Journal of Microbiology

Volume 40(1); March 2002

A Restrictive Virus Tropism, Latency and Reactivation of Pseudorabies Virus Following Irreversible Deletion of BsrI Restriction Site in the Thymidine-kinase Gene: Mohd Lila Mohd Azmi , Nazariah Allaudin Zeenathul , Abdel-Wahid Saeed Ali , Che Abdul Rahim Mohamed , Awang Isa Kamarudin; J. Microbiol. 2002;40(1):1-10.

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Abstract: At the dose of 1000 p.f.u. per mouse, 100% mortality occurred in mice inoculated with wild-type pseudorabies virus (PrV). In contrast, upon stable deletion of 10 bp nucleotides at the BsrI site within the TK gene, PrV was rendered to be completely apathogenic. The deletion also caused the virus to be less capable of replicating in respiratory as well as in nervous system tissues. Although animals were exposed to high titers of TK-deleted PrVs, the virus failed to replicate to a high titer as compared to the pathogenic parental virus. In contrast to previous studies, the deletion in the TK gene did not prevent the virus from establishing latency. Upon immunosuppression, the latent virus, however, reactivated but replicated at low titers. Interestingly, TK-deleted virus established latency and reactivation, that are occurred only in trigeminal ganglia and the cerebrum, and no other tissues involved. Following reactivation, there was no indication of virus shedding in respiratory tissues as confirmed by virus isolation and polymerase chain reaction (PCR) technique targeting at the gB gene of PrV. The non-pathogenic virus with non-shedding characteristics, upon reactivation of the latent virus, would be the important feature of a live virus vaccine candidate.

Protective Immune Reponses Induced by Non-infectious L-particles of Equine Herpesvirus Type-1: Implication of Cellular Immunity: Mohd Lila Mohd Azmi , Hugh John Field , Frazer Rixon , John McLauchlan; J. Microbiol. 2002;40(1):11-19.

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Abstract: Mice immunized with equine herpesvirus type-1 (EHV-1) L-particles showed a significant increase (p<0.05) in serum antibody titers. Upon a booster dose four weeks later, antibody titers increased significantly. Interestingly, immunization via intravenous or intramuscular route induced significantly higher (p<0.05) antibody titers. However, mice iummunized with UV-treated L-particles, virions or immunization via intranasal route induced lower antibody titers. Upon challenge inoculation with wild-type EHV-1, our data showed there was a poor correlation between antibody titers and protection against virus replication. Therefore, the role of cell-mediated immunity towards protection was investigated. As predicted, the strongest cell-mediated immunity, as measured by delayed-hypersensitivity test, was detected in mice immunized with live virus particles. The magnitude of cell-mediated immune response correlated with the efficacy of L-particles as immunizing agent. The highest efficacy, as indicated in mice immunized via intranasal route, was highly correlated with cell-mediated immunity. A similar phenomenon was also demonstrated in mice immunized intranasally with UV-treated L-particles. However, the degree of protection was reduced when mice immunized intravenously or intramuscularly with UV-treated L-particles. In conclusion, protection conferred in these animals was highly implicated by immune cells and the least by antibodies. The route of immunization and the nature of the antigen also contributed to the efficacy of L-particles as immunizing agent. In contrast to that of herpes simplex virus type 1, our data showed EHV-1 non-infectious L-particles are highly suitable for immunization of the host against EHV-1 disease.

Purification and Characterization of Extracellular Poly(3-hydroxybutyrate) Depolymerase from Penicillium simplicissimum LAR13: Jee-Sun Han , Mal-Nam Kim; J. Microbiol. 2002;40(1):20-25.

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Abstract: An extracellular PHB depolymerase was purified from P. simplicissimum LAR13 cultural medium by Sepharose CL-6B chromatography. When the fungus was grown in a basal salt medium with poly(3-hydroxybutyrate) (PHB) as the sole carbon source, PHB depolymerase production reached maximum at its stationary phase. The mycelial growth rate was higher at 37 C than at 30 C and even higher than at 25 C, However, the enzyme production was lower at 37 C than 30 C or 25 C. The isolated enzyme is composed of a single polypeptide chain with a molecular mass of about 36 kDa as determined by SDS-PAGE. The optimum conditions for the enzyme activity are pH 5.0 and 45 C. The enzyme was stable for 30 min at a temperature lower than 50 C, and stable at pH higher than 2.0 but it was unstable at pH 1.0. 1 mM Fe^2+ reduced the enzyme activity by 56% and the enzyme was inhibited almost completely by 4 mM Fe^2+ . The enzyme was partially inhibited by phenylmethylsulfonyl fluoride and was very sensitive to diazo-DL-norleucine methyl ester, dithiothreitol and mercuric ion. However, N-p-tosyl-L-lysinechloromethyl ketone, p-hydroxymercuricbenzoate and N-acetylimidazole had no influence upon its activity.

