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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Journal of Microbiology and Biotechnology
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Journal DOI :
The Korean Society for Applied Microbiology and Biotechnology
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Volume & Issues
Volume 13, Issue 6 - Dec 2003
Volume 13, Issue 5 - Oct 2003
Volume 13, Issue 4 - Aug 2003
Volume 13, Issue 3 - Jun 2003
Volume 13, Issue 2 - Apr 2003
Volume 13, Issue 1 - Feb 2003
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Membrane Hyperpolarization Increases cAMP to Induce the Initiation of Sperm Motility in Salmonid Fishes, Rainbow Trout and Masu Salmon
Kho, Kang-Hee ; Morisawa, Masaaki ; Choi, Kap-Seong ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 833~840
Sperm motility of salmonid fishes is suppressed by external
and initiated by decrease of
concentration surrounding the sperm. It was shown that the decrease in external
concentration induced not only the initiation of sperm motility, but also hyperpolarization of the plasma membrane and synthesis of cAMP in the sperm of rainbow trout, steelhead trout, and masu salmon. Inhibitors of
channels, especially voltage-dependent
channels, inhibited these three reactions, and the inhibitions were abolished by subsequent addition of a
ionophore, valinomycin, suggesting that
efflux through the
channel contributes to rapid changes in the membrane potential of sperm and cAMP synthesis, thereby resulting in the initiation of sperm motility of salmonid fishes.
Tissue Engineering of Smooth Muscle under a Mechanically Dynamic Condition
Kim, Byung-Soo ; Jeong, Sung-In ; Cho, Seung-Woo ; Nikolovski, Janeta ; Mooney, David-J. ; Lee, Soo-Hong ; Jeon, O-Ju ; Kim, Tae-Wan ; Lim, Sang-Hyun ; Hong, Yoo-Sun ; Choi, Cha-Yong ; Lee, Young-Moo ; Kim, Soo-Hyun ; Kim, Young-Ha ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 841~845
In order for engineered tissues to find clinical utility, the engineered tissues must function appropriately. However, smooth muscle (SM) tissues engineered in vitro with a conventional tissue engineering technique may not exhibit contractile functions, because smooth muscle cells (SMCs) cultured in vitro typically revert from a contractile, differentiated phenotype to a synthetic, nondifferentiated phenotype and lose their ability to contract. SMCs in vivo typically reside in mechanically dynamic environments. We hypothesized that cyclic mechanical stretch induces the features of SMCs in in vitro engineered tissues to be similar to those of SMCs in native tissues. To test the hypothesis, aortic SMCs were seeded onto elastic, three-dimensional scaffolds and cultured in vitro under a cyclic mechanical stretching condition for 4 weeks. A significant cell alignment in a direction parallel to the cyclic stretching direction was found in the SM tissues exposed to cyclic stretching. The cellular alignment and alignment direction were consistent with those of native vascular SM tissues, in which SMCs in vivo align in the radial direction (parallel to stretching direction). In control tissues (SM tissues engineered without stretching), cells randomly aligned. The expression of SM
and SM myosin heavy chain, phenotypic markers of SMCs in a contractile state, was upregulated in the stretched tissues by 2.5- and 2.0-fold, respectively, compared to SMCs in the control tissues. The cellular features of alignment and contractile phenotype of SMCs in the SM tissues engineered under a mechanically dynamic environment could allow the engineered SM tissues to exhibit contractile functions.
Differentiation of Actinomycete Genera Based on Partial rpoB Gene Sequences
Kim, Bum-Joon ; Koh, Young-Hwan ; Chun, Jong-Sik ; Kim, Chang-Jin ; Lee, Seung-Hyun ; Cho, Moon-Jae ; Hyun, Jin-Won ; Lee, Keun-Hwa ; Cha, Chang-Yong ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 846~852
rpoB DNAs (279 bp) from 34 species of 5 actinomycete genera were sequenced and a phylogenetic tree was constructed based on the sequences obtained. The genera were clearly differentiated in the rpoB tree, forming clades specific to their respective genus. In addition, 2 signature amino acid residues specific to Streptomyces were found in a multiple alignment of the deduced amino acid sequences. To empirically confirm that this rpoB gene analysis system could be used to differentiate actinomycete isolates, the proposed system was used to identify 16 actinomycete isolates from Jeju Island. All isolates were successfully differentiated into the genera Streptomyces and Micromonospora. Accordingly, this is the first report that an rpoB sequence analysis has been effectively used to differentiate actinomycete strains at the genus level.
Inhibition of Helicobacter pylori Adhesion by Acidic Polysaccharide Isolated from Artemisia capillaris
Woo, Jeung-S. ; Ha, Byung-H. ; Kim, Tae-G. ; Lim, Yoon-Gho ; Kim, Kyung-H. ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 853~858
Helicobacter pylori specifically adhere to host cells through a number of putative receptors and ligands, mainly based on carbohydrate-protein interactions. Polysaccharide fractions isolated from the leaves of Artemisia capillaris showed different inhibitory activities against H. pylori adhesion by using hemagglutination assay. Among these fractions, an acidic polysaccharide fraction FlA showed highly effective inhibitory activity, and its minimum inhibition concentration was 0.63 mg/ml. The inhibition results by the hemagglutination assay were consistent with those obtained by the enzymelinked glycosorbent assay, which was developed by the conjugation of horseradish peroxidase with fetuin, a sialic acid-containing glycoprotein which was specific to H. pylori adhesion. FlA contained the highest carbohydrate content among polysaccharide fractions, and no protein was detectable when further purified by gel filtration FPLC. Sugar composition analysis using GC revealed the highest amount of galacturonic acid among sugars, which suggests that FlA contains essentially acidic polysaccharides. Our data suggest that acidic polysaccharides may play an important role in the inhibition of H. pylori adhesion to host cells.
