Journal of Microbiology

Volume 36(1); March 1998

Genetic Manipulation of Rhabdoviruses : New Insights to Virus Replication, Transcription and Assembly: Michael A. Whitt; J. Microbiol. 1998;36(1):1-8.

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Abstract: Rhabdoviruses, together with the other members of the Rhabdoviridae family, are one of the most widely distributed groups of viruses in nature. Rhabdoviruses have been isolated from virtually all vertebrates, several different species of insects, as well as many plant (65). It is thought that insects were the original hosts for this group of viruses and that rhabdoviruses have since adapted to grow in both vertebrates and invertebrates. This adaptation undoubtedly contributed to one of the disdinguishing features of the prototypic rhabdovirus, vesicular stomatitis virus (VSV), namely the ability to replicate in most primary cell cultures and essentially all established mammalian cell lines, as well as a number of insect and amphibian cell lines. Because VSV has a broad host range, is relatively easy to grow and replicates to high titers in cell culture it has been used extensively as a model system to study many aspects of rhabdovirus entry (32, 69, 70), replication (3, 4) and assembly(36, 55, 58).

Characterization of the Two Na^+/H^+ Antiporters of Escherichia coli: Sung-Yum Seo; J. Microbiol. 1998;36(1):9-13.

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Abstract: Escherichia coli has two Na^+/H^+ antiporters which can be used to lower intracellular pH transiently elevated upon exposure to alkaline stress and to extrude intracellular Na^+. Previous studies on the propertied of the Na^+/H^+ antiporter were done, but later studies showed that E. coli has two antiporters(Pinner E., Kolter Y., Padan E., Schuldiner S. (1993) J. Biol. Chem 268, 1729~1734). The properties of each antiporter were studied in this report. Both antiporters were specific only to Na^+ and Li^+, and showed hyperbolic kinetics. K_M values of NhaA were 0.8 mM and 2,2mM for Li^+ and Na^+, respectively. K_M values of NhaB were 2.8mM and 12mM for Li^+ and Na^+, respectively. The pH effects can be summarized as follows: 1) both antiporters do not show activity at very acidic pH values; 2) NhaB seems to work in a neutral pH range; 3) NhaA seems to show activity at alkaline pH. The effect of pH on the kinetics of the antiporters was studied. V_max of NhaB remained maximum in the pH range 7.0~8.2. V_max of NhaA increases steeply in the pH range 6.5~7.8 and remained maximum thereafter up to pH 8.6. When the pH was increased, the K_M decreased sharply, especially for NhaA.

Electrophoretic Karyotypes of Fusarium oxysporum formae speciales: Min, Byung Re , Kim, Kyung Ah , Choi, Yong Keel; J. Microbiol. 1998;36(1):14-19.

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Abstract: Chromosomal DNAs from nine strains of Fusarium oxysporum formae speciales were analysed by pulsed field gel electrophoresis (PFGE). Intact chromosomal DNA were prepared from fungal protoplasts. About 7-13 chromosomal DNA bands were separated in nine Fusarium oxysporum formaespeciales, ranging in size from 0.8 to 6.7 Mb. Considering that some of the chromosomal bands represented unresolved doublets, the total genome size was estimated to vary between at least 22.3 and 45.5 Mb. Gels run under different conditions were Southern blotted and probed with randomly selected genomic DNA from Fusarium oxysporum f. sp. lilii. Electrophoretix karyotyping revealed some differences within and between Fusarium oxysporum formae speciales.

Purification and Partial Characterization of a metalloprotease in Flammulina velutipes: Shin, Hyun Hee , Choi, Hye Seon; J. Microbiol. 1998;36(1):20-25.

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Abstract: Metalloprotease was detected in the fruit body of the edible mushroom Flammulina velutipe. Inactivation of the metalloprotease reduced mycelial growth signicantly, implying that metalliprotease is important for growth. A neutral metalloprotease was purified by hydrophobic, gel filtration, and anion exchange chromatography. The M_r of the protein was determined to be 30,000 by SDS-PAGE and 33,000 by gel filtration on a Sepjadex G-150 column, indicating that it is a monomer. Its first 11 N-terminal amino acids (P-Q-V-K-T-W-D-L-A) did not show any homology with any known protein in Database(GENEBANK, Swissprot). The enzyme was inhibited by EDTA, 1, 10-phenanthroline, and phosphoamidon but not by inhibitors specific for serine, aspartate and custeine protease. Addition of Zn^2+ and Co^2+ reversed the inhibition caused by 1,10-ohenanthroline. This protease hydrolyzed human fivrinogen, fibrin, azoalbumin, and azocasein as substrates. It showed cleavage preference for hydrophobic residues among tested synthetic substrates.

