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Pseudomonasspp., such as P. fluorescensgroup, P. fragi, and P. putida, are the major psychrophilic spoilage bacteria in the food industry. Bacteriophages (phages) are a promising tool for controlling food-spoilage and food-poisoning bacteria; however, there are few reports on phages effective on food-spoilage bacteria such as Pseudomonasspp. In this study, 12 Pseudomonasphages were isolated from chicken and soil samples. Based on the host range and lytic activity at 30 °C and 4 °C and various combinations of phages, phages vB_PflP-PCS4 and vB_PflP-PCW2 were selected to prepare phage cocktails to control Pseudomonasspp. The phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 showed the strongest lytic activity and retarded regrowth of P. fluorescensand P. putidaat 30 °C, 8 °C, and 4 °C at a multiplicity of infection of 100. Nucleotide sequence analysis of the genomic DNA indicated that vB_PflP-PCS4 and vB_PflP-PCW2 phages were lytic phages of the Podoviridae family and lacked tRNA, toxin, or virulence genes. A novel endolysin gene was found in the genomic DNA of phage vB_PflP-PCS4. The results of this study suggest that the phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 is a promising tool for the biocontrol of psychrophilic food-spoilage pseudomonads during cold storage and distribution.

•EGCg upregulated transcription of genes involved in membrane transport.•EGCg downregulated transcription of genes involved in toxin production and stress response.•Staphylococcus aureuscells largely decreased membrane potential in the presence of EGCg.

A chloroplastic NADPH-dependent thioredoxin reductase gene was identified from Chlorella vulgarisand designated CvNTRC. Mature CvNTRC protein (mCvNTRC) was expressed in Escherichia coli, and it showed both NADPH-dependent thioredoxin reductase (NTR) and thioredoxin (Trx)-like dithiol-disulfide oxidoreductase activities. The transcript of CvNTRCincreased throughout 24-h hardening, whereas the encoded protein amount and total NTR activity decreased once and then increased during hardening. By in vitropull-down assay, a 21.2-kDa protein bound to mCvNTRC was isolated and identified as a 2-Cys peroxiredoxin (2-Cys Prx) based on the N-terminal sequence. These data suggest that CvNTRC is maintained at a constant level during hardening and functions as an antioxidant with 2-Cys Prx in the acquisition of freezing tolerance of Chlorella.

Proteins and genes involved in the recovery of heat-injured SalmonellaEnteritidis were investigated. SalmonellaEnter-itidis cells cultured overnight in tryptic soy broth (TSB; nonselective medium) were suspended in citric acid–disodium hydrogen phosphate buffer (pH 6). After heat treatment at 55°C for 15 min, the culturable counts measured by tryptic soy agar (TSA; nonselective medium) decreased from 108to 107CFU/ml. On the other hand, culturable counts measured by desoxycholate–hydrogen sulfite–lactose (DHL) agar (selective medium) were decreased from 108to 104CFU/ml by the same treatment. The results suggest that 99.9% of SalmonellaEnteritidis detected on TSA were injured but recoverable. When injured SalmonellaEnteritidis was incubated in TSB, the culturable count measured by TSA did not increase for 2 h, whereas that by DHL agar increased after incubation for 30 min. After incubation for 2 h, the culturable count measured by DHL agar reached a similar level with that by TSA, indicating that SalmonellaEnteritidis had recovered. The two-dimensional polyacrylamide gel electrophoresis analysis revealed that elongation factor G (FusA) and pyruvate kinase (PykF) specifically increased in the cells just after heat treatment and in the recovery cells. The levels of transcription of 86 stress-inducible genes were also investigated by reverse transcription PCR. Nineteen heat-inducible (clpB, clpX, degP, dnaJ, fkpA, ftsJ, gapA, hflB, hslJ, hslU, hslV, htpG, htrA, lon, mopA, mopB, mreB, rpoE, and ppiD), and 12 oxidative-stress and DNA damage–inducible (ahpC, ahpF, fldB, fur, grxA, dinF, katG, mutM, recA, soxR, trxC, and zwf) genes were transcribed extensively during recovery in TSB. The results obtained in this study will be used to develop the media or culture conditions that will promote recovery for the detection of food poisoning bacteria, including injured cells from food products.