Endophytic Bacillus sp. CY22 from a Balloon Flower (Platycodon grandiflorum) Produces Surfactin Isoforms
Cho, Soo-Jeong ; Hong, Su-Young ; Kim, Jin-Young ; Park, Sang-Ryeol ; Kim, Min-Keun ; Lim, Woo-Jin ; Shin, Eun-Chule ; Kim, Eun-Ju ; Cho, Yong-Un ; Yun, Han-Dae ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 859~865
Surfactin is a mixture of cyclic lipopeptides built from variants of a heptapeptide and a
fatty acid produced by several strains of Bacillus sp. Surfactin isoforms produced by endophytic Bacillus sp. CY22 from a balloon flower were isolated and characterized. It was found that the purified surfactin had three isoforms with protonated masses of m/z 1,008, 1,022, and 1,036, and different structures in combination with Na, K, Ca ions using MALDI-TOF MS, ESI-MS/MS, and ICP MS, respectively. In the MS/MS analysis, the isolated surfactin had the identical amino acid sequence (LLVDLL) and hydroxy fatty acids (with 13 to 15 carbons in length), even though isolated from different Bacillus strains. The sfp22 gene, required for producing the surfactin, consisted of an open reading frame (ORF) of 675 bp encoding 224 amino acid residues with a signal peptide of 20 amino acids. The predicted amino acid sequence of sfp22 was very similar to that of Ipa-8.
Removal of Dimethyl Sulfide in Ceramic Biofilters Immobilized with Thiobacillus thioparus TK-m
Kim, Jong-Yeon ; Kim, Byung-Woo ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 866~871
Malodorous gas of dimethyl sulfide (DMS) was biologically oxidized to sulfate by Thiobacillus thioparus TK-m (DSM5368) immobilized in/on ceramic beads. More than 99.99％ of DMS removal efficiency was obtained in a ceramic-biofilter reactor of 3.91 when the feed concentrations were about 27.5 and 55.0 mg DMS/1 at
. However, the removal efficiency of the biofilter at above
decreased to 4.5 mg DMS/(lㆍmin) which was 85％ of that at
lacZ- and aph-Based Reporter Vectors for In Vivo Expression Technology
Baek, Chang-Ho ; Kim, Kun-Soo ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 872~880
Three vectors, pSG1, 2, and 3, which facilitate in vivo expression technology (IVET) in Gram-negative bacteria, were developed. Vectors pSG1and 2 are derivatives of ColE1, and pSG3 is a derivative of an R6K replicon. These vectors contain oriT sites that allow mobilization when the RK2 Tra functions are provided in trans. These vectors contain promoterless lacZ (pl-lacZ) and promoterless aph (pl-aph) transcriptionally fused together, which allow qualitative and quantitative measurements of the expression of genes in the genome of bacterial cells. pSG1 and 3 contain gentamicin-resistance genes, and pSG2 carries a streptomycin-/spectinomycin-resistance gene, allowing for selection of recombinants generated by a single crossover between a library fragment cloned into a pSG vector and the identical region in the genome of a bacterial species from which the library fragment originated. These vectors were successfully applied to the generation of random fusions at high rates in the genomes of four representative Gram-negative bacteria. In addition, the expression level of
and the degree of resistance to kanamycin in cells with fusions generated by these vectors were found to be linearly correlated, proving that these vectors can be used for IVET.
Involvement of Growth-Promoting Rhizobacterium Paenibacillus polymyxa in Root Rot of Stored Korean Ginseng
Jeon, Yong-Ho ; Chang, Sung-Pae ; Hwang, In-Gyu ; Kim, Young-Ho ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 881~891
Paenibacillus polymyxa is a plant growth-promoting rhizobacterium (PGPR) which can be used for biological control of plant diseases. Several bacterial strains were isolated from rotten roots of Korean ginseng (Panax ginseng C. A. Meyer) that were in storage. These strains were identified as P. polymyxa, based on a RAPD analysis using a P. polymyxa-specific primer, cultural and physiological characteristics, an analysis utilizing the Biolog system, gas chromatography of fatty acid methyl esters (GC-FAME), and the 16S rDNA sequence analysis. These strains were found to cause the rot in stored ginseng roots. Twenty-six P. polymyxa strains, including twenty GBR strains, were phylogenetically classified into two groups according to the ERIC and BOX-PCR analyses and 16S rDNA sequencing, and the resulting groupings systematized to the degrees of virulence of each strain in causing root rot. In particular, highly virulent GBR strains clustered together, and this group may be considered as subspecies or biovar. The virulence of the strains seemed to be related to their starch hydrolysis enzyme activity, but not their cellulase or hemicellulase activity, since strains with reduced or no starch-hydrolytic activity showed little or no virulence. Artificial inoculation of the highly virulent strain GBR-1 onto the root surfaces of Korean ginseng resulted in small brown lesions which were sunken and confined to the outer portion of the root. Ginseng root discs inoculated in vitro or two-year-old roots grown in soil drenched with the inoculum developed significant rot only when the inoculum density was
or more colony-forming units (CFU) per ml. These results suggest that P. polymyxa might induce ginseng root rot if their population levels are high. Based on these results, it is recommended that the concentration of P. polymyxa should be monitored, when it is used as a biocontrol agent of ginseng, especially in the treatment of stored roots.
