Journal of Microbiology

Volume 34(3); September 1996

Phylogenetic study of trichaptum species based on the RFLP analysis of mitochondrial DNA: Kim, Mi Sun , jung, Hack Sung; J. Microbiol. 1996;34(3):215-219.

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Abstract: Eight strains of Trichaptunm (Polyporaceae), two strains from each species of T. abietinum, T. biforme, T. fusco-violaceum, and T. laricinum were examined to see their phylogenetic relationship by digesting mitochondrial DNAs with EcoRV, Hind III, XbaI, and PstI, and then analyzing fragmentation patterns with the methods of Nei and Li. T. abietinum, T. biforme, and T. laricinum developed an independent phylogenetic lineage, respectively, but T. fusco-violaceum FP-133997-sp showed a close relationship with two strains of T. bioforme, and T. fusco-violaceum HHB-4016-sp barely grouped with those of T. laricinum. Based on the results of the RFLP analysis of mtDNA, it is concluded that T. fusco-violaceum is under way to differentiation into two different subgroups.

Ultrastructural studies of encystment in allomyces macrogynus: Kim, Jung Soeup , Youn, Hyun Joo , Cho, Chung Won; J. Microbiol. 1996;34(3):220-224.

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Abstract: Ultrastructural organization of encysting zoospores of Allomyces macrogynus was examined using the methods of cryofixation and freeze substitution. During enxystment, obvious changes were observed at the surface of the plasma membrane and in the structure of gamma particles. Many multivesicular bodies associated with the plasma membrane were observed at early stages of encystment. After induction of encystment, vesicles were found within the gamma particles. These vesicles appeared to leave gamma particles after formaing multivesicular bodies. This study suggests that the cell wall formation during encystment is mediated by the fusion of multivesicular bodies with the plasma membrane.

Analysis of cellular fatty acid methyl esters (FAMEs) for the identification of leuconostoc strains isolated from kimchi: Lee, Jung Sook , Chun, Chang Ouk , Kim, hong Joong , Joo, Yun Jung , Lee, Hun Joo , Park, Chan Sun , Ahn, Jong Seog , Park, Yong Ha , Mheen, Tae Ickc; J. Microbiol. 1996;34(3):225-228.

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Abstract: The cellular fatty acid methyl esters (FAMEs) analysis data obtained for clusters defined at a Euclidian distance of 17.5, in the classification of lactic acid bacteria isolated from kimchi, described by Lee et al. (4), was used for the identification of 79 Leuconostoc isolates. The test strains were isolated using a selective isolation medium specific for the genus Leuconostoc. These strains were then characterized according to their fatty acid profiles. The results show that all seventy nine test strains were identified to the known Leucomostock clusters B, C, and D. Cluster B had the highest relative amount of the saturated fatty acid 16 : 0. The saturated fatty acid 16 : 0 and summed feature 9 were found as a major components in cluster C, which had a higher level of summed feature 9 than cluster B. Cluster D is characterized by the highest relative amount of the unsaturated fatty acid 18 : 1 w9c. It is suggested that FAMEs analysis can be successfully applied in the identification of lactic acid bacteria isolated from kimchi.

Direct extraction of DNA from soil for amplification of 16S rRNA gene sequences by polymerase chain reaction: Cho, Jae Chang , Lee, Dong Hun , Cho, Young Cheol , Cho, Jang Cheon , Kim, Sang Jong; J. Microbiol. 1996;34(3):229-235.

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Abstract: Microgram quantities of DNA per gram soil were recovered with SDS- based and freeze-and thaw procedures. The average DNA fragment size was > 23 Kb. This method generated minimal shearing of extracted DNA. However, the DNA extracts still contained considerable amounts of humic impurities sufficient to inhibit PCR. Several approaches were used to reduce the interferences with the PCR (use of CTAF in extraction step, Elutip-d column purification, addition of BSA to PCR buffer) to accomplish PCR with DNA extract as a template. Most of the DNA extracts were not digested completely by restriction endonuclease, and CTAB-TREATED and Elutip-d column purified DNA extracts were partially digested. Regarding as restriction enzyme digestion, all PCRs failed to amplify 16S rRNA gene fragments in the DNA extracts. In the case of DNA extracts only where BSA was added to PCR buffer, PCR was successfully conducted whether the DNA extracts were treated with CTAB or purified with columns. However, these two treatments were indispensable for humic impurity-rich DNA extracts to generate the PCR-compatible DNA samples. Direct extraction of DNA, coupled with these procedures to remove and relieve interferences by humic impurities and followed by the PCR, can be rapid and simple method for molecular microbiological study on soil microorganisms.

