement of fatty acid desaturases (FADs) in the freezing tolerance of Chlorella vulgaris IAM C-27, developed by hardening, we have isolated cDNA clones for two types of FADs from the Chlorella strain, based on the sequence information of genes for Δ12 and ω-3 FADs, respectively desaturating oleic acid (18:1) to linoleic acid (18:2) and linoleic acid (18:2) to linolenic acid (18:3). The deduced amino acid sequence of the first clone, designated CvFad2, showed about 66% similarity to the microsomal Δ12 FADs from several higher plants and this gene had Δ12 FAD activity when expressed in Saccharomyces cerevisiae. The predicted protein encoded by a second gene, designated CvFad3, showed about 60% similarity to the microsomal and plastidial ω-3 FADs from several higher plants. The features of the amino acid sequences of the C- and N-terminal regions of CvFAD3 and fatty acid analysis of polar lipids in transgenic tobacco plant expressing the CvFad3 gene suggested that this gene encodes the microsomal ω-3 FAD. Southern blot analysis showed that both genes were single-copy genes in the genome of the Chlorella strain. Different transcriptional patterns were observed with the two genes during hardening in Northern blot analysis.

Rapid detection of Salmonella spp. in foods by combination of a new selective enrichment and a sandwich ELISA using two monoclonal antibodies against dulcitol 1-phosphate dehydrogenase

A rapid-detection method was developed for food-borne dulcitol-positive Salmonella spp. in foods that involves a new preenrichment and selective enrichment system and a sandwich ELISA using two monoclonal antibodies against dulcitol 1-phosphate dehydrogenase. Preenrichment and selective enrichment were in Enterobacteriaceae enrichment mannitol (EEM) broth at 42°C for 6 h and in a new dulcitol-magnesium chloride-pyridinesulfonic acid brilliant green-novobiocin (DMPBN) medium at 42°C for 27 h, respectively. The cells were collected from the selective enrichment culture and suspended in 0.1 ml of 1 N NaOH for 2 min. The solution was neutralized with 0.1 ml of 2 M Tris-HCl buffer (pH 7.5) and the mixture was used as a sample for ELISA. The detection sensitivity of the ELISA was 105 CFU of Salmonella spp. per ml of culture. Competing non- Salmonella organisms in raw food did not interfere with the detection of Salmonella cells even when present at 107: 1 (non- Salmonella : Salmonella ratio) in food. Nonmotile Salmonella gallinarum was detected by the ELISA. The minimum detectable number of initial inoculum of Salmonella typhimurium was 0.69 CFU/25 g of raw chicken after the preenrichment in EEM broth and the selective enrichment in DMPBN medium. The present ELISA method required a total analysis time of 36 h including the preenrichment and selective enrichment periods. The ELISA method was compared with a conventional cultural method for the detection of Salmonella cells in 130 samples of raw foods. Of the samples tested, 16 were Salmonella -positive and 114 samples were negative by both methods. False-positive and false-negative results were not encountered.

Improvement of Freezing Tolerance in Transgenic Tobacco Leaves by Expressing the hiC6 Gene

A cryoprotective protein, HIC6, was expressed transgenically in tobacco, a cold-sensitive plant, and the localization of the protein within the cell as well as freezing tolerance of the transgenic tobacco was investigated. For constitutive expression of HIC6 in tobacco, its corresponding gene was subcloned into pBI121. Through the transformation with pBI121/hiC6, fifteen transgenic tobacco lines were acquired, out of which twelve lines expressed the HIC6 protein. None of the transgenic tobacco lines, however, showed significant differences in freezing tolerance from the control plants (wild-type and transformed with pBI121) at −1, −3, and −4°C, with the exception that their freezing temperature was −2°C. In order to increase the accumulation level of HIC6, pBE2113 with a stronger promoter was used. Eight lines expressed the protein out of thirteen lines transformed with pBE2113/hiC6. The accumulation levels of the protein were clearly higher in the tobacco plants transformed with pBE2113/hiC6 than in those with pBI121/hiC6. The HIC6 protein seemed to be localized in mitochondria of the transgenic tobacco plants. Freezing-tolerance test at −1 - −4°C showed that the degree of electrolyte leakage was significantly lower in the plants with pBE2113/hiC6 than in the control plants. A leaf browning observation also showed that high accumulation of HIC6 significantly suppressed injury caused by freezing to the transgenic tobacco at −3°C.

Purification and characterization of two isoforms of glucose 6-phosphate dehydrogenase (G6PDH) from Chlorella vulgaris C-27.

Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a HiTrap Blue column which showed excellent separation of the isoforms from each other. The two isoforms (G6PDH1 and G6PDH2) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH1 showed an apparent M r of 200,000 with a subunit M r of about 58,000, whereas G6PDH2 showed an apparent M r of 450,000 with a subunit M r of about 52,000. The kinetic parameters were measured and several enzymatic features of the isoforms, such as effects of metal ions on the enzyme activity, were clarified, which showed that the two isoforms were different from each other in many respects. Among the effective ions, Cd2+ showed marked stimulating effects on both isoforms. G6PDH1 and G6PDH2 seem to be a cytosolic and a chloroplastic type, respectively, as judged by their sensitivity to DTT, and also from the results of sequence similarity searches using their N-terminal and internal amino acid sequences.

Studies on frost hardiness in Chlorella ellipsoidea. V. The role of glucose and related compounds

Abstract Chlorella ellipsoidea cells at an intermediate stage in the ripening phase of the life cycle were hardened at 3°C. d -Glucose added during hardening in the dark induced a tolerance to freezing. Sucrose, d -galactose and d -fructose were also capable of increasing hardiness, but pyruvate, succinate and citrate were not. These results suggest the involvement of the pentose-phosphate cycle in the hardening process. The amounts of monosaccharide and dextrin in Chlorella cells did not change during hardening. Sucrose concentration increased during hardening in both the light and the dark in the presence of glucose. Starch concentration remained unchanged in the dark in the presence of glucose, but increased remarkably in the light. These results suggest that the hardening process involves an accumulation of sucrose, and indicate that there is no direct correlation between starch content and frost hardiness. Glucose 6-phosphate dehydrogenase activity increased greatly during the early phase of hardening; 6-phosphogluconate dehydrogenase activity also increased with hardiness. These results suggest that pentose-phosphate cycle activity occurs at a high level during hardening. Cycloheximide and oligomycin completely inhibited the hardiness increase in the dark in the presence of glucose; chloramphenicol and DCMU had no effect. These results suggest that protein synthesis on cytoplasmic ribosomes and ATP synthesis in mitochondria are involved in the process of hardening in the dark in the presence of glucose, but protein and ATP synthesis in chloroplasts are not. The inhibition of the hardiness increase by sodium dodecyl sulfate was greater in the light than in the dark in the presence of glucose. Triton X-100 inhibited completely the hardiness increase in both the light and the dark in the presence of glucose. It seems likely, therefore, that membrane alterations during hardening in the dark in the presence of glucose may occur; these alterations are different from those which occur in the light.

Rapid detection and counting of viable bacteria in vegetables and environmental water using a photon-counting TV camera

A bioluminescence assay carried out with a photon-counting TV camera was evaluated for rapid enumeration of viable bacterial counts. The test sample was filtered through a membrane filter, and the membrane filter retaining bacteria was incubated at 37 degrees C for 6 h on a filter paper soaked with nutrient broth supplemented with 0.5% NaCl. The membrane filter was then subjected to a bioluminescence reaction, and the intensity of light and numbers of light emission points on the filter were measured with a photon-counting TV camera. The light intensity measured on seven different bacteria correlated with initial viable counts; the correlation coefficient was calculated to be 0.89. The number of light emission points measured on Escherichia coli also correlated with the initial viable counts (r = 0.81) in a range from 1 to 100 CFU. Presumptive bacterial counts by the present bioluminescence assay determined on 79 samples of vegetables and 122 samples of environmental water correlated well with the viable counts obtained by the conventional plating method, with correlation coefficients of 0.87 and 0.82, respectively.

Polymerase Chain Reaction Assay for Detection of Escherichia coli O157:H7 and Escherichia coli O157:H

The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non-E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H- and E. coli O157:H7. Since the DNA sequence from base 15 to base 1,008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H- and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 x 10(0) to 3.5 x 10(2) CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42 degrees C for 18 h and for the conventional cultural method.

Application of monoclonal antibodies to dulcitol 1-phosphate dehydrogenase for rapid detection of Salmonella

Monoclonal antibodies raised against dulcitol 1-phosphate dehydrogenase of Salmonella typhimurium IFO 12529 were screened against 20 serotypes of Salmonella and 13 non- Salmonella bacteria. A sandwich-capture, enzyme-linked immunosorbent assay (sandwich ELISA) was developed for detection of Salmonella in food. The assay utilizes two monoclonal antibodies (DUI2 and DU28) which show no cross-reactions with non- Salmonella bacteria. The limit of detection of the sandwich ELISA was about 1 × 107 CPU/ml. After cultivation in a medium containing dulcitol at 37°C for 18 h followed by the sandwich ELISA. 1 CPU of Salmonella was detected. Although a high inoculum level of E. coli interfered with the detection of Salmonella , the interference was minimized by using a selective dulcitol-magnesium chloride-pyridinesulfonic acid medium for enrichment. The novel ELISA procedure detected Salmonella in chicken filtrates inoculated with 1.4 CPU/50 m1 and 1.3 × 107 CPU/50 ml of E. coli within 25 h.