High-Level Expression and Secretion of Bacillus pumilus Lipase B26 in Bacillus subtilis Chungkookjang
Lee, Mi-Hwa ; Song, Jae-Jun ; Choi, Yoon-Ho ; Hong, Seung-Pyo ; Rha, Eu-Gene ; Kim, Hyung-Kwoun ; Lee, Seung-Goo ; Poo, Har-Young ; Lee, Sang-Chul ; Seu, Young-Bae ; Sung, Moon-Hee ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 892~896
High-level expression of the lipase B26 gene from Bacillus pumilus was achieved using Bacillus subtilis Chungkookjang isolated from the Korean traditional fermented bean paste, Chungkookjang. For the secretory production of recombinant lipase B26 in a Bacillus host system, pLipB26 was constructed by ligating the lipase B26 gene into the recently designed Escherichia coli-Bacillus shuttle vector, pLipSM, and that was then transformed into B. subtilis Chungkookjang. Among the various vector, medium, and host combinations, B. subtilis Chungkookjang harboring the pLipB26 exhibited the highest lipase activity in PY medium, and B. subtilis Chungkookjang secreted two times more enzymes than B. subtilis DB 104 under the same condition. When B. subtilis Chungkookjang harboring the pLipB26 was cultured in a 5-1 jar-fermentor containing 21 of a PY medium, the maximum lipase activity (140 U/ml) and production yield (0.68 g/l) were obtained during the late exponential phase from a cell-free culture broth. Although B. subtilis Chungkookjang also secreted extracellular proteases at the late exponential phase, these results suggested the potential of B. subtilis Chungkookjang as a host for the secretory production of foreign proteins.
Phospholipase D in Guinea Pig Lung Tissue Membrane is Regulated by Cytosolic ARF Proteins
Chung, Yean-Jun ; Jeong, Jin-Rak ; Lee, Byung-Chul ; Kim, Ji-Young ; Park, Young-In ; Ro, Jai-Youl ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 897~905
Phospholipase D (PLD) and ADP-ribosylation factor (ARF) were partially purified on a series of column chromatography, and their biochemical properties were characterized to understand the regulatory mechanism of PLD activation by ARF protein in the antigen-induced immune responses in guinea pigs. Heparin Sepharose and high-Q Sepharose column chromatographies were used for the purification of PLD, and Sephadex G-25, DEAE Sephacel, Source 15 PHE (HIC), Superdex-75, and Uno-Q column chromatographies were used for the purification of ARF. The purified PLD and ARF proteins were identified with anti-rabbit PLD- or ARF-specific antibodies, showing about 64 or 85 kDa for the molecular mass of PLD and 29 or 35 kDa for the sizes of ARF. Partial cDNA of ARF3 was cloned by RT-PCR in guinea pig lung tissue and its nucleotides and amino acids were sequenced. Guinea pig ARF3 showed 92% of nucleotides sequence identity and 100% of amino acid sequence homology with human ARF3. The ARF-regulated PLD activity was measured in the oleate or ARFs-containing mixed lipid vesicles. The purified and recombinant ARF (rARF) activities were assessed with the
binding assay. The PLD activity was induced by oleate in a dose-dependent manner. The purified ARF and recombinant ARF3 increased PLD activity in guinea pig lung tissues. These data show that the activity of membrane-bound PLD can be regulated by the cytosolic ARF proteins, suggesting that ARF proteins in guinea pig lung can act as a regulatory factor in controlling the PLD activity in allergic reaction.
Functional Analysis of Spectinomycin Biosynthetic Genes from Streptomyces spectabilis ATCC 27741
Jo, You-Young ; Kim, Sun-Hee ; Yang, Young-Yell ; Kang, Choong-Min ; Sohng, Jae-Kyung ; Suh, Joo-Won ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 906~911
The function of genes related to spectinomycin biosynthesis (spcD, speA, speB, spcS2) from Streptomyces spectabilis ATCC 27741, a spectinomycin producer, was analyzed. Each gene was subcloned from a spectinomycin biosynthetic gene cluster and overexpressed in E. coli BL21 (DE3) using pET vector. After incubating each purified protein with its possible substrates, the final products were analyzed using high-performance liquid chromatography (HPLC). From these results, spcD, speA, and speB have been identified to be dTDP-glucose synthase, myo-inositol monophosphatase, and myo-inositol dehydrogenase, respectively. In addition, the results suggest that the spcS2 gene product functions downstream of the speB gene product in the biosynthetic pathway of spectinomycin. Taken together, the present study elucidates the early steps of the biosynthetic pathway for 6-deoxyhexose (6-DOH) part (actinospectose) and aminocyclitol part (actinamine) of spectinomycin.