Detection of rifampin resistance mutation and its altered nucleotide sequences in mycobacterium leprae isolated from Korean patients with leprosy: Kim, Soon Ok , Kim, Min Joo , Chae, Gue Tae , Suh, Joo Won; J. Microbiol. 1996;34(3):236-240.

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Abstract: Rifampin is the most powerful drug for treating leprosy and tuberculosis today. It inhibits initiation and elonation of RNA transcription by binding to β-subunit of RNA polymerase, leading to kill mycobacteria. We isolated one variant strain of Mycobacterium leprae from 24 Korean leprosy patients who are less susceptible to rifampin or have suffered from relapse by polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) of the rpoB gene. Direct sequencing of the rpoB to Ser-464, Arg-465, Arg-467 and Ala-468. This is the first finding on rpoB gene mutation of M. leprae from Korean patients ; moreover the mutant type was found to be different from the previously reported cases in other countries.

Structural and functional stability of the genetic recombinant plasmid pCU103 in different water environments: Kim, Chi Kyung , Kwak, Myoung Ja , Lee, Sung Gie; J. Microbiol. 1996;34(3):241-247.

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Abstract: The stbility of the genetically engineered microorganisms and their recombinant plasmids released in natural environments has been regarded as one of the molecular ecological topics. In this study, the recombinant plasmids pCU103 in which the pcbCD genes involved in biodegradation of biphenyl and 4-chlorobiphenyl were cloned in pBluescript SK(+) vector, were examined for their structural and functional stability in different waters at 15℃ by the methods of electrophoresis, Southern hybridization, quantification with fluorescent dye, and transformation. The recombinant plamids maintained their stabilities for about 30 days in sterilized distilled water (SDW), 15 days in autoclaved creek water (AW), 25 days in filtered and autoclaved non-sterible creek water (FAW), 4 days in Luria-Bertani (LB) broth, and less than one day in filtered non-sterile creek water (FW). The covalently closed circular (CCC) form of the plasmid was decreased and open circular (OC) form was increased as a function of incubation time, and then linear (L) form was produced to be ultimately degraded out. The degradation rates of the plasmid were proportionally correlated to trophic level of the water, and the biological factor such as DNases was found to be one of the most critical factors affecting structural and functional stability of the plasmid in non-sterile natural water.

Immunofluorescence localization of schizosacharomjyces pombe cdc103^+ gene product: Kim , Hyong Bai; J. Microbiol. 1996;34(3):248-254.

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Abstract: cdc103^+ gene in Schizosaccharomyces pombe which is similar to the CDC3 gene in Saccharomyces cerevisiae was cloned and sequenced. Comparison of the predicted amino acid sequences of cdc103^+ and CDC3 revealed that they share significant similarity (43% identity and 56% identity or similarity) to each other. The gene product of CDC3 S. cerevisiae is known to be a highly ordered ring of filaments that lies just inside the cytoplasmic membrane in the region of the mother-bud neck. In order to characterize the gene product of cdc103^+ in Schizosaccharomyces pombe, fusion proteins were used to generate the polyclonal antibodies specific for the gene product (cdc103p). In immunofluorescence experiments, these antibodies decorate the region of the septum formation as a double ring structure late in the cell division cycle.

Lipoppolysaccharide yields form Rhodobacter capasulatus with indirect ELISA: Yoo, Tae Eun , Lee, Hyun Soon; J. Microbiol. 1996;34(3):255-262.