Synthesis and Requirement of Escherichia coli Heat Shock Proteins GroEL and DnaK for Survival under Phenol Stress Conditions: Jeon, Taeck Joong , Lee, Kil Jae; J. Microbiol. 1998;36(1):26-33.

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Abstract: Exposure of Escherichia coli strain MC4100 to various concentrations of phenol at temperatures higher than 20℃ led to induction of stress proteins such as GroEL and DnaK, as analyzed by SDS-PAGE and Western blotting methods. The optimum range of phenol concentration for the induction of GroEL and DnaK was slightly different at each temperature of bacterial growth and phenol treatment. The level of GroEL increased as the temperatures of growth and phenol treatment were increased from 30℃ to 40℃. The level of induced FroEL was maximal in the wild type cells which had been grown and treated by 2000㎍/㎖ phenol at 40℃. In contrast to GroEL, the level of DnaK decreased as the temperatures of growth and phenol treatment were increased from 25℃ to 40℃. Dnak was maximally induced in the cells grown and exposed to 1000㎍/㎖ phenol at 25℃. In rpoH mutant cells KY1601, GroEL was not additionally induced by phenol treatment and DnaK was not even detectable under normal and phenol stress conditions. Viability of cells under the same conditions of phenol treatment showed that the phenol resistance in much more induced in wild type cells than rpoH mutant cells. These results suggest that the induction of GroEL and DnaK is required for the enhanced viability of cells under conditions of phenol stress.

Degradation of Gaseous BTX by Biofiltration with Phanerochaete chrysosporium: Oh, Young Sook , Choi, Sung Chan , Kim, Yeong Kwan; J. Microbiol. 1998;36(1):34-38.

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Abstract: Biodegradation of benzene, toluene, and o-, m-, p-xylenes(BTX) by the white rot fungus, Phanerochaete chrysosporium was studied in a biofilter. P. chrysosporium was cultured under shaking conditions on YMG growth medium and homogenized pre-grown cells were transferred to biofilter. A preliminary batch culture experiment showed that all BTX components were degraded simultaneously without any observable substrate interactions, while the rate constant was the highest for p-xylene and lowest for benzene. For the biofiltration of the BTX, the BTX compounds were individually vaporized from 3 glass bottles containing benzene, toluene, and xylenes, respectively, by applying air flow. The vaporized fluxes of the compounds were immediately taken by the air current to the biofilter through the horizontal tube at the rim of the source other than the pollutants themselves. The effect of air flow rate (0.026~0.450 l/h) on the degradation of the compounds was evaluated in the biofilter packed with glass beads. A substantially higher degradation of all the BTX compounds was observed at higher flow rates, suggesting that mass transfer is a limiting factor in the degradation process. At a flow rate of 0.026l/h, there was no substantial difference in the extent of degradation between the two support media.

Chemical midification of Cytosine Deaminase from Aspergillus fumigatus: Yu, Tae Shick , Kim, Jung , Kim, Hyun Soo; J. Microbiol. 1998;36(1):39-42.

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Abstract: Essential amino acids involved in the catalytic mechanisms of cytosine deaminase from Aspergillus fumigatus IFO 5840 were determined by chemical modification studies. The enzyme was perfectly inhibited by N-bromosuccinimide, chloramines-T, pyridoxal-5’-phosphate, and p-chloromercuribenzoate. It was strongly inhibited by phenylmethyl sulfonyl fluoride, and weakly inhibited by phenylglyoxal. The inactivation of the enzyme activity by p-CMB was reversed by sulfhydryl reagents. Furthermore, activities inhibited by chloramines-T, pyridoxal-5’-phosphate, results, we speculate that tryptophan, methionine, lysine and cysteine residues are located in ornear the active center of the cytosine deaminase, while a serine is indirectly involved I the enzyme activity.

Detection of Cholera Cells Using Surface Plasmon Resonance Sensor: Choi, Ki Bong , Youn, Hee Ju , Ha, Youn Chul , Kim, Kwang Joong , Choi, Jung Do; J. Microbiol. 1998;36(1):43-48.