Detection of Mitotic Centromere-Associated Kinesin (MCAK) During Cell-Cycle Progression of Human Jurkat T Cells Using Polyclonal Antibody Raised Against Its N- Terminal Region Overexpressed in E. coli
Jun, Do-Youn ; Rue, Seok-Woo ; Kim, Byung-Woo ; Kim, Young-Ho ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 912~918
Mitotic centromere-associated kinesin (MCAK), which is a novel kinesin with a central motor domain, is believed to playa role in mitotic segregation of chromosome during the M phase of the cell cycle. In the present study, it is shown that a rabbit polyclonal antibody has been produced using the N-terminal region (187 aa) of human MCAK expressed in E. coli as the antigen. To express the N-terminal region in E. coli, the MCAK cDNA fragment encoding N-terminal 187 aa was obtained by PCR and was then inserted into the pET 3d expression vector. Molecular mass of the N-terminal region overexpressed in the presence of IPTG was 23.2 kDa on SDS-PAGE, and the protein was insoluble and mainly localized in the inclusion body that could be easily purified from the other cellular proteins. The N-terminal region was purified by electro-elution from the gel after the inclusion body was resolved on the SDS-PAGE. The antiserum obtained after tertiary immunization with the purified protein specifically recognized HsMCAK when subjected to Western blot analysis, and showed a fluctuation of the protein level during the cell cycle of human Jurkat T cells. Synchronization of the cell-cycle progression required for recovery of cells at a specific stage of the cell cycle was performed by either hydroxyurea or nocadazole, and subsequent release from each blocking at 2, 4, and 7 h. Northern and Western analyses revealed that both mRNA and protein of HsMCAK reached a maximum level in the S phase and declined to a basal level in the G1 phase. These results indicate that a polyclonal antibody raised against the N-terminal region (187 aa) of HsMCAK, overexpressed in E. coli, specifically detects HsMCAK (81 kDa), and it can analyze the differential expression of HsMCAK protein during the cell cycle.
Effect of Lactobacillus fermentum MG590 on Alcohol Metabolism and Liver Function in Rats
Kim, Ji-Hyun ; Kim, Hyun-Jin ; Son, Jeong-Hwa ; Chun, Ho-Nam ; Yang, Jin-Oh ; Choi, Sung-Jin ; Paek, Nam-Soo ; Choi, Gyoung-Hoon ; Kim, Sung-Koo ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 919~925
Alcohol consumption has numerous health consequences for the human body. For example, heavy drinking on a daily basis causes liver diseases, and certain products such as acetaldehyde produced from alcohol metabolism are more toxic than alcohol itself. Accordingly, the current study evaluated the role of Lactobacillus fermentum MG590 to enhance the removal of the toxic effect of alcohol in alcohol metabolism. The maximum activities of the alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) by L. fermentum MG590 were observed after 6 h of culture. The production of ADH and ALDH by L. fermentum MG590 was also confirmed by SDS-PAGE. Six hours after the addition of alcohol to a culture broth of L. fermentum MG590, the alcohol concentration decreased from 7.5 to 2.7％. From an in vitro evaluation based on hepatocytes, the viability of hepatocytes in a medium containing alcohol and the cytosol of L. fermentum MG590 was higher than that in a medium containing only alcohol. From an in vivo test using SD rats fed a 22％ alcoholic drink, the blood alcohol concentration (BAC), glutamic-oxaloacetic transaminase (GOT), and glutamic-pyruvic transaminase (GPT) in the rats fed a medium containing L. fermentum MG590 were lower than those in the rats fed a medium containing only the alcohol drink. These results demonstrate that the ADH and ALDH produced by L. fermentum MG590 play an important role in detoxicating alcohol in vivo. Therefore, a fermentation broth of L. fermentum MG590 could be used as an effective alcohol detoxification drink.
Characterization of Humanized Antibody Produced by Apoptosis-Resistant CHO Cells under Sodium Butyrate-Induced Condition
Kim, No-Soo ; Chang, Kern-Hee ; Chung, Bo-Sup ; Kim, Sung-Hyun ; Kim, Jung-Hoe ; Lee, Gyun-min ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 926~936
Overexpression of human Bcl-2 protein in recombinant Chinese hamster ovary (rCHO) cells producing humanized antibody (SH2-0.32) considerably suppressed sodium butyrate (NaBu)-induced apoptosis during batch culture by using commercially available serum-free medium, which extended the culture longevity. Due to the extended culture longevity provided by the anti-apoptotic effect of Bcl-2 overexpression, the final antibody concentration of 14C6-bcl-2 culture (Bcl-2 high producer,
) was 2 times higher than that of the
culture (cells transfected with bcl-2-deficient plasmid,
) in the presence of NaBu. To determine the effect of NaBu/Bcl-2 overexpression on the molecular integrity of protein products, antibodies purified from 14C6-bcl-2 and
cultures in the presence of NaBu were characterized by using various molecular assay systems. For comparison, antibody purified from the parental rCHO cell culture (SH2-0.32) in the absence of NaBu was also characterized. No significant changes in molecular weight of antibodies could be observed by SDS-PAGE. From GlycoSep-N column analysis, it was found that the core oligosaccharide structure (
) was not affected by NaBu/Bcl-2 overexpression, while the microheterogeneity of N-linked oligosaccharide structure was slightly affected. Compared with the antibody produced in the absence of NaBu, the proportion of neutral oligosaccharides was increased from 10% (14C6-bcl-2) to 16% (
) in the presence of NaBu, which was accompanied by the reduced proportion of acidic oligosaccharides, especially of monosialylated and disialylated forms. The changes in microheterogeneous oligoformal structures of antibody in turn affected the mobility of antibody isoforms in isoelectric focusing (IEF), resulting in the occurrence of some more basic antibody isoforms produced in the presence of NaBu. However, the antigen-antibody binding properties were not changed by alteration of glycosylation pattern. The competitive enzyme-linked immunosorbent assay (ELISA) showed that the antibody produced by NaBu/Bcl-2 overexpression maintained its antigen-antibody binding properties with binding affinity of about
. Taken together, no significant effects of NaBu/Bcl-2 overexpression on the molecular integrity of antibodies, produced by using serum-free medium, could be observed by the molecular assay systems.