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Abstract: The lipopolysaccharide (LPS) yields were measured in Rhodobacter capsulatus under several conditions by the ELISA method. The purification of LPS was done by affinity chromatography of IgG coupled CNBr-activated sepharose-4B instead of ultra-centrifugation. The purity of the LPS didn't show much difference between affinity chromatography and ultra-centrifugation method, but affinity chromatography method required much fewer organisms and was more convenient. LPS yield was measured in ng units by the ELISA method. Mannitol was a better single carbon source than other sugars, but mixing two carbon sources resulted in greater LPS yields than any sugar alone. LPS yield was directly proportional to NH₄CI concentration, with optimum yields at 0.05% nitrogen. In contrest to LPS yields, which decreased at 0.005% nitrogen concentration total protein was increased 16 times. Calcium influenced LPS yields. At 0.7 mM CaCI₂, the LPS yield was 16.5 ㎍/mg DW, five times the yield without calcium.

Calcium in infectious hematopoietic necrosis virus (IHNY) infected fish cell lines: Kim, Nam Shik , Heo, Gnag Joon , Lee, Chang Hee; J. Microbiol. 1996;34(3):263-269.

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Abstract: Infection of fish cells with IHNV resulted in gradual increase in cytosolic free Ca^2+ concentration ([Ca^2+]) in CHSE, gradual decrease in [Ca^2+] in FHM, and no significant change in RTG cells. The degree of [Ca^2+] increase or decrease was dependent on the amount of infectious virus, and these [Ca^2+] variations were maximal at 16 hours after virus infection (p.i.) in both cell lines. When the fish cells were infected with inactivated IHNV, evident variation in [Ca^2+] was not observed. Thus, infectivity of IHNV appears to correlate with changes in [Ca^2+] in virus-infected cells. These IHNV-induced [Ca^2+] changes were partially blocked by cycloheximide, but not affected by cordycepin. It seems to be that virus-induced Ca^2+ variations were more related with protein synthesis than RNA synthesis. Various Ca^2+ related drugs were used in search for the mechanisms of the [Ca^2+], changes following IHNV infection of CHSE cells. Decreasing extracellular Ca^2+ concentration or blocking Ca^2+ influx from extracellular media inhibited the IHNV-induced increase in [Ca^2+], in CHSE cells. Similar results were obtained with intracellular Ca^2+ sources are important in IHNV-induced [Ca^2+] increase in CHSE cells.

Inhibition of purine nucleoside phosphorylase (PNP) in micrococcus luteus phenylglyoxal: Choi , Hye Seon; J. Microbiol. 1996;34(3):270-273.

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Abstract: Micrococcus luteus purine nucleoside phosphorylase (PNP) has been purified and characterized. The physical and kinetic properties have been described previously. Chemical modification of the enzyme was attempted to gain insight on the active site. The enzyme was inactivated in a time-dependent manner by the arginine- specific modifying reagent phenylglyoxal. There was a linear relationship between the observed rate of inactivation and the phenylglyoxal concentration. At 30℃ the bimolecular rate constant for the modification was 0.015 min^-1 mM^-1 in 50 mM NaHCO₃buffer, pH 7.5. The plot of logk versus log phenylglyoxal concentration was a strainght line with a slope enzyme. Preincuation with saturated solutions of substrates protected the enzyme from inhibition of phenylglyoxal, indicating that reactions with phenylglyoxal were directed at arginyl residues essential for the catalytic functioning of the enzyme.

Antifungal activities of peptides with the sequence 10-17 of magainin 2 at the N-termini against aspergillus fumigatus: Lee, Myung Kyu , Lee, Dong Gun , Shin, Song Yub , Lee, Sung Gu , Kang, Joo Hyun , Hahm, Kyung Soo; J. Microbiol. 1996;34(3):274-278.