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Abstract: In this report, we describe the application of colloidal gold particles for bacterial cell detection using a surface plasmon resonance(SPR) sensor. A monoclonal antibody against V. cholere was immobilized onto the gold film of a sensor chip activated with N-hydroxysuccinimide. Optimal conditions for the immunochemical reaction for cholera detection was investigated with streptavidin coated colloidal fold particles and ciotinylated monoclonal antibody against cholera cells. The sensitivity of the cholera cell assay with SPR sensor could be enhanced 1000 times by binding of anti-cholera monoclonal antibody-biotin conjugate and streptavidin coated colloidal gold suspension. From a viewpoint of sensitivity, SPR sensor is about 10 times more sensitive than ELISA. This result illustrates the feasibility of using the SPR sensor in bacterial cell detection.

A Novel Immunodominant Epitope on aouter Membrane protein F of Pseudomonas aeruginosa in Humans: Park, Wan Je , Lee, Na Gyong , Jung, Sang Bo , Ahn, Bo Young , Kim, Yu Sam , Kim, Hyun Su; J. Microbiol. 1998;36(1):49-54.

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Abstract: The outer membrane protein F(OprF) of Pseudomonas aeruginosa is known to be conserved among various P. aeruginosa strains, and has been shown to be protective against P. aeruginosa infections in animal model systems. In order to identify epitopes essential for immunogenicity and protective efficacy, we synthesized 14 peptides of the mature OprF protein and evaluated their immunofenicity in mice. Among them two peptides in the carboxy-terminus region of OprF, peptide 3(^204GAPAVAEVVRVQLDVKFD^221) and peptide 14(^305NATAEGRAINRRVE^318), elicited high titer of antibody responses in mices, and reacted strongly with anti-OprF antibodies. The peptides were also examined for reactivity with antisera from human volunteers immunized with a P. aeruginosa vaccine composed of outer membrane proteins and from patients who recovered from Pseudomons infections. Peptide 3 showed high reactivity with both antisera from the vaccines and the convalescents, comparable to that of peptide 14, which has been previously reported to afford protection against P. aeruginosa in a murine acute infection model. Substitution of three amino acids KFD in peptide 3 with AAA significantly diminished its reactivity to the antisera, indicating that the core sequence ^216LDVKFD^221 is crucial for its immunogenicity and reactivity with human antisera. These data suggest a potential use of this peptide for development of a vaccine against P. aeruginosa infection.

Rapid and Simple Purification of Biologically Active Human Hepatitis B virus Transactivator-X Proteins: Poo, Ha Ryoung , Kim, Sun Ok , Sohn, Mi Jin , Lee, Sook , Lee, Young Ik; J. Microbiol. 1998;36(1):55-58.

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Abstract: The human hepatitis B virus-X(HBV-X) was cloned into an expression vector, pET3d, containing a T7 promoter and direct expression was induced in Escherichia coli. The expressed HBV-V protein was purified to homogeneity by centrifugation, ion-exchange chromatography and gel filtration. After gel filtration, renaturation of HBV-X were performed using dialysis against serially diluted urea buffer. The biological activity of refolded HBV-X protein was confirmed by enhancement of protein/DNA complexes using gel-shift analysis.

Production and Characterization of Monoclonal Antibodies to Human Immunodeficiency Virus Type 1 gp120 Envelope Glycoprotein: Choi, Eui Yul , Ryu, Ji Yoon , Lee, Yoon , Ha, Sung Gil , Chung, So Young , Park, Sang Yeol , Nham, Sang Uk , Lee, Young Ik , Park, Jin Seu; J. Microbiol. 1998;36(1):59-65.

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Abstract: Monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoprotein gp 120(HIV-1 gp120) were produced and characterized. For immunogen recombinant gp120 polypeptide expressed in bacteria was prepared and injected into mice. From two fusion experiments, twenty hybridomas secreting monoclonal antibodies against the recombinant gp120 were initially screened by immunodot blot analysis. Among the antibodies, 15 of them showed strong reactivities with the recombinant protein expressed in bacteria in Western blot and thus it was tested if these could react with the recombinant protein expressed in insect cells. All of the 15 antibodies immunostained the protein band with varing degrees of reactivities. Next, we tested whether the antibodies recognize authentic gp120 protein expressed in mammalian cells. COS-1 cells were tranfected with the cDNA encoding gp120 protein, and the transiently ecpressed protein were analyzed with the mAbs by Western blot analysis and immunofluorescence microscopy. Six of the monoclonal antibodies reacted with the protein band of authentic gp120 expressed in mammalian cells in the Western blot, and five stained the cell periphery of the transfected COS-1 cells in immunofluorescence. The mAbs described in this study should prove to be useful tools for the biochemical, immunological and structural analysis of HIV-1 gp120 envelope glycoprotein.

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