Involvement of Organic Acid During Corrosion of Iron Coupon by Desulfovibrio desulfuricans
Park, Kyung-Ran ; Lee, Hyun-Jin ; Lee, Hong-Keum ; Kim, Yeong-Kwan ; Oh, Young-Sook ; Choi, Sung-Chan ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 937~941
Microbiologically influenced corrosion (MIC) is an electrochemical process where the participation of microorganisms initiates, facilitates, or accelerates the corrosion reaction. Sulfate-reducing bacteria (SRB) reduce sulfate to sulfide and are known to be the most destructive microorganisms in anaerobic MIC. Accordingly, the current study attempted to elucidate the mechanisms involved and the relative importance of the corrosive products in SRB-induced corrosion. The measured rate of anaerobic corrosion of iron coupons by Desulfovibrio desulfuricans was
. Direct contact between the cells and the iron coupon did not seem to be necessary for corrosion to occur, since the corrosion rate was similar (
) when the coupon was enclosed in a dialysis bag. The participation of sulfide in the corrosion process was only marginal, as the specific corrosion rate was 2.5 times higher in a sulfate-free pyruvate medium than in an
lactate medium. Acetate (18.8-22.1 mM), the end-product of pyruvate and lactate metabolism, was identified in the culture medium and thus presumed to play a major role in the corrosion process involving Desulfovibrio desulfuricans.
Phylogenetic Analysis of Phenanthrene-Degrading Sphingomonas
Han, Kyu-Dong ; Jung, Yong-Tae ; Son, Seung-Yeol ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 942~948
Soil samples were obtained from 5 sites contaminated with polycyclic aromatic hydrocarbons (PAHs). These soil samples were cultured in using phenanthrene as a sole carbon and energy source, and 36 strains of phenanthrene-degrading bacteria were isolated from 3 sites. Most of them degraded 500 ppm of phenanthrene within 8 to 10 days, and these isolates could degrade a few other PAHs other than phenanthrene. Their genotypes were determined by restriction digests of the l6S rRNA genes [amplified ribosomal DNA restriction analysis (ARDRA)]. It was found that all the phenanthrene degrading isolates were included in 4 ARDRA types, and they showed a strict site endemism. l6S rDNAs of 12 strains selected from different sites were sequenced, and they were all confirmed as Sphingomonas strains. Their l6S rDNA sequences were compared for phylogenetic analysis; their sequence showed a similar result to ARDRA typing, thus indicating that these heterotrophic soil bacteria are not regionally mixed. In addition, it was found that the microbial diversity among sampling sites could be monitored by l6S rDNA PCR-RFLP pattern alone, which is simpler and easier to perform, without l6S rDNA sequence analysis.
Isolation and Analysis of the argG Gene Encoding Argininosuccinate Synthetase from Corynebacterium glutamicum
Ko, Soon-Young ; Kim, Sei-Hyun ; Lee, Heung-Shick ; Lee, Myeong-Sok ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 949~954
The argG gene of Corynebacterium glutamicum encoding argininosuccinate synthetase (EC6345) was cloned and sequenced. The gene was cloned by heterologous complementation of an Escherichia coli arginine auxotrophic mutant (argG/sup -/). The cloned DNA fragment also complements E. coli argD, argF, and argH mutants, suggesting a clustered organization of the genes in the chromosome. The coding region of the argG gene is 1,206 nucleotides long with a deduced molecular weight of about 44 kDa, comparable with the predicted size of the expressed protein on the SDS-PAGE. Computer analysis revealed that the amino acid sequence of the argG gene product had a high similarity to that of Mycobacterium tuberculosis and Streptomyces clavuligerus. Two conserved sequence motifs within the ArgG appear to be ATP-binding sites which correspond to 2 of the 3 conserved regions found in sequences of all known argininosuccinate synthetases.