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Abstract: Two peptides, MA-inv AND MA-ME, with the sequence 10-17 of maganin 2 at their-N-termini were designed and synthesized. The peptides had higher antifungal activities against Aspergilus fumigatus without hemolytic activities. The minimal inhibition concentratory (MIC) values of both peptides against A. fumigatus were 5 ㎍/ml, whereas those of the native peptides, magainin 2 and melittin, were 10㎍/ml. At 3 ㎍/ml, MA-inv and MA-ME inhibited the mycelium growth of A. fumigatus by 94.6% and 97.3% respectively, whereas magainin 2 and melittin inhibited by 62.2% and 32.4, respectively. MA-inv showed up to 80% inhibition of (1, 3)-β-D-glucan synthase activity of A. fumigatus. The peptides also showed up to 80% inhibition of (1, 3)-β-D glucan synthase activity of A. fumigatus. The peptides also showed antifungal activities for other fungi of Aspergillus sp. However, the antibiotic activities of MA-ME against Escherichia coli, Bacillus subtilis and Fusarium oxysporum were more effective than those of MA-inv, suggesting that the C-terminal sequences of MA-inv and MA-ME may also influence their antibiotic activities. These results suggest that the N-terminal sequence of the designed peptides, KKFGKAFV, is important for their antifungal activities against A. fumigatus and their C- terminal sequences are related to the organism selectivity.

Culture conditions affecting the molecular weight distribution of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesized by alcaligenaes sp. SH-69: Yoon, Joo Seok , Park, Sang Kyu , Kim, Young Baek , Maeng, Hack Young , Rhee, Young Ha; J. Microbiol. 1996;34(3):279-283.

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Abstract: The weight average molecular weight of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesized by Alcaligenes sp. SH-69 was altered between 3.2 × 10^5 and 1.1 × 10^6 depending upon various culture conditions. It appeared that culture conditions favorable for the efficient production of copolyesters promoted the formation of higher molecular weight copolyesters. Polydispersity indices of isolated copolyesters were in the range of 1.5 to 2.5.

Fungal-sporulation suppressing substances produced by pseudomonas aeruginosa KMCS-1: Min, Bu Yong , Shim, Jae Young , Kim, Kun Woo , Lee, Jong Kyu , Yoon, Kwon Sang; J. Microbiol. 1996;34(3):284-288.

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Abstract: Among the bacteria isolated form compost piles of cattle excretion in a pasture located at the suburbs of Chunchon city, Pseudomonas aeruginosa KMCS-1 was selected for the test of antifungal substances produced. Six fractions were separated by silica gel column chromatography, and then the antifungal activity of each fraction was assayed against Escherichia coli, Bacillus subtilis, Candida albicans, Rhizopus sp., Aspergillus nidulans, Coprinus cinereus, and Pyricularia oryzae by paper disc method. Two fractions showed significant suppressive activities against A. nidulans, C. cinereus, and P. oryzae; however, their mycelial growth was not affected by neither of these fractions. Inhibitory activities of these fractions to sporulation was assayed at the concentration of 50. 25, 12. 5, and 6.25 ㎍/ml and the average inhibition rates against sporulation of A. nidulans, C. cinereus, and P. oryzae were 94.0, 98.3, and 77.9%, respectively. Further purification and analysis of active substances are now being conducted.

Characterization of azomonas agilis PY101, a cadmium-resistant strain isolated from anyang stream: You, Kyung Man , Lee, Ji Hyun , Kim, Jeong Kook , Hah, Nam Ju , Lee, Yung Nok , Park, Yong Keun; J. Microbiol. 1996;34(3):289-293.

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Abstract: A cadimium-resistant strain isolated from Anyang stream, Azomonas agilis PY101 exhibited strong resistance to 1000 ppm of cadmium ion (Cd^2+). A agilis PY101 also exhibited resistance to various antibiotics, such as amoxicillin, amplicilin, bacitracin, cefazolin, erythromycin, penicilin, tetracycline, and vancomycin. In the presence of Cd^2+, the growth of A. aglis PY101 started after an extended lag phase and produced a green-fluorescent pigment induced by cadmium. The dramatic decrease (approximately 400ppm) of concentration of cd^2+ in the culture medium during the growth phase of A. agilis PY101 was confirmed by the inductively coupled plasma-atomic emission spectrophotometer. Transmission electron microscopic analysis revealed that A. agilis PY 101 actively accumulated Cd^2+ in the cytoplasm.

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