Journal of Microbiology

Volume 41(4); December 2003

Plant Growth Promotion in Soil by Some Inoculated Microorganisms: Jong-Soo Jeon , Sang-Soo Lee , Hyoun-Young Kim , Tae-Seok Ahn , Hong-Gyu Song; J. Microbiol. 2003;41(4):271-276.

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Abstract: The inoculation of some microorganisms into a microcosm containing soil from a barren lakeside area at Lake Paro in Kangwon-do enhanced plant growth significantly. The direct and viable counts of soil bacteria and soil microbial activities measured by electron transport system assay and fluorescein diacetate hydrolysis assay were higher in inoculated soil. The plant growth promoting effect of this inoculation may be caused by phytohormone production and the solubilization of insoluble phosphates by the inoculated bacteria. Three inoculated strains of Pseudomonas fluorescens produced several plant growth promoting phytohormones, including indole-3-acetic acid (auxin), which was confirmed by thin layer chromatography and GC/MS. P. fluorescens strain B16 and M45 produced 502.4 and 206.1 mg/l of soluble phosphate from Ca3(PO4)2 and hydroxyapatite, respectively. Bacillus megaterium showed similar solubilization rates of insoluble phosphates to those of Pseudomonas spp. We believe that this plant growth promoting capability may be used for the rapid revegetation of barren or disturbed land.

Characteristics of Several Bacterial Isolates Capable of Degrading Chloroaliphatic Compounds via Hydrolytic Dechlorination: Ji-Sook Song , Dong-Hun Lee , Kyoung Lee , Chi-Kyung Kim; J. Microbiol. 2003;41(4):277-283.

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Abstract: Haloaliphatic hydrocarbons have been widely used as solvents and ingredients of pesticides and herbicides. However, when these compounds contaminate the environment, they can be very hazardous to animals and humans because of their potential toxicity and carcinogenicity. Therefore, lots of studies have been made for microbial degradation of those pollutant chemicals. In this study, 11 bacterial strains capable of degrading 1,2-dichloroethane (1,2-DCA), 2-chloropropionic acid (2-CPA), 2,3-dichloropropionic acid (2,3-DCPA), and 2-monochloroacetic acid (2-MCA) by hydrolytic dechlorination under aerobic conditions were isolated from wastewaters and rice paddy soil samples. Their morphological and biochemical characteristics and their degradation capabilities of haloaliphatic hydrocarbons were examined. On the basis of the 16S rDNA sequences, 8 different kinds of microbial species, including Pseudomonas plecoglossicida, Xanthobacter flavus, Ralstonia eutropha, were identified. All of the isolated strains can degrade MCA. In particular, strains UE-2 and UE-15 degraded 1,2-DCA, and strain CA-11 degraded 2,3-DCPA, which are hardly degraded by other strains.

Degradation of Cholesterol by Bacillus subtilis SFF34 in Flatfish during Fermentation: Kwan-Pil Kim , In-Koo Rhee , Heui-Dong Park; J. Microbiol. 2003;41(4):284-288.

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Abstract: Bacillus subtilis SFF34 degrading cholesterol was applied to reduce residual cholesterol content in fermented flatfish. When the bacterial cells were inoculated as a start culture, a maximal level (1.7 U/g) of cholesterol oxidase was obtained after 10 days, which was two times higher than that (0.8 U/g) without inoculation. Residual cholesterol contents with and without inoculation of the cells were 0.5 mg/g and 0.8 mg/g after 12 days of fermentation, respectively. Cholesterol derivatives including cholesterol- 5?, 6?-epoxide, 4-cholesten-3-one and 7?-hydroxycholesterol were detected in raw flatfish as well as fermented flatfish. Campesterol and 25-hydroxycholesterol were detected only after fermentation. However, no significant differences in their contents were observed regardless of inoculation.

Streptomyces griseus Trypsin (SGT) Has Gelatinase Activity and Its Proteolytic Activity Is Enhanced by Manganese: Won-Jae Chi , Yoon-Hee Kim , Jong-Hee Kim , Dae-Kyung Kang , Sang-Soon Kang , Joo-Won Suh , Soon-Kwang Hong; J. Microbiol. 2003;41(4):289-294.