PQQ-Dependent Organic Acid Production and Effect on Common Bean Growth by Rhizobium tropici CIAT 899
Cho, Young-Shin ; Park, Ro-Dong ; Kim, Yong-Woong ; Hwangbo, Hoon ; Jung, Woo-Jin ; Suh, Jang-Sun ; Koo, Bon-Sung ; Krishnan, Hari-B. ; Kim, Kil-Yong ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 955~959
Rhizobium tropici CIAT 899 is capable of synthesizing inactive apo-glucose dehydrogenase (GDH). To become an active holo enzyme, the GDH requires a cofactor, PQQ. When R. tropici CIAT 899 was grown in a broth culture medium containing hydroxyapatite and pyrrolo quinoline quinone (PQQ), pH decreased while the concentration of soluble P increased. The solubilization of hydroxyapatite was associated with the production of gluconic acid and 2-ketogluconic acids. The organic acid production and P solubilization were greatly enhanced when the bacterium was grown with air supply. Effect of R. tropici CIAT 899 with (CI＋PQQ) and without PQQ (CI) on the common bean growth was examined. Shoot and root weight, and N and P contents in CI＋PQQ treatment, were significantly higher than those in control and CI treatment. Nodule weight and acetylene reducing activities were also significantly higher in CI＋PQQ treatment than in other treatments.
Characterization of Endochitosanases-Producing Bacillus cereus P16
Jo, Yu-Young ; Jo, Kyu-Jong ; Jin, Yu-Lan ; Jung, Woo-Jin ; Kuk, Ju-Hee ; Kim, Kil-Yong ; Kim, Tae-Hwan ; Park, Ro-Dong ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 960~968
A bacterial isolate showing a strong endochitosanase activity was isolated from soil and then characterized. The isolate was identified and designated as Bacillus cereus P16, based on morphological and biochemical properties, assimilation tests, cellular fatty acids pattern, along with 16S rRNA gene sequence. The optimized medium for producing extracellular chitosanase in a batch culture contained 1% tryptone, 0.5% chitosan, and 1% NaCl (pH 7.0). Powder chitosan and tryptone served the best as carbon and nitrogen sources, respectively, for the chitosanase production. Chitosanase activity was the highest when culture was completed at
among various temperatures (
) tested in a shaking incubator (200 rpm). The levels of chitosanase activity in the culture fluid were 2.0 U/ml and 3.8 U/ml, respectively, when incubated in a flask for 60 h and in a jar fermenter for 24 h. The culture supernatant showed a strong liquefying activity on the soluble chitosan. The viscosity of 1% chitosan solution, that was incubated with the culture supernatant, was rapidly decreased, suggesting the secretion of endochitosanolytic enzymes by P16. The culture fluid revealed six endo-type chitosanase isozymes, two major (38 and 45 kD), and four minor (54, 65, 82, and 96 kD) forms by staining profile. The crude enzymes were very stable, and full activity was maintained for 4 weeks at
in the culture supernatant, suggesting a highly desirable stability rate for making an industrial application of the crude enzymes. The supernatant also cleaved the insoluble chitosan powder, but the hydrolysis rate was much lower. The enzymic degradation products of chitosan contained
(n=2-8). The concentration of chitosan in the reaction mixture of the crude enzyme affected the chitooligosaccharides composition of the hydrolysis products. When the higher concentration of chitosan was used, the higher degree of polymerized chitooligosaccharides were produced. By comparison with other commercial chitosanase preparations, P16 was indeed found to be a valuable enzyme source for industrial production of chitooligosaccharides from chitosan.
Role of Dipeptide at Extra Sugar-Binding Space of Thermus Maltogenic Amylase in Transglycosylation Activity
Baek, Jin-Sook ; Kim, Tae-Jip ; Kim, Young-Wan ; Cha, Hyun-Ju ; Kim, Jung-Wan ; Kim, Yong-Ro ; Lee, Sung-Joon ; Moon, Tae-Wha ; Park, Kwan-Hwa ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 969~975
Two conserved amino acid residues in the extra sugar-binding space near the catalytic site of Thermus maltogenic amylase (ThMA) were analyzed for their role in the hydrolysis and transglycosylation activity of the enzyme. Site-directed mutagenesis was carried out by replacing N33l with a lysine (N331K), E332 with a histidine (E332H), or by replacing both residues at the same time (N331K/E332H). The measured
values indicated that affinities toward all substrates tested, including starch, pullulan,
, and acarbose, were lower in all the mutants compared to that of wild-type ThMA, leading to reduced hydrolysis activity. In addition, the lower ratio of transglycosylation to hydrolysis in the mutants compared to that in the wild-type ThMA indicated that these mutants preferred hydrolysis to the transglycosylation reaction. These results demonstrated that the conserved dipeptide at 331 and 332 of ThMA is directly involved in the formation and accumulation of transfer products by accommodating acceptor sugar molecules.