A rapid and sensitive method for detection of staphylococcal enterotoxin B (SEB) in raw and dry milk samples with the use of antibody-based immunomagnetic separation (IMS) in conjunction with flow cytometry (FCM) was developed. Sheep anti-SEB immunoglobulin G (IgG) was immobilized on Dynabeads M-280. The SEB initially binds to the capturing antibody, which is bound on the magnetic beads. The rabbit anti-SEB IgG binds to the captured toxin and is further labeled with a Cy5-labeled goat anti-rabbit IgG antibody. The percentage of the beads that were fluorescent was measured by FCM. FCM was carried out for 1 min, and the data obtained were expressed as histograms for particle size (forward light scatter) and histograms for fluorescence intensity. A peak corresponding to the magnetic beads was clearly distinguished from a peak derived from contaminating particles in the sample solution. In the absence of SEB, about 10% of the beads emitted fluorescence. The percentage of fluorescent beads and the fluorescence intensity increased with increasing SEB concentrations. For this IMS-FCM assay, the lower limits of detection for SEB were estimated to be 0.01 and 0.25 ng/ml for buffer and milk samples, respectively.

Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgarisC-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 (G6PDH1and G6PDH2) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH1showed an apparent Mrof 200,000 with a subunit Mrof about 58,000, whereas G6PDH2showed an apparent Mrof 450,000 with a subunit Mrof 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. G6PDH1and G6PDH2seem 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.

The pbp3gene encoding PBP3 of Bacillus cereuswas cloned and sequenced. For this purpose, PBP3 was first purified from B. cereusts-4, and N-terminal amino acid sequences of the peptides obtained from the protease digests of the protein were analyzed. The B. cereusts-4 pbp3gene consisted of an open reading frame of 1,986 bp encoding 662 amino acid residues with a calculated molecular mass of 73,044 Da. The active site-motifs SXXK, SXN, and KTG are present at the positions 393, 452, and 590, respectively, in the deduced amino acid sequence. The pbp3structural gene was ligated into the pET17×b expression vector and pET-pbp3was constructed. A protein was produced by the cells of E. colicarrying pET-pbp3. The produced protein migrated at about 75 kDa in SDS-polyacrylamide gel and strongly reacted with biotinylated ampicillin.

In an attempt to clarify the involvement of fatty acid desaturases (FADs) in the freezing tolerance of Chlorella vulgarisIAM C-27, developed by hardening, we have isolated cDNA clones for two types of FADs from the Chlorellastrain, 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 CvFad3gene 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 Chlorellastrain. Different transcriptional patterns were observed with the two genes during hardening in Northern blot analysis.

The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coliand non–E. colibacteria 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. coliO157 strains tested. Some nonpathogenic E. coliand all pathogenic E. coliexcept E. coliO157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coliO157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coliO157:H− and E. coliO157:H7. Since the DNA sequence from base 15 to base 1008 of the PCR product seems to be specific to E. coliO157, 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. coliO157:H− and E. coliO157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coliexcept E. coliO157. All raw meats inoculated with E. coliO157:H7 at 3 × 100to 3.5 × 102CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42°C for 18 h and for the conventional cultural method.

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.

Monoclonal antibodies (MAbs) raised against Escherichia coliO6:H16 were screened against 15 strains of E. coliand 19 non–E. colibacteria. A MAb-luminescence assay using MAb-5.8, which shows no cross-reactions with non–E. colibacteria, and a photon-counting television camera were developed for rapid enumeration of E. coliO6:H16 in water. The membrane filter that retained bacteria was boiled for 5 min in a buffer and incubated with biotinylated MAb-5.8. After incubation with streptavidin-peroxidase conjugate, it was reacted with luminol-based reaction mixture. Luminous image and light intensity of the filter was recorded with a Biocell Counter. Levels of E. coliO6 higher than 7 × 103CFU were detected by the MAbluminescence assay when E. coliO6 was spotted onto the membrane filter. The sample that contained E. coliO6:H16 was filtered through a membrane filter, and the filter that retained bacteria was incubated on a filter paper soaked with nutrient broth supplemented with 0.5% NaCl at 37°C for 6 h. The number of light emission points on the filter correlated well with initial E. coliO6:H16 counts within the range of 1 to 3 × 102CFU. The correlation coefficient was 0.89.

The gene of IMP dehydrogenase of Bacillus cereusts-4, a temperature-sensitive mutant of B. cereusJCM 2152, was subcloned and its sequence was analyzed. A B. cereusts-4 DNA fragment of 2,065 bp containing the entire impdhgene and flanking regions was sequenced. The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdhsequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B. cereusts-4 impdhgene. The gene amplified by PCR was expressed in Escherichia coliusing a pET17×b expression plasmid. The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp- Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-Leu-Thr-Phe-Asp- Asp-Val-Leu-Leu-Val-Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5. These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B. cereus.

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 hiC61)and hiC12genes, 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 3.1×106times 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 ChlorellaLEA proteins.

The hiC6gene 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 1promoter. 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 hiC6gene was approximately 3.3 times (from 20 % to 66 % survival rate) higher than that of the control yeast.