Purification and Characterization of Caseinolytic Extracellular protease from Bacillus amyloliquefaciens S94: Eui-Sun Son , Jong-Il Kim; J. Microbiol. 2002;40(1):26-32.

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Abstract: From the culture supernatant of the psychrotrophic strain of Bacillus amyloliquefaciens an extracellular serine protease was purified to apparent homogeneity by successive purification steps using QAE-Sephadex, SP-Sephadex and Sephacryl S-100 column chromatography. The protease is monomeric, with a relative molecular mass of 23,000. It is inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride, but not by EDTA. The enzyme is most active at pH 9-10, and at 45 C, although it is unstable at 60 C.

Kinetic Study on the Enzymatic Production of D-Alanine from D-Aspartic Acid: Jae-Heung Lee , Moon-Hee Sung , Yeong-Joong Jeon; J. Microbiol. 2002;40(1):33-37.

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Abstract: An enzymatic reaction for the production of D-alanine from D-aspartic acid and pyruvate as substrates by a thermostable D-amino acid aminotransferase (D-AAT) was investigated at various conditions in the temperature range of 40-70 C and pH range of 6.0-9.5. The D-AAT was produced with recombinant E. coli BL21, which hosted the chimeric plasmid pTLK2 harboring the D-AAT from the novel thermophilic Bacillus sp. LK-2. The enzyme reaction was shown to follow the Ping Pong Bi Bi mechanism. The K m values for D-aspartic acid and pyruvate were 4.38 mM and 0.72 mM, respectively. It was observed that competitive inhibition by D-alanine, the product of this reaction, was evident with the inhibition constant K i value of 0.1 mM. A unique feature of this reaction scheme is that the decarboxylation of oxaloacetic acid, one of the products, spontaneously produces pyruvate. Therefore, only a catalytic amount of pyruvate is necessary for the enzyme conversion reaction to proceed. A typical time-course kinetic study showed that D-alanine up to 88 mM could be produced from 100 mM of D-aspartic acid with a molar yield of 1.0.

Cellular Responses of Pseudomonas sp. KK1 to Two-Ring Polycyclic Aromatic Hydrocarbon, Naphthalene: Hyung-Yeel Kahng; J. Microbiol. 2002;40(1):38-42.

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Abstract: The strain KK1 isolated from soil contaminated with polycyclic aromatic hydrocarbons was identified as Pseudomonas sp. based on analyses by MIDI and Biolog Identification System. Cellular and physiological responses of strain KK1 to two-ring polycyclic aromatic hydrocarbon, naphthalene were evaluated using radiorespirometry, PLFAs and sequence analysis of Rieske-type iron sulfur center of dioxygenase. KK1 was found to be able to rapidly mineralize naphthalene. Notably, KK1 cells pre-grown on phenanthrene were able to mineralize naphthalene much more rapidly than naphthalene-pregrown cells. The total cellular fatty acids of KK1 were comprised of eleven C-even and two C-odd fatty acids (fatty acids < 0.2% in abundance were not considered in this calculation). Lipids 12:0 2OH, 12:0 3OH, 16:0, 18:1 6c, 18:0 increased for naphthalene-exposed cells, while lipids 18:1 7c/15:0 iso 2OH, 17:0 cyclo, 18:1 7c, 19:0 cyclo decreased. Data from Northern hybridization using a naphthalene dixoygenase gene fragment cloned out from KK1 as a probe provided the information that naphthalene dioxygenase gene was more highly expressed in cells grown on phenanthrene than naphthalene.

Sequence Analysis and Functional Expression of the Structural and Regulatory Genes for Pyruvate Dehydrogenase Complex from Streptomyces seoulensis: Hwan Youn , Jangyul Kwak , Dong-Won Kim , Chang-Jin Lee , Yang-In Yim , Jin-Won Lee , In-Kwon Kim , Jeong-Il Yu , Hyung-Soon Yim , Sa-Ouk Kang; J. Microbiol. 2002;40(1):43-50.