Identification of Genes for Growth with Oxygen in Escherichia coli by Operon Fusion and Southern Blot Techniques
Kim, Il-Man ; Lee, Yong-Chan ; Won, Jae-Seon ; Choe, Mu-Hyeon ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 976~983
Seven Escherichia coli cells defective with aerobic growth were isolated by the insertion of
, a hybrid bacteriophage of
and Mu, which created a transcriptional fusion to lacZY. These insertion mutant cells were tested on an XG (
) medium for anaerobic expression of lacZ by fusion to a promoter. The chromosomal DNA from these strains were digested by EcoRI, and the EcoRI fragments that contained the fused gene and lacZ sequence were identified by Southern hybridization, using lacZ containing plasmid as a probe. The EcoRI fragment from each strain was cloned and sequenced. The sequence data were compared with the GenBank database. The mutated gene of three strains, CYT4, CYT5, and OS11, was found to be identical, and it was nrdAB that encoded ribonucleoside diphosphate reductase. The gene nrdAB was at min 50.5 on the Escherichia coli linkage map and 2,348,084 on the physical map, and is involved in hemAe-related reduction-oxidation reaction. OS6 and OS14 mutant strains had insertion at min 8.3 and the mutated gene was hemB. The hemB encodes 5-aminolevulinate dehydratase or porphobilinogen synthase. The OS3 mutant had insertion in cydB at min 16.6. The cydAB encodes cytochrome d oxidase. In the case of OS1, the fusion was made with sucA, the E1 component of
Purification and Identification of an Antifungal Agent from Streptomyces sp. KH-614 Antagonistic to Rice Blast Fungus, Pyricularia oryzae
Rhee, Ki-Hyeong ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 984~988
The actinomycete strain KH-6l4 possessed strong antifungal activity, especially antagonistic to the rice blast fungus, Pyricularia oryzae. Diaminopimelic acid (DAP) type and morphological and physiological characteristics, examined by scanning electron microscopy (SEM), indicated that KH-614 belonged to the genus Streptomyces. Antifungal agent produced by this strain was found to be most active, when the strain was cultured in the presence of glucose, polypeptone, and yeast extract (PY) medium for 6 days at
. Based on the spectral report data, MS and NMR, the antifungal agent was identified as cyclo(L-leucyl-L-prolyl). According to the antimicrobial activity test measured by minimal inhibitory concentration (MIC), the cyclo(1eu-pro) exhibited the activity against Candida albicans IAM 4905, Mucor ramannianus IAM6218, Rhizoctonia solani IFO 6218, Aspergilus fumigatus ATCC 42202, Glomerella cingulata IFO 9767, Trichophton mentagrophytes ATCC 18749, and Trichophyton rubrum ATCC 44766, the order of MIC values were 50, 12.5, 5, 50, 25, 5,
, respectively. Specifically, cyclo(1eu-pro) was one of the most effective elements against Pyricularia oryzae IFO 5994 with the MIC value of
, thus indicating that cyclo(leu-pro) is a potential antifungal agent.
Cloning, Expression, and Renaturation Studies of Reteplase
Zhao, Youchun ; Ge, Wang ; Kong, Young ; Zhang, Changkai ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 989~992
Recombinant human tissue plasminogen activator deletion mutein (Reteplase) is a clinically promising thrombolytic drug. Reteplase cDNA was subcloned into a bacteria expression system, and the resultant recombinant was biologically characterized. The Reteplase was expressed in Escherichia coli as an inclusion body, and the downstream processes of the Reteplase inclusion body included denaturation, renaturation, and purification. A protein disulfide isomerase (PDI) was used to assist the refolding of Reteplase, and it was found to increase the refolding rate from less than 2％ to more than 20％. The refolded Reteplase was purified through two chromatography steps, including lysine-coupled agarose affinity chromatography and then CM-sepharose cation-exchange chomatography. The purity of r-PA was analyzed by Western bolt analysis, and N-terminal amino acid and amino acid composition analyses confirmed the end-product. Reteplase showed higher thrombolytic potency in an animal thrombus model.
Optimization for Novel Glucanhydrolase Production of Lipomyces starkeyi KSM 22 by Statistical Design
PARK, JUN-SEONG ; BYUNG-HOON KIM ; JIN-HA LEE ; EUN-SEONG SEO ; KAB-SU CHO ; HYUN-JUNG PARK ; HEE-KYOUNG KANG ; SUN-KYUN YOO ; MYUNG-SUK HA ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 993~997
Response surface methodology was applied to find the optimum conditions for the production of DXAMase (containing both dextranase and amylase activities) based on the cultivation variables (pH, temperature, and agitation rate). The experimental values from the model equation conceded with predicted values in which the predicted values for dextranase and amylase activities were 2.26 and 3.52 U/ml at pH 4,
, 235 rpm, and the corresponding experimental values were 2.41 and 3.68 U/ml, respectively.
N-Acetylglycine Side Chain is Critical for the Antimicrobial Activity of Xanthostatin
Kim, Si-Kwan ; Ubukata, Makoto ; Isono, Kiyoshi ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 998~1000
This study was carried out to elucidate the mode of bacteriostatic property of xanthostatin (XS), a novel depsipeptide antibiotic with an N-acetylglycine side chain and selective antimicrobial activity against Xanthomonas spp. Two biotransformed XSs were isolated by the treatment of XS with the cell lysate of Xanthomonas campestris pv. citri, a solvent partition, preparative TLC, and HPLC. Structure determination of those two biotransformed XSs demonstrated deletion of the N-acetylglycine side chain. Noteworthily, they showed no antimicrobial activity against Xanthomonas spp. This result suggests that the N-acetylglycine side chain plays a critical role in the antimicrobial activity of XS, and that the bacteriostatic property of XS is due to susceptibility of the ester bond between the hexadepsipeptide nucleus and the N-acetylglycine side chain to hydrolytic enzyme(s) produced by Xanthomonas spp.