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Abstract: Gelatinase is a proteolytic enzyme that hydrolyzes gelatin. Gelatinolytic activity was detected from culture broths of Streptomyces griseus IFO13350 and HH1 by paper disc assays on 0.5% agar plates containing 1% gelatin. The concentrated extracellular protein from the S. griseus was analyzed by SDS polyacrylamide gel, and two proteins, with molecular weights of 30 and 28 kDa, respectively, were identified to have gelatinase activity by gelatin zymography. The protein with a molecular weight of 28 kDa was confirmed to be S. griseus trypsin (SGT). The effects of metal ions and metal chelators on the protease activity of the SGT were studied. Of the metal ions tested, only manganese was found to enhance the protease activity, 2.6 times, however, Co_2^+, Cu_2^+, and Zn_2^+, and metal chelators, such as EDTA and EGTA, inhibited the SGT activity. When the protease activity of the SGT was measured at various pHs, in the presence of 5 mM MnCl_2, its highest activity was at pH 11.0, whereas only 60% of the maximum activity was observed between pHs 4.0 and pH 6.0, and almost 80% activity between pHs 7.0 to pH 10.0. The protease activity was measured at various temperatures in the presence of 5 mM MnCl_2. The SGT was found to be stable up to 60℃ for 30 min, while only 16% of the enzyme activity remained at 60℃, and at 80℃ almost all the activity was lost. The optimal temperature for the protease activity was 50℃.

Functional Implications in Apoptosis by Interferon Inducible Gene Product 1-8D, the Binding Protein to Adenovirus Preterminal Protein: Insil Joung , Jeffrey A. Engler; J. Microbiol. 2003;41(4):295-299.

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Abstract: Adenovirus (Ad) precursor to the terminal protein (pTP) plays an essential roles in the viral DNA replication. Ad pTP serves as a primer for the synthesis of a new DNA strand during the initiation step of replication. In addition, Ad pTP forms organized spherical replication foci on the nuclear matrix (NM) and anchors the viral genome to the NM. Here we identified the interferon inducible gene product 1-8D (Inid) as a pTP binding protein by using a two-hybrid screen of a HeLa cDNA library. Of the clones obtained in this assay, nine were identical to the Inid, a 13-kDa polypeptide that shares homology with genes 1-8U and Leu-13/9-27, most of which have little known functions. The entire open reading frame (ORF) of Inid was cloned into the tetracycline inducible expression vector in order to determine the biological functions related with adenoviral infection. When Inid was introduced to the cells along with adenoviruses, fifty to sixty percent of Ad-infected cells expressing Inid had rounded morphology, which was suggestive of apoptosis. Results from the terminal deoxynucleotidyl transferase (TdT) and DNA fragmentation assays confirmed that Inid induces apoptosis in Ad-infected or in uninfected cells. The Inid binding to pTP may target the cell for apoptotic destruction as a host defense mechanism against the viral infection.

Improved Inhibition of Human Immunodeficiency Virus Type 1 Replication by Intracellular Co-overexpression of TAR and RRE Decoys in Tandem Array: Seong-Wook Lee; J. Microbiol. 2003;41(4):300-305.

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Abstract: Intracellular expression of RNA decoys, such as TAR or RRE decoy, has been previously shown to protect immune cells from human immunodeficiency virus type 1 (HIV-1) replication by inhibiting the binding of the HIV-1 regulatory protein to the authentic HIV RNA sequence. However, HIV-1 challenge experiments of primary human T cells, which express the RNA decoy, demonstrated that the cells were only transiently protected, and hence, more improved protocols for HIV-1 inhibition with the RNA decoys need to be developed. In this report, in order to develop a more effective RNA decoy, we analyzed and compared the ability of a series of RNA decoy derivatives in inhibiting HIV-1 replication in CEM cells. Using an improved tRNA cassette to express high levels of RNA decoy transcripts in cells, we found that co-expression of both TAR and RRE decoys, in the form of an aligned sequence in a single transcription cassette, much more potently blocked cells from HIV-1 than the expression of only one kind of RNA decoy. This observation will have an important implication for experiments involving optimization of clinical applications in RNA decoy-based gene therapy against HIV-1.

Isolation and Characterization of a Lymantria dispar Multinucleocapsid Nucleopolyhedrovirus Isolate in Korea: Hee Jin Shim , Jong Yul Roh , Jae Young Choi , Ming Shun Li , Soo Dong Woo , Hyun Woo Oh , Kyung Saeng Boo , Yeon Ho Je; J. Microbiol. 2003;41(4):306-311.