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Abstract: A cluster of genes encoding the pyruvate dehydrogenase complex (PDC) of Streptomyces seoulensis, a Gram-positive bacterium, was cloned and sequenced. The genes of S. seoulensis consist of four open reading frames. The first gene, lpd, which encodes a lipoamide dehydrogenase, is followed by pdhB encoding a dihydrolipoamide acetyltransferase (E2p), pdhR, a regulatory gene, and pdhA encoding a pyruvate dehydrogenase component (E1p). E1p had an unusual homodimeric subunit, which has been known only in Gram-negative bacteria. S. seoulensis E2p contains two lipoyl domains like those of humans and Streptococcus faecalis. The pdhR gene appears to be clustered with the structural genes of S. seoulensis PDC. The PdhR-overexpressed S. seoulensis showed growth retardation and the decrease of E1p, indicating that PdhR regulates the function of PDC by repressing the expression of E1p. A strain of Streptomyces lividans overexpressing S. seoulensis PdhR showed a significant decrease in the level of actinorhodin, implying a regulatory role for Streptomyces PDC in antibiotic biosynthesis.

Heavy Metal Biosorption and its Significance to Metal Tolerance of Streptomycetes: Jae-young Rho , Jae-heon Kim; J. Microbiol. 2002;40(1):51-54.

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Abstract: Heavy metal adsorptions of four streptomycetes were compared with each other. Among the test strains, Streptomyces viridochromogenes showed the most efficient metal binding activity, which was carried out by cell wall as well as freeze-dried mycelium. An order of adsorption potential (zinc > copper > lead > cadmium) was observed in single metal reactions, whereas this adsorption order was disturbed in mixed-metal reactions. The metal adsorption reactions were very fast, pH dependent and culture age-independent, suggestive of a physico-chemical reaction between cell wall components and heavy metal ions. The metal tolerant stains presented the weakest adsorbing activity, indicating that the metal biosorption was not the basis of the metal tolerance.

Diversity of Yeasts Associated with Natural Environments in Korea: Soon Gyu Hong , Kang Hyun Lee , Kyung Sook Bae; J. Microbiol. 2002;40(1):55-62.

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Abstract: Biodiversity of yeasts in various natural environments including soils, swamps and plants was investigated. By molecular identification methods based on the partial sequences of 26S rDNA, 69 isolates were assigned to 44 taxa including 27 known species. The remaining 17 taxa could potentially form new species. All of them were classified into Ascomycota, Hymenomycetes, Urediniomycetes and Ustilaginomycetes. Ascomycetous and ustilaginomycetous yeasts were generally isolated from flower samples, and hymenomycetous and urediniomycetous yeasts were generally isolated from soil samples. Distribution of yeast groups exhibited geographical variation. Yeast biodiversity of root soil also varied according to the associated plant species.

Isolation of Norfloxacin Resistant Escherichia coli from the Han River and Characterization of Resistance Mechanism: Yoosun Jung , Hyunjin Hong , Hyeran Nam , Yeonhee Lee; J. Microbiol. 2002;40(1):63-69.

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Abstract: A total of twenty-five norfloxacin resistant Escherichia coli were isolated from Joongrang-chun stream, a branch of the Han River in Seoul, Korea from May to July in 2000 and their norfloxacin resistance mechanism was characterized for target site mutation, permeability, and efflux pump. Fourteen isolates contained the same three mutations, Ser83->Leu and Asp87->Asn in GyrA and Ser90->Ile in ParC. Six isolates had Ser83->Leu and Asp87->Tyr in GyrA and Ser80->Ile in ParC while one isolate had Ser83->Leu and Val103->Ala in GyrA and Ser80->Ile in ParC. Two isolates had mutation(s) in GyrA without any mutation in ParC. Two isolates had Ser80->Arg in ParC instead of the commonly found Ser80->Ile. Every norfloxacin resistant isolate had an efflux system but the correlation between the efflux activity and MIC was not observed. The amount of OmpF for norfloxacin permeability decreased in resistant isolates compared to the susceptible strains. When amplified polymorphic DNA (RAPD) and pulse field gel electrophoresis (PFGE) were performed, these isolates showed no similarity to each other or clinical isolates.