Construction and Characterization of Multiple Heavy Metal-Resistant Phenol-Degrading Pseudomonads Strains
Yoon, Kyung-Pyo ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 1001~1007
Metal ions contamination may inhibit microorganisms involved in the biodegradation of organic compounds and affect biodegradation rates. Therefore, it is likely that bioremediation of xenobiotics-contaminated soils and waste will require inoculation with efficient biodegrading microbial communities, with capabilities of being resistant to heavy metals as well. Two different transconjugants (Pseudomonas sp. KMl2TC and P. aeruginosa TC) were constructed by conjugation experiments. Results on MIC, induction and growth inhibition strongly indicated that arsenic-resistant plasmid, pKM20, could be mobilized, and the newly acquired phenotype of pKM20 was not only expressed but also well regulated, resulting in newly acquired resistances to
. The phenol- degradation efficiencies of Pseudomonas sp. KMl2TC were maintained significantly even at high heavy metal concentrations at which these efficiencies of P. aeruginosa TC were completely impaired. The results in this study on the effects of heavy metals on phenol degradation, especially after conjugation, are the first ever reported. All the results described in this study encourage to establish a goal of making "designer biocatalysts" which could degrade certain xenobiotics in the area contaminated with multiple heavy metals.
Application of Denaturing Gradient Gel Electrophoresis to Estimate the Diversity of Commensal Thermophiles
Bae, Jin-Woo ; Kim, Joong-Jae ; Jeon, Che-Ok ; Kim, Kwang ; Song, Jae-Jun ; Lee, Seung-Goo ; Poo, Har-Young ; Jung, Chang-Min ; Park, Yong-Ha ; Sung, Moon-Hee ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 1008~1012
Symbiobacterium toebii has been reported as a thermophile exhibiting a commensal interaction with Geobacillus toebii. The distribution of the commensal thermophiles in various soils was investigated using a denaturing gradient gel electrophoresis (DGGE). Based on the DGGE analysis, the enrichment condition for the growth of Symbiobacterium sp. was found to also enrich populations of several other microbial spp. as well as Symbiobacterium sp. In the enrichment experiment, several different 16S rDNA sequences of commensal thermophiles were detected in all of the soil samples tested, indicating that commensal thermophiles are widely distributed in various soils.
Symbiobacterium toebii Sp. nov., Commensal Thermophile Isolated from Korean Compost
Sung, Moon-Hee ; Bae, Jin-Woo ; Kim, Joong-Jae ; Kim, Kwang ; Song, Jae-Jun ; Rhee, Sung-Keun ; Jeon, Che-Ok ; Choi, Yoon-Ho ; Hong, Seung-Pyo ; Lee, Seung-Goo ; Ha, Jae-Suk ; Kang, Gwan-Tae ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 1013~1017
A thermophilic nonspore-forming rod isolated from hay compost in Korea was subjected to a taxonomic study. The microorganism, designated as
, was identified as a nitrate-reducing and nonmotile bacterium. Although the strain was negatively Gram-stained, a KOH test showed that the strain
belonged to a Gram-positive species. Growth was observed between 45 and
. The optimal growth temperature and pH were
and pH 7.5, respectively. The G+C content of the genomic DNA was 65 mol% and the major quinone types were MK-6 and MK-7. A phylogenetic analysis based on 16S rDNA sequences revealed that the strain
was most closely related to Symbiobacterium thermophilum. However, the level of DNA-DNA relatedness between strain
and the type strain for Symbiobacterium thermophilum was approximately 30%. Accordingly, on the basis of the phenotypic traits and molecular systematic data, the strain
would appear to represent a new species within the genus Symbiobacterium. The type strain for the new species is named
Complete DNA Sequence and Analysis of a Cryptic Plasmid Isolated from Lactobacillus bifermentans in Kimchi
Jeon, Deok-Young ; Lee, Sun-Ho ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 1018~1020
The complete 1,486 nucleotide sequence of a cryptic plasmid separated from Lactobacillus bifermentans strain A02 isolated from Kimchi has been determined. The plasmid, designated as pA021, encodes a 33,488 Da putative Rep protein. Based on the sequence similarity, the protein shows homology with coding protein of pRS1, a previously reported plasmid of Oenococcus oeni and the replication initiation protein (Rep) of the Staphylococcal pT181 plasmid family.
Evidence That Temporally Alternative Expression of the Vibrio vulnificus Elastase Prevents Proteolytic Inactivation of Hemolysin
Rhee, Jee-Eun ; Lee, Jeong-Hyun ; Jeong, Hye-Sook ; Park, U-Ryung ; Lee, Dong-Ha ; Woo, Gun-Jo ; Miyoshi, Shin-Ichi ; Choi, Sang-Ho ;
Journal of Microbiology and Biotechnology, volume 13, issue 6, 2003, Pages 1021~1026
Numerous secreted and cell-associated virulence factors have been proposed to account for the fulminating and destructive nature of Vibrio vulnificus infections. Among the putative virulence factors are an elastase, elastolytic protease, and a cytolytic hemolysin. Effects of the elastase on the hemolysin were assessed by evaluating changes of hemolytic activities either in the presence or absence of the protease. Although hemolytic activity in the culture supernatant was lowered by the purified elastase added in vitro, the cellular level of hemolytic activity was unaffected by the mutation of vvpE encoding the elastase. Growth kinetic studies revealed that hemolysin reached its maximum level in the exponential phase of growth, and the elastase appeared at the onset of the stationary phase. These results have provided insight into the regulation of virulence factors: temporally coordinate regulation of virulence factors is essential for the overall success of the pathogen during pathogenesis.