Cephalexin, cefaclor, cefadroxil, and cefotaxime strongly inhibited sporulation of Bacillus cereusts-4 at 1 μg/ml. Cephalexin was most inhibitory on the sporulation of B. cereuswhen the antibiotic was added at 3 h after induction of sporulation by nutrient downshift technique. Examination of 4′,6-diamidino-2-phenylindole-stained cells by fluorescence-phase contrast microscopy showed that cephalexin inhibited the formation of asymmetric septum. By using [3H]penicillin, eight penicillin-binding proteins (PBPs) were detected from the cells of B. cereusts-4. Among them, four PBPs were also detected in sporulating cells. Affinity of cephalexin to PBPs were measured indirectly by competition for subsequent binding of radioactive penicillin G. Cephalexin strongly bound to PBP 4 with molecular weight of 72,000 in sporulating cells.

IMP dehydrogenase activity of B. cereusincreased parallel to cell growth in YE-EMM, where B. cereusdid not sporulate. When B. cereuswas cultured in a modified G medium, a sporulation medium, the activity reached the highest level at 6 hr and decreased thereafter. After induction of sporulation by nutritional shift down in 1/100 G medium, the enzyme activity decreased to about 5% compared with exponentially growing cells at 1 hr of resuspension. The sporulation rate of B. cereuswas over 90% in the modified G medium and 1/100 G medium. Sporulation was strongly inhibited by mycophenolic acid at 1 mM, when the drug was added at 0 and 1 hr of resuspension in 1/100 G medium.

IMP dehydrogenase was purified from a crude extract of B.cereuscells.The molecular mass of the purified enzyme was estimated to be 56 kDa by SDS-PAGE and 225 kDa by gel filtration.The optimum pH of the enzyme was about 9.5.The first seven residues at N-terminus of the enzyme was determined to be Met-Trp-Glu-Ser-Lys-Phe-Val.The enzyme showed a significant specificity for inosine nucleotides among 15 purines and pyrimidines tested, but not acted on other purines and pyrimidines including inosine.Among 11 metal ions and 3 enzyme inhibitors tested, Al3+activated the IMP dehydrogenase.The enzyme activity was strongly inhibited by Zn2+and Fe3+.

VH(heavy-chain variable region) and VL(light-chain variable region) genes were amplified by PCR from hybridomas producing MAb-11 and MAb-18 which inhibited Japanese radish acid phosphatase. Nucleotide sequencing of the V genes demonstrates that the MAbs contained similar VHand identical VLdomains. Initially, the VHand VLgenes were expressed in Escherichia colias single-chain Fv (ScFv) fragments. Fragments ScFv-11 and ScFv-18, named for MAb-11 and MAb-18, respectively, inhibited the enzyme activity to the same extent as the intact MAbs. Both of the antibody fragments widely cross-reacted with other phosphatases, including some phosphomonoesterases and phosphodiesterases from different sources. ScFv-18 also inhibited acid phosphatase from a different origin, but stimulated the activity of alkaline phosphatase from calf intestine. The PCR-amplified VHand VLgenes were subsequently expressed separately in Escherichia colias fusion products with glutathione S-transferase. The fusion proteins had little effect on Japanese radish acid phosphatase. Furthermore, a large number of recombinant ScFv fragments specific to the acid phosphatase were generated by using a bacteriophage expression system and a mouse ScFv gene library. These ScFv fragments had a range of effects on the enzyme activity, including inhibition, stimulation, and none. Among them, an ScFv fragment, designated ScFv-G7, inhibited more strongly than ScFv-11 and ScFv-18.

We isolated a cDNA corresponding to a chloroplast NADPH-dependent thioredoxin reductase gene (NTR-C), in Chlorella that is low-temperature-inducible. The obtained cDNA was 1,838 bp in length and coded for 529 amino acids. The deduced amino acid sequence showed higher homology to those of Arabidopsis and rice NTR-C, containing a thioredoxin (Trx) and a thioredoxin reductase (TR), than those of NTR-A (mitochondrial) and NTR-B (cytosolic) from various organisms, which contain only a TR domain and differ in subcellular localization. The results of enzyme assays of partially-purified mature NTR-C protein (mNTR-C), expressed in Escherichia coli with a pET-29b(+) expression vector, provided evidence that the gene included both regions. Northern blot analysis showed a remarkable increase in transcripts under low temperature, while the protein level did not significantly change when examined by using Western blotting with anti-mNTR-C antibodies. The TR activity dependent on NADPH was not enhanced by low temperature despite the substantial increase in transcripts. Based on the results of measurement of peroxiredoxin (Prx) activity and Western blotting using both an extract of Chlorella and purified mNTR-C, the Chlorella was suggested to possess a Prx that interacts with NTR-C.