Evaluation of Several Parameters of in situ Polymerase Chain Reaction (ISPCR) to Reduce the Leakage of Amplificants from Cells: Jae Yung Lee , Chung-Kyoon Auh , George W. Jordan; J. Microbiol. 2002;40(1):70-76.

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Abstract: Proviral DNAs from HIV-1-infected CD4+ T cells (Molt/LAV cells) were amplified and detected in infected individual cells using polymerase chain reaction and in situ hybridization. In this in situ PCR, three parameters were considered to achieve effective amplification and retention of amplificants inside the cells by making high molecular weight PCR products intracellularly, forming agarose matrix against the cells, and maintaining the appropriate PCR temperature profile. Over the cycles of amplification, tailed primers with complementary overhanging sequences at their 5 sides manufactured high molecular weight products by using short primary products as a repeating unit. Agarose matrix could prevent the diffusion of the amplificants from the cells. Use of Thermanox coverslip inside the PCR tube offered target cells a similar temperature profile to that of conventional PCR in solution.

A Recombinant Human GM-CSF Protein Expressed as an Inclusion form in Escherichia coli Stimulates Colony Formation and Cell Proliferation in vitro: Ah Young Lee , Jin-Kyoo Kim , Hye Kyung Chung , Eun Kyong Bae , Jung Suk Hwang , Chung Won Cho , Dong Seok Lee , Jae Yong Han , Choon-Taek Lee , Soon-; J. Microbiol. 2002;40(1):77-81.

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Abstract: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hematopoietic growth factor involved in the development of myeloid cells from bone marrow, and an activator of mature myeloid cells functioning in a variety of antimicrobial and inflammatory responses. Recently, recombinant GM-CSF is increasingly under clinical study for treatment of various diseases including cancer, infectious diseases and hematopoietic diseases as well as for an immune response modulator. In this study, we constructed a recombinant human GM-CSF (rhGM-CSF) expression plasmid with a pelB leader sequence and His.Tag under T7 promoter control. The expression construct was shown to produce a recombinant protein of 20 kDa in the 8M urea preparation, indicating the rhGM-CSF may be expressed as an insoluble inclusion form. The 20 kDa recombinant protein in 8M urea was transformed into the water-soluble form by dialysis against PBS buffer (phosphate buffered saline). The soluble rhGM-CSF protein was shown to stimulate colony formation and cell proliferation in vitro, indicating that the rhGM-CSF could be refolded into its native form to show colony stimulating activity.

Growth of Issatchenkia orientalis in Aerobic Batch and Fed-batch Cultures: Hyung Tai Shin , Yoo Beom Lim , Jong Ho Koh , Jong Yun Kim , Soon Young Baig , Jae Heung Lee; J. Microbiol. 2002;40(1):82-85.

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Abstract: The aerobic batch growth of Issatchenkia orientalis DY252 with glucose and fructose medium was investigated at 32 C and pH 5.0. Aerobic ethanol production was evident with yeast I. orientalis. A diauxic lag of about 1 h between growth on glucose and growth on ethanol during batch culture was observed. However, no diauxic growth occurred with fructose. As the incubation temperature was increased from 32 to 39 C, viability at the end of each batch culture declined significantly, from 93 to 43%. Unlike the effect of temperature, viability was not greatly affected by incubation pH, and cell yield values in a range of 0.45-0.48 were obtained. In order to overcome overflow metabolism, a fed-batch culture under glucose limitation was carried out. Compared with aerobic batch culture, about 10% improvement in cell yield was achieved with a fed-batch culture in optimal conditions.

Three Separate Pathways for the Initial Oxidation of Limonene, Biphenyl, and Phenol by Rhodococcus sp. Strain T104: Dockyu Kim , Min Jung Park , Sung-Cheol Koh , Jae-Seong So , Eungbin Kim; J. Microbiol. 2002;40(1):86-89.

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Abstract: Rhodococcus sp. strain T104, which is able to grow on either biphenyl or limonene, was found to utilize phenol as sole carbon and energy sources. Furthermore, T104 was positively identified to possess three separate pathways for the degradation of limonene, phenol, and biphenyl. The fact that biphenyl and limonene induced almost the same amount of catechol 1,2-dioxygenase activity indicates that limonene can induce both upper and lower pathways for biphenyl degradation by T104.

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