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Abstract: In Korea, a Lymantria dispar multinucleocapsid nucleopolyhedrovirus, LdMNPV-NM, was isolated and characterized from dead L. dispar larvae. The polyhedra of LdMNPV-NM were irregularly shaped with a diameter of 1.62?0.33 ?m. Numerous virions comprised of the multinucleocapsid were evident in the electron microscopic examination of the polyhedra cross sections. These polyhedra were composed of a major protein of 30 kDa. The restriction enzyme digestion patterns of LdMNPV-NM showed that this isolate had some different fragments from those of the Gypchek팜?LdMNPV isolate, although their overall profiles were similar. The deduced amino acid sequence of the enhancin gene of LdMNPV-NM showed differences when compared to previously reported enhancin genes of other LdMNPV strains. These results suggested that the LdMNPV-NM isolate from Korea was a new NPV strain and had a new enhancin gene.

Genotyping of Six Pathogenic Vibrio Species Based on RFLP of 16S rDNAs for Rapid Identification: Young-Jun Yoon , Kyung-Hwan Im , Young-Hwan Koh , Seong-Kon Kim , Jung-Wan Kim; J. Microbiol. 2003;41(4):312-319.

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Abstract: In an attempt to develop a method for rapid and accurate identification of six Vibrio species that are clinically important and most frequently detected in Korea, 16S rDNA restriction fragment length polymorphism (RFLP) of Vibrio type strains, as well as environmental isolates obtained from the Korean coastal area, was analyzed using ten restriction endonucleases. Digestion of the 16S rDNA fragments amplified by polymerase chain reaction (PCR) with the enzymes gave rise to 2~6 restriction patterns for each digestion for 47 Vibrio strains and isolates. An additional 2~3 restriction patterns were observed for five reference species, including Escherichia coli, Aeromonas hydrophila, A. salmonicida, Photobacterium phosphoreum, and Plesiomonas shigelloides. A genetic distance tree based on RFLP of the bacterial species correlated well with that based on 16S rDNA sequences. The very small 16S rDNA sequence difference (0.1%) between V. alginolyticus and V. parahaemolyticus was resolved clearly by RFLP with a genetic distance of more than 2%. RFLP variation within a species was also detected in the cases of V. parahaemolyticus, V. proteolyticus, and V. vulnificus. According to the RFLP analysis, six Vibrio and five reference species were assigned to 12 genotypes. Using three restriction endonucleases to analyze RFLP proved sufficient to identify the six pathogenic Vibrio species.

Rapid Identification of Vibrio vulnificus in Seawater by Real-Time Quantitative TaqMan PCR: Hye-Young Wang , Geon-Hyoung Lee; J. Microbiol. 2003;41(4):320-326.

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Abstract: In order to identify Vibrio vulnificus in the Yellow Sea near Gunsan, Korea during the early and late summers, the efficiency of the real-time quantitative TaqMan PCR was compared to the efficiency of the conventional PCR and Biolog identification system^TM. Primers and a probe were designed from the hemolysin/cytolysin gene sequence of V. vulnificus strains. The number of positive detections by real-time quantitative TaqMan PCR, conventional PCR, and the Biolog identification system from seawater were 53 (36.8%), 36 (25%), and 10 strains (6.9%), respectively, among 144 samples collected from Yellow Sea near Gunsan, Korea. Thus, the detection method of the real-time quantitative TaqMan PCR assay was more effective in terms of accuracy than that of the conventional PCR and Biolog system. Therefore, our results showed that the real-time TaqMan probe and the primer set developed in this study can be applied successfully as a rapid screening tool for the detection of V. vulnificus.

The Genetic Diversity Analysis of the Bacterial Community in Groundwater by Denaturing Gradient Gel Electrophoresis (DGGE): Hong-Bum Cho , Jong-Kwang Lee , Yong-Keel Choi; J. Microbiol. 2003;41(4):327-334.

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Abstract: This study employed two PCR-based 16S rDNA approaches, amplified rDNA restriction analysis (ARDRA) and denaturing gradient gel electrophoresis (DGGE), to characterize the bacterial community structure in groundwater. Samples were collected from groundwater for the use by private residences, as well as for industrial and agricultural purposes, in Ansan City. Each PCR product was obtained by PCR with eubacteria 16S rDNA and variable V3 region specific primer sets. After amplification, the 16S rDNA PCR products were digested with 4-base site specific restriction endonucleases, and the restriction pattern analyzed. The genetic diversity and similarity of the groundwater bacterial community was analyzed by eubacteria universal primer sets for the amplification of variable V3 regions of the bacterial 16S rDNA. The result of the bacterial community analysis, by ARDRA and DGGE, revealed the same pattern. The highest diversity was found in groundwater from site G1, which was used in residences. In the DGGE profile, a high intensity band was sequenced, and revealed to be Pseudomonas sp. strain P51.

A Recombinant Human [alpha]_1-Antitrypsin Variant, M_malton, Undergoes a Spontaneous Conformational Conversion into a Latent Form: Chan-Hun Jung , Hana Im; J. Microbiol. 2003;41(4):335-339.

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Abstract: Many genetic variants of [alpha]_1-antitrypsin have been associated with early onset emphysema and liver cirrhosis. However, the detailed structural basis of pathogenic [alpha]_1-antitrypsin molecules is rarely known. Here we found that a recombinant M_malton variant (Phe52-deleted) lost inhibitory activity by spontaneous conformational conversion into a more stable, inactive form under physiological conditions. Biochemical and spectroscopic data suggested that the variant converts into a reactive center loop-inserted conformation, resembling the latent form of plasminogen activator inhibitor-1.

Replacement of Thymidine Phosphorylase RNA with Group I Intron of Tetrahymena thermophila by Targeted Trans-Splicing: Young-Hee Park , Heung-Su Jung , Byung-Su Kwon , Seong-Wook Lee; J. Microbiol. 2003;41(4):340-344.

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Abstract: The group I intron from Tetrahymena thermophila has been demonstrated to employ splicing reactions with its substrate RNA in the trans configuration. Moreover, we have recently shown that the transsplicing group I ribozyme can replace HCV-specific transcripts with a new RNA that exerts anti-viral activity. In this study, we explored the potential use of RNA replacement for cancer treatment by developing trans-splicing group I ribozymes, which could replace tumor-associated RNAs with the RNA sequence attached to the 3' end of the ribozymes. Thymidine phosphorylase (TP) RNA was chosen as a target RNA because it is known as a valid cancer prognostic factor. By performing an RNA mapping strategy that is based on a trans-splicing ribozyme library, we first determined which regions of the TP RNA are accessible to ribozymes, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. Next, we assessed the ribozyme activities by comparing trans-splicing activities of several ribozymes that targeted different regions of the TP RNA. This assessment was performed to verify if the target site predicted to be accessible is truly the most accessible. The ribozyme that could target the most accessible site, identified by mapping studies, was the most active with high fidelity in vitro. Moreover, the specific trans-splicing ribozyme reacted with and altered the TP transcripts by transferring an intended 3' exon tag sequence onto the targeted TP RNA in mammalian cells with high fidelity. These results suggest that the Tetrahymena ribozyme can be utilized to replace TP RNAs in tumors with a new RNA harboring anti-cancer activity, which would revert the malignant phenotype.

Calcite Production by Bacillus amyloliquefaciens CMB01: Young Nam Lee; J. Microbiol. 2003;41(4):345-348.

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Abstract: The bio-mediated production of calcite crystals by calcinogenic bacteria has great applicable value for the restoration of deteriorated calcareous monuments, because of its high purity and coherency. An investigation of the conditions for calcite production by an alkalophilic Bacillus amyloliquefaciens CMB01 strain was made. Optimal calcite precipitation occurred when the bacterium was cultured at pH 8.0 and 30℃, and in B4 medium that consisted of 0.4% yeast extract, 0.5% glucose, and 1.5% calcium acetate. Calcium ion of the bacterially induced calcite was analyzed by an inductively coupled plasma (ICP) spectrophotometer. Optical and scanning electron microscopy (SEM) of the calcite revealed a typical rombohedral polycrystalline structure.

Plant Terpene-Induced Expression of Multiple Aromatic Ring Hydroxylation Oxygenase Genes in Rhodococcus sp. Strain T104: Byung-Hyuk Kim , Eun-Taex Oh , Jae-Seong So , Yeonghee Ahn , Sung-Cheol Koh; J. Microbiol. 2003;41(4):349-352.

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Abstract: Recent studies have shown that some of the PCB (polychlorinated biphenyl)-degraders are able to effectively degrade PCB in the presence of monoterpenes, which act as inducers for the degradation pathway. Rhodococcus sp. T104, an effective PCB degrader, has been shown to induce the degradation pathway by utilizing limonenes, cymenes, carvones, and pinenes as sole carbon sources which can be found in the natural environment. Moreover, the strain T104 proved to possess three separate oxidation pathways of limonene, biphenyl, and phenol. Of these three, the limonene can also induce the biphenyl degradation pathway. In this work, we report the presence of three distinct genes for aromatic oxygenase, which are putatively involved in the degradation of aromatic substrates including biphenyl, limonene, and phenol, through PCR amplification and denaturing gradient gel electrophoresis (DGGE). The genes were differentially expressed and well induced by limonene, cymene, and plant extract A compared to biphenyl and/or glucose. This indicates that substrate specificity must be taken into account when biodegradation of the target compounds are facilitated by the plant natural substrates.

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